U.S. patent application number 13/849330 was filed with the patent office on 2013-12-12 for renewable chemicals and fuels from oleaginous yeast.
This patent application is currently assigned to SOLAZYME, INC.. The applicant listed for this patent is SOLAZYME, INC.. Invention is credited to Anna Coragliotti, Anthony G. Day, Harrison F. Dillon, Scott Franklin, Chung-Soon Im, Donald E. Trimbur.
Application Number | 20130330790 13/849330 |
Document ID | / |
Family ID | 40094391 |
Filed Date | 2013-12-12 |
United States Patent
Application |
20130330790 |
Kind Code |
A1 |
Trimbur; Donald E. ; et
al. |
December 12, 2013 |
Renewable Chemicals and Fuels from Oleaginous Yeast
Abstract
The invention provides methods of cultivating oil-bearing
microbes using xylose alone or in combination with other
depolymerized cellulosic material. Also provided are microorganisms
comprising an exogenous gene encoding a polysaccharide degrading
enzyme, such as a cellulase, a hemicellulase, a pectinase, or a
driselase. Some methods of microbial fermentation are provided that
comprise the use of xylose and depolymerized cellulosic materials
for the production of oil-bearing microorgansims.
Inventors: |
Trimbur; Donald E.; (Palo
Alto, CA) ; Im; Chung-Soon; (Palo Alto, CA) ;
Dillon; Harrison F.; (San Mateo, CA) ; Day; Anthony
G.; (San Francisco, CA) ; Franklin; Scott; (La
Jolla, CA) ; Coragliotti; Anna; (San Francisco,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SOLAZYME, INC. |
SOUTH SAN FRANCISCO |
CA |
US |
|
|
Assignee: |
SOLAZYME, INC.
SOUTH SAN FRANCISCO
CA
|
Family ID: |
40094391 |
Appl. No.: |
13/849330 |
Filed: |
March 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13406417 |
Feb 27, 2012 |
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13849330 |
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13273179 |
Oct 13, 2011 |
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13406417 |
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12772173 |
Apr 30, 2010 |
8512999 |
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13273179 |
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12131783 |
Jun 2, 2008 |
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12772173 |
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60941581 |
Jun 1, 2007 |
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60959174 |
Jul 10, 2007 |
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60968291 |
Aug 27, 2007 |
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61024069 |
Jan 28, 2008 |
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Current U.S.
Class: |
435/134 ;
435/167; 44/388; 554/1; 585/16 |
Current CPC
Class: |
Y02E 50/30 20130101;
C12N 9/1205 20130101; C12P 21/06 20130101; C12N 9/20 20130101; Y02E
50/10 20130101; C10L 2200/0484 20130101; C12N 1/32 20130101; C10L
2270/04 20130101; C12N 1/12 20130101; Y02E 50/13 20130101; C12N
15/79 20130101; C12P 5/02 20130101; C12P 7/6445 20130101; Y02E
50/343 20130101; C10L 1/06 20130101; C12N 9/14 20130101; C12N 9/88
20130101; C12P 7/6409 20130101; C10L 2270/026 20130101; C12Y
302/01026 20130101; C07H 21/04 20130101; Y02T 50/678 20130101; C12P
7/6436 20130101; C12P 7/6463 20130101; C10L 1/026 20130101; C12N
9/0006 20130101; Y02P 20/52 20151101; C12N 9/0008 20130101; C12N
9/2431 20130101; C12N 9/16 20130101; C12N 9/2408 20130101; C12P
7/649 20130101; C11C 3/00 20130101 |
Class at
Publication: |
435/134 ;
435/167; 44/388; 585/16; 554/1 |
International
Class: |
C12P 7/64 20060101
C12P007/64; C11C 3/00 20060101 C11C003/00; C12P 5/02 20060101
C12P005/02 |
Claims
1. A method of producing a fuel or a chemical, the method
comprising the steps of: (a) heterotrophically culturing an
oleaginous yeast in the presence of a fixed carbon source, wherein:
(i) the fixed carbon source is selected from glycerol,
depolymerized cellulosic material, sucrose, molasses, glucose,
arabinose, galactose, xylose, fructose, mannose, or acetate; and
(ii) the oleaginous yeast accumulates at least 10% of its dry cell
weight as triacylglyceride; (b) isolating the triacylglyceride from
the oleaginous yeast; and (c) subjecting the isolated
triacylglyceride to a chemical reaction to produce the fuel or
chemical.
2. The method of claim 1, wherein the depolymerized cellulosic
material is selected from the group consisting of sugar cane
bagasse, sugar beet pulp, corn stover, wood chips, sawdust,
switchgrass, rice hull, corn fiber, wheat straw, rice straw, and
citrus pulp.
3. The method of claim 1, wherein the oleaginous yeast is selected
from the group consisting of Cryptococcus curvatus, Cryptococcus
terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer,
Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis,
and Yarrowia lipolytica.
4. The method of claim 1, wherein the oleaginous yeast contains at
least one exogenous gene encoding a lipid pathway enzyme.
5. The method of claim 4, wherein the lipid pathway enzyme is
selected from the group consisting of a stearoyl-ACP desaturase, a
glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA
carboxylase, an acyl carrier protein, a fatty acyl-ACP
thioesterase, and a glycerol-3 phosphate acyltransferase.
6. The method of claim 1, wherein said fuel or chemical is selected
from the group consisting of renewable diesel, fatty acid esters,
and jet fuel.
7. The method of claim 6, wherein said renewable diesel conforms to
the specifications of the ASTM D975 standard.
8. The method of claim 6, wherein said biodiesel conforms to the
specifications of the ASTM D6751 standard.
9. The method of claim 6, wherein said jet fuel conforms to the
specifications of the ASTM D1655 standard.
10. The method of claim 1, wherein the triacylglyceride is isolated
by lysing the oleaginous yeast.
11. The method of claim 10, wherein the oleaginous yeast is lysed
by mechanical means.
12. The method of claim 1, wherein the chemical reaction is
selected from the group consisting of cracking,
(hydro)deoxygenation, esterification, and isomerization.
13. The method of claim 1, further comprising distilling the fuel
or chemical.
14. A method for producing a triacylglyceride, said method
comprising the steps of culturing an oleaginous yeast in a culture
media to provide an oleaginous yeast comprising at least about 10%
triacylglyceride by dry weight; and extracting the triacylglyceride
from the oleaginous yeast.
15. The method of claim 14, wherein said oleaginous yeast is
selected from the group consisting of Cryptococcus curvatus,
Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces
lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula
gracilis, Saccharomyces cerevisiae and Yarrowia lipolytica.
16. The method of claim 14, wherein the oleaginous yeast is
cultured in media comprising a carbon source selected from the
group consisting of glucose, fructose, sucrose, lactose, galactose,
xylose, mannose, rhamnose, arabinose, glycerol, and acetate.
17. The method of claim 14, wherein the oleaginous yeast is
cultured in media comprising a carbon source selected from the
group consisting of depolymerized sugar beet pulp, black liquor,
corn starch, depolymerized cellulosic material, corn stover, sugar
beet pulp, sugar cane juice, switchgrass, milk whey, molasses,
potato, rice, sorghum, and wheat.
18. The method of claim 14, wherein said triacylglyceride is
extracted by solvent extraction.
19. A fuel or chemical produced by the method of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/406,417, filed Feb. 27, 2012, which is a continuation of
U.S. application Ser. No. 13/273,179, filed Oct. 13, 2011, which is
a continuation of U.S. application Ser. No. 12/772,173, filed Apr.
30, 2010, which is a continuation of U.S. application Ser. No.
12/131,783, filed Jun. 2, 2008, which claims the benefit under 35
U.S.C. 119(e) of U.S. Provisional Application No. 60/941,581, filed
Jun. 1, 2007, U.S. Provisional Application No. 60/959,174, filed
Jul. 10, 2007, U.S. Provisional Application No. 60/968,291, filed
Aug. 27, 2007, and U.S. Provisional Application No. 61/024,069,
filed Jan. 28, 2008, the disclosures of which are incorporated
herein by reference in their entirety for all purposes.
REFERENCE TO A SEQUENCE LISTING
[0002] This application includes an electronic sequence listing in
a file named "431494-Sequence.txt", created on Mar. 22, 2013 and
containing 28,039 bytes, which is hereby incorporated by reference
in its entirety for all purposes.
FIELD OF THE INVENTION
[0003] This disclosure relates to the production of oils, fuels,
and oleochemicals made from microorganisms. In particular, the
disclosure relates to oil-bearing microorganisms, including
microalgae, yeast and fungi, and to methods of cultivating such
microorganisms for the production of useful compounds, including
lipids, fatty acid esters, fatty acids, aldehydes, alcohols, and
alkanes, for use in industry or as an energy or food source. The
microorganisms of the invention can be selected or genetically
engineered for use in the methods or other aspects of the invention
described herein.
BACKGROUND OF THE INVENTION
[0004] Fossil fuel is a general term for buried combustible
geologic deposits of organic materials, formed from decayed plants
and animals that have been converted to crude oil, coal, natural
gas, or heavy oils by exposure to heat and pressure in the earth's
crust over hundreds of millions of years.
[0005] In common dialogue, fossil fuel, also known as mineral fuel,
is used synonymously with other hydrocarbon-containing natural
resources such as coal, oil and natural gas. The utilization of
fossil fuels has enabled large-scale industrial development and
largely supplanted water driven mills, as well as the combustion of
wood or peat for heat. Fossil fuels are a finite, non-renewable
resource.
[0006] When generating electricity, energy from the combustion of
fossil fuels is often used to power a turbine. Older generations
often used steam generated by the burning of the fuel to turn the
turbine, but in newer power plants, the gases produced by burning
of the fuel turn a gas turbine directly. With global modernization
in the 20th and 21st centuries, the thirst for energy from fossil
fuels, especially gasoline derived from oil, is one of the causes
of major regional and global conflicts.
[0007] The burning of fossil fuels by humans is the largest source
of emissions of carbon dioxide, which is one of the greenhouse
gases that allows radiative forcing and contributes to global
warming. In the United States, more than 90% of greenhouse gas
emissions come from the combustion of fossil fuels. In addition,
other air pollutants, such as nitrogen oxides, sulfur dioxide,
volatile organic compounds (VOCs), and heavy metals are
produced.
[0008] Human activity raises levels of greenhouse gases primarily
by releasing carbon dioxide from fossil fuel combustion, but other
gases, e.g., methane, are not negligible. The concentrations of
several greenhouse gases have increased over time due to human
activities, such as burning of fossil fuels and deforestation
leading to higher carbon dioxide concentrations. According to the
global warming hypothesis, greenhouse gases from industry and
agriculture have played a major role in the recently observed
global warming.
[0009] Increased demand for energy by the global economy has also
placed increasing pressure on the cost of hydrocarbons. Aside from
energy, many industries, including plastics and chemical
manufacturers, rely heavily on the availability of hydrocarbons as
a feedstock for their manufacturing processes. Cost-effective
alternatives to current sources of supply could help mitigate the
upward pressure on energy and these raw material costs.
SUMMARY OF THE INVENTION
[0010] In one aspect, the present invention is directed to a
microbe, which in various embodiments can comprise a microalgae
cell, an oleaginous yeast, or a fungus containing an exogenous gene
that encodes a protein selected from the group consisting of a
lipase, sucrose transporter, sucrose invertase, fructokinase,
polysaccharide-degrading enzyme, a fatty acyl-ACP thioesterase, a
fatty acyl-CoA/aldehyde reductase, a fatty acyl-CoA reductase, a
fatty aldehyde reductase, a fatty aldehyde decarbonylase, and an
acyl carrier protein (ACP). The microbe (e.g., microalgae cell)
can, for example, be selected from Table 1. In particular
embodiments, the cell is a species of the genus Chlorella, such as,
e.g., Chlorella fusca, Chlorella protothecoides, Chlorella
pyrenoidosa, Chlorella kessleri, Chlorella vulgaris, Chlorella
saccharophila, Chlorella sorokiniana or Chlorella ellipsoidea. In
other embodiments, the microbe is an oleaginous yeast selected from
the group consisting of Cryptococcus curvatus, Cryptococcus
terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer,
Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis,
and Yarrowia lipolytica. In still other embodiments, the microbe is
a fungus selected from the group consisting of a species of the
genus Mortierella, Mortierrla vinacea, Mortierella alpine, Pythium
debaryanum, Mucor circinelloides, Aspergillus ochraceus,
Aspergillus terreus, Pennicillium iilacinum, a species of the genus
Hensenulo, a species of the genus Chaetomium, a species of the
genus Cladosporium, a species of the genus Malbranchea, a species
of the genus Rhizopus, and a species of the genus Pythium. In other
embodiments, the invention includes expression of hydrocarbon
modification enzymes in bacterial hosts such as E. Coli and Bacilla
method of producing renewable diesel. In one embodiment, the method
comprises (a) culturing a population of microorganisms in the
presence of a fixed carbon source, wherein (i) the microorganisms
accumulate at least 10% of their dry cell weight as lipid, and (ii)
the fixed carbon source is selected from the group consisting of
glycerol, depolymerized cellulosic material, sucrose, molasses,
glucose, arabinose, galactose, xylose, fructose, arabinose,
mannose, acetate, and any combination of the foregoing, (b)
isolating lipid components from the cultured microorganisms, and
(c) subjecting the isolated lipid components to one or more
chemical reactions to generate straight chain alkanes, whereby
renewable diesel is produced.
[0011] In another aspect, the present invention is directed to a
composition of liquid hydrocarbons made according to the method
described directly above, wherein the composition conforms to the
specifications of ASTM D975.
[0012] In another aspect, the present invention is directed to a
method of producing jet fuel. In one embodiment, the method
comprises (a) culturing a population of microorganisms in the
presence of a fixed carbon source, wherein (i) the microorganisms
accumulate at least 10% of their dry cell weight as lipid, and (ii)
the fixed carbon source is selected from the group consisting of
glycerol, depolymerized cellulosic material, sucrose, glucose,
arabinose, galactose, xylose, fructose, arabinose, mannose,
acetate, and any combination of the foregoing, (b) isolating lipid
components from the cultured microorganisms, (c) subjecting the
isolated lipid components to one or more chemical reactions to
generate straight chain alkanes, (d) cracking the straight chain
alkanes, whereby jet fuel is produced.
[0013] In another aspect, the present invention is directed to a
composition of liquid hydrocarbons produced according to the method
described directly above, wherein the composition conforms to the
specifications of ASTM D1655.
[0014] In another aspect, the present invention is directed to a
microalgae or yeast cell that has been genetically engineered
and/or selected to express a lipid pathway enzyme at an altered
level compared to a wild-type cell of the same species. In some
cases, the cell produces more lipid compared to the wild-type cell
when both cells are grown under the same conditions. In some cases,
the cell has been genetically engineered and/or selected to express
a lipid pathway enzyme at a higher level than the wild-type cell.
In some cases, the lipid pathway enzyme is selected from the group
consisting of pyruvate dehydrogenase, acetyl-CoA carboxylase, acyl
carrier protein, and glycerol-3 phosphate acyltransferase. In some
cases, the cell has been genetically engineered and/or selected to
express a lipid pathway enzyme at a lower level than the wild-type
cell. In at least one embodiment in which the cell expresses the
lipid pathway enzyme at a lower level, the lipid pathway enzyme
comprises citrate synthase.
[0015] In some embodiments, the microalgae or yeast cell described
above has been genetically engineered and/or selected to express a
global regulator of fatty acid synthesis at an altered level
compared to the wild-type cell, whereby the expression levels of a
plurality of fatty acid synthetic genes are altered compared to the
wild-type cell. In some cases, the lipid pathway enzyme comprises
an enzyme that modifies a fatty acid. In some cases, the lipid
pathway enzyme is selected from a stearoyl-ACP desaturase and a
glycerolipid desaturase.
[0016] In another aspect, the present invention is directed to an
oil-producing microbe containing one or more exogenous genes,
wherein the exogenous genes encode protein(s) selected from the
group consisting of a fatty acyl-ACP thioesterase, a fatty acyl-CoA
reductase, a fatty aldehyde reductase, a fatty acyl-CoA/aldehyde
reductase, a fatty aldehyde decarbonylase, and an acyl carrier
protein. In some cases, the microbe is Chlorella protothecoides,
Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana,
Chlorella ellipsoidea, or Chlorella sp. In other cases, the microbe
is another species as described herein. In one embodiment, the
exogenous gene is in operable linkage with a promoter, which is
inducible or repressible in response to a stimulus. In some cases,
the stimulus is selected from the group consisting of an
exogenously provided small molecule, heat, cold, and light. In some
cases, the exogenous gene is expressed in a cellular compartment.
In some embodiments, the cellular compartment is selected from the
group consisting of a chloroplast and a mitochondrion.
[0017] In one embodiment, the exogenous gene encodes a fatty acid
acyl-ACP thioesterase. In some cases, the thioesterase encoded by
the exogenous gene catalyzes the cleavage of an 8 to 18-carbon
fatty acid from an acyl carrier protein (ACP). In some cases, the
thioesterase encoded by the exogenous gene catalyzes the cleavage
of a 10 to 14-carbon fatty acid from an ACP. In one embodiment, the
thioesterase encoded by the exogenous gene catalyzes the cleavage
of a 12-carbon fatty acid from an ACP.
[0018] In one embodiment, the exogenous gene encodes a fatty
acyl-CoA/aldehyde reductase. In some cases, the reductase encoded
by the exogenous gene catalyzes the reduction of a 20 to 30-carbon
fatty acyl-CoA to a corresponding primary alcohol. In some cases,
the reductase encoded by the exogenous gene catalyzes the reduction
of an 8 to 18-carbon fatty acyl-CoA to a corresponding primary
alcohol. In some cases, the reductase encoded by the exogenous gene
catalyzes the reduction of a 10 to 14-carbon fatty acyl-CoA to a
corresponding primary alcohol. In one embodiment, the reductase
encoded by the exogenous gene catalyzes the reduction of a
12-carbon fatty acyl-CoA to dodecanol.
[0019] In one embodiment, the exogenous gene encodes a fatty
acyl-CoA reductase. In some cases, the reductase encoded by the
exogenous gene catalyzes the reduction of an 8 to 18-carbon fatty
acyl-CoA to a corresponding aldehyde. In one embodiment, the
reductase encoded by the exogenous gene catalyzes the reduction of
a 12-carbon fatty acyl-CoA to dodecanal.
[0020] In at least one embodiment, the microbe of the invention
further contains one or more exogenous sucrose utilization
genes.
[0021] In another aspect, the present invention is directed to a
microbe containing two exogenous genes, wherein a first exogenous
gene encodes a fatty acyl-ACP thioesterase and a second exogenous
gene encodes a protein selected from the group consisting of a
fatty acyl-CoA reductase, a fatty acyl-CoA/aldehyde reductase, and
an acyl carrier protein. In some cases, the microbe is Chlorella
minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella
ellipsoidea, Chlorella sp., or Chlorella protothecoides. In other
cases, the microbe is another species as described herein. In some
cases, the two exogenous genes are each in operable linkage with a
promoter, which is inducible in response to a stimulus. In some
cases, each promoter is inducible in response to an identical
stimulus.
[0022] In one embodiment, the thioesterase encoded by the first
exogenous gene catalyzes the cleavage of an 8 to 18-carbon fatty
acid from an ACP. In some embodiments, the second exogenous gene
encodes a fatty acyl-CoA/aldehyde reductase which catalyzes the
reduction of an 8 to 18-carbon fatty acyl-CoA to a corresponding
primary alcohol. In some cases, the thioesterase encoded by the
first exogenous gene catalyzes the cleavage of a 10 to 14-carbon
fatty acid from an ACP, and the reductase encoded by the second
exogenous gene catalyzes the reduction of a 10 to 14-carbon fatty
acyl-CoA to the corresponding primary alcohol, wherein the
thioesterase and the reductase act on the same carbon chain length.
In one embodiment, the thioesterase encoded by the first exogenous
gene catalyzes the cleavage of a 12-carbon fatty acid from an ACP,
and the reductase encoded by the second exogenous gene catalyzes
the reduction of a 12-carbon fatty acyl-CoA to dodecanol. In some
embodiments, the second exogenous gene encodes a fatty acyl-CoA
reductase which catalyzes the reduction of an 8 to 18-carbon fatty
acyl-CoA to a corresponding aldehyde.
[0023] In some embodiments, the second exogenous gene encodes a
fatty acyl-CoA reductase, and the microbe further contains a third
exogenous gene encoding a fatty aldehyde decarbonylase. In some
cases, the thioesterase encoded by the first exogenous gene
catalyzes the cleavage of an 8 to 18-carbon fatty acid from an ACP,
the reductase encoded by the second exogenous gene catalyzes the
reduction of an 8 to 18-carbon fatty acyl-CoA to a corresponding
fatty aldehyde, and the decarbonylase encoded by the third
exogenous gene catalyzes the conversion of an 8 to 18-carbon fatty
aldehyde to a corresponding alkane, wherein the thioesterase, the
reductase, and the decarbonylase act on the same carbon chain
length.
[0024] In some embodiments, the second exogenous gene encodes an
acyl carrier protein that is naturally co-expressed with the fatty
acyl-ACP thioesterase.
[0025] In some embodiments, the second exogenous gene encodes an
acyl carrier protein, and the microbe further contains a third
exogenous gene encoding a protein selected from the group
consisting of a fatty acyl-CoA reductase and a fatty
acyl-CoA/aldehyde reductase. In some cases, the third exogenous
gene encodes a fatty acyl-CoA reductase, and the microbe further
contains a fourth exogenous gene encoding a fatty aldehyde
decarbonylase.
[0026] In another aspect, the present invention is directed to a
method of producing a molecule in a microbe population. In one
embodiment, the method comprises culturing a population of microbes
in a culture medium, wherein the microbes contain (i) a first
exogenous gene encoding a fatty acyl-ACP thioesterase, and (ii) a
second exogenous gene encoding a fatty acyl-CoA/aldehyde reductase,
and the microbes synthesize a fatty acid linked to an acyl carrier
protein (ACP), the fatty acyl-ACP thioesterase catalyzes the
cleavage of the fatty acid from the ACP to yield, through further
processing, a fatty acyl-CoA, and the fatty acyl-CoA/aldehyde
reductase catalyzes the reduction of the acyl-CoA to an
alcohol.
[0027] In one embodiment of the method of producing a molecule in a
microbe population, the microbe is Chlorella minutissima, Chlorella
emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella
sp. or Chlorella protothecoides. In other cases, the microbe is
another species of microorganism as described herein. In some
cases, the culture medium contains glycerol. In one embodiment, the
glycerol is a byproduct of a transesterification process. In some
cases, the culture medium contains glycerol and at least one other
fixed carbon source.
[0028] In one embodiment, the at least one other fixed carbon
source is sucrose. In some cases, all of the glycerol and all of
the at least one other fixed carbon source are provided to the
microbes at the beginning of fermentation. In some cases, the
glycerol and the at least one other fixed carbon source are fed to
the microbes at a predetermined rate over the course of
fermentation. In some culture methods of the invention, glycerol is
provided to the microbes in the absence of the at least one other
fixed carbon source for a first period of time, the at least one
other fixed carbon source is provided at the end of the first
period of time, and the microbes are cultured for a second period
of time in the presence of the at least one other fixed carbon
source.
[0029] In some embodiments, the exogenous genes are in operable
linkage with a promoter that is inducible in response to a first
stimulus. In some cases, the method further comprises providing the
first stimulus, and incubating the population of microbes for a
first period of time in the presence of the first stimulus to
produce an alcohol. In some cases, the method further comprises
extracting the alcohol from aqueous biomass comprising the culture
medium and the microbes.
[0030] In some embodiments, the thioesterase encoded by the first
exogenous gene catalyzes the cleavage of an 8 to 18-carbon fatty
acid from the ACP, and the reductase encoded by the second
exogenous gene catalyzes the reduction of an 8 to 18-carbon fatty
acyl-CoA to a corresponding primary alcohol, wherein the
thioesterase and the reductase act on the same carbon chain length.
In some cases, the thioesterase encoded by the first exogenous gene
catalyzes the cleavage of an 10 to 14-carbon fatty acid from the
ACP, and the reductase encoded by the second exogenous gene
catalyzes the reduction of an 10 to 14-carbon fatty acyl-CoA to a
corresponding primary alcohol, wherein the thioesterase and the
reductase act on the same carbon chain length. In one embodiment,
the thioesterase encoded by the first exogenous gene catalyzes the
cleavage of a 12-carbon fatty acid from the ACP, and the reductase
encoded by the second exogenous gene catalyzes the reduction of a
12-carbon fatty acyl-CoA to dodecanol. In some cases, the microbes
further contain a third exogenous gene encoding an acyl carrier
protein. In some embodiments, the third exogenous gene encodes an
acyl carrier protein that is naturally co-expressed with the fatty
acyl-ACP thioesterase.
[0031] In another aspect, the present invention is directed to a
method of producing a lipid molecule in a microbe population. In
one embodiment, the method comprises culturing a population of
microbes in a culture medium, wherein the microbes contain (i) a
first exogenous gene encoding a fatty acyl-ACP thioesterase, and
(ii) a second exogenous gene encoding a fatty acyl-CoA reductase,
and wherein the microbes synthesize a fatty acid linked to an acyl
carrier protein (ACP), the fatty acyl-ACP thioesterase catalyzes
the cleavage of the fatty acid from the ACP to yield, through
further processing, a fatty acyl-CoA, and the fatty acyl-CoA
reductase catalyzes the reduction of the acyl-CoA to an aldehyde.
In some cases, the microbe is Chlorella minutissima, Chlorella
emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella
sp., or Chlorella protothecoides. In other cases, the microbe is
another species of microorganism as described herein.
[0032] In some embodiments, the exogenous genes are in operable
linkage with a promoter that is inducible in response to a first
stimulus, and the method further comprises providing the first
stimulus, and incubating the population of microbes for a first
period of time in the presence of the first stimulus to produce an
aldehyde. In one embodiment, the method further comprises
extracting the aldehyde from aqueous biomass comprising the culture
medium and the population of microbes.
[0033] In some embodiments, the thioesterase encoded by the first
exogenous gene catalyzes the cleavage of an 8 to 18-carbon fatty
acid from the ACP, and the reductase encoded by the second
exogenous gene catalyzes the reduction of an 8 to 18-carbon fatty
acyl-CoA to a corresponding aldehyde, wherein the thioesterase and
the reductase act on the same carbon chain length. In some cases,
the microbes further contain a third exogenous gene encoding a
fatty aldehyde decarbonylase that catalyzes the conversion of the
aldehyde to an alkane.
[0034] In some cases, the exogenous genes are in operable linkage
with a promoter that is inducible in response to a first stimulus,
and the method further comprises providing the first stimulus, and
incubating the population of microbes for a first period of time in
the presence of the first stimulus to produce an alkane. In some
cases, the method further comprises extracting the alkane from
aqueous biomass comprising culture medium and the microbe
population.
[0035] In some cases, the thioesterase encoded by the first
exogenous gene catalyzes the cleavage of an 8 to 18-carbon fatty
acid from the ACP, the reductase encoded by the second exogenous
gene catalyzes the reduction of an 8 to 18-carbon fatty acyl-CoA to
a corresponding aldehyde, and the decarbonylase encoded by the
third exogenous gene catalyzes the conversion of an 8 to 18-carbon
aldehyde to a corresponding alkane, wherein the thioesterase, the
reductase, and the decarbonylase act on the same carbon chain
length. In some embodiments, the microbes further contain a third
exogenous gene encoding an acyl carrier protein. In some cases, the
third exogenous gene encodes an acyl carrier protein that is
naturally co-expressed with the fatty acyl-ACP thioesterase. In
some cases, the microbes further contain a fourth exogenous gene
encoding a fatty aldehyde decarbonylase that catalyzes the
conversion of the aldehyde to an alkane.
[0036] In some methods, the culture medium contains glycerol. In
one embodiment, the glycerol is a byproduct of a
transesterification process. In some cases, the culture medium
contains glycerol and at least one other fixed carbon source. In
one embodiment, the at least one other fixed carbon source is
sucrose. In some cases, all of the glycerol and all of the at least
one other fixed carbon source are provided to the microbes at the
beginning of fermentation. In some cases, the glycerol and the at
least one other fixed carbon source are fed to the microbes at a
predetermined rate over the course of fermentation. In one
embodiment, glycerol is provided to the microbes in the absence of
the at least one other fixed carbon source for a first period of
time, the at least one other fixed carbon source is provided at the
end of the first period of time, and the microbes are cultured for
a second period of time in the presence of the at least one other
fixed carbon source.
[0037] In another aspect, the present invention is directed to a
method of producing a fatty acid molecule having a specified carbon
chain length in a microbe population. In one embodiment, the method
comprises culturing a population of lipid-producing microbes in a
culture medium, wherein the microbes contain an exogenous gene
encoding a fatty acyl-ACP thioesterase having an activity specific
to a carbon chain length, and wherein the microbes synthesize a
fatty acid linked to an acyl carrier protein (ACP) and the
thioesterase catalyzes the cleavage of the fatty acid from the ACP
when the fatty acid has been synthesized to the specific carbon
chain length. In some cases, the microbe is Chlorella minutissima,
Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea,
Chlorella sp., or Chlorella protothecoides. In other cases, the
microbe is another species of microorganism as described
herein.
[0038] In some embodiments, the exogenous gene is in operable
linkage with a promoter that is inducible in response to a first
stimulus, and the method further comprises providing the first
stimulus, and incubating the population of microbes for a period of
time in the presence of the first stimulus. In some cases, the
method further comprises extracting the fatty acid from aqueous
biomass comprising culture medium and the microbe population.
[0039] In some cases, the microbes further contain a second
exogenous gene encoding an acyl carrier protein. In some
embodiments, the second exogenous gene encodes an acyl carrier
protein that is naturally co-expressed with the fatty acyl-ACP
thioesterase. In one embodiment, the acyl-ACP thioesterase
catalyzes the cleavage of an 8 to 18-carbon fatty acid from the
ACP.
[0040] In some cases, the culture medium contains glycerol. In one
embodiment, the glycerol is a byproduct of a transesterification
process. In some embodiments, the culture medium contains glycerol
and at least one other fixed carbon source. In one embodiment, the
at least one other carbon source is sucrose. In some cases, all of
the glycerol and all of the at least one other fixed carbon source
are provided to the microbes at the beginning of fermentation. In
some cases, the glycerol and the at least one other fixed carbon
source are fed to the microbes at a predetermined rate over the
course of fermentation. In one embodiment, glycerol is provided to
the microbes in the absence of the at least one other fixed carbon
source for a first period of time, the at least one other fixed
carbon source is provided at the end of the first period of time,
and the microbes are cultured for a second period of time in the
presence of the at least one other fixed carbon source.
[0041] In another aspect, the present invention is directed to a
microalgae cell containing an exogenous gene, wherein the exogenous
gene encodes a protein selected from the group consisting of a
lipase, a sucrose transporter, a sucrose invertase, a fructokinase,
or a polysaccharide-degrading enzyme. In some cases, the cell is
Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana,
Chlorella ellipsoidea, Chlorella sp., or Chlorella protothecoides.
In other cases, the cell is another species of microalgae as
described herein.
[0042] In some cases, the exogenous gene is in operable linkage
with a promoter. In some cases, the promoter is inducible or
repressible in response to a stimulus. In various embodiments, the
stimulus is selected from the group consisting of an exogenously
provided small molecule, heat, cold, and light. In some cases, the
exogenous gene is expressed in a cellular compartment. In some
embodiments, the cellular compartment is selected from the group
consisting of a chloroplast and a mitochondrion.
[0043] In some cases, the gene encodes a lipase that has at least
70% amino acid identity with a lipase selected from Table 9. In one
embodiment, the lipase is novozym-435. In one embodiment, the
polysaccharide-degrading enzyme is endogenous to a Chlorella
virus.
[0044] In another aspect, the present invention is directed to a
microalgae cell containing two exogenous genes, wherein a first
exogenous gene encodes a lipase and a second exogenous gene encodes
a polysaccharide-degrading enzyme. In some cases, the exogenous
genes are each in operable linkage with a promoter. In some cases,
the exogenous genes are each in operable linkage with promoters
that are inducible in response to a stimulus. In some cases, the
exogenous genes are each in operable linkage with promoters that
are inducible in response to the same stimulus. In some cases, the
exogenous genes are each in operable linkage with a promoter that
is inducible in response to at least one stimulus that does not
induce the other promoter.
[0045] In another aspect, the present invention is directed to a
method of manufacturing a lipid molecule in a microbe. In one
embodiment, the method comprises (a) culturing the microbe for a
first period of time sufficient to increase the cell density,
wherein the microbe contains (i) an exogenous gene encoding a
lipase, and/or (ii) an exogenous gene encoding a
polysaccharide-degrading enzyme, wherein the exogenous gene(s) are
in operable linkage with a promoter that is inducible in response
to a stimulus, (b) providing the stimulus, and (c) incubating the
microbe for a second period of time in the presence of the
stimulus.
[0046] In another aspect, the present invention is directed to a
method of manufacturing a lipid molecule in a microbe. In one
embodiment, the method comprises (a) culturing a lipid-producing
microbe for a first period of time sufficient to increase the cell
density, (b) providing a virus capable of infecting and lysing the
microbe when in direct contact with the microbe, and (c) incubating
the microbe for a second period of time to produce lysed aqueous
biomass. In one embodiment, the method further comprises extracting
lipid molecules from the lysed aqueous biomass.
[0047] In another aspect, the present invention is directed to a
microalgae cell containing an exogenous gene, wherein the exogenous
gene encodes a cofactor for a lipid pathway enzyme or encodes a
protein that participates in the synthesis of the cofactor.
[0048] In another aspect, the present invention is directed to a
method of culturing a lipid-producing microbe. In one embodiment,
the method comprises culturing the microbe in the presence of a
sufficient amount of one or more cofactor(s) for a lipid pathway
enzyme to increase microbial lipid yield over microbial lipid yield
in the absence of said one or more cofactors. In some cases, the
one or more cofactors is a vitamin required by one or more lipid
pathway enzymes. In one embodiment, the one or more cofactors is
biotin. In some cases, the one or more cofactors is/are provided by
including in the culture a microbe that has been genetically
engineered to produce the one or more cofactors.
[0049] In another aspect, the present invention is directed to a
method of fermenting a microorganism, which comprises providing a
mixture comprising glucose and xylose as an energy source to the
microorganism. In one embodiment, the mixture further comprises
lignin. In one embodiment, the mixture further comprises at least
one species of furfural. In some cases, the mixture is
depolymerized cellulosic material. In some cases, the mixture
further comprises as least one sucrose utilization enzyme. In one
embodiment, the mixture comprises a sucrose invertase.
[0050] In some cases, the microorganism is selected from the group
consisting of Bracteococcus minor, Chlorella ellipsoidea, Chlorella
kessleri, Chlorella luteoviridis, Bracteococcus medionucleatus,
Chlorella minutissima, Chlorella ovalis, Chlorella protothecoides,
Chlorella saccharophila, Chlorella sorokiniana, Chlorella sp.,
Chlorella vulgaris, Parachlorella kessleri, Prototheca moriformis,
and Pseudochlorella aquatica. In other cases, the microorganism is
another species of microorganism as described herein. In some
cases, the microorganism has been genetically engineered to express
an exogenous gene encoding at least one lipid modification enzyme,
hydrocarbon modification enzyme, or sucrose utilization enzyme.
[0051] In another aspect, the present invention is directed to a
method of culturing a microalgae, which comprises culturing the
microalgae in a culture medium including a feedstock comprising at
least one carbon substrate selected from the group consisting of a
cellulosic material, a 5-carbon sugar, a 6-carbon sugar, and
acetate. In some cases, the carbon substrate is glucose and the
microalgae is of a genus selected from the group consisting of
Chlorella, Parachlorella, Pseudochlorella, Bracteococcus,
Prototheca and Scenedesmus. In some cases, the carbon substrate is
xylose and the microalgae is of a genus selected from the group
consisting of Chlorella, Pseudochlorella, and Prototheca. In some
cases, the carbon substrate is sucrose and the microalgae is of a
genus selected from the group consisting of Chlorella, and
Bracteococcus. In some cases, the carbon substrate is fructose and
the microalgae is of a genus selected from the group consisting of
Chlorella, Parachlorella, Prototheca, and Scenedesmus. In some
cases, the carbon substrate is arabinose and the microalgae is
Chlorella sp. In some cases, the carbon substrate is mannose and
the microalgae is of a genus selected from the group consisting of
Chlorella, Parachlorella, Bracteococcus, Prototheca, and
Scenedesmus. In some cases, the carbon substrate is galactose and
the microalgae is of a genus selected from the group consisting of
Bracteococcus, Parachlorella, Chlorella, Pseudochlorella,
Bracteococcus, and Prototheca. In some cases, the carbon substrate
is acetate and the microalgae is of a genus selected from the group
consisting of Chlorella, Parachlorella, and Prototheca.
[0052] In one embodiment, the culture medium further includes at
least one sucrose utilization enzyme. In some cases, the microalgae
has been genetically engineered to express an exogenous gene
encoding at least one lipid modification enzyme, hydrocarbon
modification enzyme, or sucrose utilization enzyme. In some cases,
the culture medium includes a sucrose invertase.
[0053] In another aspect, the present invention is directed to a
method of culturing microalgae comprising placing a plurality of
microalgae cells in the presence of depolymerized cellulosic
material. In some cases, the microalgae are cultured in the
presence of an additional fixed carbon source selected from the
group consisting of glycerol, sucrose, glucose, arabinose,
galactose, xylose, fructose, arabinose, mannose, acetate, and any
combination of the foregoing. In one embodiment, the microalgae are
cultured in the presence of at least one sucrose utilization
enzyme.
[0054] In some cases, the microalgae is selected from a species of
the genus Bracteococcus, a species of the genus Chlorella, a
species of the genus Parachlorella, a species of the genus
Prototheca, or a species of the genus Pseudochlorella. In some
cases, the microalgae is selected from Bracteococcus minor,
Chlorella ellipsoidea, Chlorella kessleri, Chlorella luteoviridis,
Bracteococcus medionucleatus, Chlorella minutissima, Chlorella
ovalis, Chlorella protothecoides, Chlorella saccharophila,
Chlorella sorokiniana, Chlorella sp., Chlorella vulgaris,
Parachlorella kessleri, Prototheca moriformis, and Pseudochlorella
aquatica. In other cases, the microalgae is another species of
microalgae as described herein.
[0055] In some embodiments, the microalgae has been genetically
engineered to express an exogenous gene encoding at least one lipid
modification enzyme, hydrocarbon modification enzyme, or sucrose
utilization enzyme. In one embodiment, the at least one sucrose
utilization enzyme is a sucrose invertase. In some cases, the at
least one lipid modification enzyme is selected from a stearoyl-ACP
desaturase, a glycerolipid desaturase, a pyruvate dehydrogenase, an
acetyl-CoA carboxylase, and a glycerol-3 phosphate acyltransferase.
In some cases, the at least one hydrocarbon modification enzyme is
selected from a fatty acyl-ACP thioesterase, a fatty acyl-CoA
reductase, a fatty aldehyde reductase, a fatty acyl-CoA/aldehyde
reductase, a fatty aldehyde decarbonylase, and an acyl carrier
protein.
[0056] In another aspect, the present invention is directed to a
method of culturing a lipid-producing microbe, the method
comprising culturing the microbe in the presence of acetic acid and
the absence of a fixed nitrogen source. In some cases, the microbe
is cultured in the presence of a sufficient amount of acetic acid
to increase microbial lipid yield over microbial lipid yield in the
absence of acetic acid, wherein culture conditions are otherwise
the same between the two cultures.
[0057] In another aspect, the present invention is directed to a
microbial culture containing a population of microorganisms and a
culture medium comprising glucose, xylose, and a molecule selected
from the group consisting of lignin and a species of furfural. In
some cases, the microorganisms are selected from Bracteococcus
minor, Chlorella ellipsoidea, Chlorella kessleri, Chlorella
luteoviridis, Bracteococcus medionucleatus, Chlorella minutissima,
Chlorella ovalis, Chlorella protothecoides, Chlorella
saccharophila, Chlorella sorokiniana, Chlorella sp., Chlorella
vulgaris, Parachlorella kessleri, Prototheca moriformis, and
Pseudochlorella aquatica. In other cases, the microorganisms are
another species of microorganism as described herein.
[0058] In another aspect, the present invention is directed to a
method of cultivating microalgae. In one embodiment, the method
comprises (a) providing a microalgae cell capable of performing
heterotrophic growth, (b) placing the microalgae cell in growth
media, wherein the growth media comprises depolymerized cellulosic
material, and (c) incubating the microalgae for a period of time
sufficient to allow the cell to grow.
[0059] In another aspect, the present invention is directed to a
method of biodiesel manufacturing. In one embodiment, the method
comprises (a) culturing a lipid-producing microorganism in a first
microbial culture, (b) recovering lipid from the biomass produced
by the first microbial culture, (c) subjecting the lipid to
transesterification to produce fatty acid ester(s) and glycerol,
and (d) adding the glycerol to a second microbial culture. In some
cases, the first and second microbial cultures are cultures of the
same species of microorganism. In some cases, the second microbial
culture comprises microorganisms selected from the group consisting
of Parachlorella kessleri, Chlorella protothecoides, Bracteococcus
medionucleatus, Prototheca moriformis, Chlorella minutissima,
Chlorella sp., and Chlorella sorokiniana. In other cases, the
second microbial culture comprises another species of microorganism
as described herein.
[0060] In another aspect, the present invention is directed to a
method of fermentation comprising culturing a microorganism in the
presence of glycerol and at least one other fixed carbon source. In
some cases, the glycerol and the at least one other fixed carbon
source are provided to the microorganism simultaneously at a
predetermined ratio. In some cases, all of the glycerol and the at
least one other fixed carbon source are provided to the
microorganism at the beginning of the fermentation. In some cases,
all of the glycerol and the at least one other fixed carbon source
are fed to the microorganism at a predetermined rate over the
course of the fermentation. In one embodiment of the method,
glycerol is provided to the microorganism in the absence of the at
least one other fixed carbon source for a first period of time, the
at least one other fixed carbon source is provided at the end of
the first period of time, and the microorganism is cultured for a
second period of time in the presence of the at least one other
fixed carbon source. In one embodiment, the at least one other
fixed carbon source is fed to the microorganism at a predetermined
rate during the second period of time. In some cases, all of the at
least one other fixed carbon source is provided to the
microorganism at the end of the first period of time. In one
embodiment of the method, the at least one other fixed carbon
source is provided to the microorganism in the absence of glycerol
for a first period of time, glycerol is provided at the end of the
first period of time, and the microorganism is cultured for a
second period of time in the presence of glycerol. In one
embodiment, the glycerol is a byproduct of a transesterification
process. In one embodiment, the glycerol is acidulated. In another
embodiment, the glycerol is non-acidulated. In some cases, the at
least one other fixed carbon source is glucose. In some cases, the
at least one other fixed carbon source is depolymerized cellulosic
material. In one embodiment, the at least one other fixed carbon
source is sucrose.
[0061] In another aspect, the present invention is directed to a
fermentor comprising a population of microorganisms, glycerol, and
at least one sugar selected from the group consisting of xylose,
glucose, and sucrose. In one embodiment, the glycerol is a
byproduct of a lipid transesterification process. In some cases,
the microorganisms are selected from the group consisting of
Parachlorella kessleri, Chlorella protothecoides, Bracteococcus
medionucleatus, Prototheca moriformis, Chlorella minutissima,
Chlorella sp., and Chlorella sorokiniana. In other cases, the
microorganisms are another species as described herein.
[0062] In another aspect, the present invention is directed to a
method of fermenting a microorganism. In one embodiment, the method
comprises providing byproduct glycerol from a transesterification
process as a sole source of fixed carbon energy. In one embodiment,
no light energy is provided to the microorganism. In another
embodiment, light energy is provided to the microorganism. In some
cases, the microorganism is selected from Parachlorella kessleri,
Chlorella protothecoides, Bracteococcus medionucleatus, Prototheca
moriformis, Chlorella minutissima, Chlorella sp., and Chlorella
sorokiniana. In other cases, the microorganism is another species
as described herein.
[0063] In another aspect, the present invention is directed to a
microorganism containing an exogenous sucrose utilization gene. In
one embodiment, the gene encodes a sucrose transporter. In one
embodiment, the gene encodes a sucrose invertase. In one
embodiment, the gene encodes a fructokinase. In some cases, the
microorganism is a species selected from the group consisting of
Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana,
Chlorella ellipsoidea, Chlorella sp., or Chlorella protothecoides.
In other cases, the microorganism is another species as described
herein.
[0064] In another aspect, the present invention is directed to a
cell of the species Chlorella protothecoides, Chlorella emersonii,
or Chlorella minutissima wherein the cell contains an exogenous
gene. In some cases, the exogenous gene encodes a protein selected
from the group consisting of a sucrose transporter, a sucrose
invertase, a lipid modification enzyme, a hydrocarbon modification
enzyme and a fructokinase. In some embodiments, the protein is a
sucrose invertase secreted into the extracellular space. In some
embodiments, the protein is a sucrose invertase targeted to the
cytoplasm.
[0065] In another aspect, the present invention is directed to a
microbial culture containing a population of microorganisms, and a
culture medium comprising (i) sucrose and (ii) a sucrose invertase
enzyme.
[0066] In another aspect, the present invention is directed to a
microbial culture containing a population of microorganisms, and a
culture medium comprising (i) molasses and (ii) a sucrose invertase
enzyme.
[0067] In another aspect, the present invention is directed to a
microbial culture containing a population of microorganisms, and a
culture medium comprising (i) sucrose, (ii) lignin, and (iii) a
sucrose invertase enzyme.
[0068] In the various microbial cultures described above, the
microorganisms contain at least one exogenous sucrose utilization
gene. In some embodiments, the sucrose utilization gene encodes a
sucrose transporter, a sucrose invertase, a hexokinase, a
glucokinase, or a fructokinase. In one embodiment, the sucrose
invertase enzyme is a secrectable sucrose invertase enzyme encoded
by an exogenous sucrose invertase gene expressed by the population
of microorganisms. In some cases, the microorganisms contain at
least one exogenous gene encoding a lipid pathway enzyme or a
hydrocarbon modification enzyme.
[0069] In another aspect, the present invention is directed to a
nucleic acid comprising a cDNA encoding a sucrose utilization gene,
and a cDNA encoding a protein conferring resistance to the
antibiotic hygromycin or the antibiotic G418.
[0070] In embodiments of the various methods, compositions, cells,
microorganisms, microbes, microbial cultures, fermentors, and the
like, described above, the microorganism or microbe can be a
microalgae, an oleaginous yeast, a fungus, or a bacterium, unless
otherwise specified. In some cases, the microorganism is selected
from the group consisting of the microalgae listed in Table 1. In
some cases, the microorganism is a species of the genus Chlorella.
In some cases, the microorganism is selected from the group
consisting of Chlorella anitrata, Chlorella antarctica, Chlorella
aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella
desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella
fusca, Chlorella fusca var. vacuolate, Chlorella glucotropha,
Chlorella infusionum, Chlorella infusionum var. Actophila,
Chlorella infusionum var. Auxenophila, Chlorella kessleri,
Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis,
Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella
minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella
parva, Chlorella photophila, Chlorella pringsheimii, Chlorella
protothecoides, Chlorella regularis, Chlorella regularis var.
minima, Chlorella regularis var. umbricata, Chlorella reisiglii,
Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea,
Chlorella sauna, Chlorella simplex, Chlorella sorokiniana,
Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora,
Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris,
Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis,
Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris
f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella
xanthella, and Chlorella zofingiensis. In some cases, the
microorganism is an oleaginous yeast selected from the group
consisting of Cryptococcus curvatus, Cryptococcus terricolus,
Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis
vernalis, Rhodotorula glutinis, Rhodotorula gracilis, and Yarrowia
lipolytica. In some cases, the microorganism is a fungus selected
from the group consisting of a species of the genus Mortierella,
Mortierrla vinacea, Mortierella alpine, Pythium debaryanum, Mucor
circinelloides, Aspergillus ochraceus, Aspergillus terreus,
Pennicillium iilacinum, a species of the genus Hensenulo, a species
of the genus Chaetomium, a species of the genus Cladosporium, a
species of the genus Malbranchea, a species of the genus Rhizopus,
and a species of the genus Pythium.
[0071] In the various embodiments described above, the
microorganism can contain at least one exogenous sucrose
utilization gene. In some cases, the sucrose utilization gene
encodes a sucrose transporter, a sucrose invertase, a hexokinase, a
glucokinase, or a fructokinase.
[0072] In the various embodiments described above, the
microorganism can contain at least one exogenous gene encoding a
lipid pathway enzyme. In some cases, the lipid pathway enzyme is
selected from the group consisting of a stearoyl-ACP desaturase, a
glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA
carboxylase, an acyl carrier protein, and a glycerol-3 phosphate
acyltransferase.
[0073] In the various embodiments described above, the
microorganism can contain at least one exogenous gene encoding a
hydrocarbon modification enzyme. In some cases, the hydrocarbon
modification enzyme is selected from the group consisting of a
fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehyde reductase, a
fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty
aldehyde decarbonylase, and/or an acyl carrier protein.
[0074] Any two or more of the various embodiments described above
can be combined together to produce additional embodiments
encompassed within the scope of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] FIG. 1 shows dry cell weight per liter of multiple species
and strains of Chlorella when cultured in the presence of various
types of glycerol with and without additional glucose.
[0076] FIG. 2 shows dry cell weight per liter of multiple species
and strains of Chlorella when cultured in the presence of various
types of glycerol with additional glucose.
[0077] FIG. 3 shows relative lipid concentration of cultures of
multiple species and strains of Chlorella when cultured in the
presence of various types of glycerol with additional glucose.
[0078] FIG. 4 shows lipid concentration of cultures of multiple
species and strains of Chlorella when cultured in the presence of
various types of glycerol with and without additional glucose.
[0079] FIG. 5 shows lipid as a percent of dry cell weight of two
species and strains of Chlorella when cultured in the presence of
various types of glycerol with additional glucose, wherein glycerol
is added sequentially after glucose.
[0080] FIG. 6 shows lipid as a percent of dry cell weight of two
species and strains of Chlorella when cultured in the presence of
various types of glycerol with additional glucose.
[0081] FIG. 7 shows relative lipid concentration of cultures of
multiple species and strains of Chlorella when cultured in the
presence of 2% glucose and 1% glucose+1% reagent grade
glycerol.
[0082] FIG. 8 shows lipid as a percent of dry cell weight of
multiple species and strains of Chlorella when cultured in the
presence of glucose with and without reagent grade glycerol,
wherein glycerol is added sequentially or in combination with
glucose.
[0083] FIG. 9 shows relative lipid concentration of cultures of
multiple species and strains of Chlorella when cultured in the
presence of various types of glycerol with additional glucose,
wherein glycerol is added sequentially or in combination with
glucose.
[0084] FIG. 10 shows dry cell weight per liter of multiple species
and strains of Chlorella when cultured in the presence of various
types of glycerol with additional glucose, wherein glycerol is
added sequentially or in combination with glucose.
[0085] FIG. 11(a) shows lipid as a percent of dry cell weight of
Spirulina platensis when cultured in the presence of glucose,
reagent grade glycerol, non-acidulated biodiesel byproduct
glycerol, and a combination of glycerol and glucose.
[0086] FIG. 11(b) shows lipid as a percent of dry cell weight of
Navicula pelliculosa when cultured in the presence of various types
of glycerol and in the presence of combinations of glycerol and
glucose.
[0087] FIG. 12(a) shows lipid as a percent of dry cell weight of
Scenedesmus armatus when cultured in the presence of various types
of glycerol and in the presence of a combination of glycerol and
glucose.
[0088] FIG. 12(b) shows dry cell weight per liter of Scenedesmus
armatus when cultured in the presence of various types of glycerol
and in the presence of a combination of biodiesel byproduct
glycerol and glucose.
[0089] FIG. 13 shows dry cell weight per liter of Navicula
pelliculosa when cultured in the presence of various types of
glycerol and in the presence of a combination of non-acidulated
biodiesel byproduct glycerol and glucose.
[0090] FIG. 14 shows dry cell weight per liter of Scenedesmus
armatus and Navicula pelliculosa when cultured in the presence of
acidulated and non-acidulated biodiesel byproduct glycerol with
additional glucose, wherein glycerol is added sequentially or in
combination with glucose.
[0091] FIG. 15 shows a synergistic effect of a combination of
xylose and glucose on growth of Chlorella compared to xylose or
glucose alone.
[0092] FIG. 16 shows genotyping of Chlorella protothecoides
transformants containing an exogenous gene.
[0093] FIG. 17 shows codon usage of Chlorella protothecoides.
[0094] FIG. 18 shows codon usage of D. sauna and Chlorella
pyrenoidosa.
[0095] FIG. 19 shows (a) reagent grade glycerol; (b) non-acidulated
biodiesel byproduct glycerol; and (c) acidulated biodiesel
byproduct glycerol, all of which were used in experiments described
in the Examples.
[0096] FIG. 20 shows growth of Chlorella protothecoides on glucose
and fructose.
[0097] FIG. 21 shows growth of Chlorella fusca on 1% sucrose.
[0098] FIG. 22 shows growth of Chlorella kessleri on 1%
sucrose.
[0099] FIG. 23 shows dry cell weight per liter of Chlorella
protothecoides when cultured in the presence of glucose, sucrose,
or one of several molasses samples (designated BS1, BS2 and HTM) in
the presence or absence of a sucrose invertase.
[0100] FIG. 24 shows growth of Chlorella protothecoides when
cultured in the presence of glucose, sucrose, or one of several
molasses samples (designated BS1, BS2 and HTM) in the presence or
absence of a sucrose invertase as measured by relative cell
density.
[0101] FIG. 25 shows an illustration of various plasmid constructs
of yeast invertase (SUC2) with three different promoters
(designated CMV, CV and HUP1) as well as restriction sites useful
for subcloning.
[0102] FIG. 26 shows genotyping of Chlorella protothecoides
transformants selected on sucrose in the dark containing an
exogenous sucrose invertase gene.
[0103] FIG. 27 shows genotyping of Chlorella protothecoides cells
transformed with a gene encoding a secreted sucrose invertase from
S. cerevisiae.
[0104] FIG. 28 shows genotyping of Chlorella minutissima and
Chlorella emersonii cells transformed with a gene encoding a
secreted sucrose invertase from S. cerevisiae.
[0105] FIG. 29 illustrates a gas chromatograph generated by
analysis of a renewable diesel product produced in accordance with
the methods of the present invention, and described in Example
27.
[0106] FIG. 30 illustrates a boiling point distribution plot for a
renewable diesel product produced in accordance with the methods of
the presence invention, and described in Example 27.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0107] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs. The following
references provide one of skill with a general definition of many
of the terms used in this invention: Singleton et al., Dictionary
of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge
Dictionary of Science and Technology (Walker ed., 1988); The
Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer
Verlag (1991); and Hale & Marham, The Harper Collins Dictionary
of Biology (1991). As used herein, the following terms have the
meanings ascribed to them unless specified otherwise.
[0108] As used with reference to a nucleic acid, "active in
microalgae" refers to a nucleic acid that is functional in
microalgae. For example, a promoter that has been used to drive an
antibiotic resistance gene to impart antibiotic resistance to a
transgenic microalgae is active in microalgae. Examples of
promoters active in microalgae are promoters endogenous to certain
algae species and promoters found in plant viruses.
[0109] An "acyl carrier protein" or "ACP" is a protein which binds
a growing acyl chain during fatty acid synthesis as a thiol ester
at the distal thiol of the 4'-phosphopantetheine moiety and
comprises a component of the fatty acid synthase complex. The
phrase "naturally co-expressed" with reference to an acyl carrier
protein in conjunction with a fatty acyl-ACP thioesterase means
that the ACP and the thioesterase are co-expressed naturally in a
tissue or organism from which they are derived, e.g., because the
genes encoding the two enzymes are under the control of a common
regulatory sequence or because they are expressed in response to
the same stimulus.
[0110] An "acyl-CoA molecule" or "acyl-CoA" is a molecule
comprising an acyl moiety covalently attached to coenzyme A through
a thiol ester linkage at the distal thiol of the
4'-phosphopantetheine moiety of coenzyme A.
[0111] "Axenic" means a culture of an organism that is free from
contamination by other living organisms.
[0112] "Biodiesel" is a biologically produced fatty acid alkyl
ester suitable for use as a fuel in a diesel engine.
[0113] The term "biomass" refers to material produced by growth
and/or propagation of cells. Biomass may contain cells and/or
intracellular contents as well as extracellular material.
Extracellular material includes, but is not limited to, compounds
secreted by a cell.
[0114] "Bioreactor" means an enclosure or partial enclosure in
which cells are cultured, optionally in suspension.
[0115] As used herein, a "catalyst" refers to an agent, such as a
molecule or macromolecular complex, capable of facilitating or
promoting a chemical reaction of a reactant to a product without
becoming a part of the product. A catalyst thus increases the rate
of a reaction, after which, the catalyst may act on another
reactant to form the product. A catalyst generally lowers the
overall activation energy required for the reaction such that it
proceeds more quickly or at a lower temperature. Thus a reaction
equilibrium may be more quickly attained. Examples of catalysts
include enzymes, which are biological catalysts, heat, which is a
non-biological catalyst, and metal catalysts used in fossil oil
refining processes.
[0116] "Cellulosic material" means the products of digestion of
cellulose, including glucose and xylose, and optionally additional
compounds such as disaccharides, oligosaccharides, lignin,
furfurals and other compounds. Nonlimiting examples of sources of
cellulosic material include sugar caner bagasses, sugar beet pulp,
corn stover, wood chips, sawdust and switchgrass.
[0117] The term "co-culture", and variants thereof such as
"co-cultivate", refer to the presence of two or more types of cells
in the same bioreactor. The two or more types of cells may both be
microorganisms, such as microalgae, or may be a microalgal cell
cultured with a different cell type. The culture conditions may be
those that foster growth and/or propagation of the two or more cell
types or those that facilitate growth and/or proliferation of one,
or a subset, of the two or more cells while maintaining cellular
growth for the remainder.
[0118] The term "cofactor" is used herein to refer to any molecule,
other than the substrate, that is required for an enzyme to carry
out its enzymatic activity.
[0119] As used herein, "complementary DNA" ("cDNA") is a DNA
representation of mRNA, usually obtained by reverse transcription
of messenger RNA (mRNA) or amplification (e.g., via polymerase
chain reaction ("PCR")).
[0120] The term "cultivated", and variants thereof, refer to the
intentional fostering of growth (increases in cell size, cellular
contents, and/or cellular activity) and/or propagation (increases
in cell numbers via mitosis) of one or more cells by use of
intended culture conditions. The combination of both growth and
propagation may be termed proliferation. The one or more cells may
be those of a microorganism, such as microalgae. Examples of
intended conditions include the use of a defined medium (with known
characteristics such as pH, ionic strength, and carbon source),
specified temperature, oxygen tension, carbon dioxide levels, and
growth in a bioreactor. The term does not refer to the growth or
propagation of microorganisms in nature or otherwise without direct
human intervention, such as natural growth of an organism that
ultimately becomes fossilized to produce geological crude oil.
[0121] As used herein, the term "cytolysis" refers to the lysis of
cells in a hypotonic environment. Cytolysis is caused by excessive
osmosis, or movement of water, towards the inside of a cell
(hyperhydration). The cell cannot withstand the osmotic pressure of
the water inside, and so it explodes.
[0122] As used herein, the terms "expression vector" or "expression
construct" refer to a nucleic acid construct, generated
recombinantly or synthetically, with a series of specified nucleic
acid elements that permit transcription of a particular nucleic
acid in a host cell. The expression vector can be part of a
plasmid, virus, or nucleic acid fragment. Typically, the expression
vector includes a nucleic acid to be transcribed operably linked to
a promoter.
[0123] "Exogenous gene" refers to a nucleic acid transformed into a
cell. A transformed cell may be referred to as a recombinant cell,
into which additional exogenous gene(s) may be introduced. The
exogenous gene may be from a different species (and so
heterologous), or from the same species (and so homologous)
relative to the cell being transformed. In the case of a homologous
gene, it occupies a different location in the genome of the cell
relative to the endogenous copy of the gene. The exogenous gene may
be present in more than one copy in the cell. The exogenous gene
may be maintained in a cell as an insertion into the genome or as
an episomal molecule.
[0124] "Exogenously provided" describes a molecule provided to the
culture media of a cell culture.
[0125] As used herein, a "fatty acyl-ACP thioesterase" is an enzyme
that catalyzes the cleavage of a fatty acid from an acyl carrier
protein (ACP) during lipid synthesis.
[0126] As used herein, a "fatty acyl-CoA/aldehyde reductase" is an
enzyme that catalyzes the reduction of an acyl-CoA molecule to a
primary alcohol.
[0127] As used herein, a "fatty acyl-CoA reductase" is an enzyme
that catalyzes the reduction of an acyl-CoA molecule to an
aldehyde.
[0128] As used herein, a "fatty aldehyde decarbonylase" is an
enzyme that catalyzes the conversion of a fatty aldehyde to an
alkane.
[0129] As used herein, a "fatty aldehyde reductase" is an enzyme
that catalyzes the reduction of an aldehyde to a primary
alcohol.
[0130] "Fixed carbon source" means molecule(s) containing carbon,
preferably organic, that are present at ambient temperature and
pressure in solid or liquid form.
[0131] "Fungus," as used herein, means heterotrophic organisms
characterized by a chitinous cell wall from the kingdom of
fungi.
[0132] "Homogenate" means biomass that has been physically
disrupted.
[0133] As used herein, "hydrocarbon" refers to: (a) a molecule
containing only hydrogen and carbon atoms wherein the carbon atoms
are covalently linked to form a linear, branched, cyclic, or
partially cyclic backbone to which the hydrogen atoms are attached;
or (b) a molecule that only primarily contains hydrogen and carbon
atoms and that can be converted to contain only hydrogen and carbon
atoms by one to four chemical reactions. Nonlimiting examples of
the latter include hydrocarbons containing an oxygen atom between
one carbon and one hydrogen atom to form an alcohol molecule, as
well as aldehydes containing a single oxygen atom. Methods for the
reduction of alcohols to hydrocarbons containing only carbon and
hydrogen atoms are well known. Another example of a hydrocarbon is
an ester, in which an organic group replaces a hydrogen atom (or
more than one) in an oxygen acid. The molecular structure of
hydrocarbon compounds varies from the simplest, in the form of
methane (CH.sub.4), which is a constituent of natural gas, to the
very heavy and very complex, such as some molecules such as
asphaltenes found in crude oil, petroleum, and bitumens.
Hydrocarbons may be in gaseous, liquid, or solid form, or any
combination of these forms, and may have one or more double or
triple bonds between adjacent carbon atoms in the backbone.
Accordingly, the term includes linear, branched, cyclic, or
partially cyclic alkanes, alkenes, lipids, and paraffin. Examples
include propane, butane, pentane, hexane, octane, triolein, and
squalene.
[0134] "Hydrocarbon modification enzyme" refers to an enzyme that
alters the covalent structure of a hydrocarbon. Examples of
hydrocarbon modification enzymes include a lipase, a fatty acyl-ACP
thioesterase, a fatty acyl-CoA/aldehyde reductase, a fatty acyl-CoA
reductase, a fatty aldehyde reductase, and a fatty aldehyde
decarbonylase. Compounds produced by the enzymatic activity of
hydrocarbon modification enzymes, including fatty acids, alcohols,
aldehydes, alkanes, or other compounds derived therefrom are
referred to herein interchangeably as hydrocarbons or lipids.
[0135] The term "hydrogen:carbon ratio" refers to the ratio of
hydrogen atoms to carbon atoms in a molecule on an atom-to-atom
basis. The ratio may be used to refer to the number of carbon and
hydrogen atoms in a hydrocarbon molecule. For example, the
hydrocarbon with the highest ratio is methane CH.sub.4 (4:1).
[0136] "Hydrophobic fraction" refers to the portion, or fraction,
of a material that is more soluble in a hydrophobic phase in
comparison to an aqueous phase. A hydrophobic fraction is
substantially insoluble in water and usually non-polar.
[0137] As used herein, the phrase "increase lipid yield" refers to
an increase in the productivity of a microbial culture by, for
example, increasing dry weight of cells per liter of culture,
increasing the percentage of cells that constitute lipid, or
increasing the overall amount of lipid per liter of culture volume
per unit time.
[0138] An "inducible promoter" is one that mediates transcription
of an operably linked gene in response to a particular
stimulus.
[0139] As used herein, the phrase "in operable linkage" refers to a
functional linkage between two sequences, such a control sequence
(typically a promoter) and the linked sequence. A promoter is in
operable linkage with an exogenous gene if it can mediate
transcription of the gene.
[0140] The term "in situ" means "in place" or "in its original
position". For example, a culture may contain a first microalgae
secreting a catalyst and a second microorganism secreting a
substrate, wherein the first and second cell types produce the
components necessary for a particular chemical reaction to occur in
situ in the co-culture without requiring further separation or
processing of the materials.
[0141] A "limiting concentration of a nutrient" is a concentration
in a culture that limits the propagation of a cultured organism. A
"non-limiting concentration of a nutrient" is a concentration that
supports maximal propagation during a given culture period. Thus,
the number of cells produced during a given culture period is lower
in the presence of a limiting concentration of a nutrient than when
the nutrient is non-limiting. A nutrient is said to be "in excess"
in a culture, when the nutrient is present at a concentration
greater than that which supports maximal propagation.
[0142] As used herein, a "lipase" is a water-soluble enzyme that
catalyzes the hydrolysis of ester bonds in water-insoluble, lipid
substrates. Lipases catalyze the hydrolysis of lipids into
glycerols and fatty acids.
[0143] As used herein, a "lipid pathway enzyme" is any enzyme that
plays a role in lipid metabolism, i.e., either lipid synthesis,
modification, or degradation. This term encompasses proteins that
chemically modify lipids, as well as carrier proteins.
[0144] "Lipids" are a class of hydrocarbon that are soluble in
nonpolar solvents (such as ether and chloroform) and are relatively
or completely insoluble in water. Lipid molecules have these
properties because they consist largely of long hydrocarbon tails
which are hydrophobic in nature. Examples of lipids include fatty
acids (saturated and unsaturated); glycerides or glycerolipids
(such as monoglycerides, diglycerides, triglycerides or neutral
fats, and phosphoglycerides or glycerophospholipids); nonglycerides
(sphingolipids, sterol lipids including cholesterol and steroid
hormones, prenol lipids including terpenoids, fatty alcohols,
waxes, and polyketides); and complex lipid derivatives
(sugar-linked lipids, or glycolipids, and protein-linked lipids).
"Fats" are a subgroup of lipids called "triacylglycerides."
[0145] As used herein, the term "lysate" refers to a solution
containing the contents of lysed cells.
[0146] As used herein, the term "lysis" refers to the breakage of
the plasma membrane and optionally the cell wall of a biological
organism sufficient to release at least some intracellular content,
often by mechanical, viral or osmotic mechanisms that compromise
its integrity.
[0147] As used herein, the term "lysing" refers to disrupting the
cellular membrane and optionally the cell wall of a biological
organism or cell sufficient to release at least some intracellular
content.
[0148] "Microalgae" means a eukarytotic microbial organism that
contains a chloroplast, and optionally that is capable of
performing photosynthesis, or a prokaryotic microbial organism
capable of performing photosynthesis. Microalgae include obligate
photoautotrophs, which cannot metabolize a fixed carbon source as
energy, as well as heterotrophs, which can live solely off of a
fixed carbon source. Microalgae can refer to unicellular organisms
that separate from sister cells shortly after cell division, such
as Chlamydomonas, and can also refer to microbes such as, for
example, Volvox, which is a simple multicellular photosynthetic
microbe of two distinct cell types. "Microalgae" can also refer to
cells such as Chlorella and Dunaliella. "Microalgae" also includes
other microbial photosynthetic organisms that exhibit cell-cell
adhesion, such as Agmenellum, Anabaena, and Pyrobotrys.
"Microalgae" also includes obligate heterotrophic microorganisms
that have lost the ability to perform photosynthesis, such as
certain dinoflagellate algae species.
[0149] The terms "microorganism" and "microbe" are used
interchangeably herein to refer to microscopic unicellular
organisms.
[0150] "Oleaginous yeast," as used herein, means yeast that can
naturally accumulate more than 10% of its dry cell weight as lipid
or can do so as a result of genetic engineering. Oleaginous yeast
includes organisms such as Yarrowia lipolytica, as well as
engineered strains of yeast such as Saccharomyces cerevisiae that
have been engineered to accumulate more than 10% of their dry cell
weight as lipid.
[0151] As used herein, the term "osmotic shock" refers to the
rupture of cells in a solution following a sudden reduction in
osmotic pressure. Osmotic shock is sometimes induced to release
cellular components of such cells into a solution.
[0152] "Photobioreactor" refers to a container, at least part of
which is at least partially transparent or partially open, thereby
allowing light to pass through, in which one or more microalgae
cells are cultured. Photobioreactors may be closed, as in the
instance of a polyethylene bag or Erlenmeyer flask, or may be open
to the environment, as in the instance of an outdoor pond.
[0153] As used herein, a "polysaccharide-degrading enzyme" refers
to any enzyme capable of catalyzing the hydrolysis, or
depolymerization, of any polysaccharide. For example, cellulases
catalyze the hydrolysis of cellulose.
[0154] "Polysaccharides" (also called "glycans") are carbohydrates
made up of monosaccharides joined together by glycosidic linkages.
Cellulose is an example of a polysaccharide that makes up certain
plant cell walls. Cellulose can be depolymerized by enzymes to
yield monosaccharides such as xylose and glucose, as well as larger
disaccharides and oligosaccharides.
[0155] "Port", in the context of a bioreactor, refers to an opening
in the bioreactor that allows influx or efflux of materials such as
gases, liquids, and cells. Ports are usually connected to tubing
leading from the photobioreactor.
[0156] A "promoter" is defined as an array of nucleic acid control
sequences that direct transcription of a nucleic acid. As used
herein, a promoter includes necessary nucleic acid sequences near
the start site of transcription, such as, in the case of a
polymerase II type promoter, a TATA element. A promoter also
optionally includes distal enhancer or repressor elements, which
can be located as much as several thousand base pairs from the
start site of transcription.
[0157] As used herein, the term "recombinant" when used with
reference, e.g., to a cell, or nucleic acid, protein, or vector,
indicates that the cell, nucleic acid, protein or vector, has been
modified by the introduction of an exogenous nucleic acid or
protein or the alteration of a native nucleic acid or protein, or
that the cell is derived from a cell so modified. Thus, e.g.,
recombinant cells express genes that are not found within the
native (non-recombinant) form of the cell or express native genes
that are otherwise abnormally expressed, under expressed or not
expressed at all. By the term "recombinant nucleic acid" herein is
meant nucleic acid, originally formed in vitro, in general, by the
manipulation of nucleic acid, e.g., using polymerases and
endonucleases, in a form not normally found in nature. In this
manner, operably linkage of different sequences is achieved. Thus
an isolated nucleic acid, in a linear form, or an expression vector
formed in vitro by ligating DNA molecules that are not normally
joined, are both considered recombinant for the purposes of this
invention. It is understood that once a recombinant nucleic acid is
made and reintroduced into a host cell or organism, it will
replicate non-recombinantly, i.e., using the in vivo cellular
machinery of the host cell rather than in vitro manipulations;
however, such nucleic acids, once produced recombinantly, although
subsequently replicated non-recombinantly, are still considered
recombinant for the purposes of the invention. Similarly, a
"recombinant protein" is a protein made using recombinant
techniques, i.e., through the expression of a recombinant nucleic
acid as depicted above.
[0158] As used herein, the term "renewable diesel" refers to
alkanes (such as C:10:0, C12:0, C:14:0, C16:0 and C18:0) produced
through hydrogenation and deoxygenation of lipids.
[0159] As used herein, the term "sonication" refers to a process of
disrupting biological materials, such as a cell, by use of sound
wave energy.
[0160] "Species of furfural" refers to 2-Furancarboxaldehyde or a
derivative thereof which retains the same basic structural
characteristics.
[0161] As used herein, "stover" refers to the dried stalks and
leaves of a crop remaining after a grain has been harvested.
[0162] A "sucrose utilization gene" is a gene that, when expressed,
aids the ability of a cell to utilize sucrose as an energy source.
Proteins encoded by a sucrose utilization gene are referred to
herein as "sucrose utilization enzymes" and include sucrose
transporters, sucrose invertases, and hexokinases such as
glucokinases and fructokinases.
[0163] "Wastewater" is watery waste which typically contains
washing water, laundry waste, faeces, urine and other liquid or
semi-liquid wastes. It includes some forms of municipal waste as
well as secondarily treated sewage.
[0164] For sequence comparison to determine percent nucleotide or
amino acid identity, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a
sequence comparison algorithm, test and reference sequences are
input into a computer, subsequence coordinates are designated, if
necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0165] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by visual
inspection (see generally Ausubel et al., supra).
[0166] Another example algorithm that is suitable for determining
percent sequence identity and sequence similarity is the BLAST
algorithm, which is described in Altschul et al., J. Mol. Biol.
215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (at the web address www.ncbi.nlm.nih.gov). This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra.). These initial neighborhood
word hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. For
identifying whether a nucleic acid or polypeptide is within the
scope of the invention, the default parameters of the BLAST
programs are suitable. The BLASTN program (for nucleotide
sequences) uses as defaults a word length (W) of 11, an expectation
(E) of 10, M=5, N=-4, and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a word length
(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring
matrix. The TBLATN program (using protein sequence for nucleotide
sequence) uses as defaults a word length (W) of 3, an expectation
(E) of 10, and a BLOSUM 62 scoring matrix. (see Henikoff &
Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
[0167] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul,
Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
II. General
[0168] The invention is premised in part on the insight that
certain microorganisms can be used to produce oils, fuels, and
other hydrocarbon or lipid compositions economically and in large
quantities for use in the transportation fuel, petrochemical
industry, and/or food and cosmetic industries, among other
applications. Suitable microorganisms include microalgae,
oleaginous yeast, and fungi. A preferred genus of microalgae for
use in the invention is the lipid-producing microalgae Chlorella.
Transesterification of lipids yields long-chain fatty acid esters
useful as biodiesel. Other enzymatic and chemical processes can be
tailored to yield fatty acids, aldehydes, alcohols, alkanes, and
alkenes. The present application describes methods for genetic
modification of multiple species and strains of microorganisms,
including Chlorella and similar microbes to provide organisms
having characteristics that facilitate the production of lipid
suitable for conversion into oils, fuels, and oleochemicals. In
some applications, renewable diesel, jet fuel, or other hydrocarbon
compounds are produced. The present application also describes
methods of cultivating microalgae for increased productivity and
increased lipid yield, and/or for more cost-effective production of
the compositions described herein.
[0169] In particular embodiments, the present application describes
genetically engineering strains of microalgae with one or more
exogenous genes. For example, microalgae that produce high levels
of triacylglycerides (TAGs) suitable for biodiesel can be
engineered to express a lipase, which can facilitate
transesterification of microalgal TAGs. The lipase can optionally
be expressed using an inducible promoter, so that the cells can
first be grown to a desirable density in a fermentor and then
harvested, followed by induction of the promoter to express the
lipase, optionally in the presence of sufficient alcohol to drive
conversion of TAGs to fatty acid esters.
[0170] Some microalgal lipid is sequestered in cell membranes and
other non-aqueous parts of the cell. Therefore, to increase the
yield of the transesterification reaction, it can be beneficial to
lyse the cells to increase the accessibility of the lipase to the
lipid. Cell disruption can be performed, for example, mechanically,
through addition of pressurized steam, or by employing a virus that
lyses the microalgae cells, expressing a gene to produce a lytic
protein in the cell, or treating the culture with an agent that
lyses microalgae cells. Steam treatment of microalgae for cell
disruption is described, for example, in U.S. Pat. No.
6,750,048.
[0171] Also disclosed herein is the genetic engineering of
microalgae that produce high levels of TAGs to express a gene that
lyses microalgae cells, such as for example, a gene from a lytic
virus. This gene can be expressed using an inducible promoter, so
that the cells can first be grown to a desirable density in a
fermentor and then harvested, followed by induction of the promoter
to express the gene to lyse the cells. A gene encoding a
polysaccharide-degrading enzyme, for example, can be expressed to
lyse the cells.
[0172] Optionally, the lipase can be expressed in an intracellular
compartment, where it remains separate from the majority of the
microalgal lipid until transesterification. Generally, it is
preferable to carry out transesterification after water has been
substantially removed from the preparation and/or an excess of
alcohol has been added. Lipases can use water, as well as alcohol,
as a substrate in transesterification. With water, the lipid is
conjugated to a hydroxyl moiety to produce a polar fatty acid,
rather than an ester. With an alcohol, such as methanol, the lipid
is conjugated to a methyl group, producing a non-polar fatty acid
ester, which is typically preferable for a transportation fuel. To
limit exposure of the lipase to microalgal lipid until conditions
are suitable for transesterification to produce fatty acid esters,
the lipase can be expressed, for example, in the chloroplast,
mitochondria, or other cellular organelle. This compartmentalized
expression results in sequestration of the lipase from the majority
of the cellular lipid until after the cells have been
disrupted.
[0173] In other particular embodiments, the present application
describes genetically engineering strains of microalgae, oleaginous
yeast, bacteria, or fungi with one or more exogenous genes to
produce various hydrocarbon compounds. For example, microalgae that
would naturally, or through genetic modification, produce high
levels of lipids can be engineered (or further engineered) to
express an exogenous fatty acyl-ACP thioesterase, which can
facilitate the cleavage of fatty acids from the acyl carrier
protein (ACP) during lipid synthesis. These fatty acids can be
recovered or, through further enzymatic processing within the cell,
yield other hydrocarbon compounds. Optionally, the fatty acyl-ACP
thioesterase can be expressed from a gene operably linked to an
inducible promoter, and/or can be expressed in an intracellular
compartment.
[0174] The fatty acyl-ACP thioesterase can be chosen based on its
specificity for a growing (during fatty acid synthesis) fatty acid
having a particular carbon chain length. For example, the fatty
acyl-ACP thioesterase can have a specificity for a carbon chain
length ranging from 8 to 34 carbon atoms, preferably from 8 to 18
carbon atoms, and more preferably from 10 to 14 carbon atoms. A
specificity for a fatty acid with 12 carbon atoms is most
preferred.
[0175] Further, the invention provides genetically engineered
strains of microalgae to express two or more exogenous genes, such
as, for example, a lipase and a lytic gene, e.g., one encoding a
polysaccharide-degrading enzyme. One or both genes can be expressed
using an inducible promoter, which allows the relative timing of
expression of these genes to be controlled to enhance the lipid
yield and conversion to fatty acid esters. The invention also
provides vectors and methods for engineering lipid-producing
microbes to metabolize sucrose, which is an advantageous trait
because it allows the engineered cells to convert sugar cane or
other feedstocks into lipids appropriate for production of oils,
fuels, oleochemicals and the like.
[0176] In other embodiments, the invention provides genetically
engineered strains of microbes (e.g., microalgae, oleaginous yeast,
bacteria, or fungi) that express two or more exogenous genes, such
as, for example, a fatty acyl-ACP thioesterase and a fatty
acyl-CoA/aldehyde reductase, the combined action of which yields an
alcohol product. The invention further provides other combinations
of exogenous genes, including without limitation, a fatty acyl-ACP
thioesterase and a naturally co-expressed acyl carrier protein to
generate length-specific fatty acids, or a fatty acyl-ACP
thioesterase and a fatty acyl-CoA reductase to generate aldehydes.
The invention also provides for the combination of a fatty acyl-ACP
thioesterase, a fatty acyl-CoA reductase, and a fatty aldehyde
decarbonylase to generate alkanes or alkenes. One or more of the
exogenous genes can be expressed using an inducible promoter.
[0177] The invention provides further modifications of microalgae,
for example to provide microalgae with desired growth
characteristics and/or to enhance the amount and/or quality of
lipids produced. For example, microalgae can be engineered to
increase carbon flux into the lipid pathway and/or modify the lipid
pathway to beneficially alter the proportions or properties of
lipid produced by the cells.
[0178] This application discloses genetically engineering strains
of microalgae to express two or more exogenous genes, one encoding
a transporter of a fixed carbon source (such as sucrose) and a
second encoding a sucrose invertase enzyme. The resulting
fermentable organisms produce hydrocarbons at lower manufacturing
cost than what has been obtainable by previously known methods of
biological hydrocarbon production. The insertion of the two
exogenous genes described above can be combined with the disruption
of polysaccharide biosynthesis through directed and/or random
mutagenesis, which steers ever greater carbon flux into hydrocarbon
production. Individually and in combination, trophic conversion,
engineering to alter hydrocarbon production and treatment with
exogenous enzymes alter the hydrocarbon composition produced by a
microorganism. The alteration can be a change in the amount of
hydrocarbons produced, the amount of one or more hydrocarbon
species produced relative to other hydrocarbons, and/or the types
of hydrocarbon species produced in the microorganism. For example,
microalgae can be engineered to produce a higher amount and/or
percentage of TAGs.
III. Oil- or Lipid-Producing Microorganisms
[0179] Any species of organism that produces suitable lipid or
hydrocarbon can be used, although microorganisms that naturally
produce high levels of suitable lipid or hydrocarbon are preferred.
Production of hydrocarbons by microorganisms is reviewed by Metzger
et al. Appl Microbiol Biotechnol (2005) 66: 486-496 and A Look Back
at the U.S. Department of Energy's Aquatic Species Program:
Biodiesel from Algae, NREL/TP-580-24190, John Sheehan, Terri
Dunahay, John Benemann and Paul Roessler (1998).
[0180] Considerations affecting the selection of microorganisms for
use in the invention include, in addition to production of suitable
lipids or hydrocarbons for production of oils, fuels, and
oleochemicals: (1) high lipid content as a percentage of cell
weight; (2) ease of growth; (3) ease of genetic engineering; and
(4) ease of biomass processing. In particular embodiments, the
wild-type or genetically engineered microorganism yields cells that
are at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, or at least 70% or more lipid. Preferred
organisms grow heterotrophically (on sugars in the absence of
light) or can be engineered to do so using, for example, methods
disclosed herein. The ease of transformation and availability of
selectable markers and promoters, constitutive and/or inducible,
that are functional in the microorganism affect the ease of genetic
engineering. Processing considerations can include, for example,
the availability of effective means for lysing the cells.
[0181] A. Algae
[0182] In one embodiment of the present invention, the
microorganism is a microalgae. Nonlimiting examples of microalgae
that can be used in accordance with the present invention can be
found in Table 1.
TABLE-US-00001 TABLE 1 Examples of microalgae. Achnanthes
orientalis, Agmenellum, Amphiprora hyaline, Amphora coffeiformis,
Amphora coffeiformis linea, Amphora coffeiformis punctata, Amphora
coffeiformis taylori, Amphora coffeiformis tenuis, Amphora
delicatissima, Amphora delicatissima capitata, Amphora sp.,
Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia
hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus
sudeticus, Bracteococcus minor, Bracteococcus medionucleatus,
Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros
muelleri subsalsum, Chaetoceros sp., Chlorella anitrata, Chlorella
Antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella
capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella
emersonii, Chlorella fusca, Chlorella fusca var. vacuolata,
Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum
var. actophila, Chlorella infusionum var. auxenophila, Chlorella
kessleri, Chlorella lobophora (strain SAG 37.88), Chlorella
luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella
luteoviridis var. lutescens, Chlorella miniata, Chlorella
minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella
ovalis, Chlorella parva, Chlorella photophila, Chlorella
pringsheimii, Chlorella protothecoides (including any of UTEX
strains 1806, 411, 264, 256, 255, 250, 249, 31, 29, 25), Chlorella
protothecoides var. acidicola, Chlorella regularis, Chlorella
regularis var. minima, Chlorella regularis var. umbricata,
Chlorella reisiglii, Chlorella saccharophila, Chlorella
saccharophila var. ellipsoidea, Chlorella salina, Chlorella
simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica,
Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris,
Chlorella vulgaris f. tertia, Chlorella vulgaris var. autotrophica,
Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris,
Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var.
vulgaris f. viridis, Chlorella xanthella, Chlorella zofingiensis,
Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum
infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp.,
Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii,
Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana,
Cyclotella sp., Dunaliella sp., Dunaliella bardawil, Dunaliella
bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella
minuta, Dunaliella parva, Dunaliella peircei, Dunaliella
primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella
tertiolecta, Dunaliella viridis, Dunaliella tertiolecta,
Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena,
Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa
sp., Gloeothamnion sp., Hymenomonas sp., Isochrysis aff. galbana,
Isochrysis galbana, Lepocinclis, Micractinium, Micractinium (UTEX
LB 2614), Monoraphidium minutum, Monoraphidium sp., Nannochloris
sp., Nannochloropsis salina, Nannochloropsis sp., Navicula
acceptata, Navicula biskanterae, Navicula pseudotenelloides,
Navicula pelliculosa, Navicula saprophila, Navicula sp.,
Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia
alexandrina, Nitzschia communis, Nitzschia dissipata, Nitzschia
frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia
intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia
pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia
quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva,
Oocystis pusilla, Oocystis sp., Oscillatoria limnetica,
Oscillatoria sp., Oscillatoria subbrevis, Parachlorella kessleri,
Pascheria acidophila, Pavlova sp., Phagus, Phormidium, Platymonas
sp., Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis
sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca
portoricensis, Prototheca moriformis, Prototheca zopfii,
Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcus
opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium,
Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus
sp., Tetraedron, Tetraselmis sp., Tetraselmis suecica,
Thalassiosira weissflogii, and Viridiella fridericiana
[0183] 1. Chlorella
[0184] In a preferred embodiment of the present invention, the
microorganism is of the genus Chlorella, preferably, Chlorella
protothecoides, Chlorella ellipsoidea, Chlorella minutissima, or
Chlorella emersonii.
[0185] Chlorella is a genus of single-celled green algae, belonging
to the phylum Chlorophyta. It is spherical in shape, about 2 to 10
.mu.m in diameter, and is without flagella. Some species of
Chlorella are naturally heterotrophic.
[0186] Chlorella, particularly Chlorella protothecoides, is a
preferred microorganism for use in the invention because of its
high composition of lipid, particularly long-chain lipid suitable
for biodiesel. In addition, this microalgae grows heterotrophically
and can be genetically engineered as demonstrated in the Examples
herein.
[0187] In a preferred embodiment of the present invention, the
microorganism used for expression of a transgene is of the genus
Chlorella, preferably, Chlorella protothecoides, Chlorella
minutissima, or Chlorella emersonii. Examples of expression of
transgenes in, e.g., Chlorella, can be found in the literature (see
for example Current Microbiology Vol. 35 (1997), pp. 356-362; Sheng
Wu Gong Cheng Xue Bao. 2000 July; 16(4):443-6; Current Microbiology
Vol. 38 (1999), pp. 335-341; Appl Microbiol Biotechnol (2006) 72:
197-205; Marine Biotechnology 4, 63-73, 2002; Current Genetics
39:5, 365-370 (2001); Plant Cell Reports 18:9, 778-780, (1999);
Biologia Plantarium 42(2): 209-216, (1999); Plant Pathol. J 21(1):
13-20, (2005)). Also see Examples herein. Other lipid-producing
microalgae can be engineered as well, including prokaryotic
Microalgae (see Kalscheuer st al., Applied Microbiology and
Biotechnology, Volume 52, Number 4/October, 1999).
[0188] 2. Identification of Chlorella Species
[0189] Species of Chlorella for use in the invention can be
identified by amplification of certain target regions of the
genome. For example, identification of a specific Chlorella species
or strain can be achieved through amplification and sequencing of
nuclear and/or chloroplast DNA using primers and methodology using
any region of the genome, for example using the methods described
in Wu et al., Bot. Bull. Acad. Sin. (2001) 42:115-121
Identification of Chlorella spp. isolates using ribosomal DNA
sequences. Well established methods of phylogenetic analysis, such
as amplification and sequencing of ribosomal internal transcribed
spacer (ITS1 and ITS2 rDNA), 18S rRNA, and other conserved genomic
regions can be used by those skilled in the art to identify species
of not only Chlorella, but other hydrocarbon and lipid producing
organisms capable of using the methods disclosed herein. For
examples of methods of identification and classification of algae
also see for example Genetics, 2005 August; 170(4): 1601-10 and
RNA, 2005 April; 11(4):361-4.
[0190] B. Oleaginous Yeast
[0191] In one embodiment of the present invention, the
microorganism is an oleaginous yeast. Nonlimiting examples of
oleaginous yeast that can be used in accordance with the present
invention can be found in Table 2.
TABLE-US-00002 TABLE 2 Examples of oleaginous yeast. Cryptococcus
curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi,
Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis,
Rhodotorula gracilis, and Yarrowia lipolytica
[0192] C. Other Fungi
[0193] In one embodiment of the present invention, the
microorganism is a fungus. Nonlimiting examples of fungi that can
be used in accordance with the present invention can be found in
Table 3.
TABLE-US-00003 TABLE 3 Examples of fungi. Mortierella, Mortierrla
vinacea, Mortierella alpine, Pythium debaryanum, Mucor
circinelloides, Aspergillus ochraceus, Aspergillus terreus,
Pennicillium iilacinum, Hensenulo, Chaetomium, Cladosporium,
Malbranchea, Rhizopus, and Pythium
[0194] D. Bacteria
[0195] In one embodiment of the present invention, the
microorganism is a bacterium.
[0196] Examples of expression of exogenous genes in bacteria, such
as E. coli, are well known; see for example Molecular Cloning: A
Laboratory Manual, Sambrook et al. (3d edition, 2001, Cold Spring
Harbor Press).
IV. Methods of Culturing Microorganisms
[0197] Microrganisms are cultured both for purposes of conducting
genetic manipulations and for subsequent production of hydrocarbons
(e.g., lipids, fatty acids, aldehydes, alcohols, and alkanes). The
former type of culture is conducted on a small scale and initially,
at least, under conditions in which the starting microorganism can
grow. For example, if the starting microorganism is a
photoautotroph the initial culture is conducted in the presence of
light. The culture conditions can be changed if the microorganism
is evolved or engineered to grow independently of light. Culture
for purposes of hydrocarbon production is usually conducted on a
large scale. Preferably a fixed carbon source is present. The
culture can also be exposed to light some or all of the time.
[0198] Microalgae can be cultured in liquid media. The culture can
be contained within a bioreactor. Optionally, the bioreactor does
not allow light to enter. Alternatively, microalgae can also be
cultured in photobioreactors that contain a fixed carbon source and
allow light to strike the cells. Exposure of microalgae cells to
light, even in the presence of a fixed carbon source that the cells
transport and utilize (i.e., mixotrophic growth), nonetheless
accelerates growth compared to culturing cells in the dark. Culture
condition parameters can be manipulated to optimize total
hydrocarbon production, the combination of hydrocarbon species
produced, and/or production of a hydrocarbon species. In some
instances it is preferable to culture cells in the dark, such as,
for example, when using extremely large (40,000 liter and higher)
fermentors that do not allow light to strike the culture.
[0199] Microalgal culture media typically contains components such
as a fixed nitrogen source, trace elements, optionally a buffer for
pH maintenance, and phosphate. Other components can include a fixed
carbon source such as acetate or glucose, and salts such as sodium
chloride, particularly for seawater microalgae. Examples of trace
elements include zinc, boron, cobalt, copper, manganese, and
molybdenum in, for example, the respective forms of ZnCl.sub.2,
H.sub.3BO.sub.3, CoCl.sub.2.6H.sub.2O, CuCl.sub.2.2H.sub.2O,
MnCl.sub.2.4H.sub.2O and
(NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O.
[0200] For organisms able to grow on a fixed carbon source, the
fixed carbon source can be, for example, glucose, fructose,
sucrose, galactose, xylose, mannose, rhamnose, N-acetylglucosamine,
glycerol, floridoside, and/or glucuronic acid. The one or more
carbon source(s) can be supplied at a concentration of at least
about 50 .mu.M, at least about 100 .mu.M, at least about 500 .mu.M,
at least about 5 mM, at least about 50 mM, and at least about 500
mM, of one or more exogenously provided fixed carbon source(s).
Some microalgae species can grow by utilizing a fixed carbon source
such as glucose or acetate in the absence of light. Such growth is
known as heterotrophic growth. For Chlorella protothecoides, for
example, heterotrophic growth results in high production of biomass
and accumulation of high lipid content in cells.
[0201] Some microorganisms naturally grow on or can be engineered
to grow on a fixed carbon source that is a heterogeneous source of
compounds such as municipal waste, secondarily treated sewage,
wastewater, and other sources of fixed carbon and other nutrients
such as sulfates, phosphates, and nitrates. The sewage component
serves as a nutrient source in the production of hydrocarbons, and
the culture provides an inexpensive source of hydrocarbons.
[0202] Other culture parameters can also be manipulated, such as
the pH of the culture media, the identity and concentration of
trace elements and other media constituents.
[0203] A. Photosynthetic Growth
[0204] Microalgae can be grown in the presence of light. The number
of photons striking a culture of microalgae cells can be
manipulated, as well as other parameters such as the wavelength
spectrum and ratio of dark:light hours per day. Microalgae can also
be cultured in natural light, as well as simultaneous and/or
alternating combinations of natural light and artificial light. For
example, microalgae of the genus Chlorella can be cultured under
natural light during daylight hours and under artificial light
during night hours.
[0205] The gas content of a photobioreactor to grow microorganisms
like microalgae can be manipulated. Part of the volume of a
photobioreactor can contain gas rather than liquid. Gas inlets can
be used to pump gases into the photobioreactor. Any gas can be
pumped into a photobioreactor, including air, air/CO.sub.2
mixtures, noble gases such as argon and others. The rate of entry
of gas into a photobioreactor can also be manipulated. Increasing
gas flow into a photobioreactor increases the turbidity of a
culture of microalgae. Placement of ports conveying gases into a
photobioreactor can also affect the turbidity of a culture at a
given gas flow rate. Air/CO.sub.2 mixtures can be modulated to
generate optimal amounts of CO.sub.2 for maximal growth by a
particular organism. Microalgae grow significantly faster in the
light under, for example, 3% CO.sub.2/97% air than in 100% air. 3%
CO.sub.2/97% air is approximately 100-fold more CO.sub.2 than found
in air. For example, air:CO.sub.2 mixtures of about 99.75%
air:0.25% CO.sub.2, about 99.5% air:0.5% CO.sub.2, about 99.0%
air:1.00% CO.sub.2, about 98.0% air:2.0% CO.sub.2, about 97.0%
air:3.0% CO.sub.2, about 96.0% air:4.0% CO.sub.2, and about 95.00%
air:5.0% CO.sub.2 can be infused into a bioreactor or
photobioreactor.
[0206] Microalgae cultures can also be subjected to mixing using
devices such as spinning blades and impellers, rocking of a
culture, stir bars, infusion of pressurized gas, and other
instruments.
[0207] Photobioreactors can have ports allowing entry of gases,
solids, semisolids and liquids into the chamber containing the
microalgae. Ports are usually attached to tubing or other means of
conveying substances. Gas ports, for example, convey gases into the
culture. Pumping gases into a photobioreactor can serve to both
feed cells CO.sub.2 and other gases and to aerate the culture and
therefore generate turbidity. The amount of turbidity of a culture
varies as the number and position of gas ports is altered. For
example, gas ports can be placed along the bottom of a cylindrical
polyethylene bag. Microalgae grow faster when CO.sub.2 is added to
air and bubbled into a photobioreactor. For example, a 5%
CO.sub.2:95% air mixture is infused into a photobioreactor
containing Botryococcus cells (see for example J Agric Food Chem.
2006 Jun. 28; 54(13):4593-9; J Biosci Bioeng. 1999; 87(6):811-5;
and J Nat. Prod. 2003 June; 66(6):772-8).
[0208] Photobioreactors can be exposed to one or more light sources
to provide microalgae with light as an energy source via light
directed to a surface of the photobioreactor. Preferably the light
source provides an intensity that is sufficient for the cells to
grow, but not so intense as to cause oxidative damage or cause a
photoinhibitive response. In some instances a light source has a
wavelength range that mimics or approximately mimics the range of
the sun. In other instances a different wavelength range is used.
Photobioreactors can be placed outdoors or in a greenhouse or other
facility that allows sunlight to strike the surface. Preferred
photon intensities for species of the genus Botryococcus are
between 25 and 500 .mu.m.sup.-2 s.sup.-1 (see for example
Photosynth Res. 2005 June; 84(1-3):21-7).
[0209] Photobioreactors preferably have one or more ports that
allow media entry. It is not necessary that only one substance
enter or leave a port. For example, a port can be used to flow
culture media into the photobioreactor and then later can be used
for sampling, gas entry, gas exit, or other purposes. In some
instances a photobioreactor is filled with culture media at the
beginning of a culture and no more growth media is infused after
the culture is inoculated. In other words, the microalgal biomass
is cultured in an aqueous medium for a period of time during which
the microalgae reproduce and increase in number; however quantities
of aqueous culture medium are not flowed through the
photobioreactor throughout the time period. Thus in some
embodiments, aqueous culture medium is not flowed through the
photobioreactor after inoculation.
[0210] In other instances culture media can be flowed though the
photobioreactor throughout the time period during which the
microalgae reproduce and increase in number. In some embodiments
media is infused into the photobioreactor after inoculation but
before the cells reach a desired density. In other words, a
turbulent flow regime of gas entry and media entry is not
maintained for reproduction of microalgae until a desired increase
in number of said microalgae has been achieved.
[0211] Photobioreactors preferably have one or more ports that
allow gas entry. Gas can serve to both provide nutrients such as
CO.sub.2 as well as to provide turbulence in the culture media.
Turbulence can be achieved by placing a gas entry port below the
level of the aqueous culture media so that gas entering the
photobioreactor bubbles to the surface of the culture. One or more
gas exit ports allow gas to escape, thereby preventing pressure
buildup in the photobioreactor. Preferably a gas exit port leads to
a "one-way" valve that prevents contaminating microorganisms from
entering the photobioreactor. In some instances cells are cultured
in a photobioreactor for a period of time during which the
microalgae reproduce and increase in number, however a turbulent
flow regime with turbulent eddies predominantly throughout the
culture media caused by gas entry is not maintained for all of the
period of time. In other instances a turbulent flow regime with
turbulent eddies predominantly throughout the culture media caused
by gas entry can be maintained for all of the period of time during
which the microalgae reproduce and increase in number. In some
instances a predetermined range of ratios between the scale of the
photobioreactor and the scale of eddies is not maintained for the
period of time during which the microalgae reproduce and increase
in number. In other instances such a range can be maintained.
[0212] Photobioreactors preferably have at least one port that can
be used for sampling the culture. Preferably a sampling port can be
used repeatedly without altering compromising the axenic nature of
the culture. A sampling port can be configured with a valve or
other device that allows the flow of sample to be stopped and
started. Alternatively a sampling port can allow continuous
sampling. Photobioreactors preferably have at least one port that
allows inoculation of a culture. Such a port can also be used for
other purposes such as media or gas entry.
[0213] B. Heterotrophic Growth
[0214] As an alternative to photosynthetic growth of
microorganisms, as described above, some microorganisms can be
cultured under heterotrophic growth conditions in which a fixed
carbon source provides energy for growth and lipid
accumulation.
[0215] In one heterotrophic culture method in accordance with the
invention, the cost of biodiesel production, crude, partially
purified, or purified glycerol produced as a byproduct of lipid
transesterification can be employed as a feedstock for fermenting,
for example, lipid-producing microbial cultures. Thus, the
invention encompasses culturing a microbe (e.g., a microalgae) in a
first microbial culture; recovering microbial lipid from the
culture; subjecting the microbial lipid to transesterification to
produce fatty acid ester(s) and glycerol, as described above; and
adding the glycerol to a second microbial culture as a feedstock.
The first and second microbial cultures can, but need not, be
cultures of the same microbe. If desired, a continuous system can
be devised whereby glycerol produced from the lipid recovered from
a culture can be fed back into the same culture.
[0216] The invention provides significantly improved culture
parameters incorporating the use of glycerol for fermentation of
multiple genera of both eukaryotic and prokaryotic microbes,
including microbes of the genera Chlorella, Navicula, Scenedesmus,
and Spirulina. As the Examples demonstrate, microbes of extremely
divergent evolutionary lineages, including Chlorella, Navicula,
Scenedesmus, and Spirulina as well as cultures of multiple distinct
Chlorella species and strains grow very well on not only purified
reagent-grade glycerol, but also on acidulated and non-acidulated
glycerol byproduct from biodiesel transesterification. In some
instances microalgae, such as Chlorella strains, undergo cell
division faster in the presence of glycerol than in the presence of
glucose. In these instances, two-stage growth processes in which
cells are first fed glycerol to rapidly increase cell density, and
are then fed glucose to accumulate lipids can improve the
efficiency with which lipids are produced. The use of the glycerol
byproduct of the transesterification process provides significant
economic advantages when put back into the production process.
Other feeding methods are provided as well, such as mixtures of
glycerol and glucose. Feeding such mixtures also captures the same
economic benefits. In addition, the invention provides methods of
feeding alternative sugars to microalgae such as sucrose in various
combinations with glycerol. These benefits provided by the
invention have been demonstrated herein on microbes from extremely
divergent evolutionary lineages, including both prokaryotes and
eukaryotes, demonstrating the utility of the invention for
microbial fermentation.
[0217] Standard methods for the growth and propagation of Chlorella
protothecoides are known (see for example Miao and Wu, J.
Biotechnology, 2004, 11:85-93 and Miao and Wu, Biosource Technology
(2006) 97:841-846). The invention also provides novel growth
conditions for Chlorella. For example, multiple species of
Chlorella and multiple strains within a species can be grown in the
presence of glycerol, including glycerol byproduct from biodiesel
transesterification.
[0218] For hydrocarbon production, cells, including recombinant
cells of the invention described herein, are preferably cultured or
fermented in large quantities. The culturing may be in large liquid
volumes, such as in suspension cultures as an example. Other
examples include starting with a small culture of cells which
expand into a large biomass in combination with cell growth and
propagation as well as hydrocarbon production. Bioreactors or steel
fermentors can be used to accommodate large culture volumes. A
fermentor similar those used in the production of beer and/or wine
is suitable, as are extremely large fermentors used in the
production of ethanol.
[0219] Appropriate nutrient sources for culture in a fermentor are
provided. These include raw materials such as one or more of the
following: a fixed carbon source such as glucose, corn starch,
depolymerized cellulosic material, sucrose, sugar cane, sugar beet,
lactose, milk whey, or molasses; a fat source, such as fats or
vegetable oils; a nitrogen source, such as protein, soybean meal,
cornsteep liquor, ammonia (pure or in salt form), nitrate or
nitrate salt, or molecular nitrogen; and a phosphorus source, such
as phosphate salts. Additionally, a fermentor allows for the
control of culture conditions such as temperature, pH, oxygen
tension, and carbon dioxide levels. Optionally, gaseous components,
like oxygen or nitrogen, can be bubbled through a liquid culture.
Other Starch (glucose) sources such as wheat, potato, rice, and
sorghum. Other carbon sources include process streams such as
technical grade glycerol, black liquor, organic acids such as
acetate, and molasses. Carbon sources can also be provided as a
mixture, such as a mixture of sucrose and depolymerized sugar beet
pulp.
[0220] A fermentor can be used to allow cells to undergo the
various phases of their growth cycle. As an example, an inoculum of
hydrocarbon-producing cells can be introduced into a medium
followed by a lag period (lag phase) before the cells begin growth.
Following the lag period, the growth rate increases steadily and
enters the log, or exponential, phase. The exponential phase is in
turn followed by a slowing of growth due to decreases in nutrients
and/or increases in toxic substances. After this slowing, growth
stops, and the cells enter a stationary phase or steady state,
depending on the particular environment provided to the cells.
[0221] Hydrocarbon production by cells disclosed herein can occur
during the log phase or thereafter, including the stationary phase
wherein nutrients are supplied, or still available, to allow the
continuation of hydrocarbon production in the absence of cell
division.
[0222] Preferably, microorganisms grown using conditions described
herein and known in the art comprise at least about 20% by weight
of lipid, preferably at least about 40% by weight, more preferably
at least about 50% by weight, and most preferably at least about
60% by weight.
[0223] A surprising discovery is that multiple species, and
multiple strains within a species of Chlorella perform better in
the presence of glycerol byproduct from transesterification than in
an equivalent amount of reagent grade glycerol. Glycerol byproduct
from transesterification usually contains residual methanol and
other contaminants in addition to glycerol. For example, FIGS. 1-6
demonstrate that strains of Chlorella protothecoides and Chlorella
kessleri exhibit better productivity on acidulated and
non-acidulated glycerol byproduct from lipid transesterification
reactions than when grown on pure reagent grade glycerol. Other
microbes, such as Scenedesmus and Navicula microalgae can also
perform better in the presence of glycerol byproduct from
transesterification than in an equivalent amount of reagent grade
glycerol.
[0224] Dry Cell Weight per Liter: FIG. 1 demonstrates that dry cell
weight was higher on biodiesel glycerol byproduct than on pure
glycerol, and this trend held true when the cells were grown in
glycerol by itself or in combination with glucose. FIG. 2 shows the
same trends with additional strains of Chlorella. FIG. 12(b)
demonstrates that dry cell weight per liter of Scenedesmus armatus
is higher on acidulated and non-acidulated biodiesel byproducts
glycerol than on pure reagent grade glycerol. FIG. 13 demonstrates
that dry cell weight per liter of Navicula pelliculosa is higher on
non-acidulated biodiesel byproduct glycerol than on pure reagent
grade glycerol.
[0225] Lipid Content per liter: FIGS. 3 and 4 demonstrates that
with multiple species of Chlorella and multiple strains within a
species of Chlorella, lipid levels per liter are higher when the
cells are cultured in the presence of biodiesel glycerol byproduct
than when cultured in the presence of equivalent concentrations of
pure reagent grade glycerol.
[0226] Lipid as a Percentage of Cell Weight: FIGS. 5 and 6
demonstrate that multiple species of Chlorella and multiple strains
within a species of Chlorella accumulate a higher percentage of dry
cell weight as lipid when cultured in the presence of biodiesel
glycerol byproduct than when cultured in the presence of equivalent
concentrations of pure reagent grade glycerol. FIG. 11 demonstrates
that both Spirulina platensis and Navicula pelliculosa can
accumulate a higher percentage of dry cell weight as lipid when
cultured in the presence of biodiesel glycerol byproduct than when
cultured in the presence of equivalent concentrations of pure
reagent grade glycerol. FIG. 12(a) demonstrates that Scenedesmus
armatus can accumulate a higher percentage of dry cell weight as
lipid when cultured in the presence of biodiesel glycerol byproduct
than when cultured in the presence of equivalent concentrations of
pure reagent grade glycerol.
[0227] Another surprising result is that multiple species of
microbes, including microalgae such as Chlorella and multiple
strains within a species of Chlorella, and other microalgae such as
Scenedesmus, Navicula, and Spirulina exhibit better characteristics
as biodiesel producers in the presence of mixtures of glycerol and
glucose than in the presence of only glucose.
[0228] Lipid Content per liter: FIG. 7 demonstrates that Chlorella
can accumulate higher levels of lipid per liter of culture in the
presence of 1% glycerol/1% glucose than in the presence of 2%
glucose.
[0229] Dry Cell Weight per Liter: FIG. 12(b) demonstrates that dry
cell weight per liter of Scenedesmus armatus is higher when
cultured in the presence of 1% biodiesel byproduct glycerol/1%
glucose than in the presence of 2% glucose. FIG. 13 demonstrates
that dry cell weight per liter of Navicula pelliculosa is higher
when cultured in the presence of 1% biodiesel byproduct glycerol/1%
glucose than in the presence of 2% glucose.
[0230] Lipid as a Percentage of Cell Weight: FIG. 8 demonstrates
that Chlorella can accumulate a higher percentage of dry cell
weight as lipid when cultured in the presence of an equal
concentration (weight percent) mixture of glycerol and glucose than
when cultured in the presence of only glucose. FIG. 11(a)
demonstrates that Spirulina platensis can accumulate a higher
percentage of dry cell weight as lipid when cultured in the
presence of an equal concentration (weight percent) mixture of
biodiesel byproduct glycerol and glucose than when cultured in the
presence of only glucose. FIG. 11(b) demonstrates that Navicula
pelliculosa can accumulate a higher percentage of dry cell weight
as lipid when cultured in the presence of an equal concentration
(weight percent) mixture of reagent grade glycerol and glucose, as
well as biodiesel byproduct glycerol and glucose, than when
cultured in the presence of only glucose. FIG. 12(b) demonstrates
that Scenedesmus armatus can accumulate a higher percentage of dry
cell weight as lipid when cultured in the presence of an equal
concentration (weight percent) mixture of biodiesel byproduct
glycerol and glucose than when cultured in the presence of only
glucose.
[0231] An additional and unexpected discovery is that adding
glycerol and glucose to microbes, including microalgae such as
Chlorella, Scenedesmus, and Navicula sequentially rather than as a
single batch mixture of glycerol and glucose can generate
additional yield gains. This attribute of multiple species of
Chlorella and multiple strains within a species of Chlorella was
tested in the presence of both biodiesel glycerol byproduct and
reagent grade glycerol.
[0232] Lipid as a Percentage of Cell Weight: FIG. 8 demonstrates
that Chlorella can accumulate a higher percentage of dry cell
weight as lipid when glycerol is added to a culture for a first
period of time, followed by addition of glucose and continued
culturing for a second period of time, than when the same
quantities of glycerol and glucose are added together at the
beginning of the experiment.
[0233] Lipid Content per liter: FIG. 9 shows Chlorella exhibiting
higher levels of lipid per liter of culture in when glycerol and
glucose are added sequentially than when the same quantities of
glycerol and glucose are added together at the beginning of the
experiment. This trend was observed when acidulated biodiesel
byproduct glycerol, non-acidulated biodiesel byproduct glycerol, or
reagent grade glycerol was used.
[0234] Dry Cell Weight per Liter: FIG. 10 demonstrates four
different strains of Chlorella of two different species
accumulating higher dry cell weight per liter of culture when
glycerol and glucose are added sequentially than when the same
quantities of glycerol and glucose are added together at the
beginning of the experiment. This trend was observed when
acidulated biodiesel byproduct glycerol, non-acidulated biodiesel
byproduct glycerol, or reagent grade glycerol was used. FIGS. 14(a)
and (b) demonstrates that both Scenedesmus armatus and Navicula
pelliculosa can exhibit increases in dry cell weight per liter when
biodiesel byproduct glycerol only is added to a culture for a first
period of time, followed later by addition of glucose, compared to
adding identical amounts of glycerol and glucose at the beginning
of the fermentation.
[0235] Three different markers of productivity (dry cell weight per
liter, grams per liter of lipid, and percentage of dry cell weight
as lipid) in microbial lipid production are improved by the use of
biodiesel byproduct and temporal separation of carbon sources. The
invention therefore provides novel methods of generating higher
quantities of lipid per unit time in multiple species of microbes
from highly divergent areas of the evolutionary tree, including
both prokaryotes and eukaryotes. The methods of manufacturing
lipids and hydrocarbons disclosed herein using glycerol are not
limited to microalgae, but can be used with any microbe capable of
utilizing glycerol as an energy source.
[0236] In an alternate heterotrophic growth method in accordance
with the present invention, microorganisms can be cultured using
depolymerized cellulosic biomass as a feedstock. Cellulosic biomass
(e.g., stover, such as corn stover) is inexpensive and readily
available; however, attempts to use this material as a feedstock
for yeast have failed. In particular, such feedstock have been
found to be inhibitory to yeast growth, and yeast cannot use the
5-carbon sugars produced from cellulosic materials (e.g., xylose
from hemi-cellulose). By contrast, microalgae can grow on processed
cellulosic material. Accordingly, the invention provides a method
of culturing a microalgae in the presence of a cellulosic material
and/or a 5-carbon sugar. Cellulosic materials generally
include:
TABLE-US-00004 Component Percent Dry Weight Cellulose 40-60%
Hemicellulose 20-40% Lignin 10-30%
[0237] Suitable cellulosic materials include residues from
herbaceous and woody energy crops, as well as agricultural crops,
i.e., the plant parts, primarily stalks and leaves, not removed
from the fields with the primary food or fiber product. Examples
include agricultural wastes such as sugarcane bagasse, rice hulls,
corn fiber (including stalks, leaves, husks, and cobs), wheat
straw, rice straw, sugar beet pulp, citrus pulp, citrus peels;
forestry wastes such as hardwood and softwood thinnings, and
hardwood and softwood residues from timber operations; wood wastes
such as saw mill wastes (wood chips, sawdust) and pulp mill waste;
urban wastes such as paper fractions of municipal solid waste,
urban wood waste and urban green waste such as municipal grass
clippings; and wood construction waste. Additional cellulosics
include dedicated cellulosic crops such as switchgrass, hybrid
poplar wood, and miscanthus, fiber cane, and fiber sorghum.
Five-carbon sugars that are produced from such materials include
xylose.
[0238] Surprisingly, some species of Chlorella have been shown
herein to exhibit higher levels of productivity when cultured on a
combination of glucose and xylose than when cultured on either
glucose or xylose alone. This synergistic effect provides a
significant advantage in that it allows cultivation of Chlorella on
combinations of xylose and glucose, such as cellulosic material,
and is shown in FIG. 15.
[0239] In still another alternative heterotrophic growth method in
accordance with the present invention, which itself may optionally
be used in combination with the methods described above, sucrose,
produced by example from sugar cane or sugar beet, is used as a
feedstock. As described in greater detail in the section entitled
"Microbe Engineering" below, lipid production can be facilitated or
made more efficient through the engineering of microbes such as
Chlorella, to utilize sucrose as a carbon source. For example,
expression of a sucrose transporter and a sucrose invertase allows
Chlorella to transport sucrose into the cell from the culture media
and hydrolyze sucrose to yield glucose and fructose. Optionally, a
fructokinase can be expressed as well in instances where endogenous
hexokinase activity is insufficient for maximum phosphorylation of
fructose. Examples of suitable sucrose transporters are Genbank
accession numbers CAD91334, CAB92307, and CAA53390. Examples of
suitable sucrose invertases are Genbank accession numbers CAB95010,
NP.sub.--012104 and CAA06839. Examples of suitable fructokinases
are Genbank accession numbers P26984, P26420 and CAA43322. Vectors
for transformation of microalgae, including Chlorella, encoding one
or more of such genes can be designed as described herein.
[0240] Secretion of a sucrose invertase can obviate the need for
expression of a transporter that can transport sucrose into the
cell. This is because a secreted invertase catalyzes the conversion
of a molecule of sucrose into a molecule of glucose and a molecule
of fructose, both of which can be transported and utilized by
microbes disclosed herein. For example, expression of a sucrose
invertase (such as SEQ ID NO:14) with a secretion signal (such as
that of SEQ ID NO:15 (from yeast), SEQ ID NO:16 (from higher
plants), SEQ ID NO:17 (eukaryotic consensus secretion signal), and
SEQ ID NO:18 (combination of signal sequence from higher plants and
eukaryotic consensus) generates invertase activity outside the
cell. See Hawkins et al., Current Microbiology Vol. 38 (1999), pp.
335-341 for examples of secretion signals active in Chlorella.
Expression of such a protein, as enabled by the genetic engineering
methodology disclosed herein, allows cells already capable of
utilizing extracellular glucose as an energy source to utilize
sucrose as an extracellular energy source. In cells such as
Chlorella protothecoides, Chlorella minutissima, and Chlorella
emersonii which as demonstrated herein can use both extracellular
fructose and extracellular glucose as an energy source, secretion
of an invertase can provide the sole catalytic activity necessary
for use of sucrose as an efficient, inexpensive energy source.
[0241] For example, as shown in FIG. 26, Chlorella protothecoides
can be engineered with a sucrose invertase gene under the
regulatory control of one of three promoters (Cauliflower mosaic
virus 35S promoter (CMV), Chlorella virus promoter (CV), or
Chlorella HUP1 promoter (HUP1)). The sucrose invertase gene used in
this example comprises a modification to the S. cerevisiae SUC2
gene to optimize for C. protothecoides codon usage. The cDNA and
amino acid sequences of the optimized gene correspond to SEQ ID
NO:8 and SEQ ID NO:19, respectively. An illustration of the plasmid
constructs used in the transformation is shown in FIG. 25.
Expression of a secretable sucrose invertase, such as that
described herein, permits the use of molasses, sugar cane juice,
and other sucrose-containing feedstocks for cell fermentation.
[0242] Similarly, FIGS. 27 and 28 show the results of
transformation of Chlorella protothecoides, and Chlorella
minutissima and Chlorella emersonii, respectively, with the sucrose
invertase gene from S. cerevisiae under the control of the CMV
promoter.
[0243] The growth potential of microorganisms expressing an
exogenous secretable sucrose invertase is illustrated by the
addition of an invertase to the culture medium of Chlorella
protothecoides, as described in further detail in the Examples.
FIGS. 23 and 24 illustrate the surprising result that Chlorella
cells grow as well on waste molasses from sugar cane processing as
they do on pure reagent-grade glucose; the use of this low-value
waste product of sugar cane processing can provide significant cost
savings in the production of hydrocarbons and other oils. Molasses
contains lignin and other cellulosic waste products that poison
many microorganisms and retard their growth, however it was
discovered that Chlorella cells thrive in the presence of such
poisons. FIGS. 23-24 show the growth of cells on three unique
sources of molasses (designated BS1, BS2 and HTM), as compared to
growth on glucose or sucrose in the presence or absence of an
extracellular sucrose invertase.
[0244] Alternatively, a sucrose invertase can also be expressed
intracellularly in cells that express a sucrose transporter, as
well as in cells that express any carbohydrate transporter that
allows sucrose to enter the cell.
[0245] A foreign gene was transformed into and expressed in
Chlorella protothecoides, as described in Example 12. Expression of
sucrose utilization genes can be accomplished using the same or
similar methodology and vector design.
[0246] Bioreactors can be employed for use in heterotrophic growth
methods. As will be appreciated, provisions made to make light
available to the cells in photosynthetic growth methods are
unnecessary when using a fixed-carbon source in the heterotrophic
growth methods described herein.
[0247] The specific examples of process conditions and
heterotrophic growth methods described herein can be combined in
any suitable manner to improve efficiencies of microbial growth and
lipid production. In addition, the invention includes the selection
and/or genetic engineering of microbes, such as microalgae, to
produce microbes that are even more suitable for use in the
above-described methods. For example, the microbes having a greater
ability to utilize any of the above-described feedstocks for
increased proliferation and/or lipid (e.g., fatty acid) production
are within the scope of the invention.
[0248] C. Mixotrophic Growth
[0249] Mixotrophic growth is the use of both light and fixed carbon
source(s) as energy sources for cells to grow and produce
hydrocarbons. Mixotrophic growth can be conducted in a
photobioreactor. Microalgae can be grown and maintained in closed
photobioreactors made of different types of transparent or
semitransparent material. Such material can include Plexiglas.RTM.
enclosures, glass enclosures, bags made from substances such as
polyethylene, transparent or semitransparent pipes, and other
materials. Microalgae can be grown and maintained in open
photobioreactors such as raceway ponds, settling ponds, and other
non-enclosed containers.
[0250] D. Growth Media
[0251] Microorganisms useful in accordance with the methods of the
present invention are found in various locations and environments
throughout the world. As a consequence of their isolation from
other species and their resulting evolutionary divergence, the
particular growth medium for optimal growth and generation of lipid
and/or hydrocarbon constituents can be difficult to predict. In
some cases, certain strains of microorganisms may be unable to grow
on a particular growth medium because of the presence of some
inhibitory component or the absence of some essential nutritional
requirement required by the particular strain of microorganism.
[0252] Solid and liquid growth media are generally available from a
wide variety of sources, and instructions for the preparation of
particular media that is suitable for a wide variety of strains of
microorganisms can be found, for example, online at
http://www.utex.org/, a site maintained by the University of Texas
at Austin for its culture collection of algae (UTEX). For example,
various fresh water and salt water media include those shown in
Table 4, below.
TABLE-US-00005 TABLE 4 Exemplary Algal Media. Fresh Water Media
Salt Water Media 1/2 CHEV Diatom Medium 1% F/2 1/3 CHEV Diatom
Medium 1/2 Enriched Seawater Medium 1/5 CHEV Diatom Medium 1/2
Erdschreiber Medium 1:1 DYIII/PEA + Gr+ 1/2 Soil + Seawater Medium
2/3 CHEV Diatom Medium 1/3 Soil + Seawater Medium 2X CHEV Diatom
Medium 1/4 ERD Ag Diatom Medium 1/4 Soil + Seawater Medium Allen
Medium 1/5 Soil + Seawater Medium BG11-1 Medium 2/3 Enriched
Sewater Medium Bold 1NV Medium 20% Allen + 80% ERD Bold 3N Medium
2X Erdschreiber's Medium Botryococcus Medium 2X Soil + Seawater
Medium Bristol Medium 5% F/2 Medium CHEV Diatom Medium 5/3 Soil +
Seawater Agar Medium Chu's Medium Artificial Seawater Medium CR1
Diatom Medium BG11-1 + .36% NaCl Medium CR1+ Diatom Medium BG11-1 +
1% NaCl Medium CR1-S Diatom Medium Bold 1NV:Erdshreiber (1:1)
Cyanidium Medium Bold 1NV:Erdshreiber (4:1) Cyanophycean Medium
Bristol-NaCl Medium Desmid Medium Dasycladales Seawater Medium
DYIII Medium Enriched Seawater Medium Euglena Medium Erdschreiber's
Medium HEPES Medium ES/10 Enriched Seawater Medium J Medium ES/2
Enriched Seawater Medium Malt Medium ES/4 Enriched Seawater Medium
MES Medium F/2 Medium Modified Bold 3N Medium F/2 + NH4 Modified
COMBO Medium LDM Medium N/20 Medium Modified 2 X CHEV Ochromonas
Medium Modified 2 X CHEV + Soil P49 Medium Modified Artificial
Seawater Medium Polytomella Medium Modified CHEV Proteose Medium
Porphridium Medium Snow Algae Media Soil + Seawater Medium Soil
Extract Medium SS Diatom Medium Soilwater: BAR Medium Soilwater:
GR- Medium Soilwater: GR-/NH4 Medium Soilwater: GR+ Medium
Soilwater: GR+/NH4 Medium Soilwater: PEA Medium Soilwater: Peat
Medium Soilwater: VT Medium Spirulina Medium Tap Medium Trebouxia
Medium Volvocacean Medium Volvocacean-3N Medium Volvox Medium
Volvox-Dextrose Medium Waris Medium Waris + Soil Extract Medium
[0253] In a particular example, a medium suitable for culturing
Chlorella protothecoides (UTEX 31) comprises Proteose Medium. This
medium is suitable for axenic cultures, and a 1 L volume of the
medium (pH .about.6.8) can be prepared by addition of 1 g of
proteose peptone to 1 liter of Bristol Medium. Bristol medium
comprises 2.94 mM NaNO.sub.3, 0.17 mM CaCl.sub.2.2H.sub.2O, 0.3 mM
MgSO.sub.4.7H.sub.2O, 0.43 mM, 1.29 mM KH.sub.2PO.sub.4, and 1.43
mM NaCl in an aqueous solution. For 1.5% agar medium, 15 g of agar
can be added to 1 L of the solution. The solution is covered and
autoclaved, and then stored at a refrigerated temperature prior to
use.
[0254] Other suitable media for use with the methods of the
invention can be readily identified by consulting the URL
identified above, or by consulting other organizations that
maintain cultures of microorganisms, such as SAG, CCAP, or CCALA.
SAG refers to the Culture Collection of Algae at the University of
Gottingen (Gottingen, Germany), CCAP refers to the culture
collection of algae and protozoa managed by the Scottish
Association for Marine Science (Scotland, United Kingdom), and
CCALA refers to the culture collection of algal laboratory at the
Institute of Botany (T{hacek over (r)}ebo{hacek over (n)}, Czech
Republic).
[0255] E. Increasing Yield of Lipids
[0256] Process conditions can be adjusted to increase the yield of
lipids suitable for a particular use and/or to reduce production
cost. For example, in certain embodiments, a microbe (e.g., a
microalgae) is cultured in the presence of a limiting concentration
of one or more nutrients, such as, for example, carbon and/or
nitrogen, phosphorous, or sulfur, while providing an excess of
fixed carbon energy such as glucose. Nitrogen limitation tends to
increase microbial lipid yield over microbial lipid yield in a
culture in which nitrogen is provided in excess. In particular
embodiments, the increase in lipid yield is at least about: 10%,
20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500%. The
microbe can be cultured in the presence of a limiting amount of a
nutrient for a portion of the total culture period or for the
entire period. In particular embodiments, the nutrient
concentration is cycled between a limiting concentration and a
non-limiting concentration at least twice during the total culture
period.
[0257] To increase lipid yield, acetic acid can be employed in the
feedstock for a lipid-producing microbe (e.g., a microalgae).
Acetic acid feeds directly into the point of metabolism that
initiates fatty acid synthesis (i.e., acetyl-CoA); thus providing
acetic acid in the culture can increase fatty acid production.
Generally, the microbe is cultured in the presence of a sufficient
amount of acetic acid to increase microbial lipid yield, and/or
microbial fatty acid yield, specifically, over microbial lipid
(e.g., fatty acid) yield in the absence of acetic acid.
[0258] In another embodiment, lipid yield is increased by culturing
a lipid-producing microbe (e.g., microalgae) in the presence of one
or more cofactor(s) for a lipid pathway enzyme (e.g., a fatty acid
synthetic enzyme). Generally, the concentration of the cofactor(s)
is sufficient to increase microbial lipid (e.g., fatty acid) yield
over microbial lipid yield in the absence of the cofactor(s). In a
particular embodiment, the cofactor(s) are provided to the culture
by including in the culture a microbe (e.g., microalgae) containing
an exogenous gene encoding the cofactor(s). Alternatively,
cofactor(s) may be provided to a culture by including a microbe
(e.g., microalgae) containing an exogenous gene that encodes a
protein that participates in the synthesis of the cofactor. In
certain embodiments, suitable cofactors include any vitamin
required by a lipid pathway enzyme, such as, for example: biotin,
pantothenate. Genes encoding cofactors suitable for use in the
invention or that participate in the synthesis of such cofactors
are well known and can be introduced into microbes (e.g.,
microalgae), using contructs and techniques such as those described
above.
V. Lipid Pathway Engineering
[0259] In some embodiments of the present invention, microorganisms
of the present invention are modified to alter the properties
and/or proportions of lipids produced and/or to increase carbon
flux into lipids. The pathway can further, or alternatively, be
modified to alter the properties and/or proportions of various
hydrocarbon molecules produced through enzymatic processing of
lipids.
[0260] A. Alteration of Properties or Proportions of Lipids or
Hydrocarbons Produced
[0261] In the case of microalgae, some wild-type cells already have
good growth characteristics but do not produce the desired types or
quantities of lipids. Examples include Pyrobotrys, Phormidium,
Agmenellum, Carteria, Lepocinclis, Pyrobotrys, Nitzschia,
Lepocinclis, Anabaena, Euglena, Spirogyra, Chlorococcum,
Tetraedron, Oscillatoria, Phagus, and Chlorogonium, which have the
desirable growth characteristic of growing in municipal sewage or
wastewater. Such cells, as well as species of Chlorella and other
microbes, can be engineered to have improved lipid production
characteristics. Desired characteristics include optimizing lipid
yield per unit volume and/or per unit time, carbon chain length
(e.g., for biodiesel production or for industrial applications
requiring hydrocarbon feedstock), reducing the number of double or
triple bonds, optionally to zero, removing or eliminating rings and
cyclic structures, and increasing the hydrogen:carbon ratio of a
particular species of lipid or of a population of distinct lipid.
In addition, microalgae that produce appropriate hydrocarbons can
also be engineered to have even more desirable hydrocarbon outputs.
Examples of such microalgae include species of the genus
Chlorella.
[0262] 1. Regulation of Enzymes that Control Branch Points in Fatty
Acid Synthesis
[0263] In particular embodiments, one or more key enzymes that
control branch points in metabolism to fatty acid synthesis can be
up-regulated or down-regulated to improve lipid production.
Up-regulation can be achieved, for example, by transforming cells
with expression constructs in which a gene encoding the enzyme of
interest is expressed, e.g., using a strong promoter and/or
enhancer elements that increase transcription. Such constructs can
include a selectable marker such that the transformants can be
subjected to selection, which can result in amplification of the
construct and an increase in the expression level of the encoded
enzyme. Examples of enzymes suitable for up-regulation according to
the methods of the invention include pyruvate dehydrogenase, which
plays a role in converting pyruvate to acetyl-CoA (examples, some
from microalgae, include Genbank accession numbers NP.sub.--415392;
AAA53047; Q1XDM1; and CAF05587). Up-regulation of pyruvate
dehydrogenase can increase production of acetyl-CoA, and thereby
increase fatty acid synthesis. Acetyl-CoA carboxylase catalyzes the
initial step in fatty acid synthesis. Accordingly, this enzyme can
be up-regulated to increase production of fatty acids (examples,
some from microalgae, include Genbank accession numbers BAA94752;
AAA75528; AAA81471; YP.sub.--537052; YP.sub.--536879;
NP.sub.--045833; and BAA57908). Fatty acid production can also be
increased by up-regulation of acyl carrier protein (ACP), which
carries the growing acyl chains during fatty acid synthesis
(examples, some from microalgae, include Genbank accession numbers
A0T0F8; P51280; NP.sub.--849041; YP.sub.--874433).
Glycerol-3-phosphate acyltransferase catalyzes the rate-limiting
step of fatty acid synthesis. Up-regulation of this enzyme can
increase fatty acid production (examples, some from microalgae,
include Genbank accession numbers AAA74319; AAA33122; AAA37647;
P44857; and AB094442). The preceding proteins are candidates for
expression in microalge, including species of the genus
Chlorella.
[0264] Down-regulation of an enzyme of interest can achieved using,
e.g., antisense, catalytic RNA/DNA, RNA interference (RNA;),
"knock-out," "knock-down," or other mutagenesis techniques. Enzyme
expression/function can also be inhibited using intrabodies.
Examples of enzymes suitable for down-regulation according to the
methods of the invention include citrate synthase, which consumes
acetyl-CoA as part of the tricarboxylic acid (TCA) cycle.
Down-regulation of citrate synthase can force more acetyl-CoA into
the fatty acid synthetic pathway.
[0265] 2. Modulation of Global Regulators of Fatty Acid Synthetic
Genes
[0266] Global regulators modulate the expression of the genes of
the fatty acid biosynthetic pathways. Accordingly, one or more
global regulators of fatty acid synthesis can be up- or
down-regulated, as appropriate, to inhibit or enhance,
respectively, the expression of a plurality of fatty acid synthetic
genes and, ultimately, to increase lipid production. Examples
include sterol regulatory element binding proteins (SREBPs), such
as SREBP-1a and SREBP-1c (for examples see Genbank accession
numbers NP.sub.--035610 and Q9WTN3). Global regulators can be up-
or down-regulated, for example, as described above with respect to
regulation of control point enzymes.
[0267] 3. Regulation of Hydrocarbon Modification Enzymes
[0268] The methods of the invention also include transforming cells
with one or more genes encoding hydrocarbon modification enzymes
such as, for example, a fatty acyl-ACP thioesterase (see examples
in Table 5 with accession numbers), a fatty acyl-CoA/aldehyde
reductase (see examples in Table 6 with accession numbers), a fatty
acyl-CoA reductase (see examples in Table 7 with accession
numbers), a fatty aldehyde decarbonylase (see examples in Table 8
with accession numbers), a fatty aldehyde reductase, or a squalene
synthase gene (see GenBank Accession number AF205791). In some
embodiments, genes encoding a fatty acyl-ACP thioesterase and a
naturally co-expressed acyl carrier protein may be transformed into
a cell, optionally with one or more genes encoding other
hydrocarbon modification enzymes. In other embodiments, the ACP and
the fatty acyl-ACP thioesterase may have an affinity for one
another that imparts an advantage when the two are used together in
the microbes and methods of the present invention, irrespective of
whether they are or are not naturally co-expressed in a particular
tissue or organism. Thus, the present invention contemplates both
naturally co-expressed pairs of these enzymes as well as those that
share an affinity for interacting with one another to facilitate
cleavage of a length-specific carbon chain from the ACP.
[0269] In still other embodiments, an exogenous gene encoding a
desaturase can be transformed into the cell in conjunction with one
or more genes encoding other hydrocarbon modification enzymes in
order to provide modifications with respect to hydrocarbon
saturation. Stearoyl-ACP desaturase (see, e.g., GenBank Accession
numbers AAF15308; ABM45911; and AAY86086), for example, catalyzes
the conversion of stearoyl-ACP to oleoyl-ACP. Up-regulation of this
gene can increase the proportion of monounsaturated fatty acids
produced by a cell; whereas down-regulation can reduce the
proportion of monounsaturates. Similarly, the expression of one or
more glycerolipid desaturases can be controlled to alter the ratio
of unsaturated to saturated fatty acids such as .omega.-6 fatty
acid desaturase, .omega.-3 fatty acid desaturase, or
.omega.-6-oleate desaturase. In some embodiments, the desaturase
can be selected with reference to a desired carbon chain length,
such that the desaturase is capable of making location specific
modifications within a specified carbon-length substrate, or
substrates having a carbon-length within a specified range.
[0270] In particular embodiments, microbes of the present invention
are genetically engineered to express one or more exogenous genes
selected from a fatty acyl-ACP thioesterase, a fatty
acyl-CoA/aldehyde reductase, a fatty acyl-CoA reductase, a fatty
aldehyde reductase, a fatty aldehyde decarbonylase, or a naturally
co-expressed acyl carrier protein. Suitable expression methods are
described above with respect to the expression of a lipase gene,
including, among other methods, inducible expression and
compartmentalized expression.
[0271] Without intending to be bound by any particular theory or
cellular mechanism, a fatty acyl-ACP thioesterase cleaves a fatty
acid from an acyl carrier protein (ACP) during lipid synthesis.
Through further enzymatic processing, the cleaved fatty acid is
then combined with a coenzyme to yield an acyl-CoA molecule. This
acyl-CoA is the substrate for the enzymatic activity of a fatty
acyl-CoA reductase to yield an aldehyde, as well as for a fatty
acyl-CoA/aldehyde reductase to yield an alcohol. The aldehyde
produced by the action of the fatty acyl-CoA reductase identified
above is the substrate for further enzymatic activity by either a
fatty aldehyde reductase to yield an alcohol, or a fatty aldehyde
decarbonylase to yield an alkane or alkene.
[0272] The enzymes described directly above have a specificity for
acting on a substrate which includes a specific number of carbon
atoms. For example, a fatty acyl-ACP thioesterase may have a
specificity for cleaving a fatty acid having 12 carbon atoms from
the ACP. In some embodiments, the ACP and the length-specific
thioesterase may have an affinity for one another that makes them
particularly useful as a combination (e.g., the exogenous ACP and
thioesterase genes may be naturally co-expressed in a particular
tissue or organism from which they are derived). Therefore, in
various embodiments, the microbe can contain an exogenous gene that
encodes a protein with specificity for catalyzing an enzymatic
activity (e.g., cleavage of a fatty acid from an ACP, reduction of
an acyl-CoA to an aldehyde or an alcohol, or conversion of an
aldehyde to an alkane) with regard to the number of carbon atoms
contained in the substrate. The enzymatic specificity can, in
various embodiments, be for a substrate having from 8 to 34 carbon
atoms, preferably from 8 to 18 carbon atoms, and more preferably
from 10 to 14 carbon atoms. The most preferred specificity is for a
substrate having 12 carbon atoms. In other embodiments the
specificity can be for 20 to 30 carbon atoms.
[0273] Fatty acyl-ACP thioesterases suitable for use with the
microbes and methods of the invention include, without limitation,
those listed in Table 5.
TABLE-US-00006 TABLE 5 Fatty acyl-ACP thioesterases and GenBank
accession numbers. Umbellularia californica fatty acyl-ACP
thioesterase (GenBank #AAC49001) Cinnamomum camphora fatty acyl-ACP
thioesterase (GenBank #Q39473) Umbellularia californica fatty
acyl-ACP thioesterase (GenBank #Q41635) Myristica fragrans fatty
acyl-ACP thioesterase (GenBank #AAB71729) Myristica fragrans fatty
acyl-ACP thioesterase (GenBank #AAB71730) Elaeis guineensis fatty
acyl-ACP thioesterase (GenBank #ABD83939) Elaeis guineensis fatty
acyl-ACP thioesterase (GenBank #AAD42220) Populus tomentosa fatty
acyl-ACP thioesterase (GenBank #ABC47311) Arabidopsis thaliana
fatty acyl-ACP thioesterase (GenBank #NP_172327) Arabidopsis
thaliana fatty acyl-ACP thioesterase (GenBank #CAA85387)
Arabidopsis thaliana fatty acyl-ACP thioesterase (GenBank
#CAA85388) Gossypium hirsutum fatty acyl-ACP thioesterase (GenBank
#Q9SQI3) Cuphea lanceolata fatty acyl-ACP thioesterase (GenBank
#CAA54060) Cuphea hookeriana fatty acyl-ACP thioesterase (GenBank
#AAC72882) Cuphea calophylla subsp. mesostemon fatty acyl-ACP
thioesterase (GenBank #ABB71581) Cuphea lanceolata fatty acyl-ACP
thioesterase (GenBank #CAC19933) Elaeis guineensis fatty acyl-ACP
thioesterase (GenBank #AAL15645) Cuphea hookeriana fatty acyl-ACP
thioesterase (GenBank #Q39513) Gossypium hirsutum fatty acyl-ACP
thioesterase (GenBank #AAD01982) Vitis vinifera fatty acyl-ACP
thioesterase (GenBank #CAN81819) Garcinia mangostana fatty acyl-ACP
thioesterase (GenBank #AAB51525) Brassica juncea fatty acyl-ACP
thioesterase (GenBank #ABI18986) Madhuca longifolia fatty acyl-ACP
thioesterase (GenBank #AAX51637) Brassica napus fatty acyl-ACP
thioesterase (GenBank #ABH11710) Oryza sativa (indica
cultivar-group) fatty acyl-ACP thioesterase (GenBank #EAY86877)
Oryza sativa (japonica cultivar-group) fatty acyl-ACP thioesterase
(GenBank #NP_001068400) Oryza sativa (indica cultivar-group) fatty
acyl-ACP thioesterase (GenBank #EAY99617) Cuphea hookeriana fatty
acyl-ACP thioesterase (GenBank #AAC49269)
[0274] Fatty acyl-CoA/aldehyde reductases suitable for use with the
microbes and methods of the invention include, without limitation,
those listed in Table 6.
TABLE-US-00007 TABLE 6 Fatty acyl-CoA/aldehyde reductases listed by
GenBank accession numbers. AAC45217, YP_047869, BAB85476,
YP_001086217, YP_580344, YP_001280274, YP_264583, YP_436109,
YP_959769, ZP_01736962, ZP_01900335, ZP_01892096, ZP_01103974,
ZP_01915077, YP_924106, YP_130411, ZP_01222731, YP_550815,
YP_983712, YP_001019688, YP_524762, YP_856798, ZP_01115500,
YP_001141848, NP_336047, NP_216059, YP_882409, YP_706156,
YP_001136150, YP_952365, ZP_01221833, YP_130076, NP_567936,
AAR88762, ABK28586, NP_197634, CAD30694, NP_001063962, BAD46254,
NP_001030809, EAZ10132, EAZ43639, EAZ07989, NP_001062488, CAB88537,
NP_001052541, CAH66597, CAE02214, CAH66590, CAB88538, EAZ39844,
AAZ06658, CAA68190, CAA52019, and BAC84377
[0275] Fatty acyl-CoA reductases suitable for use with the microbes
and methods of the invention include, without limitation, those
listed in Table 7.
TABLE-US-00008 TABLE 7 Fatty acyl-CoA reductases listed by GenBank
accession numbers. NP_187805, ABO14927, NP_001049083, CAN83375,
NP_191229, EAZ42242, EAZ06453, CAD30696, BAD31814, NP_190040,
AAD38039, CAD30692, CAN81280, NP_197642, NP_190041, AAL15288, and
NP_190042
[0276] Fatty aldehyde decarbonylases suitable for use with the
microbes and methods of the invention include, without limitation,
those listed in Table 8.
TABLE-US-00009 TABLE 8 Fatty aldehyde decarbonylases listed by
GenBank accession numbers. NP_850932, ABN07985, CAN60676, AAC23640,
CAA65199, AAC24373, CAE03390, ABD28319, NP_181306, EAZ31322,
CAN63491, EAY94825, EAY86731, CAL55686, XP_001420263, EAZ23849,
NP_200588, NP_001063227, CAN83072, AAR90847, and AAR97643
[0277] Combinations of naturally co-expressed fatty acyl-ACP
thioesterases and acyl carrier proteins are suitable for use with
the microbes and methods of the invention.
[0278] Additional examples of hydrocarbon modification enzymes
include amino acid sequences contained in, referenced in, or
encoded by nucleic acid sequences contained or referenced in, any
of the following U.S. Pat. Nos.: 6,610,527; 6,451,576; 6,429,014;
6,342,380; 6,265,639; 6,194,185; 6,114,160; 6,083,731; 6,043,072;
5,994,114; 5,891,697; 5,871,988; 6,265,639, and further described
in GenBank Accession numbers: AA018435; ZP.sub.--00513891; Q38710;
AAK60613; AAK60610; AAK60611; NP.sub.--113747; CAB75874; AAK60612;
AAF20201; BAA11024; AF205791; and CAA03710.
[0279] Other suitable enzymes for use with the microbes and the
methods of the invention include those that have at least 70% amino
acid identity with one of the proteins listed in Tables 5-8, and
that exhibit the corresponding desired enzymatic activity (e.g.,
cleavage of a fatty acid from an acyl carrier protein, reduction of
an acyl-CoA to an aldehyde or an alcohol, or conversion of an
aldehyde to an alkane). In additional embodiments, the enzymatic
activity is present in a sequence that has at least about 75%, at
least about 80%, at least about 85%, at least about 90%, at least
about 95%, or at least about 99% identity with one of the above
described sequences, all of which are hereby incorporated by
reference as if fully set forth.
[0280] The hydrocarbon modification enzymes described above are
useful in the production of various hydrocarbons from a microbe
(e.g., a microalgae, an oleaginous yeast, or a fungus) or
population of microbes, whereby a fatty acyl-ACP thioesterase
cleaves a fatty acid from an acyl carrier protein (ACP) during
lipid synthesis. Through further enzymatic processing, the cleaved
fatty acid is then combined with a coenzyme to yield an acyl-CoA
molecule. This acyl-CoA is the substrate for the enzymatic activity
of a fatty acyl-CoA reductase to yield an aldehyde, as well as for
a fatty acyl-CoA/aldehyde reductase to yield an alcohol. The
aldehyde produced by the action of the fatty acyl-CoA reductase
identified above is the substrate for further enzymatic activity by
either a fatty aldehyde reductase to yield an alcohol, or a fatty
aldehyde decarbonylase to yield an alkane or alkene.
[0281] The hydrocarbon modification enzymes have a specificity for
acting on a substrate which includes a specific number of carbon
atoms. For example, a fatty acyl-ACP thioesterase may have a
specificity for cleaving a fatty acid having 12 carbon atoms from
the ACP. Therefore, in various embodiments, the microbe can contain
an exogenous gene that encodes a protein with specificity for
catalyzing an enzymatic activity (e.g., cleavage of a fatty acid
from an ACP, reduction of an acyl-CoA to an aldehyde or an alcohol,
or conversion of an aldehyde to an alkane) with regard to the
number of carbon atoms contained in the substrate. The enzymatic
specificity can, in various embodiments, be for a substrate having
from 8 to 34 carbon atoms, preferably from 8 to 18 carbon atoms,
and more preferably from 10 to 14 carbon atoms. The most preferred
specificity is for a substrate having 12 carbon atoms. In other
embodiments the specificity can be for 20 to 30 carbon atoms.
[0282] In some embodiments, fatty acids, or the corresponding
primary alcohols, aldehydes, alkanes or alkenes, generated by the
methods described herein, contain at least about 8, at least about
10, at least about 12, at least about 14, at least about 16, at
least about 18, at least about 20, at least about 22, at least
about 24, at least about 26, at least about 28, at least about 30,
at least about 32, or at least about 34 carbon atoms or more.
Preferred fatty acids for the produciton of biodiesel, renewable
diesel, or jet fuel, or the corresponding primary alcohols,
aldehydes, alkanes and alkenes, for industrial applications contain
at least about 8 carbon atoms or more. In certain embodiments, the
above fatty acids, as well as the other corresponding hydrocarbon
molecules, are saturated (with no carbon-carbon double or triple
bonds); mono unsaturated (single double bond); poly unsturated (two
or more double bonds); are linear (not cyclic); and/or have little
or no branching in their structures.
[0283] By selecting the desired combination of exogenous genes to
be expressed, one can tailor the product generated by the microbe,
which may then be extracted from the aqueous biomass. For example,
the microbe can contain: (i) an exogenous gene encoding a fatty
acyl-ACP thioesterase; and, optionally, (ii) a naturally
co-expressed acyl carrier protein or an acyl carrier protein
otherwise having affinity for the fatty acyl-ACP thioesterase (or
conversely); and, optionally, (iii) an exogenous gene encoding a
fatty acyl-CoA/aldehyde reductase or a fatty acyl-CoA reductase;
and, optionally, (iv) an exogenous gene encoding a fatty aldehyde
reductase or a fatty aldehyde decarbonylase. The microbe, when
cultured as described below, synthesizes a fatty acid linked to an
ACP and the fatty acyl-ACP thioesterase catalyzes the cleavage of
the fatty acid from the ACP to yield, through further enzymatic
processing, a fatty acyl-CoA molecule. When present, the fatty
acyl-CoA/aldehyde reducatase catalyzes the reduction of the
acyl-CoA to an alcohol. Similarly, the fatty acyl-CoA reductase,
when present, catalyzes the reduction of the acyl-CoA to an
aldehyde. In those embodiments in which an exogenous gene encoding
a fatty acyl-CoA reductase is present and expressed to yield an
aldehyde product, a fatty aldehyde reductase, encoded by the third
exogenous gene, catalyzes the reduction of the aldehyde to an
alcohol. Similarly, a fatty aldehyde decarbonylase catalyzes the
conversion of the aldehyde to an alkane or an alkene, when
present.
[0284] Genes encoding such enzymes can be obtained from cells
already known to exhibit significant lipid production such as
Chlorella protothecoides. Genes already known to have a role in
lipid production, e.g., a gene encoding an enzyme that saturates
double bonds, can be transformed individually into recipient cells.
However, to practice the invention it is not necessary to make a
priori assumptions as to which genes are required. A library of DNA
containing different genes, such as cDNAs from a good
lipid-production organism, can be transformed into recipient cells.
The cDNA is preferably in operable linkage with a promoter active
in microalgae. Different recipient microalgae cells transformed by
a library receive different genes from the library. Transformants
having improved lipid production are identified though screening
methods known in the art, such as, for example, HPLC, gas
chromatography, and mass spectrometry methods of hydrocarbon
analysis (for examples of such analysis, see Biomass and Bioenergy
Vol. 6. No. 4. pp. 269-274 (1994); Experientia 38; 47-49 (1982);
and Phytochemistry 65 (2004) 3159-3165). These transformants are
then subjected to further transformation with the original library
and/or optionally interbred to generate a further round of
organisms having improved lipid production. General procedures for
evolving whole organisms to acquire a desired property are
described in, e.g., U.S. Pat. No. 6,716,631. Such methods entail,
e.g., introducing a library of DNA fragments into a plurality of
cells, whereby at least one of the fragments undergoes
recombination with a segment in the genome or an episome of the
cells to produce modified cells. The modified cells are then
screened for modified cells that have evolved toward acquisition of
the desired function. Vectors and methods for transformation are
analogous to those discussed in connection with expression of
lipase genes.
[0285] Furthermore, subtractive libraries can be used to identify
genes whose transcription is induced under different conditions,
especially conditions employed in culturing microorganisms for
biodiesel production, or for the production of hydrocarbons useful
as a feedstock for industrial applications. Subtractive libraries
contain nucleotide sequences reflecting the differences between two
different samples. Such libraries are prepared by procedures that
include the steps of denaturing and hybridizing populations of
polynucleotides (e.g., mRNA, cDNA, amplified sequences) from each
sample. Sequences common to both samples hybridize and are removed,
leaving the sequences that differ between the samples. In this
manner, sequences that are induced under particular conditions can
be identified. This technique can be employed, for example, to
identify genes useful for increasing lipid (e.g., fatty acid)
production and, in particular, lipid production under any desired
culture conditions. The subtractive hybridization technique can
also be employed to identify promoters, e.g., inducible promoters,
useful in expression constructs according to the invention.
[0286] Thus, for example, subtractive libraries can be prepared
from microorganism cultures grown autotrophically (in the light
without a fixed carbon source) or heterotrophically (in the dark in
the presence of a fixed carbon source). In particular,
heterotrophic genes may be induced during dark growth in the
presence of a fixed carbon source and may therefore be present in a
library generated by subtracting sequences from autotrophic cells
from sequences from dark heterotrophic cells. Subtractive libraries
can also be prepared from cultures to which a particular carbon
substrate, such as glucose, has been added to identify genes that
play a role in metabolizing the substrate. Subtractive libraries
prepared from cultures grown in the presence of excess versus
limited nitrogen can be used to identify genes that control cell
division as opposed to hydrocarbon accumulation production. The
preparation of a subtractive library from a culture to which lipids
(e.g., fatty acids) have been added can help identify genes whose
overexpression increases fatty acid production. More specifically,
the addition of fatty acids to a culture of cells that can use the
added fatty acids will lead to the down-regulation of fatty acid
synthetic genes to down-regulate fatty acid production. The
overexpression of one or more such genes will have the opposite
effect.
[0287] B. Increased Carbon Flux into Lipid Pathway
[0288] Some microalgae produce significant quantities of non-lipid
metabolites, such as, for example, polysaccharides. Because
polysaccharide biosynthesis can use a significant proportion of the
total metabolic energy available to cells, mutagenesis of
lipid-producing cells followed by screening for reduced or
eliminated polysaccharide production generates novel strains that
are capable of producing higher yields of lipids.
[0289] The phenol: sulfuric acid assay detects carbohydrates (see
Hellebust, Handbook of Phycological Methods, Cambridge University
Press, 1978; and Cuesta G., et al., J Microbiol Methods. 2003
January; 52(1):69-73). The 1,6 dimethylmethylene blue assay detects
anionic polysaccharides. (see for example Braz J Med Biol Res. 1999
May; 32(5):545-50; Clin Chem. 1986 November; 32(11):2073-6).
[0290] Polysaccharides can also be analyzed through methods such as
HPLC, size exclusion chromatography, and anion exchange
chromatography (see for example Prosky L, Asp N, Schweizer T F,
DeVries J W & Furda I (1988) Determination of insoluble,
soluble and total dietary fiber in food and food products:
Interlaboratory study. Journal of the Association of Official
Analytical Chemists 71, 1017.+-.1023; Int J Biol Macromol. 2003
November; 33(1-3):9-18). Polysaccharides can also be detected using
gel electrophoresis (see for example Anal Biochem. 2003 Oct. 15;
321(2):174-82; Anal Biochem. 2002 Jan. 1; 300(1):53-68).
Vi. Methods of Recovering Lipids and Hydrocarbons
[0291] Hydrocarbons (e.g., lipids, fatty acids, aldehydes,
alcohols, and alkanes) produced by cells of the invention can be
harvested, or otherwise collected, by any convenient means. For
example, hydrocarbons secreted from cells can be centrifuged to
separate the hydrocarbons in a hydrophobic layer from contaminants
in an aqueous layer and optionally from any solid materials as a
precipitate in after centrifugation. Material containing cell or
cell fractions can be treated with proteases to degrade
contaminating proteins before or after centrifugation. In some
instances the contaminating proteins are associated, possibly
covalently, to hydrocarbons or hydrocarbon precursors which form
hydrocarbons upon removal of the protein. In other instances the
hydrocarbon molecules are in a preparation that also contains
proteins. Proteases can be added to hydrocarbon preparations
containing proteins to degrade proteins (for example, the protease
from Streptomyces griseus can be used (SigmaAldrich catalog number
P5147). After digestion, the hydrocarbons are preferably purified
from residual proteins, peptide fragments, and amino acids. This
purification can be accomplished, for example, by methods listed
above such as centrifugation and filtration.
[0292] Extracellular hydrocarbons can also be extracted in vivo
from living microalgae cells which are then returned to a
bioreactor by exposure of the cells, in an otherwise sterile
environment, to a non-toxic extraction solvent, followed by
separation of the living cells and the hydrophobic fraction of
extraction solvent and hydrocarbons, wherein the separated living
cells are then returned to a culture container such as a stainless
steel fermentor or photobioreactor (see Biotechnol Bioeng. 2004
Dec. 5; 88(5):593-600 and Biotechnol Bioeng. 2004 Mar. 5;
85(5):475-81).
[0293] Hydrocarbons can also be isolated by whole cell extraction.
The cells are first disrupted, as described in the section entitled
"Lysing Cells", and then intracellular and cell membrane/cell
wall-associated hydrocarbons as well as extracellular hydrocarbons
can be collected from the whole cell mass, such as by use of
centrifugation as described above.
[0294] Various methods are available for separating hydrocarbons
and lipids from cellular lysates produced by the above methods. For
example, hydrocarbons can be extracted with a hydrophobic solvent
such as hexane (see Frenz et al. 1989, Enzyme Microb. Technol.,
11:717). Hydrocarbons can also be extracted using liquefaction (see
for example Sawayama et al. 1999, Biomass and Bioenergy 17:33-39
and Inoue et al. 1993, Biomass Bioenergy 6(4):269-274); oil
liquefaction (see for example Minowa et al. 1995, Fuel
74(12):1735-1738); and supercritical CO.sub.2 extraction (see for
example Mendes et al. 2003, Inorganica Chimica Acta
356:328-334).
[0295] Miao and Wu describe a protocol of the recovery of
microalgal lipid from a culture of Chlorella prototheocoides in
which the cells were harvested by centrifugation, washed with
distilled water and dried by freeze drying. The resulting cell
powder was pulverized in a mortor and then extracted with n-hexane.
Miao and Wu, Biosource Technology (2006) 97:841-846.
[0296] A. Lysing Cells
[0297] Intracellular lipids and hydrocarbons produced in
microorganisms are, in some embodiments, extracted after lysing the
cells of the microorganism. Once extracted, the lipids and/or
hydrocarbons can be further refined to produce oils, fuels, or
oleochemicals.
[0298] After completion of culturing, the microorganisms can be
separated from the fermentation broth. Optionally, the separation
is effected by centrifugation to generate a concentrated paste.
Centrifugation does not remove significant amounts of intracellular
water from the microorganisms and is not a drying step. The biomass
can then be washed with a washing solution (e.g., DI water) to get
rid of the fermentation broth and debris. Optionally, the washed
microbial biomass may also be dried (oven dried, luyophilized,
etc.) prior to cell disruption. Alternatively, cells can be lysed
without separation from some or all of the fermentation broth when
the fermentation is complete. For example, the cells can be at a
ratio of less than 1:1 v:v cells to extracellular liquid when the
cells are lysed.
[0299] Microorganisms containing a lipid and/or hydrocarbon can be
lysed to produce a lysate. As detailed herein, the step of lysing a
microorganism (also referred to as cell lysis) can be achieved by
any convenient means, including heat-induced lysis, adding a base,
adding an acid, using enzymes such as proteases and polysaccharide
degradation enzymes such as amylases, using ultrasound, mechanical
lysis, using osmotic shock, infection with a lytic virus, and/or
expression of one or more lytic genes. Lysis is performed to
release intracellular molecules which have been produced by the
microorganism. Each of these methods for lysing a microorganism can
be used as a single method or in combination simultaneously or
sequentially.
[0300] The extent of cell disruption can be observed by microscopic
analysis. Using one or more of the methods described herein,
typically more than 70% cell breakage is observed. Preferably, cell
breakage is more than 80%, more preferably more than 90% and most
preferred about 100%.
[0301] In particular embodiments, the microorganism is lysed after
growth, for example to increase the exposure of cellular lipid
and/or hydrocarbon for extraction or further processing. The timing
of lipase expression (e.g., via an inducible promoter) or cell
lysis can be adjusted to optimize the yield of lipids and/or
hydrocarbons. Below are described a number of lysis techniques.
These techniques can be used individually or in combination.
[0302] 1. Heat-Induced Lysis
[0303] In a preferred embodiment of the present invention, the step
of lysing a microorganism comprises heating of a cellular
suspension containing the microorganism. In this embodiment, the
fermentation broth containing the microorganisms (or a suspension
of microorganisms isolated from the fermentation broth) is heated
until the microorganisms, i.e., the cell walls and membranes of
microorganisms degrade or breakdown. Typically, temperatures
applied are at least 50.degree. C. Higher temperatures, such as, at
least 30.degree. C. at least 60.degree. C., at least 70.degree. C.,
at least 80.degree. C., at least 90.degree. C., at least
100.degree. C., at least 110.degree. C., at least 120.degree. C.,
at least 130.degree. C. or higher are used for more efficient cell
lysis.
[0304] Lysing cells by heat treatment can be performed by boiling
the microorganism. Alternatively, heat treatment (without boiling)
can be performed in an autoclave. The heat treated lysate may be
cooled for further treatment.
[0305] Cell disruption can also be performed by steam treatment,
i.e., through addition of pressurized steam. Steam treatment of
microalgae for cell disruption is described, for example, in U.S.
Pat. No. 6,750,048.
[0306] 2. Lysis Using a Base
[0307] In another preferred embodiment of the present invention,
the step of lysing a microorganism comprises adding a base to a
cellular suspension containing the microorganism.
[0308] The base should be strong enough to hydrolyze at least a
portion of the proteinaceous compounds of the microorganisms used.
Bases which are useful for solubilizing proteins are known in the
art of chemistry. Exemplary bases which are useful in the methods
of the present invention include, but are not limited to,
hydroxides, carbonates and bicarbonates of lithium, sodium,
potassium, calcium, and mixtures thereof. A preferred base is KOH.
Base treatment of microalgae for cell disruption is described, for
example, in U.S. Pat. No. 6,750,048.
[0309] 3. Acidic Lysis
[0310] In another preferred embodiment of the present invention,
the step of lysing a microorganism comprises adding an acid to a
cellular suspension containing the microorganism. Acid lysis can be
effected using an acid at a concentration of 10-500 mN or
preferably 40-160 nM. Acid lysis is preferably performed at above
room temperature (e.g., at 40-160.degree., and preferably a
temperature of 50-130.degree.. For moderate temperatures (e.g.,
room temperature to 100.degree. C. and particularly room
temperature to 65.degree., acid treatment can usefully be combined
with sonication or other cell disruption methods.
[0311] 4. Lysing Cells Using Enzymes
[0312] In another preferred embodiment of the present invention,
the step of lysing a microorganism comprises lysing the
microorganism by using an enzyme. Preferred enzymes for lysing a
microorganism are proteases and polysaccharide-degrading enzymes
such as hemicellulase (e.g., hemicellulase from Aspergillus niger;
Sigma Aldrich, St. Louis, Mo.; #H2125), pectinase (e.g., pectinase
from Rhizopus sp.; Sigma Aldrich, St. Louis, Mo.; #P2401), Mannaway
4.0 L (Novozymes), cellulase (e.g., cellulose from Trichoderma
viride; Sigma Aldrich, St. Louis, Mo.; #C9422), and driselase
(e.g., driselase from Basidiomycetes sp.; Sigma Aldrich, St. Louis,
Mo.; #D9515.
[0313] a) Cellulases
[0314] In a preferred embodiment of the present invention, a
cellulase for lysing a microorganism is a polysaccharide-degrading
enzyme, optionally from Chlorella or a Chlorella virus.
[0315] b) Proteases
[0316] Proteases such as Streptomyces griseus protease,
chymotrypsin, proteinase K, proteases listed in Degradation of
Polylactide by Commercial Proteases, Oda Y et al., Journal of
Polymers and the Environment, Volume 8, Number 1, January 2000, pp.
29-32(4), and other proteases can be used to lyse microorganisms.
Other proteases that can be used include Alcalase 2.4 FG
(Novozymes) and Flavourzyme 100 L (Novozymes).
[0317] c) Combinations
[0318] Any combination of a protease and a polysaccharide-degrading
enzyme can also be used, including any combination of the preceding
proteases and polysaccharide-degrading enzymes.
[0319] 5. Lysing Cells Using Ultrasound
[0320] In another preferred embodiment of the present invention,
the step of lysing a microorganism is performed by using
ultrasound, i.e., sonication. Thus, cells can also by lysed with
high frequency sound. The sound can be produced electronically and
transported through a metallic tip to an appropriately concentrated
cellular suspension. This sonication (or ultrasonication) disrupts
cellular integrity based on the creation of cavities in cell
suspension.
[0321] 6. Mechanical Lysis
[0322] In another preferred embodiment of the present invention,
the step of lysing a microorganism is performed by mechanical
lysis. Cells can be lysed mechanically and optionally homogenized
to facilitate hydrocarbon (e.g., lipid) collection. For example, a
pressure disrupter can be used to pump a cell containing slurry
through a restricted orifice valve. High pressure (up to 1500 bar)
is applied, followed by an instant expansion through an exiting
nozzle. Cell disruption is accomplished by three different
mechanisms: impingement on the valve, high liquid shear in the
orifice, and sudden pressure drop upon discharge, causing an
explosion of the cell. The method releases intracellular
molecules.
[0323] Alternatively, a ball mill can be used. In a ball mill,
cells are agitated in suspension with small abrasive particles,
such as beads. Cells break because of shear forces, grinding
between beads, and collisions with beads. The beads disrupt the
cells to release cellular contents. Cells can also be disrupted by
shear forces, such as with the use of blending (such as with a high
speed or Waring blender as examples), the french press, or even
centrifugation in case of weak cell walls, to disrupt cells.
[0324] 7. Lysing Cells By Osmotic Shock (Cytolysis)
[0325] In another preferred embodiment of the present invention,
the step of lysing a microorganism is performed by applying an
osmotic shock.
[0326] 8. Infection With A Lytic Virus
[0327] In a preferred embodiment of the present invention, the step
of lysing a microorganism comprises infection of the microorganism
with a lytic virus. A wide variety of viruses are known to lyse
microorganisms suitable for use in the present invention, and the
selection and use of a particular lytic virus for a particular
microorganism is within the level of skill in the art.
[0328] For example, paramecium bursaria chlorella virus (PBCV-1) is
the prototype of a group (family Phycodnaviridae, genus
Chlorovirus) of large, icosahedral, plaque-forming, double-stranded
DNA viruses that replicate in, and lyse, certain unicellular,
eukaryotic chlorella-like green algae. Accordingly, any susceptible
microalgae can be lysed by infecting the culture with a suitable
chlorella virus. Methods of infecting species of Chlorella with a
chlorella virus are known. See for example Adv. Virus Res. 2006;
66:293-336; Virology, 1999 Apr. 25; 257(1):15-23; Virology, 2004
Jan. 5; 318(1):214-23; Nucleic Acids Symp. Ser. 2000;(44):161-2; J.
Virol. 2006 March; 80(5):2437-44; and Annu. Rev. Microbiol. 1999;
53:447-94.
[0329] 9. Autolysis (Expression of a Lytic Gene)
[0330] In another preferred embodiment of the present invention,
the step of lysing a microorganism comprises autolysis. In this
embodiment, a microorganism according to the invention is
genetically engineered to produce a lytic protein that will lyse
the microorganism. This lytic gene can be expressed using an
inducible promoter so that the cells can first be grown to a
desirable density in a fermentor, followed by induction of the
promoter to express the lytic gene to lyse the cells. In one
embodiment, the lytic gene encodes a polysaccharide-degrading
enzyme.
[0331] In certain other embodiments, the lytic gene is a gene from
a lytic virus. Thus, for example, a lytic gene from a Chlorella
virus can be expressed in an algal cell of the genus Chlorella,
such as C. protothecoides.
[0332] Suitable expression methods are described herein with
respect to the expression of a lipase gene. Expression of lytic
genes is preferably done using an inducible promoter, such as a
promoter active in microalgae that is induced by a stimulus such as
the presence of a small molecule, light, heat, and other stimuli.
Lytic genes from chlorella viruses are known. For example, see
Virology 260, 308-315 (1999); FEMS Microbiology Letters 180 (1999)
45-53; Virology 263, 376-387 (1999); and Virology 230, 361-368
(1997).
[0333] B. Extraction of Lipids and Hydrocarbons
[0334] Lipids and hydrocarbons generated by the microorganisms of
the present invention can be recovered by extraction with an
organic solvent. In some cases, the preferred organic solvent is
hexane. Typically, the organic solvent is added directly to the
lysate without prior separation of the lysate components. In one
embodiment, the lysate generated by one or more of the methods
described above is contacted with an organic solvent for a period
of time sufficient to allow the lipid and/or hydrocarbon components
to form a solution with the organic solvent. In some cases, the
solution can then be further refined to recover specific desired
lipid or hydrocarbon components. Hexane extraction methods are well
known in the art.
VII. Methods of Processing Lipids and Hydrocarbons
[0335] A. Enzymatic Modification
[0336] Hydrocarbons (e.g., lipids, fatty acids, aldehydes,
alcohols, and alkanes) produced by cells as described herein can be
modified by the use of one or more enzymes, including a lipase, as
described above. When the hydrocarbons are in the extracellular
environment of the cells, the one or more enzymes can be added to
that environment under conditions in which the enzyme modifies the
hydrocarbon or completes its synthesis from a hydrocarbon
precursor. Alternatively, the hydrocarbons can be partially, or
completely, isolated from the cellular material before addition of
one or more catalysts such as enzymes. Such catalysts are
exogenously added, and their activity occurs outside the cell or in
vitro.
[0337] B. Thermal and Other Catalytic Modification
[0338] Hydrocarbons produced by cells in vivo, or enzymatically
modified in vitro, as described herein can be optionally further
processed by conventional means. The processing can include
"cracking" to reduce the size, and thus increase the
hydrogen:carbon ratio, of hydrocarbon molecules. Catalytic and
thermal cracking methods are routinely used in hydrocarbon and
triglyceride oil processing. Catalytic methods involve the use of a
catalyst, such as a solid acid catalyst. The catalyst can be
silica-alumina or a zeolite, which result in the heterolytic, or
asymmetric, breakage of a carbon-carbon bond to result in a
carbocation and a hydride anion. These reactive intermediates then
undergo either rearrangement or hydride transfer with another
hydrocarbon. The reactions can thus regenerate the intermediates to
result in a self-propagating chain mechanism. Hydrocarbons can also
be processed to reduce, optionally to zero, the number of
carbon-carbon double, or triple, bonds therein. Hydrocarbons can
also be processed to remove or eliminate a ring or cyclic structure
therein. Hydrocarbons can also be processed to increase the
hydrogen:carbon ratio. This can include the addition of hydrogen
("hydrogenation") and/or the "cracking" of hydrocarbons into
smaller hydrocarbons.
[0339] Thermal methods involve the use of elevated temperature and
pressure to reduce hydrocarbon size. An elevated temperature of
about 800.degree. C. and pressure of about 700 kPa can be used.
These conditions generate "light," a term that is sometimes used to
refer to hydrogen-rich hydrocarbon molecules (as distinguished from
photon flux), while also generating, by condensation, heavier
hydrocarbon molecules which are relatively depleted of hydrogen.
The methodology provides homolytic, or symmetrical, breakage and
produces alkenes, which may be optionally enzymatically saturated
as described above.
[0340] Catalytic and thermal methods are standard in plants for
hydrocarbon processing and oil refining. Thus hydrocarbons produced
by cells as described herein can be collected and processed or
refined via conventional means. See Hillen et al. (Biotechnology
and Bioengineering, Vol. XXIV:193-205 (1982)) for a report on
hydrocracking of microalgae-produced hydrocarbons. In alternative
embodiments, the fraction is treated with another catalyst, such as
an organic compound, heat, and/or an inorganic compound. For
processing of lipids into biodiesel, a transesterification process
is used as described in Section IV herein.
[0341] Hydrocarbons produced via methods of the present invention
are useful in a variety of industrial applications. For example,
the production of linear alkylbenzene sulfonate (LAS), an anionic
surfactant used in nearly all types of detergents and cleaning
preparations, utilizes hydrocarbons generally comprising a chain of
10-14 carbon atoms. See, for example, U.S. Pat. Nos. 6,946,430;
5,506,201; 6,692,730; 6,268,517; 6,020,509; 6,140,302; 5,080,848;
and 5,567,359. Surfactants, such as LAS, can be used in the
manfacture of personal care compositions and detergents, such as
those described in U.S. Pat. Nos. 5,942,479; 6,086,903; 5,833,999;
6,468,955; and 6,407,044.
Viii. Methods of Producing Fuels Suitable for Use in Diesel
Vehicles and Jet Engines
[0342] Increasing interest is directed to the use of hydrocarbon
components of biological origin in fuels, such as biodiesel,
renewable diesel, and jet fuel, since renewable biological starting
materials that may replace starting materials derived from fossil
fuels are available, and the use thereof is desirable. There is an
urgent need for methods for producing hydrocarbon components from
biological materials. The present invention fulfills this need by
providing methods for production of biodiesel, renewable diesel,
and jet fuel using the lipids generated by the methods described
herein as a biological material to produce biodiesel, renewable
diesel, and jet fuel.
[0343] Traditional diesel fuels are petroleum distillates rich in
paraffinic hydrocarbons. They have boiling ranges as broad as
370.degree. to 780.degree. F., which are suitable for combustion in
a compression ignition engine, such as a diesel engine vehicle. The
American Society of Testing and Materials (ASTM) establishes the
grade of diesel according to the boiling range, along with
allowable ranges of other fuel properties, such as cetane number,
cloud point, flash point, viscosity, aniline point, sulfur content,
water content, ash content, copper strip corrosion, and carbon
residue. Technically, any hydrocarbon distillate material derived
from biomass or otherwise that meets the appropriate ASTM
specification can be defined as diesel fuel (ASTM D975), jet fuel
(ASTM D1655), or as biodiesel (ASTM D6751).
[0344] After extraction, lipid and/or hydrocarbon components
recovered from the microbial biomass described herein can be
subjected to chemical treatment to manufacture a fuel for use in
diesel vehicles and jet engines.
[0345] A. Biodiesel
[0346] Biodiesel is a liquid which varies in color--between golden
and dark brown--depending on the production feedstock. It is
practically immiscible with water, has a high boiling point and low
vapor pressure. Biodiesel refers to a diesel-equivalent processed
fuel for use in diesel-engine vehicles. Biodiesel is biodegradable
and non-toxic. An additional benefit of biodiesel over conventional
diesel fuel is lower engine wear.
[0347] Typically, biodiesel comprises C14-C18 alkyl esters. Various
processes convert biomass or a lipid produced and isolated as
described herein to diesel fuels. A preferred method to produce
biodiesel is by transesterification of a lipid as described herein.
A preferred alkyl ester for use as biodiesel is a methyl ester or
ethyl ester.
[0348] Biodiesel produced by a method described herein can be used
alone or blended with conventional diesel fuel at any concentration
in most modern diesel-engine vehicles. When blended with
conventional diesel fuel (petroleum diesel), biodiesel may be
present from about 0.1% to about 99.9%. Much of the world uses a
system known as the "B" factor to state the amount of biodiesel in
any fuel mix. For example, fuel containing 20% biodiesel is labeled
B20. Pure biodiesel is referred to as B 100.
[0349] Biodiesel can also be used as a heating fuel in domestic and
commercial boilers. Existing oil boilers may contain rubber parts
and may require conversion to run on biodiesel. The conversion
process is usually relatively simple, involving the exchange of
rubber parts for synthetic parts due to biodiesel being a strong
solvent. Due to its strong solvent power, burning biodiesel will
increase the efficiency of boilers.
[0350] Biodiesel can be used as an additive in formulations of
diesel to increase the lubricity of pure Ultra-Low Sulfur Diesel
(ULSD) fuel, which is advantageous because it has virtually no
sulfur content.
[0351] Biodiesel is a better solvent than petrodiesel and can be
used to break down deposits of residues in the fuel lines of
vehicles that have previously been run on petrodiesel.
[0352] 1. Production Of Biodiesel
[0353] Biodiesel can be produced by transesterification of
triglycerides contained in oil-rich biomass. Thus, in another
aspect of the present invention a method for producing biodiesel is
provided. In a preferred embodiment, the method for producing
biodiesel comprises the steps of (a) cultivating a lipid-containing
microorganism using methods disclosed herein (b) lysing a
lipid-containing microorganism to produce a lysate, (c) isolating
lipid from the lysed microorganism, and (d) transesterifying the
lipid composition, whereby biodiesel is produced.
[0354] Methods for growth of a microorganism, lysing a
microorganism to produce a lysate, treating the lysate in a medium
comprising an organic solvent to form a heterogeneous mixture and
separating the treated lysate into a lipid composition have been
described above and can also be used in the method of producing
biodiesel.
[0355] Lipid compositions can be subjected to transesterification
to yield long-chain fatty acid esters useful as biodiesel.
Preferred transesterification reactions are outlined below and
include base catalyzed transesterification and transesterification
using recombinant lipases.
[0356] In a base-catalyzed transesterification process, the
triacylglycerides are reacted with an alcohol, such as methanol or
ethanol, in the presence of an alkaline catalyst, typically
potassium hydroxide. This reaction forms methyl or ethyl esters and
glycerin (glycerol) as a byproduct.
[0357] a). General Chemical Process
[0358] Animal and plant oils are typically made of triglycerides
which are esters of free fatty acids with the trihydric alcohol,
glycerol. In transesterification, the glycerol in a
triacylglyceride (TAG) is replaced with a short-chain alcohol such
as methanol or ethanol. A typical reaction scheme is as
follows:
##STR00001##
[0359] In this scheme, the alcohol is deprotonated with a base to
make it a stronger nucleophile. Commonly, ethanol or methanol is
used in vast excess (up to 50-fold). Normally, this reaction will
proceed either exceedingly slowly or not at all. Heat, as well as
an acid or base can be used to help the reaction proceed more
quickly. The acid or base are not consumed by the
transesterification reaction, thus they are not reactants but
catalysts. Almost all biodiesel has been produced using the
base-catalyzed technique as it requires only low temperatures and
pressures and produces over 98% conversion yield (provided the
starting oil is low in moisture and free fatty acids).
[0360] b). Using Recombinant Lipases
[0361] Transesterification has also been carried out experimentally
using an enzyme, such as a lipase instead of a base.
Lipase-catalyzed transesterification can be carried out, for
example, at a temperature between the room temperature and
80.degree. C., and a mole ratio of the TAG to the lower alcohol of
greater than 1:1, preferably about 3:1.
[0362] Lipases suitable for use in transesterification include, but
are not limited to, those listed in Table 9. Other examples of
lipases useful for transesterification are found in, e.g. U.S. Pat.
Nos. 4,798,793; 4,940,845 5,156,963; 5,342,768; 5,776,741 and
WO89/01032.
TABLE-US-00010 TABLE 9 Lipases suitable for use in
transesterification. Aspergillus niger lipase ABG73614, Candida
antarctica lipase B (novozym-435) CAA83122, Candida cylindracea
lipase AAR24090, Candida lipolytica lipase (Lipase L; Amano
Pharmaceutical Co., Ltd.), Candida rugosa lipase (e.g., Lipase-OF;
Meito Sangyo Co., Ltd.), Mucor miehei lipase (Lipozyme IM 20),
Pseudomonas fluorescens lipase AAA25882, Rhizopus japonicas lipase
(Lilipase A-10FG) Q7M4U7_1, Rhizomucor miehei lipase B34959,
Rhizopus oryzae lipase (Lipase F) AAF32408, Serratia marcescens
lipase (SM Enzyme) ABI13521, Thermomyces lanuginosa lipase
CAB58509, Lipase P (Nagase ChemteX Corporation), and Lipase QLM
(Meito Sangyo Co., Ltd., Nagoya, Japan)
[0363] One challenge to using a lipase for the production of fatty
acid esters suitable for biodiesel is that the price of lipase is
much higher than the price of sodium hydroxide (NaOH) used by the
strong base process. This challenge has been addressed by using an
immobilized lipase, which can be recycled. However, the activity of
the immobilized lipase must be maintained after being recycled for
a minimum number of cycles to allow a lipase-based process to
compete with the strong base process in terms of the production
cost. Immobilized lipases are subject to poisoning by the lower
alcohols typically used in transesterification. U.S. Pat. No.
6,398,707 (issued Jun. 4, 2002 to Wu et al.) describes methods for
enhancing the activity of immobilized lipases and regenerating
immobilized lipases having reduced activity.
[0364] In particular embodiments, a recombinant lipase is expressed
in the same microorganisms that produce the lipid on which the
lipase acts. Suitable recombinant lipases include those listed
above in Table 9 and/or having GenBank Accession numbers listed
above in Table 9, or a polypeptide that has at least 70% amino acid
identity with one of the lipases listed above in Table 9 and that
exhibits lipase activity. In additional embodiments, the enzymatic
activity is present in a sequence that has at least about 75%, at
least about 80%, at least about 85%, at least about 90%, at least
about 95%, or at least about 99% identity with one of the above
described sequences, all of which are hereby incorporated by
reference as if fully set forth. DNA encoding the lipase and
selectable marker is preferably codon-optimized cDNA. Methods of
recoding genes for expression in microalgae are described in U.S.
Pat. No. 7,135,290.
[0365] 2. Standards
[0366] The common international standard for biodiesel is EN 14214.
ASTM D6751 is the most common biodiesel standard referenced in the
United States and Canada. Germany uses DIN EN 14214 and the UK
requires compliance with BS EN 14214.
[0367] Basic industrial tests to determine whether the products
conform to these standards typically include gas chromatography,
HPLC, and others. Biodiesel meeting the quality standards is very
non-toxic, with a toxicity rating (LD.sub.50) of greater than 50
mL/kg.
[0368] B. Renewable Diesel
[0369] Renewable diesel comprises alkanes, such as C16:0 and C18:0
and thus, are distinguishable from biodiesel. High quality
renewable diesel conforms to the ASTM D975 standard.
[0370] The lipids produced by the methods of the present invention
can serve as feedstock to produce renewable diesel. Thus, in
another aspect of the present invention, a method for producing
renewable diesel is provided. Renewable diesel can be produced by
at least three processes: hydrothermal processing (hydrotreating);
hydroprocessing; and indirect liquefaction. These processes yield
non-ester distillates. During these processes, triacylglycerides
produced and isolated as described herein, are converted to
alkanes.
[0371] In a preferred embodiment, the method for producing
renewable diesel comprises (a) cultivating a lipid-containing
microorganism using methods disclosed herein (b) lysing the
microorganism to produce a lysate, (c) isolating lipid from the
lysed microorganism, and (d) deoxygenating and hydrotreating the
lipid to produce an alkane, whereby renewable diesel is produced.
Lipids suitable for manufacturing renewable diesel can be obtained
via extraction from microbial biomass using an organic solvent such
as hexane, or via other methods, such as those described in U.S.
Pat. No. 5,928,696.
[0372] In some methods, the microbial lipid is first cracked in
conjunction with hydrotreating to reduce carbon chain length and
saturate double bonds, respectively. The material is then
isomerized, also in conjunction with hydrotreating. The naptha
fraction can then be removed through distillation, followed by
additional distillation to vaporize and distill components desired
in the diesel fuel to meet a D975 standard while leaving components
that are heavier than desired for meeting a D 975 standard.
Hydrotreating, hydrocracking, deoxygenation and isomerization
methods of chemically modifying oils, including triglyceride oils,
are well known in the art. See for example European patent
applications EP1741768 (A1); EP1741767 (A1); EP1682466 (A1);
EP1640437 (A1); EP1681337 (A1); EP1795576 (A1); and U.S. Pat. Nos.
7,238,277; 6,630,066; 6,596,155; 6,977,322; 7,041,866; 6,217,746;
5,885,440; 6,881,873.
[0373] 1. Hydrotreating
[0374] In a preferred embodiment of the method for producing
renewable diesel, treating the lipid to produce an alkane is
performed by hydrotreating of the lipid composition. In
hydrothermal processing, typically, biomass is reacted in water at
an elevated temperature and pressure to form oils and residual
solids. Conversion temperatures are typically 300.degree. to
660.degree. F., with pressure sufficient to keep the water
primarily as a liquid, 100 to 170 standard atmosphere (atm).
Reaction times are on the order of 15 to 30 minutes. After the
reaction is completed, the organics are separated from the water.
Thereby a distillate suitable for diesel is produced.
[0375] 2. Hydroprocessing
[0376] A renewable diesel, referred to as "green diesel," can be
produced from fatty acids by traditional hydroprocessing
technology. The triglyceride-containing oils can be hydroprocessed
either as co-feed with petroleum or as a dedicated feed. The
product is a diesel fuel that conforms with the ASTM D975
specification. Thus, in another preferred embodiment of the method
for producing renewable diesel, treating the lipid composition to
produce an alkane is performed by hydroprocessing of the lipid
composition.
[0377] In some methods of making renewable diesel, the first step
of treating a triglyceride is hydroprocessing to saturate double
bonds, followed by deoxygenation at elevated temperature in the
presence of hydrogen and a catalyst. In some methods, hydrogenation
and deoxygenation occur in the same reaction. In other methods
deoxygenation occurs before hydrogenation. Isomerization is then
optionally performed, also in the presence of hydrogen and a
catalyst. Naphtha components are preferably removed through
distillation. For examples, see U.S. Pat. Nos. 5,475,160
(hydrogenation of triglycerides); 5,091,116 (deoxygenation,
hydrogenation and gas removal); 6,391,815 (hydrogenation); and
5,888,947 (isomerization).
[0378] Petroleum refiners use hydroprocessing to remove impurities
by treating feeds with hydrogen. Hydroprocessing conversion
temperatures are typically 300.degree. to 700.degree. F. Pressures
are typically 40 to 100 atm. The reaction times are typically on
the order of 10 to 60 minutes.
[0379] Solid catalysts are employed to increase certain reaction
rates, improve selectivity for certain products, and optimize
hydrogen consumption.
[0380] Hydrotreating and hydroprocessing ultimately lead to a
reduction in the molecular weight of the feed. In the case of
triglyceride-containing oils, the triglyceride molecule is reduced
to four hydrocarbon molecules under hydroprocessing conditions: a
propane molecule and three heavier hydrocarbon molecules, typically
in the C8 to C18 range.
[0381] 3. Indirect Liquefaction
[0382] A traditional ultra-low sulfur diesel can be produced from
any form of biomass by a two-step process. First, the biomass is
converted to a syngas, a gaseous mixture rich in hydrogen and
carbon monoxide. Then, the syngas is catalytically converted to
liquids. Typically, the production of liquids is accomplished using
Fischer-Tropsch (FT) synthesis. This technology applies to coal,
natural gas, and heavy oils. Thus, in yet another preferred
embodiment of the method for producing renewable diesel, treating
the lipid composition to produce an alkane is performed by indirect
liquefaction of the lipid composition.
[0383] C. Jet Fuel
[0384] The annual U.S. usage of jet fuel in 2006 was about 21
billion gallons (about 80 billion liters). Aeroplane fuel is clear
to straw colored. The most common fuel is an unleaded/paraffin
oil-based fuel classified as Aeroplane A-1, which is produced to an
internationally standardized set of specifications. Aeroplane fuel
is a mixture of a large number of different hydrocarbons, possibly
as many as a thousand or more. The range of their sizes (molecular
weights or carbon numbers) is restricted by the requirements for
the product, for example, freezing point or smoke point.
Kerosone-type Aeroplane fuel (including Jet A and Jet A-1) has a
carbon number distribution between about 8 and 16 carbon numbers.
Wide-cut or naphta-type Aeroplane fuel (including Jet B) typically
has a carbon number distribution between about 5 and 15
carbons.
[0385] Both Aeroplanes (Jet A and jet B) may contain a number of
additives. Useful additives include, but are not limited to,
antioxidants, antistatic agents, corrosion inhibitors, and fuel
system icing inhibitor (FSII) agents. Antioxidants prevent gumming
and usually, are based on alkylated phenols, for example, AO-30,
AO-31, or AO-37. Antistatic agents dissipate static electricity and
prevent sparking. Stadis 450 with dinonylnaphthylsulfonic acid
(DINNSA) as the active ingredient, is an example. Corrosion
inhibitors, e.g., DCI-4A is used for civilian and military fuels
and DCI-6A is used for military fuels. FSII agents, include, e.g.,
Di-EGME.
[0386] A solution is blending algae fuels with existing jet fuel.
The present invention provides such a solution. The lipids produced
by the methods of the present invention can serve as feedstock to
produce jet fuel. Thus, in another aspect of the present invention,
a method for producing jet fuel is provided. Herewith two methods
for producing jet fuel from the lipids produced by the methods of
the present invention are provided: fluid catalytic cracking (FCC);
and hydrodeoxygenation (HDO).
[0387] 1. Fluid Catalytic Cracking
[0388] Fluid Catalytic Cracking (FCC) is one method which is used
to produce olefins, especially propylene from heavy crude
fractions. There are reports in the literature that vegetable oils
such as canola oil could be processed using FCC to give a
hydrocarbon stream useful as a gasoline fuel.
[0389] The lipids produced by the method of the present invention
can be converted to olefins. The process involves flowing the
lipids produced through an FCC zone and collecting a product stream
comprised of olefins, which is useful as a jet fuel. The lipids
produced are contacted with a cracking catalyst at cracking
conditions to provide a product stream comprising olefins and
hydrocarbons useful as jet fuel.
[0390] In a preferred embodiment, the method for producing jet fuel
comprises (a) cultivating a lipid-containing microorganism using
methods disclosed herein, (b) lysing the lipid-containing
microorganism to produce a lysate, (c) isolating lipid from the
lysate, and (d) treating the lipid composition, whereby jet fuel is
produced.
[0391] In a preferred embodiment of the method for producing a jet
fuel, the lipid composition can be flowed through a fluid catalytic
cracking zone, which, in one embodiment, may comprise contacting
the lipid composition with a cracking catalyst at cracking
conditions to provide a product stream comprising C.sub.2-C.sub.5
olefins.
[0392] In certain embodiments of this method it may be desirable to
remove any contaminants that may be present in the lipid
composition. Thus, prior to flowing the lipid composition through a
fluid catalytic cracking zone, the lipid composition is pretreated.
Pretreatment may involve contacting the lipid composition with an
ion-exchange resin. The ion exchange resin is an acidic ion
exchange resin, such as Amberlyst.TM.-15 and can be used as a bed
in a reactor through which the lipid composition is flowed, either
upflow or downflow. Other pretreatments may include mild acid
washes by contacting the lipid composition with an acid, such as
sulfuric, acetic, nitric, or hydrochloric acid. Contacting is done
with a dilute acid solution usually at ambient temperature and
atmospheric pressure.
[0393] The lipid composition, optionally pretreated, is flowed to
an FCC zone where the hydrocarbonaceous components are cracked to
olefins. Catalytic cracking is accomplished by contacting the lipid
composition in a reaction zone with a catalyst composed of finely
divided particulate material. The reaction is catalytic cracking,
as opposed to hydrocracking, and is carried out in the absence of
added hydrogen or the consumption of hydrogen. As the cracking
reaction proceeds, substantial amounts of coke are deposited on the
catalyst. The catalyst is regenerated at high temperatures by
burning coke from the catalyst in a regeneration zone.
Coke-containing catalyst, referred to herein as "coked catalyst",
is continually transported from the reaction zone to the
regeneration zone to be regenerated and replaced by essentially
coke-free regenerated catalyst from the regeneration zone.
Fluidization of the catalyst particles by various gaseous streams
allows the transport of catalyst between the reaction zone and
regeneration zone. Methods for cracking hydrocarbons, such as those
of the lipid composition described herein, in a fluidized stream of
catalyst, transporting catalyst between reaction and regeneration
zones, and combusting coke in the regenerator are well known by
those skilled in the art of FCC processes. Exemplary FCC
applications and catalysts useful for cracking the lipid
composition to produce C.sub.2-C.sub.5 olefins are described in
U.S. Pat. Nos. 6,538,169, 7,288,685, which are incorporated in
their entirety by reference.
[0394] In one embodiment, cracking the lipid composition of the
present invention, takes place in the riser section or,
alternatively, the lift section, of the FCC zone. The lipid
composition is introduced into the riser by a nozzle resulting in
the rapid vaporization of the lipid composition. Before contacting
the catalyst, the lipid composition will ordinarily have a
temperature of about 149.degree. C. to about 316.degree. C.
(300.degree. F. to 600.degree. F.). The catalyst is flowed from a
blending vessel to the riser where it contacts the lipid
composition for a time of abort 2 seconds or less.
[0395] The blended catalyst and reacted lipid composition vapors
are then discharged from the top of the riser through an outlet and
separated into a cracked product vapor stream including olefins and
a collection of catalyst particles covered with substantial
quantities of coke and generally referred to as "coked catalyst."
In an effort to minimize the contact time of the lipid composition
and the catalyst which may promote further conversion of desired
products to undesirable other products, any arrangement of
separators such as a swirl arm arrangement can be used to remove
coked catalyst from the product stream quickly. The separator, e.g.
swirl arm separator, is located in an upper portion of a chamber
with a stripping zone situated in the lower portion of the chamber.
Catalyst separated by the swirl arm arrangement drops down into the
stripping zone. The cracked product vapor stream comprising cracked
hydrocarbons including light olefins and some catalyst exit the
chamber via a conduit which is in communication with cyclones. The
cyclones remove remaining catalyst particles from the product vapor
stream to reduce particle concentrations to very low levels. The
product vapor stream then exits the top of the separating vessel.
Catalyst separated by the cyclones is returned to the separating
vessel and then to the stripping zone. The stripping zone removes
adsorbed hydrocarbons from the surface of the catalyst by
counter-current contact with steam.
[0396] Low hydrocarbon partial pressure operates to favor the
production of light olefins. Accordingly, the riser pressure is set
at about 172 to 241 kPa (25 to 35 psia) with a hydrocarbon partial
pressure of about 35 to 172 kPa (5 to 25 psia), with a preferred
hydrocarbon partial pressure of about 69 to 138 kPa (10 to 20
psia). This relatively low partial pressure for hydrocarbon is
achieved by using steam as a diluent to the extent that the diluent
is 10 to 55 wt-% of lipid composition and preferably about 15 wt-%
of lipid composition. Other diluents such as dry gas can be used to
reach equivalent hydrocarbon partial pressures.
[0397] The temperature of the cracked stream at the riser outlet
will be about 510.degree. C. to 621.degree. C. (950.degree. F. to
1150.degree. F.). However, riser outlet temperatures above
566.degree. C. (1050.degree. F.) make more dry gas and more
olefins. Whereas, riser outlet temperatures below 566.degree. C.
(1050.degree. F.) make less ethylene and propylene. Accordingly, it
is preferred to run the FCC process at a preferred temperature of
about 566.degree. C. to about 630.degree. C., preferred pressure of
about 138 kPa to about 240 kPa (20 to 35 psia). Another condition
for the process is the catalyst to lipid composition ratio which
can vary from about 5 to about 20 and preferably from about 10 to
about 15.
[0398] In one embodiment of the method for producing a jet fuel,
the lipid composition is introduced into the lift section of an FCC
reactor. The temperature in the lift section will be very hot and
range from about 700.degree. C. (1292.degree. F.) to about
760.degree. C. (1400.degree. F.) with a catalyst to lipid
composition ratio of about 100 to about 150. It is anticipated that
introducing the lipid composition into the lift section will
produce considerable amounts of propylene and ethylene.
[0399] Gas and liquid hydrocarbon products produced can be analyzed
by gas chromatography, HPLC, etc.
[0400] 2. Hydrodeoxygenation
[0401] In another embodiment of the method for producing a jet fuel
using the lipid composition or the lipids produced as described
herein, the structure of the lipid composition or the lipids is
broken by a process referred to as hydrodeoxygenation (HDO).
[0402] HDO means removal of oxygen by means of hydrogen, that is,
oxygen is removed while breaking the structure of the material.
Olefinic double bonds are hydrogenated and any sulphur and nitrogen
compounds are removed. Sulphur removal is called
hydrodesulphurization (HDS). Pretreatment and purity of the raw
materials (lipid composition or the lipids) contribute to the
service life of the catalyst.
[0403] Generally in the HDO/HDS step, hydrogen is mixed with the
feed stock (lipid composition or the lipids) and then the mixture
is passed through a catalyst bed as a co-current flow, either as a
single phase or a two phase feed stock. After the HDO/MDS step, the
product fraction is separated and passed to a separate isomerzation
reactor. An isomerization reactor for biological starting material
is described in the literature (F1 100 248) as a co-current
reactor.
[0404] The process for producing a fuel by hydrogenating a
hydrocarbon feed, e.g., the lipid composition or the lipids herein,
can also be performed by passing the lipid composition or the
lipids as a co-current flow with hydrogen gas through a first
hydrogenation zone, and thereafter the hydrocarbon effluent is
further hydrogenated in a second hydrogenation zone by passing
hydrogen gas to the second hydrogenation zone as a counter-current
flow relative to the hydrocarbon effluent. Exemplary HDO
applications and catalysts useful for cracking the lipid
composition to produce C.sub.2-C.sub.5 olefins are described in
U.S. Pat. No. 7,232,935, which is incorporated in its entirety by
reference.
[0405] Typically, in the hydrodeoxygenation step, the structure of
the biological component, such as the lipid composition or lipids
herein, is decomposed, oxygen, nitrogen, phosphorus and sulphur
compounds, and light hydrocarbons as gas are removed, and the
olefinic bonds are hydrogenated. In the second step of the process,
i.e. in the so-called isomerization step, isomerzation is carried
out for branching the hydrocarbon chain and improving the
performance of the paraffin at low temperatures.
[0406] In the first step i.e. HDO step of the cracking process,
hydrogen gas and the lipid composition or lipids herein which are
to be hydrogenated are passed to a HDO catalyst bed system either
as co-current or counter-current flows, said catalyst bed system
comprising one or more catalyst bed(s), preferably 1-3 catalyst
beds. The HDO step is typically operated in a co-current manner. In
case of a HDO catalyst bed system comprising two or more catalyst
beds, one or more of the beds may be operated using the
counter-current flow principle.
[0407] In the HDO step, the pressure varies between 20 and 150 bar,
preferably between 50 and 100 bar, and the temperature varies
between 200 and 500.degree. C., preferably in the range of
300-400.degree. C.
[0408] In the HDO step, known hydrogenation catalysts containing
metals from Group VII and/or VIB of the Periodic System may be
used. Preferably, the hydrogenation catalysts are supported Pd, Pt,
Ni, NiMo or a CoMo catalysts, the support being alumina and/or
silica. Typically, NiMo/Al.sub.2O.sub.3 and CoMo/Al.sub.2O.sub.3
catalysts are used.
[0409] Prior to the HDO step, the lipid composition or lipids
herein may optionally be treated by prehydrogenation under milder
conditions thus avoiding side reactions of the double bonds. Such
prehydrogenation is carried out in the presence of a
prehydrogenation catalyst at temperatures of 50 400.degree. C. and
at hydrogen pressures of 1 200 bar, preferably at a temperature
between 150 and 250.degree. C. and at a hydrogen pressure between
10 and 100 bar. The catalyst may contain metals from Group VIII
and/or VIB of the Periodic System. Preferably, the prehydrogenation
catalyst is a supported Pd, Pt, Ni, NiMo or a CoMo catalyst, the
support being alumina and/or silica.
[0410] A gaseous stream from the HDO step containing hydrogen is
cooled and then carbon monoxide, carbon dioxide, nitrogen,
phosphorus and sulphur compounds, gaseous light hydrocarbons and
other impurities are removed therefrom. After compressing, the
purified hydrogen or recycled hydrogen is returned back to the
first catalyst bed and/or between the catalyst beds to make up for
the withdrawn gas stream. Water is removed from the condensed
liquid. The liquid is passed to the first catalyst bed or between
the catalyst beds.
[0411] After the HDO step, the product is subjected to an
isomerization step. It is substantial for the process that the
impurities are removed as completely as possible before the
hydrocarbons are contacted with the isomerization catalyst. The
isomerization step comprises an optional stripping step, wherein
the reaction product from the HDO step may be purified by stripping
with water vapour or a suitable gas such as light hydrocarbon,
nitrogen or hydrogen. The optional stripping step is carried out in
counter-current manner in a unit upstream of the isomerization
catalyst, wherein the gas and liquid are contacted with each other,
or before the actual isomerization reactor in a separate stripping
unit utilizing counter-current principle.
[0412] After the stripping step the hydrogen gas and the
hydrogenated lipid composition or lipids herein, and optionally an
n-paraffin mixture, are passed to a reactive isomerization unit
comprising one or several catalyst bed(s). The catalyst beds of the
isomerization step may operate either in co-current or
counter-current manner.
[0413] It is important for the process that the counter-current
flow principle is applied in the isomerization step. In the
isomerization step this is done by carrying out either the optional
stripping step or the isomerization reaction step or both in
counter-current manner.
[0414] The isomerization step and the HDO step may be carried out
in the same pressure vessel or in separate pressure vessels.
Optional prehydrogenation may be carried out in a separate pressure
vessel or in the same pressure vessel as the HDO and isomerization
steps.
[0415] In the isomerzation step, the pressure varies in the range
of 20 150 bar, preferably in the range of 20 100 bar, the
temperature being between 200 and 500.degree. C., preferably
between 300 and 400.degree. C.
[0416] In the isomerization step, isomerization catalysts known in
the art may be used. Suitable isomerization catalysts contain
molecular sieve and/or a metal from Group VII and/or a carrier.
Preferably, the isomerization catalyst contains SAPO-11 or SAPO41
or ZSM-22 or ZSM-23 or ferrierite and Pt, Pd or Ni and
Al.sub.2O.sub.3 or SiO.sub.2. Typical isomerization catalysts are,
for example, Pt/SAPO-11/Al.sub.2O.sub.3, Pt/ZSM-22/Al.sub.2O.sub.3,
Pt/ZSM-23/Al.sub.2O.sub.3 and Pt/SAPO-11/SiO.sub.2.
[0417] As the product, a high quality hydrocarbon component of
biological origin, useful as a diesel fuel or a component thereof,
is obtained, the density, cetane number and performance at low
temperate of said hydrocarbon component being excellent.
IX. Microbe Engineering
[0418] As noted above, in certain embodiments of the present
invention it is desirable to genentically modify a microorganism to
enhance lipid production, modify the properties or proportions of
components generated by the microorganism, or to improve or provide
de novo growth characteristics on a variety of feedstock materials.
Chlorella, particularly Chlorella protothecoides, Chlorella
minutissima, Chlorella sorokiniana, Chlorella ellipsoidea,
Chlorella sp., and Chlorella emersonii are preferred microorganisms
for use in the genetic engineering methods described herein,
although other Chlorella species as well as other varieties of
microorganisms can be used.
[0419] Promoters, cDNAs, and 3'UTRs, as well as other elements of
the vectors, can be generated through cloning techniques using
fragments isolated from native sources (see for example Molecular
Cloning: A Laboratory Manual, Sambrook et al. (3d edition, 2001,
Cold Spring Harbor Press; and U.S. Pat. No. 4,683,202).
Alternatively, elements can be generated synthetically using known
methods (see for example Gene. 1995 Oct. 16; 164(1):49-53).
[0420] A. Codon-Optimization for Expression
[0421] DNA encoding a polypeptide to be expressed in a
microorganism, e.g., a lipase and selectable marker are preferably
codon-optimized Cdna. Methods of recoding genes for expression in
microalgae are described in U.S. Pat. No. 7,135,290. Additional
information for codon optimization is available, e.g., at the codon
usage database of GenBank. As non-limiting examples, codon usage in
Chlorella pyrenoidosa, Dunaliella salina, and Chlorella
protothecoides are shown in Tables 10, 11, and 12,
respectively.
TABLE-US-00011 TABLE 10 Codon usage in Chlorella pyrenoidosa. Phe
UUU 39 (0.82) Ser UCU 50 (1.04) UUC 56 (1.18) UCC 60 (1.25) Leu UUA
10 (0.20) UCA 6 (0.96) UUG 46 (0.91) UCG 43 (0.89) Tyr UAU 15
(0.59) Cys UGU 46 (0.77) UAC 36 (1.41) UGC 73 (1.23) ter UAA 9
(0.00) ter UGA 43 (0.00) ter UAG 15 (0.00) Trp UGG 69 (1.00) Leu
CUU 49 (0.97) Pro CCU 80 (0.98) CUC 73 (1.45) CCC 88 (1.08) CUA 22
(0.44) CCA 93 (1.14) CUG 103 (2.04) CCG 65 (0.80) His CAU 50 (0.88)
Arg CGU 39 (0.76) CAC 3 (1.12) CGC 63 (1.23) Gln CAA 59 (0.84) CGA
46 (0.90) CAG 2 (1.16) CGG 47 (0.92) Ile AUU 24 (0.69) Thr ACU 32
(0.67) AUC 61 (1.76) ACC 76 (1.60) AUA 19 (0.55) ACA 41 (0.86) Met
AUG 42 (1.00) ACG 41 (0.86) Asn AAU 26 (0.75) Ser AGU 23 (0.48) AAC
3 (1.25) AGC 67 (1.39) Lys AAA 32 (0.54) Arg AGA 51 (1.00) AAG 86
(1.46) AGG 61 (1.19) Val GUU 36 (0.75) Ala GCU 57 (0.79) GUC 54
(1.13) GCC 97 (1.34) GUA 30 (0.63) GCA 89 (1.23) GUG 71 (1.49) GCG
47 (0.65) Asp GAU 60 (0.95) Gly GGU 35 (0.60) GAC 66 (1.05) GGC 78
(1.33) Glu GAA 41 (0.68) GGA 54 (0.92) GAG 80 (1.32) GGG 67
(1.15)
TABLE-US-00012 TABLE 11 Preferred codon usage in Dunaliella salina.
TTC (Phe) TAC (Tyr) TGC (Cys) TAA (Stop) TGG (Trp) CCC (Pro) CAC
(His) CGC (Arg) CTG (Leu) CAG (Gln) ATC (Ile) ACC (Thr) AAC (Asn)
AGC (Ser) ATG (Met) AAG (Lys) GCC (Ala) GAC (Asp) GGC (Gly) GTG
(Val) GAG (Glu)
TABLE-US-00013 TABLE 12 Preferred codon usage in Chlorella
protothecoides. TTC (Phe) TAC (Tyr) TGC (Cys) TGA (Stop) TGG (Trp)
CCC (Pro) CAC (His) CGC (Arg) CTG (Leu) CAG (Gln) ATC (Ile) ACC
(Thr) GAC (Asp) TCC (Ser) ATG (Met) AAG (Lys) GCC (Ala) AAC (Asn)
GGC (Gly) GTG (Val) GAG (Glu)
[0422] B. Promoters
[0423] Many promoters are active in microalgae, including promoters
that are endogenous to the algae being transformed, as well as
promoters that are not endogenous to the algae being transformed
(i.e., promoters from other algae, promoters from higher plants,
and promoters from plant viruses or algae viruses). Exogenous
and/or endogenous promoters that are active in microalgae, and
antibiotic resistance genes functional in microalgae are described
by e.g., Curr Microbiol. 1997 December; 35(6):356-62 (Chlorella
vulgaris); Mar Biotechnol (NY). 2002 January; 4(1):63-73 (Chlorella
ellipsoidea); Mol Gen Genet. 1996 Oct. 16; 252(5):572-9
(Phaeodactylum tricornutum); Plant Mol. Biol. 1996 April;
31(1):1-12 (Volvox carteri); Proc Natl Acad Sci USA. 1994 Nov. 22;
91(24):11562-6 (Volvox carteri); Falciatore A, Casotti R, Leblanc
C, Abrescia C, Bowler C, PMID: 10383998, 1999 May; 1(3):239-251
(Laboratory of Molecular Plant Biology, Stazione Zoologica, VIIIa
Comunale, 1-80121 Naples, Italy) (Phaeodactylum tricornutum and
Thalassiosira weissflogii); Plant Physiol. 2002 May; 129(1):7-12.
(Porphyridium sp.); Proc Natl Acad Sci USA. 2003 Jan. 21;
100(2):438-42. (Chlamydomonas reinhardtii); Proc Natl Acad Sci USA.
1990 February; 87(3):1228-32. (Chlamydomonas reinhardtii); Nucleic
Acids Res. 1992 Jun. 25; 20(12):2959-65; Mar Biotechnol (NY). 2002
January; 4(1):63-73 (Chlorella); Biochem Mol Biol Int. 1995 August;
36(5):1025-35 (Chlamydomonas reinhardtii); J. Microbiol. 2005
August; 43(4):361-5 (Dunaliella); Yi Chuan Xue Bao. 2005 April;
32(4):424-33 (Dunaliella); Mar Biotechnol (NY). 1999 May;
1(3):239-251. (Thalassiosira and Phaedactylum); Koksharova, Appl
Microbiol Biotechnol 2002 February; 58(2):123-37 (various species);
Mol Genet Genomics. 2004 February; 271(1):50-9 (Thermosynechococcus
elongates); J. Bacteriol. (2000), 182, 211-215; FEMS Microbiol
Lett. 2003 Apr. 25; 221(2):155-9; Plant Physiol. 1994 June;
105(2):635-41; Plant Mol. Biol. 1995 December; 29(5):897-907
(Synechococcus PCC 7942); Mar Pollut Bull. 2002; 45(1-12):163-7
(Anabaena PCC 7120); Proc Natl Acad Sci USA. 1984 March;
81(5):1561-5 (Anabaena (various strains)); Proc Natl Acad Sci USA.
2001 Mar. 27; 98(7):4243-8 (Synechocystis); Wirth, Mol Gen Genet.
1989 March; 216(1):175-7 (various species); Mol Microbiol, 2002
June; 44(6):1517-31 and Plasmid, 1993 September; 30(2):90-105
(Fremyella diplosiphon); Hall et al. (1993) Gene 124: 75-81
(Chlamydomonas reinhardtii); Gruber et al. (1991). Current Micro.
22: 15-20; Jarvis et al. (1991) Current Genet. 19: 317-322
(Chlorella); for additional promoters see also table 1 from U.S.
Pat. No. 6,027,900).
[0424] The promoter used to express an exogenous gene can be the
promoter naturally linked to that gene or can be a heterologous
gene. Some promoters are active in more than one species of
microalgae. Other promoters are species-specific. Preferred
promoters include promoters such as RBCS2 from Chlamydomonas
reinhardtii and viral promoters, such as cauliflower mosaic virus
(CMV) and chlorella virus, which have been shown to be active in
multiple species of microalgae (see for example Plant Cell Rep.
2005 March; 23(10-11):727-35; J. Microbiol. 2005 August;
43(4):361-5; Mar Biotechnol (NY). 2002 January; 4(1):63-73). In
other embodiments, the Botryococcus malate dehydrogenase promoter,
such a nucleic acid comprising any part of SEQ ID NO:3, or the
Chlamydomonas reinhardtii RBCS2 promoter (SEQ ID NO:4) can be used.
Optionally, at least 10, 20, 30, 40, 50, or 60 nucleotides or more
of these sequences containing a promoter are used. Preferred
promoters endogenous to species of the genus Chlorella are SEQ ID
NO:1 and SEQ ID NO:2.
[0425] Preferred promoters useful for expression of exogenous genes
in Chlorella are listed in the sequence listing of this
application, such as the promoter of the Chlorella HUP1 gene (SEQ
ID NO:1) and the Chlorella ellipsoidea nitrate reductase promoter
(SEQ ID NO:2). Chlorella virus promoters can also be used to
express genes in Chlorella, such as SEQ ID NOs: 1-7 of U.S. Pat.
No. 6,395,965. Additional promoters active in Chlorella can be
found, for example, in Biochem Biophys Res Commun. 1994 Oct. 14;
204(1):187-94; Plant Mol. Biol. 1994 October; 26(1):85-93;
Virology. 2004 Aug. 15; 326(1):150-9; and Virology. 2004 Jan. 5;
318(1):214-23.
[0426] C. Selectable Markers
[0427] Any of a wide variety of selectable markers can be employed
in a transgene construct useful for transforming Chlorella.
Examples of suitable selectable markers include the nitrate
reductase gene, the hygromycin phosphotransferase gene (HPT), the
neomycin phosphotransferase gene, and the ble gene, which confers
resistance to phleomycin. Methods of determining sensitivity of
microalgae to antibiotics are well known. For example, Mol Gen
Genet. 1996 Oct. 16; 252(5):572-9.
[0428] More specifically, Dawson et al. (1997), Current
Microbiology 35:356-362 (incorporated by reference herein in its
entirety), described the use of the nitrate reductase (NR) gene
from Chlorella vulgaris as a selectable marker for NR-deficient
Chlorella sorokiniana mutants. Kim et al. (2002), Mar. Biotechnol.
4:63-73 (incorporated by reference herein in its entirety),
disclosed the use of the HPT gene as a selectable marker for
transforming Chorella ellipsoidea. Huang et al. (2007), Appl.
Microbiol. Biotechnol. 72:197-205 (incorporated by reference herein
in its entirety), reported on the use of Sh ble as a selectable
marker for Chlorella sp. DT.
[0429] D. Inducible Expression
[0430] The present invention also provides for the use of an
inducible promoter to express a gene of interest. In particular,
the use of an inducible promoter to express a lipase gene permits
production of the lipase after growth of the microorganism when
conditions have been adjusted, if necessary, to enhance
transesterification, for example, after disruption of the cells,
reduction of the water content of the reaction mixture, and/or
addition sufficient alcohol to drive conversion of TAGs to fatty
acid esters.
[0431] Inducible promoters useful in the invention include those
that mediate transcription of an operably linked gene in response
to a stimulus, such as an exogenously provided small molecule (e.g,
glucose, as in SEQ ID NO:1), temperature (heat or cold), light,
etc. Suitable promoters can activate transcription of an
essentially silent gene or upregulate, preferably substantially,
transcription of an operably linked gene that is transcribed at a
low level. In the latter case, the level of transcription of the
lipase preferably does not significantly interfere with the growth
of the microorganism in which it is expressed.
[0432] Expression of transgenes in Chlorella can be performed
inducibly through promoters such as the promoter that drives the
Chlorella hexose transporter gene (SEQ ID NO:1). This promoter is
strongly activated by the presence of glucose in the culture
media.
[0433] E. Expression of Two or More Exogenous Genes
[0434] Further, a genetically engineered microorganism, such as a
microalgae, may comprise and express two or more exogenous genes,
such as, for example, a lipase and a lytic gene, e.g., one encoding
a polysaccharide-degrading enzyme. One or both genes can be
expressed using an inducible promoter, which allows the relative
timing of expression of these genes to be controlled to enhance the
lipid yield and conversion to fatty acid esters. Expression of the
two or more exogenous genes may be under control of the same
inducible promoter or under control of a different inducible
promoters. In the latter situation, expression of a first exogenous
gene can be induced for a first period of time (during which
expression of a second exogenous gene may or may not be induced)
and expression of a second exogenous gene can be induced for a
second period of time (during which expression of a first exogenous
gene may or may not be induced). Provided herein are vectors and
methods for engineering lipid-producing microbes to metabolize
sucrose, which is an advantageous trait because it allows the
engineered cells to convert sugar cane feedstocks into lipids.
[0435] Also provided herein are genetically engineered strains of
microbes (e.g., microalgae, oleaginous yeast, bacteria, or fungi)
that express two or more exogenous genes, such as, for example, a
fatty acyl-ACP thioesterase and a fatty acyl-CoA/aldehyde
reductase, the combined action of which yields an alcohol product.
Further provided are other combinations of exogenous genes,
including without limitation, a fatty acyl-ACP thioesterase and a
fatty acyl-CoA reductase to generate aldehydes. In addition, this
application provides for the combination of a fatty acyl-ACP
thioesterase, a fatty acyl-CoA reductase, and a fatty aldehyde
decarbonylase to generate alkanes. One or more of the exogenous
genes can be expressed using an inducible promoter.
[0436] Examples of further modifications suitable for use in the
present invention are include genetically engineering strains of
microalgae to express two or more exogenous genes, one encoding a
transporter of a fixed carbon source (such as sucrose) and a second
encoding a sucrose invertase enzyme. The resulting fermentable
organisms produce hydrocarbons at lower manufacturing cost than
what has been obtainable by previously known methods of biological
hydrocarbon production. Insertion of the two exogenous genes
described above can be combined with the disruption of
polysaccharide biosynthesis through directed and/or random
mutagenesis, which steers ever greater carbon flux into hydrocarbon
production. Individually and in combination, trophic conversion,
engineering to alter hydrocarbon production and treatment with
exogenous enzymes alter the hydrocarbon composition produced by a
microorganism. The alteration can be a change in the amount of
hydrocarbons produced, the amount of one or more hydrocarbon
species produced relative to other hydrocarbons, and/or the types
of hydrocarbon species produced in the microorganism. For example,
microalgae can be engineered to produce a higher amount and/or
percentage of TAGs.
[0437] F. Compartmentalized Expression
[0438] The present invention also provides for compartmentalized
expression of a gene of interest. In particular, it can be
advantageous, in particular embodiments, to target expression of
the lipase to one or more cellular compartments, where it is
sequestered from the majority of cellular lipids until initiation
of the transesterification reaction. Preferred organelles for
targeting are chloroplasts, mitochondria, and endoplasmic
reticulum.
[0439] 1. Expression in Chloroplasts
[0440] In one embodiment of the present invention, the expression
of a polypeptide in a microorganism is targeted to chloroplasts.
Methods for targeting expression of a heterologous gene to the
chloroplast are known and can be employed in the present invention.
Methods for targeting foreign gene products into chloroplasts are
described in Shrier et al., EMBO J. (1985) 4:25 32. See also Tomai
et al. Gen. Biol. Chem. (1988) 263:15104 15109 and U.S. Pat. No.
4,940,835 for the use of transit peptides for translocating nuclear
gene products into the chloroplast. Methods for directing the
transport of proteins to the chloroplast are also reviewed in
Kenauf TIBTECH (1987) 5:40 47. Chloroplast targeting sequences
endogenous to Chlorella are known, such as genes in the Chlorella
nuclear genome that encode proteins that are targeted to the
chloroplast; see for example GenBank Accession numbers AY646197 and
AF499684.
[0441] Wageningen UR--Plant Research International sells an
IMPACTVECTOR1.4 vector, which uses the secretion signal of the
Chrysanthemum morifolium small subunit protein to deliver a
heterologous protein into the chloroplast stroma (cytoplasmic)
environment, shuttling across a double membrane system. The protein
is fused to the first 11 amino acids of the mature rubisco protein
in order to allow proper processing of the signal peptide (Wong et
al., Plant Molecular Biology 20: 81-93 (1992)). The signal peptide
contains a natural intron from the RbcS gene.
[0442] In another approach, the chloroplast genome is genetically
engineered to express the heterologous protein. Stable
transformation of chloroplasts of Chlamydomonas reinhardtii (a
green alga) using bombardment of recipient cells with high-velocity
tungsten microprojectiles coated with foreign DNA has been
described. See, for example, Boynton et al., Science (1988) 240:
1534 1538; Blowers et al. Plant Cell (1989) 1:123 132 and Debuchy
et al., EMBO J. (1989) .delta.: 2803 2809. The transformation
technique, using tungsten microprojectiles, is described by Klein
et al., Nature (London) (1987) 7:70 73. Other methods of
chloroplast transformation for both plants and microalgae are
known. See for example U.S. Pat. Nos. 5,693,507; 6,680,426; and
Plant Physiol. 2002 May; 129(1):7-12; and Plant Biotechnol J. 2007
May; 5(3):402-12.
[0443] As described in U.S. Pat. No. 6,320,101 (issued Nov. 20,
2001 to Kaplan et al.; which is incorporated herein by reference),
cells can be chemically treated so as to reduce the number of
chloroplasts per cell to about one. Then, the heterologous nucleic
acid can be introduced into the cells via particle bombardment with
the aim of introducing at least one heterologous nucleic acid
molecule into the chloroplasts. The heterologous nucleic acid is
selected such that it is integratable into the chloroplast's genome
via homologous recombination which is readily effected by enzymes
inherent to the chloroplast. To this end, the heterologous nucleic
acid includes, in addition to a gene of interest, at least one
nucleic acid sequence that is derived from the chloroplast's
genome. In addition, the heterologous nucleic acid typically
includes a selectable marker. Further details relating to this
technique are found in U.S. Pat. Nos. 4,945,050 and 5,693,507 which
are incorporated herein by reference. A polypeptide can thus be
produced by the protein expression system of the chloroplast.
[0444] U.S. Pat. No. 7,135,620 (issued Nov. 14, 2006 to Daniell et
al.; incorporated herein by reference) describes chloroplast
expression vectors and related methods. Expression cassettes are
DNA constructs including a coding sequence and appropriate control
sequences to provide for proper expression of the coding sequence
in the chloroplast. Typical expression cassettes include the
following components: the 5' untranslated region from a
microorganism gene or chloroplast gene such as psbA which will
provide for transcription and translation of a DNA sequence
encoding a polypeptide of interest in the chloroplast; a DNA
sequence encoding a polypeptide of interest; and a translational
and transcriptional termination region, such as a 3' inverted
repeat region of a chloroplast gene that can stabilize RNA of
introduced genes, thereby enhancing foreign gene expression. The
cassette can optionally include an antibiotic resistance gene.
[0445] Typically, the expression cassette is flanked by convenient
restriction sites for insertion into an appropriate genome. The
expression cassette can be flanked by DNA sequences from
chloroplast DNA to facilitate stable integration of the expression
cassette into the chloroplast genome, particularly by homologous
recombination. Alternatively, the expression cassette may remain
unintegrated, in which case, the expression cassette typically
includes a chloroplast origin of replication, which is capable of
providing for replication of the heterologous DNA in the
chloroplast.
[0446] The expression cassette generally includes a promoter region
from a gene capable of expression in the chloroplast. The promoter
region may include promoters obtainable from chloroplast genes,
such as the psbA gene from spinach or pea, or the rbcL and atpB
promoter region from maize and Rrna promoters. Examples of
promoters are described in Hanley-Bowdoin and Chua, TIBS (1987)
12:67 70; Mullet et al., Plant Molec Biol. (1985) 4: 39 54;
Hanley-Bowdoin (1986) PhD. Dissertation, the Rockefeller
University; Krebbers et al., Nucleic Acids Res. (1982) 10: 4985
5002; Zurawaki et al., Nucleic Acids Res. (1981) 9:3251 3270; and
Zurawski et al., Proc. Nat'l Acad. Sci. U.S.A. (1982) 79: 7699
7703. Other promoters can be identified and the relative strength
of promoters so identified evaluated, by placing a promoter of
interest 5' to a promoterless marker gene and observing its
effectiveness relative to transcription obtained from, for example,
the promoter from the psbA gene, a relatively strong chloroplast
promoter. The efficiency of heterologus gene expression
additionally can be enhanced by any of a variety of techniques.
These include the use of multiple promoters inserted in tandem 5'
to the heterologous gente, for example a double psbA promoter, the
addition of enhancer sequences and the like.
[0447] Numerous promoters active in the Chlorella chloroplast can
be used for expression of exogenous genes in the Chlorella
chloroplast, such as those found in GenBank accession number
NC.sub.--001865 (Chlorella vulgaris chloroplast, complete
genome),
[0448] Where it is desired to provide for inducible expression of
the heterologous gene, an inducible promoter and/or a 5'
untranslated region containing sequences which provide for
regulation at the level of transcription and/or translation (at the
3' end) may be included in the expression cassette. For example,
the 5' untranslated region can be from a gene wherein expression is
regulatable by light. Similarly, 3' inverted repeat regions could
be used to stabilize RNA of heterologous genes. Inducible genes may
be identified by enhanced expression in response to a particular
stimulus of interest and low or absent expression in the absence of
the stimulus. For example, a light-inducible gene can be identified
where enhanced expression occurs during irradiation with light,
while substantially reduced expression or no expression occurs in
low or no light. Light regulated promoters from green microalgae
are known (see for example Mol Genet Genomics. 2005 December;
274(6):625-36).
[0449] The termination region which is employed will be primarily
one of convenience, since the termination region appears to be
relatively interchangeable among chloroplasts and bacteria. The
termination region may be native to the transcriptional initiation
region, may be native to the DNA sequence of interest, or may be
obtainable from another source. See, for example, Chen and Orozco,
Nucleic Acids Res. (1988) 16:8411.
[0450] The expression cassettes may be transformed into a plant
cell of interest by any of a number of methods. These methods
include, for example, biolistic methods (See, for example, Sanford,
Trends In Biotech. (1988) 6:299 302, U.S. Pat. No. 4,945,050;
electroporation (Fromm et al., Proc. Nat'l. Acad. Sci. (USA) (1985)
82:5824 5828); use of a laser beam, microinjection or any other
method capable of introducing DNA into a chloroplast.
[0451] Additional descriptions of chloroplast expression vectors
suitable for use in microorganisms such as microalgae are found in
U.S. Pat. Nos. 7,081,567 (issued Jul. 25, 2006 to Xue et al.);
6,680,426 (issued Jan. 20, 2004 to Daniell et al.); and 5,693,507
(issued Dec. 2, 1997 to Daniell et al.).
[0452] Proteins expressed in the nuclear genome of Chlorella can be
targeted to the chloroplast using chloroplast targeting signals.
Chloroplast targeting sequences endogenous to Chlorella are known,
such as genes in the Chlorella nuclear genome that encode proteins
that are targeted to the chloroplast; see for example GenBank
Accession numbers AY646197 and AF499684. Proteins can also be
expressed in the Chlorella chloroplast by insertion of genes
directly into the chloroplast genome. Chloroplast transformation
typically occurs through homologous recombination, and can be
performed if chloroplast genome sequences are known for creation of
targeting vectors (see for example the complete genome sequence of
a Chlorella chloroplast; Genbank accession number NC.sub.--001865).
See previous sections herein for details of chloroplast
transformation.
[0453] 2. Expression in Mitochondria
[0454] In another embodiment of the present invention, the
expression of a polypeptide in a microorganism is targeted to
mitochondria. Methods for targeting foreign gene products into
mitochnodria (Boutry et al. Nature (London) (1987) 328:340 342)
have been described, including in green microalgae (see for example
Mol Gen Genet. 1993 January; 236(2-3):235-44).
[0455] For example, an expression vector encoding a suitable
secretion signal can target a heterologus protein to the
mitochondrion. The IMPACTVECTOR1.5 vector, from Wageningen
UR--Plant Research International, uses the yeast CoxIV secretion
signal, which was shown to deliver proteins in the mitochondrial
matrix. The protein is fused to the first 4 amino acids of the
yeast CoxIV protein in order to allow proper processing of the
signal peptide (Kohler et al. Plant J 11: 613-621 (1997)). Other
mitochondrial targeting sequences are known, including those
functional in green microalgae. For example, see FEBS Lett. 1990
Jan. 29; 260(2):165-8; and J Biol. Chem. 2002 Feb. 22;
277(8):6051-8.
[0456] Proteins expressed in the nuclear genome of Chlorella can be
targeted to the mitochondria using mitochondrial targeting signals.
See previous sections herein for details of mitochondrial protein
targeting and transformation.
[0457] 3. Expression in Endoplasmic Reticulum
[0458] In another embodiment of the present invention, the
expression of a polypeptide in a microorganism is targeted to the
endoplasmic reticulum. The inclusion of an appropriate retention or
sorting signal in an expression vector ensure that proteins are
retained in the endoplasmic reticulum (ER) and do not go downstream
into Golgi. For example, the IMPACTVECTOR1.3 vector, from
Wageningen UR--Plant Research International, includes the well
known KDEL retention or sorting signal. With this vector, ER
retention has a practical advantage in that it has been reported to
improve expression levels 5-fold or more. The main reason for this
appears to be that the ER contains lower concentrations and/or
different proteases responsible for post-translational degradation
of expressed proteins than are present in the cytoplasm. ER
retention signals functional in green microalgae are known. For
example, see Proc Natl Acad Sci USA. 2005 Apr. 26;
102(17):6225-30.
[0459] G. Transformation
[0460] Cells can be transformed by any suitable technique
including, e.g., biolistics, electroporation, glass bead
transformation and silicon carbide whisker transformation. See,
e.g., Examples herein.
[0461] Any convenient technique for introducing a transgene into
Chorella can be employed in the present invention. Dawson et al.
(1997) (supra) described the use of micro-projectile bombardment to
introduce the nitrate reductase (NR) gene from Chlorella vulgaris
into NR-deficient Chlorella sorokiniana mutants, resulting in
stable transformants. Briefly, 0.4 micron tungsten beads were
coated with plasmid; 3.times.10.sup.7 C. sorokiniana cells were
spread in the center third of a non-selective agar plate and
bombarded with the PDS-1000/He Biolistic Particle Delivery.RTM.
system (Bio-Rad).
[0462] A preferred method for introducing a transgene into
Chlorella is the method described by Kim et al. (2002), Mar.
Biotechnol. 4:63-73. Kim reports the transformation of Chorella
ellipsoidea protoplasts using CaCl.sub.2 and polyethylene glycol
(PEG). In particular, protoplasts were prepared by growing C.
ellipsoidea cells to a density of 1-2.times.10.sup.8/Ml. Cells were
recovered and washed by centrifugation for 5 minutes at 1600 g and
resuspended in 5 Ml of phosphate buffer (Ph 6.0) containing 0.6 M
sorbitol, 0.6 M mannitol, 4% (weight/volume) cellulose
(Calbiochem), 2% (weight/volume) macerase (Calbiochem), and 50
units pectinase (Sigma). The cell suspension was incubated at
25.degree. C. for 16 hours in the dark with gentle shaking. The
resultant protoplasts were recovered by centrifugation at 400 g for
5 minutes. The pellet was gently resuspended in 5 Ml of f/2 medium
containing 0.6 M sorbitol and 0.6 M mannitol and centrifuged at 400
g for 5 minutes. This pellet was resuspended in 1 Ml of 0.6 M
sorbitol/mannitol solution containing 50 mMCaCl.sub.2. Then, 5 mg
of transgene DNA was added, along with 25 .mu.g calf thymus DNA
(Sigma), to 10.sup.7-10.sup.8 protoplasts in 0.4 Ml. After 15
minutes at room temperature, 200 .mu.L at of PNC (40% polyethylene
glycol 4000, 0.8 M NaCl, 50 Mm CaCl.sub.2) was added and mixed
gently for 30 minutes at room temperature. After this, 0.6 Ml of
f/2 medium supplemented with 0.6 M sorbitol/mannitol solution, 1%
yeast extract and 1% glucose was added, and the transformed cells
were incubated at 25.degree. C. for 12 hours in the dark for cell
wall regeneration. A similar method was used by Huang et al. (2007)
(supra) to introduce a transgene encoding mercuric reductase into
Chlorella sp. DT.
[0463] Electorporation has also been employed to transform
Chorella. As reported by Maruyama et al. (2004), Biotechnology
Techniques 8:821-826 (incorporated by reference herein in its
entirety), this technique was used to introduce a transgene into
protoplasts of Chlorella saccharophila c-211-1a prepared from the
cells in the stationary phase. Transient expression of the
introduced plasmid was observed under a field strength of between
600 and 900 V/cm, and a pulse duration of around 400 ms, where high
membrane permeability to 70-kDa FITC-dextran was ascertained.
[0464] Examples of expression of transgenes in Chlorella can be
found in the literature (see for example Current Microbiology Vol.
35 (1997), pp. 356-362; Sheng Wu Gong Cheng Xue Bao. 2000 July;
16(4):443-6; Current Microbiology Vol. 38 (1999), pp. 335-341; Appl
Microbiol Biotechnol (2006) 72: 197-205; Marine Biotechnology 4,
63-73, 2002; Current Genetics 39:5, 365-370 (2001); Plant Cell
Reports 18:9, 778-780, (1999); Biologia Plantarium 42(2): 209-216,
(1999); Plant Pathol. J 21(1): 13-20, (2005)). Also see Examples
herein.
[0465] Examples of expression of transgenes in oleaginous yeast
(e.g., Yarrowia lipolytica) can be found in the literature (see,
for example, Bordes et al., J Microbiol Methods, June 27 (2007)).
Examples of expression of transgenes in fungi (e.g., Mortierella
alpine, Mucor circinelloides, and Aspergillus ochraceus) can also
be found in the literature (see, for example, Microbiology, July;
153(Pt. 7):2013-25 (2007); Mol Genet Genomics, June; 271(5):595-602
(2004); Curr Genet, March; 21(3):215-23 (1992); Current
Microbiology, 30(2):83-86 (1995); Sakuradani, NISR Research Grant,
"Studies of Metabolic Engineering of Useful Lipid-producing
Microorganisms" (2004); and PCT/JP2004/012021). Examples of
expression of exogenous genes in bacteria such as E. coli are well
known; see for example Molecular Cloning: A Laboratory Manual,
Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press.
[0466] Vectors for transformation of microorganisms in accordance
with the present invention can be prepared by known techniques
familiar to those skilled in the art. The nucleotide sequence of
the construct used for transformation of multiple Chlorella species
corresponds to SEQ ID NO:25. In one embodiment, an exemplary vector
design for expression of a lipase gene in a microorganism such as a
microalgae contains a gene encoding a lipase in operable linkage
with a promoter active in microalgae. Alternatively, if the vector
does not contain a promoter in operable linkage with the gene of
interest, the gene can be transformed into the cells such that it
becomes operably linked to an endogenous promoter at the point of
vector integration. The promoterless method of transformation has
been proven to work in microalgae (see for example Plant Journal
14:4, (1998), pp. 441-447). The vector can also contain a second
gene that encodes a protein that, e.g., imparts resistance to an
antibiotic or herbicide, i.e., a selectable marker. Optionally, one
or both gene(s) is/are followed by a 3' untranslated sequence
containing a polyadenylation signal. Expression cassettes encoding
the two genes can be physically linked in the vector or on separate
vectors. Co-transformation of microalgae can also be used, in which
distinct vector molecules are simultaneously used to transform
cells (see for example Protist 2004 December; 155(4):381-93). The
transformed cells can be optionally selected based upon the ability
to grow in the presence of the antibiotic or other selectable
marker under conditions in which cells lacking the resistance
cassette would not grow.
[0467] All references cited herein, including patents, patent
applications, and publications, are hereby incorporated by
reference in their entireties, whether previously specifically
incorporated or not. The publications mentioned herein are cited
for the purpose of describing and disclosing reagents,
methodologies and concepts that may be used in connection with the
present invention. Nothing herein is to be construed as an
admission that these references are prior art in relation to the
inventions described herein.
[0468] Although this invention has been described in connection
with specific embodiments thereof, it will be understood that it is
capable of further modifications. This application is intended to
cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth.
X. EXAMPLES
[0469] The following examples are offered to illustrate, but not to
limit, the claimed invention.
Example 1
[0470] Chlorella strains from the University of Texas culture
collection were tested for growth on glycerol and glucose. The
following Chlorella species and strains were cultured: Chlorella
kessleri (strains 263, 397, 398, 2228); Chlorella sorokiniana
(strains 1663, 1665, 1669, 1671, 1810); Chlorella saccharophila
(2911; 2469); Chlorella protothecoides (31, 249, 250, 264). Each
strain was inoculated from solid media into 25 ml liquid base media
(2 g/L yeast extract, 2.94 mM NaNO.sub.3, 0.17 mM
CaCl.sub.2.2H.sub.2O, 0.3 mM MgSO.sub.4.7H.sub.2O, 0.4 mM
K.sub.2HPO.sub.4, 1.28 mM KH.sub.2PO.sub.4, 0.43 mM NaCl) and grown
shaking at 27.degree. C. for 72 hours under a light intensity of 75
.mu.Em.sup.-2s.sup.-1. These cultures were used to inoculate each
strain to a final density of 1.times.10.sup.5 cells per ml into
24-well plates containing 2 ml of (a) base media only; (b) base
media plus 0.1% glucose; and (c) base media plus 0.5% reagent grade
glycerol (EM Science, catalog #GX0185-6). Plates were placed in the
dark and grown for 72 hours shaking at 27.degree. C. Samples of
each strain grown in the three conditions were diluted 1.9:1 in
distilled H2O and absorbance was read at 600 nm in a Molecular
Devices SpectraMax 340PC. All strains exhibited growth in the
presence of glucose and glycerol compared to only base media.
Example 2
Strains and Media
[0471] Chlorella protothecoides #1 (STRAIN 250), #2 (STRAIN 264)
and Chlorella kessleri #1 (STRAIN 398) were obtained from the
Culture Collection of Algae at the University of Texas (Austin,
Tex., USA). The stock cultures were maintained on modified Proteose
medium. Modified Proteose medium consisted (g/L) of 0.25 g
NaNO.sub.3, 0.09 g K.sub.2HPO.sub.4, 0.175 g KH.sub.2PO.sub.4 0.025
g, 0.025 g CaCl.sub.2.2H.sub.2O, 0.075 g MgSO.sub.4.7H.sub.2O, and
2 g yeast extract per liter. Glycerol wastes from biodiesel
production (acidulated glycerol (AG) and non-acidulated glycerol
(NAG)) were obtained from Imperial Western Products (Selma, Calif.,
USA). "Pure" or "reagent grade" glycerol was from EM Science (a
division of Merck KGA), catalog #GX0185-6.
[0472] Experimental design and Growth Measurement: For each strain,
1 ml of following different media was prepared in 24-well
plates.
[0473] 1. Proteose+1% pure glycerol
[0474] 2. Proteose+1% acidulated glycerol
[0475] 3. Proteose+1% non-acidulated glycerol
[0476] 4. Proteose+1% pure glycerol+1% glucose (added after 72
hr)
[0477] 5. Proteose+1% acidulated glycerol+1% glucose (added after
72 hr)
[0478] 6. Proteose+1% non-acidulated glycerol+1% glucose (added
after 72 hr)
[0479] Each strain was inoculated to different media to
5.times.10.sup.5 cells/ml concentration. The cultures were kept in
dark and were agitated by orbital shaker from Labnet (Berkshire,
UK) at 430 rpm. After 72 hr of initial growth, 1% (w/v) glucose was
added to samples #4, 5, and 6 and cultured another 24 hr. To
measure dry cell weight, 1 ml of each culture was pelleted by
centrifugation at 5,000 rpm for 5 min in an Eppendorf 5415C
centrifuge. After removing supernatant, cell pellets were frozen at
-80.degree. C. and lyophilized in a lab scale freeze dryer
(Labconco, Mo., USA). Results are shown in FIG. 1.
Example 3
[0480] Strains and Media:
[0481] Chlorella protothecoides #1 (STRAIN 250), #3 (STRAIN 249)
and Chlorella kessleri #2 (strain 397) were obtained from the
Culture Collection of Algae at the University of Texas (Austin,
Tex., USA). The stock cultures were maintained on modified Proteose
medium (see EXAMPLE 2).
[0482] Experimental Design and Growth Measurement:
[0483] For each strain, 1 ml of following different media was
prepared in 24-well plates.
[0484] 1. Proteose+1% pure glycerol+1% glucose
[0485] 2. Proteose+1% acidulated glycerol+1% glucose
[0486] 3. Proteose+1% non-acidulated glycerol+1% glucose
[0487] Each strain was inoculated to different media to
5.times.10.sup.5 cells/ml concentration. The cultures were kept in
dark and agitated by orbital shaker from Labnet (Berkshire, UK) at
430 rpm. After 96 hr, cell growth was measured for dry cell weight
(see EXAMPLE 2). Results are shown in FIG. 2.
Example 4
[0488] Strains and Media:
[0489] Chlorella protothecoides #3 (STRAIN 249), #4 (STRAIN 31),
and Chlorella kessleri #2 (STRAIN 397) were obtained from the
Culture Collection of Alga at the University of Texas (Austin,
Tex., USA). The stock cultures were maintained on modified Proteose
medium (see EXAMPLE 2)
[0490] Experimental Design and Lipid Assay:
[0491] For each strain, 1 ml of following different media was
prepared in 24-well plates.
[0492] 1. Proteose+1% pure glycerol+1% glucose
[0493] 2. Proteose+1% acidulated glycerol+1% glucose
[0494] 3. Proteose+1% non-acidulated glycerol+1% glucose
[0495] Each strain was inoculated to media containing different
glycerols (pure, acidulated, or non-acidulated) to 5.times.10.sup.5
cells/ml concentration. The cultures were kept in dark and agitated
by orbital shaker from Labnet (Berkshire, UK) at 430 rpm. After 96
hr, lipid contents were measured. To measure the amount of lipid
content in cells, 100 .mu.A of cultures were collected and washed
once with same volume of media. To each tube, 5 .mu.A of washed
cells and 200 .mu.A of sulfuric acid 18 M were added. The tubes
were incubated at 90.degree. C. in a water bath for 30 min, and 1
ml of phosphoric acid-vanillin reagent was added to the tubes and
incubated at 37.degree. C. for 15 min. To prepare the phosphoric
acid-vanillin reagent, 0.12 g of vanillin was added to 20 ml of
water, and the volume adjusted to 100 ml with 85% phosphoric acid.
The optical density at 530 nm was read in a glass cuvette against a
reference tube with 5 .mu.l water as sample. Results are shown in
FIG. 3.
Example 5
[0496] Strains and Media:
[0497] Chlorella protothecoides #2 (STRAIN 264) and Chlorella
kessleri #1 (STRAIN 398) were obtained from the Culture Collection
of Alga at the University of Texas (Austin, Tex., USA). The stock
cultures were maintained on modified Proteose medium (see EXAMPLE
2).
[0498] Experimental Design and Lipid Assay:
[0499] For each strain, 1 ml of the following different media was
prepared in 24-well plates.
[0500] 1. Proteose+1% pure glycerol
[0501] 2. Proteose+1% non-acidulated glycerol
[0502] 3. Proteose+1% pure glycerol+1% glucose (added after 72
hr)
[0503] 4. Proteose+1% non-acidulated glycerol+1% glucose (added
after 72 hr)
[0504] Each strain was inoculated to media containing different
glycerols (pure or non-acidulated) to 5.times.10.sup.5 cells/ml
concentration. The cultures were kept in dark and agitated by
orbital shaker from Labnet (Berkshire, UK) at 430 rpm. After 72 hr
of initial growth, 1% glucose was added to sample #3 and #4 and
cultured another 24 hr. Lipid contents were measured in all samples
(see EXAMPLE 4). The optical density at 600 nm was also measured to
check for non-specific absorbance and subtracted from O.D. 530 nm
to calculate the amount of lipid. The reference curve is composed
of Triolein dissolved in chloroform ranging from 1 to 10 .mu.g.
Results are shown in FIG. 4.
Example 6
[0505] Strains and Media:
[0506] Chlorella protothecoides #3 (STRAIN 249) and Chlorella
kessleri #2 (STRAIN 397) were obtained from the Culture Collection
of Alga at the University of Texas (Austin, Tex., USA). The stock
cultures were maintained on modified Proteose medium (see EXAMPLE
2).
[0507] Experimental design and lipid assay: For each strain, 1 ml
of following different media was prepared in 24-well plates.
[0508] 1. Proteose+1% pure glycerol+1% glucose (added after 72
hr)
[0509] 2. Proteose+1% acidulated glycerol+1% glucose (added after
72 hr)
[0510] 3. Proteose+1% non-acidulated glycerol+1% glucose (added
after 72 hr)
[0511] Each strain was inoculated to media containing different
glycerols (pure, acidulated, or non-acidulated) to 5.times.10.sup.5
cells/ml concentration. The cultures were kept in dark and agitated
by orbital shaker from Labnet (Berkshire, UK) at 430 rpm. After 72
hr of initial growth, 1% glucose was added and cultured another 24
hr. Dried cell-weight and lipid content were measured in all
samples (see EXAMPLES 2 and 5). The lipid percentage was calculated
from total lipid amount divided by dried cell weight. Results are
shown in FIG. 5.
Example 7
[0512] Strains and Media:
[0513] Chlorella protothecoides #2 (STRAIN 264) and Chlorella
kessleri #1 (STRAIN 398) were obtained from the Culture Collection
of Alga at the University of Texas (Austin, Tex., USA). The stock
cultures were maintained on modified Proteose medium (see EXAMPLE
2).
[0514] Experimental Design and Lipid Assay:
[0515] For each strain, 1 ml of following different media was
prepared in 24-well plates.
[0516] 1. Proteose+1% pure glycerol+1% glucose (added after 72
hr)
[0517] 2. Proteose+1% non-acidulated glycerol+1% glucose (added
after 72 hr)
[0518] Each strain was inoculated media containing either 1% pure
or 1% non-acidulated glycerol to 5.times.10.sup.5 cells/ml
concentration. The cultures were kept in dark and agitated by
orbital shaker from Labnet (Berkshire, UK) at 430 rpm. After 72 hr
of initial growth, 1% glucose was added and cultured another 24 hr.
Dried cell-weight and lipid content were measured in all samples
(see EXAMPLE 1 and 4). The lipid percentage was calculated from
total lipid amount divided by dried cell weight. Results are shown
in FIG. 6.
Example 8
[0519] Strains and Media:
[0520] Chlorella protothecoides #1 (STRAIN 250), #4 (STRAIN 31) and
Chlorella kessleri #2 (STRAIN 397) were obtained from the Culture
Collection of Alga at the University of Texas (Austin, Tex., USA).
The stock cultures were maintained on modified Proteose medium (see
EXAMPLE 2)
[0521] Experimental Design and Lipid Assay:
[0522] For each strain, 1 ml of following different media was
prepared in 24-well plates.
[0523] 1. Proteose+2% glucose
[0524] 2. Proteose+1% glycerol+1% glucose
[0525] Each strain was inoculated to different media to
5.times.10.sup.5 cells/ml concentration. The cultures were kept in
dark and agitated by orbital shaker from Labnet (Berkshire, UK) at
430 rpm. After 96 hr of initial growth, lipid contents were
measured (see EXAMPLE 5). Results are shown in FIG. 7.
Example 9
[0526] Strains and Media:
[0527] Chlorella protothecoides #3 (STRAIN 249), #4 (STRAIN 31) and
Chlorella kessleri #1 (STRAIN 398) were obtained from the Culture
Collection of Alga at the University of Texas (Austin, Tex., USA).
The stock cultures were maintained on modified Proteose medium (see
EXAMPLE 2).
[0528] Experimental Design and Lipid Assay:
[0529] For each strain, 1 ml of following different media was
prepared in 24-well plates.
[0530] 1. Proteose+2% glucose
[0531] 2. Proteose+1% glycerol+1% glucose
[0532] 3. Proteose+1% glycerol+1% glucose (added after 72 hr)
[0533] Each strain was inoculated to different media to
5.times.10.sup.5 cells/ml concentration. The cultures were kept in
dark and agitated by orbital shaker from Labnet (Berkshire, UK) at
430 rpm. After 72 hr of initial growth, 1% (w/v) glucose was added
to #3 media and cultured another 24 hr. Dried cell-weight and lipid
contents were measured in all samples (see EXAMPLES 2 and 5). The
lipid percentage was calculated from total lipid amount divided by
dried cell weight. Results are shown in FIG. 8.
Example 10
[0534] Strains and Media:
[0535] Chlorella protothecoides #1 (STRAIN 250), #3 (STRAIN 249),
and Chlorella kessleri #2 (STRAIN 397) were obtained from the
Culture Collection of Alga at the University of Texas (Austin,
Tex., USA). The stock cultures were maintained on modified Proteose
medium (see EXAMPLE 2).
[0536] Experimental Design and Lipid Assay:
[0537] For each strain, 1 ml of following different media was
prepared in 24-well plates.
[0538] 1. Proteose+1% pure glycerol+1% glucose
[0539] 2. Proteose+1% pure glycerol+1% glucose (added after 72
hr)
[0540] 3. Proteose+1% acidulated glycerol+1% glucose
[0541] 4. Proteose+1% acidulated glycerol+1% glucose (added after
72 hr)
[0542] 5. Proteose+1% non-acidulated glycerol+1% glucose
[0543] 6. Proteose+1% non-acidulated glycerol+1% glucose (added
after 72 hr)
[0544] Each strain was inoculated to different media to
5.times.10.sup.5 cells/ml concentration. The cultures were kept in
dark and agitated by orbital shaker from Labnet (Berkshire, UK) at
430 rpm. After 72 hr of initial growth, 1% (w/v) glucose was added
to #2, #4, and #6 media and cultured another 24 hr. Lipid contents
were measured in all samples (see EXAMPLE 4). Results are shown in
FIG. 9.
Example 11
[0545] Strains and Media:
[0546] Chlorella protothecoides #1 (STRAIN 250), #3 (STRAIN 249),
#4 (STRAIN 31) and Chlorella kessleri #2 (STRAIN 397) were obtained
from the Culture Collection of Alga at the University of Texas
(Austin, Tex., USA). The stock cultures were maintained on modified
Proteose medium (see EXAMPLE 2).
[0547] Experimental Design and Lipid Assay:
[0548] For each strain, 1 ml of following different media was
prepared in 24-well plates.
[0549] 1. Proteose+1% pure glycerol+1% glucose
[0550] 2. Proteose+1% pure glycerol+1% glucose (added after 72
hr)
[0551] 3. Proteose+1% acidulated glycerol+1% glucose
[0552] 4. Proteose+1% acidulated glycerol+1% glucose (added after
72 hr)
[0553] 5. Proteose+1% non acidulated glycerol+1% glucose
[0554] 6. Proteose+1% non acidulated glycerol+1% glucose (added
after 72 hr)
[0555] Each strain was inoculated to different media to
5.times.10.sup.5 cells/ml concentration. The cultures were kept in
dark and agitated by orbital shaker from Labnet (Berkshire, UK) at
430 rpm. After 72 hr of initial growth, 1% (w/v) glucose was added
to #2, #4, and #6 media and cultured another 24 hr. Dried
cell-weight was measured in all samples (see EXAMPLE 2). Results
are shown in FIG. 10.
Example 12
Vector Construction
[0556] A BamHI-SacII fragment containing the CMV promoter, a
hygromycin resistance cDNA, and a CMV 3' UTR (SEQ ID NO:5, a
subsequence of the pCAMBIA1380 vector, Cambia, Can berra,
Australia) was cloned into the BamHI and SacII sites of pBluescript
and is referred to herein as pHyg.
Biolistic Transformation of Chlorella
[0557] S550d gold carriers from Seashell Technology were prepared
according to the protocol from manufacturer. Linearized pHyg
plasmid (20 .mu.g) was mixed with 50 .mu.l of binding buffer and 60
.mu.l (30 mg) of S550d gold carriers and incubated in ice for 1
min. Precipitation buffer (100 .mu.l) was added, and the mixture
was incubated in ice for another 1 min. After vortexing, DNA-coated
particles were pelleted by spinning at 10,000 rpm in an Eppendorf
5415C microfuge for 10 seconds. The gold pellet was washed once
with 500 .mu.l of cold 100% ethanol, pelleted by brief spinning in
the microfuge, and resuspended with 50 .mu.l of ice-cold ethanol.
After a brief (1-2 sec) sonication, 10 .mu.l of DNA-coated
particles were immediately transferred to the carrier membrane.
[0558] Chlorella protothecoides culture (Univeristy of Texas
Culture Collection 250) was grown in proteose medium (2 g/L yeast
extract, 2.94 mM NaNO.sub.3, 0.17 mM CaCl.sub.2.2H.sub.2O, 0.3 mM
MgSO4.7H2O, 0.4 mM K2HPO4, 1.28 mM KH2PO4, 0.43 mM NaCl) on a
gyratory shaker under continuous light at 75 .mu.mol photons
m.sup.-2 sec.sup.-1 until it reached a cell density of
2.times.10.sup.6 cells/ml. The cells were harvested, washed once
with sterile distilled water, and resuspended in 50 .mu.l of
medium. 1.times.10.sup.7 cells were spread in the center third of a
non-selective proteose media plate. The cells were bombarded with
the PDS-1000/He Biolistic Particle Delivery system (Bio-Rad).
Rupture disks (1100 and 1350 psi) were used, and the plates were
placed 9 and 12 cm below the screen/macrocarrier assembly. The
cells were allowed to recover at 25.degree. C. for 12-24 h. Upon
recovery, the cells were scraped from the plates with a rubber
spatula, mixed with 100 .mu.l of medium and spread on hygromycin
contained plates (200 .mu.g/ml). After 7-10 days of incubation at
25.degree. C., colonies representing transformed cells were visible
on the plates from 1100 and 1350 psi rupture discs and from 9 and
12 cm distances. Colonies were picked and spotted on selective agar
plates for a second round of selection.
Transformation of Chlorella by Electroporation
[0559] Chlorella protothecoides culture was grown in proteose
medium on a gyratory shaker under continuous light at 75 .mu.mol
photons m.sup.-2 sec.sup.-1 until it reached a cell density of
2.times.10.sup.6 cells/ml. The cells were harvested, washed once
with sterile distilled water, and resuspended in a tris-phosphate
buffer (20m M Tris-HCl, pH 7.0; 1 mM potassium phosphate)
containing 50 mM sucrose to a density of 4.times.10.sup.8 cells/ml.
About 250 .mu.l cell suspension (1.times.10.sup.8 cells) was placed
in a disposable electroporation cuvette of 4 mm gap. To the cell
suspension, 5 .mu.g of linearized pHyg plasmid DNA and 200 .mu.g of
carrier DNA (sheared salmon sperm DNA) was added. The
electroporation cuvette was then incubated in a water bath at
16.degree. C. for 10 minutes. An electrical pulse (1100 V/cm) was
then applied to the cuvette at a capacitance of 25 .mu.F (no shunt
resistor was used for the electroporation) using a Gene Pulser II
(Bio-Rad Labs, Hercules, Calif.) electroporation apparatus. The
cuvette was then incubated at room temperature for 5 minutes,
following which the cell suspension was transferred to 50 ml of
proteose media, and shaken on a gyratory shaker for 2 days.
Following recovery, the cells were harvested by centrifugation at
low speed, resuspended in proteose media, and plated at low density
on plates supplemented with 200 .mu.g/ml hygromycin. The plates
were incubated under continuous light at 75 mmol photons m.sup.-2
sec.sup.-1. Transformants appeared as colonies in 1-2 weeks.
Colonies were picked and spotted on selective agar plates for a
second round of selection.
Genotyping
[0560] A subset of colonies that survived a second round of
selection were cultured in small volume and harvested. Pellets of
approximately 5-10 uL volume were resuspended in 50 uL of 10 mM
NaEDTA by vortexing and then incubated at 100.degree. C. for 10.
The tubes were then vortexed briefly and sonicated for 10 seconds,
then centifuged at 12,000.times.g for 1 minute. 2 uL of supernatant
as template was used in a 50 uL PCR reaction. Primers used for
genotyping were SEQ ID NO:6 and SEQ ID NO:7. PCR conditions were as
follows: 95.degree. C. 5 min.times.1 cycle; 95.degree. C. 30
sec-58.degree. C. 30 sec-72.degree. C. 1 min 30 sec.times.35
cycles; 72.degree. C. 10 min.times.1 cycle. The expected 992 bp
fragment was found in 6 of 10 colonies from the biolistic method
and from a single electroporation colony. A lower sized,
nonspecific band was present in all lanes. Results are shown in
FIG. 16. To confirm the identity of the amplified 992 bp fragment,
two biolistic bands and the electroporation band were excised from
the gel and individually sequenced. The sequence of all three bands
corresponded to the expected 992 bp fragment. (DNA ladder:
Bionexus.RTM. All Purpose Hi-Lo.RTM. DNA ladder catalog
#BN2050).
Example 13
[0561] Strains and Media:
[0562] (a) Spirulina platensis (UTEX 2340) and (b) Navicula
pelliculosa (UTEX 667) were obtained from the Culture Collection of
Algae at the University of Texas (Austin, Tex., USA). The stock
culture of Spirulina was maintained in Spirulina medium and
Navicula was maintained in soil extract medium (SEM). Spirulina
medium consisted of 162 mM NaHCO.sub.3, 38 mM Na.sub.2CO.sub.3, 1.9
mM K.sub.2HPO.sub.4, 29 mM NaNO.sub.3, 5.75 mM K.sub.2SO.sub.4,
17.1 mM NaCl, 0.8 mM MgSO.sub.4.7H.sub.2O, 0.25 mM
CaCl.sub.2.2H.sub.2O, 2 mM Na.sub.2EDTA, 0.36 mM
FeCl.sub.3.6H.sub.2O, 0.21 mM MnCl.sub.2.4H.sub.2O, 0.037 mM
ZnCl.sub.2, 0.0085 mM CoCl.sub.2.6H.sub.2O, 0.017 mM
NaMoO.sub.4.2H.sub.2O, 0.78 .mu.M CuSO.sub.4.5H.sub.2O, 0.15 .mu.M
ZnSO.sub.4.7H.sub.2O, 10 .mu.M H.sub.3BO.sub.3, and 0.001 mM
Vitamin B.sub.12. Soil extract medium consisted of 2.94 mM
NaNO.sub.3, 0.17 mMCaCl.sub.2.2H.sub.2O, 0.3 mM
MgSO.sub.4.7H.sub.2O, 0.43 mM K.sub.2HPO.sub.4, 1.29 mM
KH.sub.2PO.sub.4, 0.43 mM NaCl, and soil extract. Glycerol wastes
from biodiesel production (acidulated glycerol (AG) and
non-acidulated glycerol (NAG)) were obtained from Imperial Western
Products (Selma, Calif., USA).
[0563] Experimental Design and Growth Measurement:
[0564] For each strain, 1 ml of following different media was
prepared in 24-well plates.
[0565] (a)
[0566] 7. Spirulina medium+2% glucose
[0567] 8. Spirulina medium+2% reagent grade glycerol
[0568] 9. Spirulina medium+2% non-acidulated glycerol
[0569] 10. Spirulina medium+1% non-acidulated glycerol+1%
glucose
[0570] (b)
[0571] 1. SEM+2% glucose
[0572] 2. SEM+2% reagent grade glycerol
[0573] 3. SEM+1% reagent grade glycerol+1% glucose
[0574] 4. SEM+2% acidulated glycerol
[0575] 5. SEM+1% acidulated glycerol+1% glucose
[0576] 6. SEM+2% non-acidulated glycerol
[0577] 7. SEM+1% non-acidulated glycerol+1% glucose
[0578] Each strain was inoculated to different media to
5.times.10.sup.5 cells/ml concentration. The cultures were kept in
dark and agitated by orbital shaker from Labnet (Berkshire, UK) at
430 rpm. After 96 hr, lipid contents were measured. To measure the
amount of lipid content in cells, 100 .mu.l of cultures were
collected and washed once with same volume of media. To each tube,
5 .mu.l of washed cells and 200 .mu.l of sulfuric acid 18 M were
added. The tubes were incubated at 90.degree. C. water bath for 30
min, and 1 ml of phosphoric acid-vanillin reagent were added to the
tubes and incubated at 37.degree. C. for 15 min. To prepare the
phosphoric acid-vanillin reagent, 0.12 g of vanillin was added to
20 ml of water, and the volume adjusted to 100 ml with 85%
phosphoric acid. The optical density at 530 nm was read in a glass
cuvette against a reference tube with 5 .mu.l water as sample. The
reference curve is composed of Triolein dissolved in chloroform
ranging from 1 to 10 .mu.g.
[0579] To measure dried cell-weight, 0.5 ml of each culture was
pelleted by centrifugation at 5000 rpm for 5 min After removing
supernatant, cell pellets were frozen at -80.degree. C. and dried
overnight in a Freeze Dry system (Labconco, Mo., USA). The lipid
percentage was calculated from total lipid amount divided by dried
cell weight. Results are shown in FIG. 11.
Example 14
[0580] Strains and Media:
[0581] Scenedesmus armatus (UTEX 2552) was obtained from the
Culture Collection of Algae at the University of Texas (Austin,
Tex., USA). The stock cultures were maintained on modified Proteose
medium. Modified Proteose medium consisted (g/L) of 0.25 g
NaNO.sub.3, 0.09 g K.sub.2HPO.sub.4, 0.175 g KH.sub.2PO.sub.4 0.025
g, 0.025 g CaCl.sub.2.2H.sub.2O, 0.075 g MgSO.sub.4.7H.sub.2O, and
2 g yeast extract per liter.
[0582] Experimental Design and Growth and Lipid Measurement:
[0583] For each growth condition, 1 ml of following different media
was prepared in 24-well plates.
[0584] (a), (b)
[0585] 1. Proteose+2% glucose
[0586] 2. Proteose+2% glycerol
[0587] 3. Proteose+2% acidulated glycerol
[0588] 4. Proteose+2% non-acidulated glycerol
[0589] 5. Proteose+1% non-acidulated glycerol+1% glucose
[0590] Scenedesmus armatus (UTEX 2552) was inoculated to different
media to 5.times.10.sup.5 cells/ml concentration. The cultures were
kept in dark and agitated by orbital shaker from Labnet (Berkshire,
UK) at 430 rpm. After 96 hr, cell growth was measured by dried
cell-weight, and lipid content was measured by phosphor-vanillin
assay. (see EXAMPLE 13). The lipid percentage was calculated from
total lipid amount divided by dried cell weight. Results are shown
in FIG. 12.
Example 15
[0591] Strains and Media:
[0592] Navicula pelliculosa (UTEX 667) was obtained from the
Culture Collection of Algae at the University of Texas (Austin,
Tex., USA). The stock cultures were maintained on soil extract
medium (see EXAMPLE 13)
[0593] Experimental Design and Growth Measurement:
[0594] For each growth condition, 1 ml of following different media
was prepared in 24-well plates.
[0595] 1. SEM+2% glucose
[0596] 2. SEM+2% glycerol
[0597] 3. SEM+2% acidulated glycerol
[0598] 4. SEM+1% acidulated glycerol+1% glucose
[0599] 5. SEM+2% non-acidulated glycerol
[0600] 6. SEM+1% non-acidulated glycerol+1% glucose
[0601] Navicula pelliculosa (UTEX 667) was inoculated to media
containing glucose or different glycerols (pure, acidulated, or
non-acidulated) to 5.times.10.sup.5 cells/ml concentration. The
cultures were kept in dark and agitated by orbital shaker from
Labnet (Berkshire, UK) at 430 rpm. After 96 hr, cell growth was
measured by dried cell-weight (see EXAMPLE 13). Results are shown
in FIG. 13.
Example 16
[0602] Strains and Media:
[0603] Scenedesmus armatus (UTEX 2552) and Navicula pelliculosa
(UTEX 667) were obtained from the Culture Collection of Algae at
the University of Texas (Austin, Tex., USA). The stock cultures
were maintained on modified Proteose medium for Scenedesmus armatus
and soil extract medium for Navicula pelliculosa (see EXAMPLE
1).
[0604] Experimental Design and Growth Measurement:
[0605] For each strain, 1 ml of following different media was
prepared in 24-well plates.
[0606] Scenedesmus armatus
[0607] 5. Proteose+1% acidulated glycerol+1% glucose
[0608] 6. Proteose+1% acidulated glycerol+1% glucose (added after
72 hr)
[0609] Navicula pelliculosa
[0610] 1. SEM+1% acidulated glycerol+1% glucose
[0611] 2. SEM+1% acidulated glycerol+1% glucose (added after 72
hr)
[0612] Each strain was inoculated to media to 5.times.10.sup.5
cells/ml concentration. The cultures were kept in dark and agitated
by orbital shaker from Labnet (Berkshire, UK) at 430 rpm. After 72
hr of initial growth, 1% glucose was added to sample #2 and
cultured another 24 hr. Cell growth was measured by dried
cell-weight (see EXAMPLE 13). Results are shown in FIG. 14.
Example 17
[0613] Strains and Media:
[0614] Chlorella protothecoides (UTEX 31) was obtained from the
Culture Collection of Algae at the University of Texas (Austin,
Tex., USA). The stock cultures were maintained on modified Proteose
medium (see EXAMPLE 1)
[0615] Experimental Design:
[0616] For each condition, 1 ml of following different media was
prepared in 24-well plates.
[0617] 4. Proteose
[0618] 5. Proteose+0.5% glucose
[0619] 6. Proteose+0.5% xylose
[0620] 7. Proteose+0.25% glucose+0.25% xylose
[0621] Chlorella protothecoides #4 (UTEX 31) was inoculated to
media containing different sugars (glucose, or xylose) to
3.times.10.sup.5 cells/ml concentration. The cultures were kept in
dark and agitated by orbital shaker from Labnet (Berkshire, UK) at
430 rpm. After 72 hr of growth, cell growth was measured by
counting cell numbers of each culture. Results are shown in FIG.
15.
Example 18
[0622] Chlorella protothecoides strains #1, #3, and #4 were
obtained from the Culture Collection of Algae at the University of
Texas (Austin, Tex., USA). The stock cultures were maintained on
modified Proteose medium (see EXAMPLE 1). For each condition, 1 ml
of following different media was prepared in 24-well plates.
[0623] 1. Proteose
[0624] 2. Proteose+1% glucose
[0625] 3. Proteose+1% fructose
[0626] Each strain was inoculated to media containing different
sugars (glucose, or fructose) to 1.times.10.sup.6 cells/ml
concentration. The cultures were kept in dark and agitated by
orbital shaker from Labnet (Berkshire, UK) at 430 rpm. After 96 hr
of growth, cell density was measured by counting cell numbers of
each culture. Results are shown in FIG. 20.
Example 19
[0627] Chlorella on Sucrose
[0628] Materials and Methods:
[0629] Chlorella protothecoides (UTEX 249) was inoculated into
three 50 ml flasks of Proteose media with 1% sucrose (2.94 mM
NaNO.sub.3, 0.428 mM K.sub.2HPO.sub.4, 1.28 mM KH.sub.2PO.sub.4,
0.427 mM NaCl, 0.17 mM CaCl.sub.2-2H.sub.2O, 0.3 mM
Mg50.sub.4-7H.sub.2O, proteose peptone 1 g/L) to a final cell
density of 4.times.10.sup.5 cells per ml. Invertase (Sigma #14504)
was added to two of the cultures at 0.01 U/ml and 0.05 U/ml. All
three cultures were grown in the dark for .about.60 hrs shaking at
150 rpm.
[0630] Results:
[0631] Final cell counts were performed on all three cultures after
.about.60 hrs of shaking in the dark. The control flask reached
4.4.times.10.sup.5 cells per ml while the 0.01 U/ml and 0.05 U/ml
flasks reached cell densities of 1.times.10.sup.8 and
3.times.10.sup.8 respectively. Each flask was checked for
contamination at the end of the experiment by microscopic analysis
and all were clean.
Example 20
Chlorella Strains Growing on Sucrose
[0632] Cultures of Chlorella kessleri ((a) UTEX 397 and (b) UTEX
398) and Chlorella fusca ((a) UTEX 251 and (b) UTEX 1801) were
inoculated from autotrophic liquid cultures into 10 ml of
Proteose+1% sucrose media in 50 ml flasks at 1.times.10.sup.6
cells/ml. Control cultures were also inoculated at the same density
with only Proteose media. Cultures were grown at 28.degree. C. in
the dark shaking at 250 rpm for 7 days, at which point cell density
was measured by hemocytometer. As shown in FIGS. 21-22, all four
strains grew on sucrose compared to the initial cell density and
the proteose-only control.
Example 21
Chlorella protothecoides Growth on Molasses with a Sucrose
Invertase
[0633] Preparation of Chlorella Cells for Inoculation:
[0634] A 10 ml liquid culture of Chlorella was started taking the
inoculum from a solid Proteose plate. The cultures were grown in
light for approximately 2 days at 26.degree. C. Growth was measured
using an optical densitomer (OD) at 750 nm and by determining dry
cell weights.
[0635] Preparation of Molasses and Sugar Stock Solutions:
[0636] A 5% stock solution was prepared with glucose, sucrose and
three different molasses samples (labeled BS1, BS2 and HTM)
obtainted from the commercial processing of sugarcane into sugar,
as shown in the following Table 13. The pH of all stocks was
verified to be in the range of 6-6.6, and the stocks were then
autoclaved.
TABLE-US-00014 TABLE 13 Molasses and sugar solutions. 5% sugar
dil.in 100 mls Molasses % Sugar grams or mls HTM 78.72 6.4 BS1 (FL)
44.25 11.3 BS2 (AU) 51.55 9.7 Sucrose 100 5 Glucose 100 5
[0637] Preparation of Invertase Solution:
[0638] A 40 units/ml stock solution of invertase was prepared by
reconstituting 1 mg of a 400 unit/mg Invertase (Sigma) in 10
milliliters of distilled water.
[0639] Experimental Conditions and Setup:
[0640] 10 ml cultures were prepared, each consisting of 1% final
molasses/sugar concentration, 0.05 units/ml Invertase, and
1.0.times.10.sup.6 cells per ml of Chlorella protothecoides in a
base Protease media. The cultures were numbered as follows: (1)
media only control; (2) 1% HTM; (3) 1% BS1; (4) 1% BS2; (5) 1%
glucose; and (6) 1% sucrose. A similar control set was also
prepared without the addition of Invertase. The cultures were grown
in darkness for five days shaking at 250 rpm at 28.degree. C.
[0641] Results:
[0642] Growth of the Chlorella protothecoides cells was evaluated
following the five days of incubation on the respective feedstock
in darkness. As shown in FIGS. 23-24, the cells can be grown on
molasses in the presence of a sucrose invertase with yields
comparable to that of growth on pure reagent-grade glucose.
Example 22
[0643] Genetic Engineering of Chlorella protothecoides to Express
an Exogenous Sucrose Invertase
[0644] Strains and Media:
[0645] Chlorella protothecoides (UTEX 250) was obtained from the
Culture Collection of Alga at the University of Texas (Austin,
Tex., USA). The stock cultures were maintained on modified Proteose
medium. Modified Proteose medium consists of 0.25 g NaNO.sub.3,
0.09 g K.sub.2HPO.sub.4, 0.175 g KH.sub.2PO.sub.4 0.025 g, 0.025 g
CaCl.sub.2.2H.sub.2O, 0.075 g MgSO.sub.4.7H.sub.2O, and 2 g yeast
extract per liter (g/L).
[0646] Plasmid Construction:
[0647] To express the secreted form of invertase in Chlorella
protothecoides, a Saccharomyces cerevisiae SUC2 gene was placed
under the control of three different promoters: Cauliflower mosaic
virus 35S promoter (CMV), Chlorella virus promoter (NC-1A), and
Chlorella HUP1 promoter. A yeast SUC2 gene was synthesized to
accommodate codon usage optimized for C. protothecoides and
includes a signal sequence required for directing extracellular
secretion of invertase. Each construct was built in pBluescript
KS+, and EcoRI/AscI, AscI/XhoI, and XhoI/BamHI sites were
introduced to each promoter, invertase gene, and CMV 3'UTR,
respectively, by PCR ampilication using specific primers. Purified
PCR products were cloned sequentially. An illustration of the final
constructs is shown in FIG. 25.
[0648] Transformation of Chlorella protothecoides:
[0649] A Chlorella protothecoides culture was grown in modified
Proteose medium on a gyratory shaker under continuous light at 75
.mu.mol photons m.sup.-2 sec.sup.-1 till it reached a cell density
of 6.times.10.sup.6 cells/ml.
[0650] For biolistic transformation, S550d gold carriers from
Seashell Technology were prepared according to the protocol from
the manufacturer. Briefly, a linearized construct (20 .mu.g) by
BsaI was mixed with 50 .mu.l of binding buffer and 60 .mu.l (3 mg)
of S550d gold carriers and incubated in ice for 1 mM Precipitation
buffer (100 .mu.l) was added, and the mixture was incubated in ice
for another 1 min. After mild vortexing, DNA-coated particles were
pelleted by spinning at 10,000 rpm in an Eppendorf microfuge for 10
seconds. The gold pellet was washed once with 500 .mu.l of cold
100% ethanol, pelleted by brief spinning in the microfuge, and
resuspended with 50 .mu.l of ice-cold ethanol. After a brief (1-2
sec) sonication, 10 .mu.l of DNA-coated particles were immediately
transferred to the carrier membrane. The cells were harvested,
washed once with sterile distilled water, resuspended in 50 .mu.l
of medium (1.times.10.sup.7 cells), and were spread in the center
third of a non-selective Proteous plate. The cells were bombarded
with the PDS-1000/He Biolistic Particle Delivery system (Bio-Rad).
Rupture disks (1100 and 1350 psi) were used, and the plates were
placed 9-12 cm below the screen/macrocarrier assembly. The cells
were allowed to recover at 25.degree. C. for 12-24 hours. Upon
recovery, the cells were scraped from the plates with a rubber
spatula, mixed with 100 .mu.l of medium and spread on modified
Proteose plates with 1% sucrose. After 7-10 days of incubation at
25.degree. C. in the dark, colonies representing transformed cells
were visible on the plates.
[0651] For transformation with electroporation, cells were
harvested, washed once with sterile distilled water, and
resuspended in a Tris-phosphate buffer (20 mM Tris-HCl, pH 7.0; 1
mM potassium phosphate) containing 50 mM sucrose to a density of
4.times.10.sup.8 cells/ml. About 250 .mu.l cell suspension
(1.times.10.sup.8 cells) was placed in a disposable electroporation
cuvette of 4 mm gap. To the cell suspension, 5 .mu.g of linearized
plasmid DNA and 200 .mu.g of carrier DNA (sheared salmon sperm DNA)
were added. The electroporation cuvette was then incubated in an
ice water bath at 16.degree. C. for 10 min. An electrical pulse
(1100 V/cm) was then applied to the cuvette at a capacitance of 25
.mu.F (no shunt resistor was used for the electroporation) using a
Gene Pulser II (Bio-Rad Labs, Hercules, Calif.) electroporation
apparatus. The cuvette was then incubated at room temperature for 5
minutes, following which the cell suspension was transferred to 50
ml of modified Proteose media, and shaken on a gyratory shaker for
2 days. Following recovery, the cells were harvested at low speed
(4000 rpm), resuspended in modified Proteose media, and plated out
at low density on modified Proteose plates with 1% sucrose. After
7-10 days of incubation at 25.degree. C. in the dark, colonies
representing transformed cells were visible on the plates.
[0652] Screening Transformants and Genotyping:
[0653] The colonies were picked from dark grown-modified Proteose
plates with 1% sucrose, and approximately the same amount of cells
were transferred to 24 well-plates containing 1 ml of modified
Proteose liquid media with 1% sucrose. The cultures were kept in
dark and agitated by orbital shaker from Labnet (Berkshire, UK) at
430 rpm for 5 days.
[0654] To verify the presence of the invertase gene introduced in
Chlorella transformants, DNA of each transformant was isolated and
amplified with a set of gene-specific primers (CMV construct:
forward primer (CAACCACGTCTTCAAAGCAA) (SEQ ID NO:6)/reverse primer
(TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO:9), CV constructs: forward primer
(TTGTCGGAATGTCATATCAA) (SEQ ID NO:10)/reverse primer
(TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO:11), and HUP1 construct: forward
primer (AACGCCTTTGTACAACTGCA) (SEQ ID NO:12)/reverse primer
(TCCGGTGTGTTGTAAGTCCA) (SEQ ID NO:13)). For quick DNA isolation, a
volume of cells (approximately 5-10 uL in size) were resuspended in
50 uL of 10 mM Na-EDTA. The cell suspension was incubated at
100.degree. C. for 10 min and sonicated for 10 sec. After
centrifugation at 12000 g for 1 mM, 3 uL of supernatant was used
for the PCR reaction. PCR amplification was performed in the DNA
thermal cycler (Perkin-Elmer GeneAmp 9600). The reaction mixture
(50 uL) contained 3 uL extracted DNA, 100 pmol each of the
respective primers described above, 200 uM dNTP, 0.5 units of Taq
DNA polymerase (NEB), and Taq DNA polymerase buffer according to
the manufacturer's instructions. Denaturation of DNA was carried
out at 95.degree. C. for 5 mM for the first cycle, and then for 30
sec. Primer annealing and extension reactions were carried out at
58.degree. C. for 30 sec and 72.degree. C. for 1 min respectively.
The PCR products were then visualized on 1% agarose gels stained
with ethidium bromide. FIG. 26 shows the PCR genotype results of C.
protothecoides transformants using the gene-specific primers
identified above. Arrows show the expected size of the PCR product,
and stars represent DNA samples from each transformant showing the
PCR product matched to the expected size (V: Vector only, WT:
wild-type).
[0655] Growth in Liquid Culture:
[0656] After five days growth in darkness, the genotype-positive
transformants showed growth on minimal liquid Proteose media+1%
sucrose in darkness, while wild-type cells showed no growth in the
same media in darkness.
Example 23
[0657] Transformation of Algal Strains with a Secreted Invertase
Derived from S. cerevisiae
[0658] Secreted Invertase:
[0659] A gene encoding a secreted sucrose invertase (Gen Bank
Accession no. NP.sub.--012104 from Saccharomyces cerevisiae) was
synthesized de-novo as a 1599 bp Asc I-Xho fragment that was
subsequently sub-cloned into a pUC19 derivative possessing the
Cauliflower Mosaic Virus 35S promoter and 3' UTR as EcoR I/Asc I
and Xho/Sac I cassettes, respectively.
[0660] Growth of Algal Cells:
[0661] Media used in these experiments was liquid base media (see
Example 1) and solid base media (+1.5% agarose) containing fixed
carbon in the form of sucrose or glucose (as designated) at 1%
final concentration. The strains used in this experiment did not
grow in the dark on base media in the absence of an additional
fixed carbon source. Species were struck out on plates, and grown
in the dark at 28.degree. C. Single colonies were picked and used
to inoculate 500 mL of liquid base media containing 1% glucose and
allowed to grow in the dark until mid-log phase, measuring cell
counts each day. Each of the following strains had been previously
tested for growth on sucrose in the dark as a sole carbon source
and exhibited no growth, and were thus chosen for transformation
with a secreted invertase: (1) Chlorella protothecoides (UTEX 31);
(2) Chlorella minutissima (UTEX 2341); and (3) Chlorella emersonii
(CCAP 211/15).
[0662] Transformation of Algal Cells via Particle Bombardment:
[0663] Sufficient culture was centrifuged to give approximately
1-5.times.10.sup.8 total cells. The resulting pellet was washed
with base media with no added fixed carbon source. Cells were
centrifuged again and the pellet was resuspended in a volume of
base media sufficient to give 5.times.10.sup.7 to 2.times.10.sup.8
cells/ml. 250-1000 .mu.l of cells were then plated on solid base
media supplemented with 1% sucrose and allowed to dry onto the
plate in a sterile hood. Plasmid DNA was precipitated onto gold
particles according to the manufacturer's recommendations (Seashell
Technology, La Jolla, Calif.). Transformations were carried out
using a BioRad PDS He-1000 particle delivery system using 1350 psi
rupture disks with the macrocarrier assembly set at 9 cm from the
rupture disk holder. Following transformations, plates were
incubated in the dark at 28.degree. C. All strains generated
multiple transformant colonies. Control plates transformed with no
invertase insert, but otherwise prepared in an identical fashion,
contained no colonies.
[0664] Analysis of Chlorella Protothecoides Transformants:
[0665] Genomic DNA was extracted from Chlorella protothecoides wild
type cells and transformant colonies as follows: Cells were
resuspended in 100 ul extraction buffer (87.5 mM Tris Cl, pH 8.0,
50 mM NaCl, 5 mM EDTA, pH 8.0, 0.25% SDS) and incubated at
60.degree. C., with occasional mixing via inversion, for 30
minutes. For PCR, samples were diluted 1:100 in 20 mM Tris Cl, pH
8.0.
[0666] Genotyping was done on genomic DNA extracted from WT, the
transformants and plasmid DNA. The samples were genotyped for the
marker gene. Primers 2383 (5' CTGACCCGACCTATGGGAGCGCTCTTGGC 3')
(SEQ ID NO:20) and 2279 (5' CTTGACTTCCCTCACCTGGAATTTGTCG 3') (SEQ
ID NO:21) were used in this genotyping PCR. The PCR profile used
was as follows: 94.degree. C. denaturation for 5 min; 35 cycles of
94.degree. C.-30 sec, 60.degree. C.-30 sec, 72.degree. C.-3 min;
72.degree. C.-5 min. A band of identical size was amplified from
the positive controls (plasmid) and two transformants of Chlorella
protothecoides (UTEX 31), as shown in FIG. 27.
[0667] Analysis of Chlorella minutissima and Chlorella emersonii
transformants: Genomic DNA was extracted from Chlorella WT and the
tranformants as follows: Cells were resuspended in 100 ul
extraction buffer (87.5 mM Tris Cl, pH 8.0, 50 mM NaCl, 5 mM EDTA,
pH 8.0, 0.25% SDS) and incubated at 60.degree. C., with occasional
mixing via inversion, for 30 minutes. For PCR, samples were diluted
1:100 in 20 mM Tris Cl, pH 8.0. Genotyping was done on genomic DNA
extracted from WT, the transformants and plasmid DNA. The samples
were genotyped for the marker gene. Primers 2336 (5'
GTGGCCATATGGACTTACAA 3') (SEQ ID NO:22) and 2279 (5'
CTTGACTTCCCTCACCTGGAATTTGTCG 3') (SEQ ID NO:21) were designated
primer set 2 (1215 bp expected product), while primers 2465 (5'
CAAGGGCTGGATGAATGACCCCAATGGACTGTGGTACGACG 3') (SEQ ID NO:23) and
2470 (5' CACCCGTCGTCATGTTCACGGAGCCCAGTGCG 3') (SEQ ID NO:24) were
designated primer set 4 (1442 bp expected product). The PCR profile
used was as follows: 94.degree. C. denaturation for 2 mM; 29 cycles
of 94.degree. C.-30 sec, 60.degree. C.-30 sec, 72.degree. C.-1 min,
30 sec; 72.degree. C.-5 min. A plasmid control containing the
secreted invertase was used as a PCR control. FIG. 28 shows the
transformation of the Chlorella minutissima (UTEX 2341) and
Chlorella emersonii (CCAP 211/15) species of microalgae with the
gene encoding a secreted invertase.
[0668] The sequence of the invertase construct corresponds to SEQ
ID NO:25.
Example 24
Growth of Algal Strains Compared to S. cerevisiae on a Cellulosic
Feedstock Prepared with Celluclast
[0669] Strains and Culture Conditions:
[0670] Algal strains used in this study are listed in Table 14
below, and were grown in Proteose media with exogenously provided
cellulosic material and in some cases additional fixed carbon in
the form of glucose. Twenty four algal strains were used in this
study, including five different genera encompassing eleven
different species of Chlorella, two of Parachlorella and
Prototheca, and one each of Bracteococcus and Pseudochlorella.
Saccharomyces cerevisiae (strain PJ-69-4A) was grown in YPD media
(per liter, 10 g Bacto-yeast extract, 20 g Bacto peptone and 20 g
glucose). Both algae and yeast were grown at 28.degree. C. in the
dark. Growth of these strains on Proteose media in the dark in the
absence of cellulosic material or other additional fixed carbon
either did not occur or was extremely minimal.
[0671] Liberation of Glucose from Cellulosic Material via Enzymatic
Depolymerization Treatment:
[0672] Wet, exploded corn stover material was prepared by the
National Renewable Energy Laboratory (Golden, Colo.) by cooking
corn stover in a 1.4% sulfuric acid solution and dewatering the
resultant slurry. Using a Mettler Toledo Moisture analyzer, the dry
solids in the wet corn stover were determined to be 24%. A 100 g
wet sample was resuspended in deionized water to a final volume of
420 ml and the pH was adjusted to 4.8 using 10 N NaOH. Celluclast
(Novozymes) (a cellulase) was added to a final concentration of 4%
and the resultant slurry incubated with shaking at 50.degree. C.
for 72 hours. The pH of this material was then adjusted to 7.5 with
NaOH (negligible volume change), filter sterilized through a 0.22
um filter and stored at -20.degree. C. A sample was reserved for
determination of glucose concentration using a hexokinase based kit
from Sigma, as described below.
[0673] Determination of Glucose Concentration Liberated by
Celluclast Treatment of Wet Corn Stover:
[0674] Glucose concentrations were determined using Sigma Glucose
Assay Reagent #G3293. Samples, treated as outlined above, were
diluted 400 fold and 40 .mu.l was added to the reaction. The corn
stover cellulosic preparation was determined to contain
approximately 23 g/L glucose.
TABLE-US-00015 TABLE 14 Algal strains grown on cellulosic
feedstock. Genus/Species Source/Designation Bracteococcus minor
UTEX 66 Chlorella ellipsoidea SAG 2141 Chlorella kessleri UTEX 1808
Chlorella kessleri UTEX 397 Chlorella emersonii CCAP 211/15
Chlorella luteoviridis SAG 2133 Chlorella luteoviridis SAG 2198
Chlorella luteoviridis SAG 2214 Chlorella luteoviridis UTEX 22
Bracteococcus medionucleatus UTEX 1244 Chlorella minutissima CCALA
20024 Chlorella minutissima UTEX 2341 Chlorella ovalis CCAP 211/21A
Chlorella protothecoides CCAP 211/8d Chlorella protothecoides UTEX
250 Chlorella saccharophila UTEX 2911 Chlorella sorokiniana UTEX
1230 Chlorella sp. SAG 241.80 Chlorella vulgaris CCAP 211/11C
Parachlorella kessleri SAG 12.80 Parachlorella kessleri SAG 27.87
Prototheca moriformis UTEX 1441 Prototheca moriformis UTEX 1434
Pseudochlorella aquatica SAG 2149
[0675] In Table 14, and as used herein, UTEX refers to the culture
collection of algae at the University of Texas (Austin, Tex., USA),
SAG refers to the Culture Collection of Algae at the University of
Gottingen (Gottingen, Germany), CCAP refers to the culture
collection of algae and protozoa managed by the Scottish
Association for Marine Science (Scotland, United Kingdom) and CCALA
refers to the culture collection of algal laboratory at the
Institute of Botany (T{hacek over (r)}ebo{hacek over (n)}, Czech
Republic).
[0676] Determination of Growth on Cellulosic Material:
[0677] After enzymatic treatment and saccharification of cellulose
to glucose, xylose, and other monosaccharide sugars, the material
prepared above was evaluated as a feedstock for the growth of 24
algal strains or S. cerevisiae in Proteose media or YPD media
respectively. Proteose media was made up to a final glucose
concentration of 23 g/L (the final concentration of glucose
generated via cellulolytic treatment of corn stover), as was YPD
for growth of S. cerevisiae, by adding varying amounts of pure
glucose and/or depolymerized cellulosic material. Varying
concentrations of cellulosic material were included, providing 0,
12.5, 25, 50 and 100% of the 23 g/L glucose in each media, the
components of which are shown in Table 15 below. One ml of the
appropriate media was added to wells of a 24 well plate. S.
cerevisiae grown heterotrophically at 28.degree. C. in YPD served
as inoculum (20 ul) for the yeast wells. Twenty microliters of
inoculum for the 24 algal strains was furnished by alga cells grown
mixotrophically in Proteose media containing 20 g/L glucose.
TABLE-US-00016 TABLE 15 Cellulosic feedstock preparations. Vol.
Vol. Vol. of of 100% of 100% Vol. Proteose Cellulosics Cellul- of
YPD Media Made up osics 23.1 g/L 23.1 g/L to Proteose Made up Final
Glucose Glucose Media to YPD Volume Percent (ml) (ml) (ml) (ml)
(ml) Cellulosics 0 1 0 0 1 0 0 0.875 0.125 0 1 12.5 0 0.75 0.25 0 1
25 0 0.5 0.5 0 1 50 0 0 1 0 1 100 1 0 0 0 1 0 0.875 0 0 0.125 1
12.5 0.75 0 0 0.25 1 25 0.5 0 0 0.5 1 50 0 0 0 1 1 100
[0678] Table 15 shows the volumes of depolymerized corn stover
modified to contain Proteose or YPD media components that were
added to Proteose or YPD media respectively to yield medias
containing the indicated percentage of cellulosics. Medias were
prepared in order to obtain a final glucose concentration of 23 g/L
in all cases. Volume of media prior to the addition of either 20
.mu.l of mid-log phase grown yeast or alga cells was 1 ml. Cells
were incubated two days in the dark on the varying concentrations
of cellulosic feedstocks at 28.degree. C. with shaking (300 rpm).
Growth was assessed by measurement of absorbance at 750 nm in a UV
spectrophotometer. Surprisingly, all strains grew on the cellulosic
material prepared with Celluclast, including media conditions in
which 100% of fermentable sugar was cellulosic-derived.
Example 25
Growth of 24 Algal Strains and S. cerevisiae on Various Cellulosic
Feedstocks Prepared with Accellerase 1000.TM. and
Celluclast.TM.
[0679] Strains and Culture Conditions:
[0680] Algal strains used in this Example are listed in Table 14
(above) and were grown in Proteose media plus additional fixed
carbon in the form of depolymerized cellulosic material and/or pure
glucose. Saccharomyces cerevisiae (strain pJ69-4-a) was grown in
YPD media plus additional fixed carbon in the form of depolymerized
cellulosic material and/or pure glucose. Both algae and yeast were
grown at 28.degree. C. in the dark.
[0681] Liberation of Glucose from Cellulosic Material via Enzymatic
Depolymerization Treatment:
[0682] Wet, exploded corn stover material was prepared by the
National Renewable Energy Laboratory (Golden, Colo.) by cooking
corn stover in a 1.4% sulfuric acid solution and dewatering the
resultant slurry. Switchgrass was also prepared by The National
Renewable Energy Laboratory (Golden, Colo.) utilizing the same
method as for corn stover. Sugar beet pulp, generated via pectinase
treatment, was supplied by Atlantic Biomass, Inc. of Frederick, Md.
Using a Mettler Toledo Moisture analyzer, the dry solids were 24%
in the wet corn stover, 26% in switch grass and 3.5% in sugar beet
pulp. A 100 g wet sample of corn stover or switchgrass was
resuspended in deionized water to a final volume of 420 ml and the
pH adjusted to 4.8 using 10 N NaOH. For beet pulp, 8.8 grams dry
solids were brought to 350 ml with deionized water and pH adjusted
to 4.8 with 10N NaOH. For all feed stocks, Accellerase 1000.TM.
(Genencor) (a cellulase enzyme complex) was used at a ratio of 0.25
ml enzyme per gram dry biomass. Samples were incubated with
agitation (110 rpm) at 50.degree. C. for 72 hours. The pH of this
material was then adjusted to 7.5 with NaOH (negligible volume
change), filter sterilized through a 0.22 um filter and used in
growth experiments outlined below. A sample was reserved for
determination of glucose concentration using a hexokinase-based kit
from Sigma, as described below. The same set of cellulosic
feedstocks were also prepared using Celluclast.TM. (Novozymes) (a
cellulase) as described in the previous example.
[0683] Determination of Glucose Concentrations in Various
Cellulosic Feedstocks Treated with Accellerase 1000:
[0684] Glucose concentrations were determined using Sigma Glucose
Assay Reagent #G3293. Samples, treated as outlined above, were
diluted 400 fold and 40 ul was added to the reaction. The
cellulosic preparations from corn stover, switch grass and beet
pulp were determined to contain approximately 23.6, 17.1 and 13.7
g/L glucose, respectively.
[0685] Determination of Growth on Cellulosic Material:
[0686] After enzymatic depolymerization of cellulosic sources to
glucose, xylose, and other monosaccharides, the materials prepared
above were evaluated as feedstocks for the growth of the 24 algal
strains listed in the Table 14 and S. cerevisiae in Proteose or YPD
medias respectively. The medias were designed to contain a
consistent concentration of glucose while varying the amount of
cellulosic material derived from corn stover, switchgrass or beet
pulp. A first set of Proteose and YPD media contained 23.6 g/L pure
glucose, while a second set of media contained depolymerized corn
stover, switchgrass and beet pulp, each of which contained 23.6 g/L
glucose. The switchgrass and beet pulp medias were supplemented
with 6.5 and 9.9 g/L pure glucose to normalize glucose in all
cellulosic medias at 23.6 g/L. One ml of the appropriate media was
added to wells of a 24 well plate. S. cerevisiae grown
heterotrophically at 28.degree. C. in YPD served as inoculum (20
ul) for the yeast wells. Twenty microliters of inoculum for the 24
algal strains was furnished by alga cells grown mixotrophically in
Proteose media containing 20 g/L glucose. all cells were incubated
two days in the dark on the varying concentrations of cellulosic
feedstocks at 28.degree. C. with shaking (300 rpm). Growth was
assessed by measurement of absorbance at 750 nm in a UV
spectrophotometer. Surprisingly, all algae strains grew on the corn
stover, switchgrass and beet pulp material prepared with
Accellerase 1000.TM. or Celluclast.TM., including media conditions
in which 100% of fermentable sugar was cellulosic-derived. Under no
combination of cellulosic feedstock and depolymerization enzyme did
S. cerevisiae outperform growth on an equivalent amount of pure
glucose, indicating that inhibitors to yeast growth in the
cellulosic material made a major impact on the productivity of the
fermentation. Combinations of algae strains, depolymerization
enzymes and feedstocks with 100% cellulosic-derived monosaccharides
that outperformed 100% pure glucose are shown in Table 16.
TABLE-US-00017 TABLE 16 Combinations of algae, enzymes, and
feedstocks. Feedstock and Source/ Depolymerization Enzyme
Genus/Species Designation corn stover/celluclast .TM. Bracteococcus
minor UTEX 66 beet pulp/accellerase .TM. Chlorella ellipsoidea SAG
2141 switchgrass/accellerase .TM. Chlorella kessleri UTEX 252 beet
pulp/accellerase .TM. Chlorella kessleri UTEX 397
switchgrass/accellerase .TM. Chlorella luteoviridis SAG 2133 beet
pulp/accellerase .TM. Chlorella luteoviridis SAG 2133
switchgrass/accellerase .TM. Chlorella luteoviridis UTEX 22 beet
pulp/accellerase .TM. Chlorella luteoviridis UTEX 22 corn
stover/accellerase .TM. Chlorella luteoviridis UTEX 22 beet
pulp/accellerase .TM. Chlorella protothecoides UTEX 250 beet
pulp/accellerase .TM. Chlorella sp. SAG 241.80 beet
pulp/accellerase .TM. Parachlorella kessleri SAG 12.80
switchgrass/accellerase .TM. Prototheca moriformis UTEX 1441 beet
pulp/accellerase .TM. Prototheca moriformis UTEX 1441 corn
stover/accellerase .TM. Prototheca moriformis UTEX 1441 corn
stover/celluclast .TM. Prototheca moriformis UTEX 1441 corn
stover/accellerase .TM. Prototheca moriformis UTEX 1434 beet
pulp/accellerase .TM. Pseudochlorella aquatica SAG 2149
Example 26
Carbon Utilization Screens
[0687] Strains and Culture Conditions:
[0688] Seed cultures of the various strains of microalgae
identified below were started as 1 ml liquid cultures in 24 well
plates and were grown autotrophically for 48 hours in light,
agitating at .about.350 rpm. Plates were setup with 1.5%
agarose-based solid Proteose media containing 1% of glucose,
glycerol, xylose, sucrose, fructose, arabinose, mannose, galactose,
or acetate as the sole fixed carbon source. For each strain, 5
.mu.l of culture from the autotrophic 24 well plate was spotted
onto the solid media. Plates were incubated for 7 days in the dark
at 28.degree. C. and examined for growth compared to a control
plate containing no additional fixed carbon. Growth was observed
for each of the species tested with each respective feedstock, as
shown in Table 17 below. Growth of these strains on Proteose media
in the dark in the absence of additional fixed carbon either did
not occur or was extremely minimal.
TABLE-US-00018 TABLE 17 Algal species grown on various fixed-carbon
feedstocks. Fixed Source/ Carbon Source Genus/Species Designation
Glucose Chlorella protothecoides UTEX 250 Glucose Chlorella
kessleri UTEX 397 Glucose Chlorella sorokiniana UTEX 2805 Glucose
Parachlorella kessleri SAG 12.80 Glucose Pseudochlorella aquatica
SAG 2149 Glucose Chlorella reisiglii CCAP 11/8 Glucose
Bracteococcus medionucleatus UTEX 1244 Glucose Prototheca stagnora
UTEX 1442 Glucose Prototheca moriformis UTEX 1434 Glucose
Prototheca moriformis UTEX 1435 Glucose Scenedesmus rubescens CCAP
232/1 Glycerol Parachlorella kessleri SAG 12.80 Glycerol Chlorella
protothecoides CCAP 211/8d Glycerol Bracteococcus medionucleatus
UTEX 1244 Glycerol Prototheca moriformis UTEX 288 Glycerol
Prototheca moriformis UTEX 1435 Glycerol Chlorella minutissima UTEX
2341 Glycerol Chlorella sp. CCAP 211/61 Glycerol Chlorella
sorokiniana UTEX 1663 Xylose Chlorella luteoviridis SAG 2133 Xylose
Chlorella ellipsoidea SAG 2141 Xylose Pseudochlorella aquatica SAG
2149 Xylose Chlorella sp. CCAP 211/75 Xylose Prototheca moriformis
UTEX 1441 Xylose Prototheca moriformis UTEX 1435 Sucrose Chlorella
saccharophila UTEX 2469 Sucrose Chlorella luteoviridis UTEX 22
Sucrose Chlorella sp. UTEX EE102 Sucrose Chlorella luteoviridis SAG
2198 Sucrose Bracteococcus medionucleatus UTEX 1244 Sucrose
Chlorella minutissima CCALA 20024 Fructose Chlorella kessleri UTEX
398 Fructose Chlorella trebouxiodes SAG 3.95 Fructose Parachlorella
kessleri SAG 27.87 Fructose Chlorella luteoviridis SAG 2214
Fructose Chlorella protothecoides UTEX 31 Fructose Chlorella
protothecoides UTEX 250 Fructose Chlorella reisiglii CCAP 11/8
Fructose Chlorella protothecoides CCAP 211/8d Fructose Prototheca
moriformis UTEX 1435 Fructose Scenedesmus rubescens CCAP 232/1
Arabinose Chlorella sp. CCAP 211/75 Mannose Chlorella kessleri UTEX
263 Mannose Chlorella saccharophila UTEX 2911 Mannose Parachlorella
kessleri SAG 12.80 Mannose Chlorella sp. SAG 241.80 Mannose
Chlorella angustoellipsoidea SAG 265 Mannose Chlorella ellipsoidea
SAG 2141 Mannose Chlorella protothecoides UTEX 250 Mannose
Chlorella emersonii CCAP 211/15 Mannose Bracteococcus minor UTEX 66
Mannose Prototheca stagnora UTEX 1442 Mannose Prototheca moriformis
UTEX 1439 Mannose Chlorella cf. minutissima CCALA 20024 Mannose
Scenedesmus rubescens CCAP 232/1 Galactose Bracteococcus minor UTEX
66 Galactose Parachlorella kessleri SAG 14.82 Galactose
Parachlorella beijerinckii SAG 2046 Galactose Chlorella
protothecoides UTEX 25 Galactose Chlorella sorokiniana UTEX 1602
Galactose Parachlorella kessleri SAG 12.80 Galactose
Pseudochlorella aquatica SAG 2149 Galactose Chlorella luteoviridis
SAG 2214 Galactose Chlorella ellipsoidea CCAP 211/42 Galactose
Chlorella ellipsoidea CCAP 211/50 Galactose Chlorella
protothecoides UTEX 250 Galactose Chlorella protothecoides UTEX 264
Galactose Bracteococcus medionucleatus UTEX 1244 Galactose
Prototheca moriformis UTEX 1439 Galactose Prototheca moriformis
UTEX 1441 Galactose Chlorella kessleri CCALA 252 Acetate Chlorella
sorokiniana UTEX 1230 Acetate Chlorella sorokiniana UTEX 1810
Acetate Chlorella luteoviridis UTEX 22 Acetate Parachlorella
kessleri SAG 12.80 Acetate Parachlorella kessleri SAG 27.87 Acetate
Chlorella sp. SAG 241.80 Acetate Chlorella luteoviridis SAG 2214
Acetate Chlorella protothecoides UTEX 31 Acetate Chlorella
protothecoides UTEX 411 Acetate Chlorella ellipsoidea CCAP 211/42
Acetate Chlorella ovalis CCAP 211/21A Acetate Chlorella
protothecoides CCAP 211/8d Acetate Prototheca stagnora UTEX 1442
Acetate Chlorella protothecoides UTEX 250 Acetate Chlorella
sorokiniana CCALA 260 Acetate Chlorella vulgaris CCAP 211/79
Acetate Parachlorella kessleri SAG 14.82
Example 27
Production of Renewable Diesel
[0689] Cell Production:
[0690] An F-Tank batch of Chlorella protothecoides (UTEX 250)
(about 1,200 gallons) was used to generate biomass for extraction
processes. The batch (#ZA07126) was allowed to run for 100 hours,
while controlling the glucose levels at 16 g/L, after which time
the corn syrup feed was terminated. Residual glucose levels dropped
to <0 g/L two hours later. This resulted in a final age of 102
hours. The final broth volume was 1,120 gallons. Both in-process
contamination checks and a thorough analysis of a final broth
sample failed to show any signs of contamination. The fermentation
broth was centrifuged and drum dried. Drum dried cells were
resuspended in hexane and homogenized at approximately 1000 bar.
Hexane extraction was then performed using standard methods, and
the resulting algal triglyceride oil was determined to be free of
residual hexane.
[0691] Production of Renewable Diesel:
[0692] The algal triglyceride oil had a lipid profile of
approximately 3% C18:0, 71% C18:1, 15% C18:2, 1% C18:3, 8% C16:0,
and 2% other components. The oil was first subjected to
hydrocracking, resulting in an approximate 20% yield loss to water
and gases. Hydroisomerization was then performed, with an
approximate 10% loss in yield to gases. A first distillation was
then performed to remove the naptha fraction, leaving the desired
product. Approximately 20% of the material was lost to naptha in
this first distillation. A second distillation was then performed
at a temperature sufficient to remove fractions necessary to meet
the ASTM D975 specification but leave a bottom fraction that did
not meet the 90% point for a D975 distillation. Approximately 30%
of the material was left in the bottom fraction in the second
distillation. The resulting material was then tested for all ASTM
D975 specifications.
[0693] FIGS. 29 and 30 illustrate a gas chromatograph and a boiling
point distribution plot, respectively, of the final renewable
diesel product produced by the method of the invention. Table 18
shows the boiling point distribution of the resulting renewable
diesel product, and Table 19 shows the results of an analysis of
the final product for compliance with the ASTM D975
specifications.
TABLE-US-00019 TABLE 18 Boiling point distribution of renewable
diesel product. Recovered BP mass % .degree. C. IBP 150.4 1.0 163.6
2.0 173.4 3.0 175.2 4.0 188.0 5.0 194.8 6.0 196.6 7.0 197.8 8.0
207.4 9.0 210.0 10.0 214.4 11.0 216.6 12.0 217.6 13.0 221.4 14.0
227.6 15.0 229.8 16.0 233.2 17.0 235.8 18.0 236.8 19.0 240.2 20.0
245.6 21.0 248.0 22.0 250.2 23.0 253.6 24.0 255.2 25.0 256.8 26.0
261.8 27.0 264.6 28.0 266.2 29.0 268.2 30.0 271.0 31.0 272.4 32.0
273.4 33.0 276.2 34.0 280.0 35.0 282.4 36.0 285.2 37.0 287.8 38.0
289.6 39.0 291.8 40.0 294.2 41.0 295.8 42.0 296.8 43.0 297.6 44.0
298.4 45.0 299.2 46.0 299.8 47.0 300.6 48.0 301.2 49.0 302.0 50.0
302.6 51.0 303.2 52.0 303.8 53.0 304.4 54.0 304.8 55.0 305.2 56.0
305.6 57.0 306.0 58.0 306.4 59.0 306.6 60.0 307.0 61.0 307.4 62.0
307.6 63.0 308.0 64.0 308.4 65.0 308.8 66.0 309.2 67.0 309.6 68.0
310.2 69.0 310.6 70.0 311.2 71.0 311.6 72.0 312.2 73.0 313.0 74.0
313.6 75.0 314.2 76.0 314.8 77.0 315.4 78.0 315.8 79.4 316.4 80.0
317.0 81.0 317.6 82.0 318.4 83.0 319.0 84.0 319.6 85.0 320.2 86.0
320.8 87.0 321.2 88.0 321.8 89.0 322.2 90.0 322.4 91.0 322.8 92.0
323.2 93.0 324.4 94.0 326.8 95.0 329.4 96.0 333.6 97.0 339.4 98.0
346.2 99.0 362.8 FBP 401.4
TABLE-US-00020 TABLE 19 Analytical report for renewable diesel
product using D975 specifications. Method Number Test Description
Results Units D93A Flash Point (PMCC) 70 .degree. C. D2709 Water
and Sediment 0 Vol % D86 Distillation 90% (Recovered) 301.0/573.9
.degree. C./.degree. F. D445 Kinematic Viscosity @ 40.0.degree. C.
(104.0.degree. F.) 2.868 mm2/sec D482 Ash <0.001 Wt % D5453
Sulfur 2.4 ppm D130 Copper Corrosion 3 hours @ 50.degree. C. 1b
D613 Cetane Number *** >65 D976 Calculated Cetane Index 71.2
D2500 Cloud Point -3 .degree. C. D524 Ramsbottom 10% Carbon Residue
0.02 Wt % D97 Pour Point -3 .degree. C. D2274 Total Insolubles
(Oxidation Stability) 40 Hour Test *** 4.0 mg/100 mL D4052 Density
@ 15.0.degree. C. (59.0.degree. F.) 793.8 kg/m.sup.3 D4176-1
Appearance by Visual inspection (Lab) Clear and Bright-Pass Visual
D4176-1 Appearance by Visual inspection (Lab) Free Water-Pass
Visual D4176-1 Appearance by Visual inspection (Lab)
Particulates-Pass Visual D1500 ASTM Color L 0.5 D664 Acid Number
<0.10 mg KOH/g D6079 Lubricity (Wear Scar) 405 .mu.m
Sequence CWU 1
1
2511187DNAArtificialSynthetic HUP promoter from Chlorella
(subsequence of GenBank accession number X55349) 1gatcagacgg
gcctgacctg cgagataatc aagtgctcgt aggcaaccaa ctcagcagct 60gcttggtgtt
gggtctgcag gatagtgttg cagggcccca aggacagcag gggaacttac
120accttgtccc cgacccagtt ttatggagtg cattgcctca agagcctagc
cggagcgcta 180ggctacatac ttgccgcacc ggtatgaggg gatatagtac
tcgcactgcg ctgtctagtg 240agatgggcag tgctgcccat aaacaactgg
ctgctcagcc atttgttggc ggaccattct 300gggggggcca gcaatgcctg
actttcgggt agggtgaaaa ctgaacaaag actaccaaaa 360cagaatttct
tcctccttgg aggtaagcgc aggccggccc gcctgcgccc acatggcgct
420ccgaacacct ccatagctgt aagggcgcaa acatggccgg actgttgtca
gcactctttc 480atggccatac aaggtcatgt cgagattagt gctgagtaag
acactatcac cccatgttcg 540attgaagccg tgacttcatg ccaacctgcc
cctgggcgta gcagacgtat gccatcatga 600ccactagccg acatgcgctg
tcttttgcca ccaaaacaac tggtacaccg ctcgaagtcg 660tgccgcacac
ctccgggagt gagtccggcg actcctcccc ggcgggccgc ggccctacct
720gggtagggtc gccatacgcc cacgaccaaa cgacgcagga ggggattggg
gtagggaatc 780ccaaccagcc taaccaagac ggcacctata ataataggtg
gggggactaa cagccctata 840tcgcaagctt tgggtgccta tcttgagaag
cacgagttgg agtggctgtg tacggtcgac 900cctaaggtgg gtgtgccgca
gcctgaaaca aagcgtctag cagctgcttc tataatgtgt 960cagccgttgt
gtttcagtta tattgtatgc tattgtttgt tcgtgctagg gtggcgcagg
1020cccacctact gtggcgggcc attggttggt gcttgaattg cctcaccatc
taaggtctga 1080acgctcactc aaacgccttt gtacaactgc agaactttcc
ttggcgctgc aactacagtg 1140tgcaaaccag cacatagcac tcccttacat
cacccagcag tacaaca 118721414DNAArtificialSynthetic Chlorella
ellipsoidea nitrate reductase promoter from AY307383 2cgctgcgcac
cagggccgcc agctcgctga tgtcgctcca aatgcggtcc cccgattttt 60tgttcttcat
cttctccacc ttggtggcct tcttggccag ggccttcagc tgcatgcgca
120cagaccgttg agctcctgat cagcatcctc aggaggccct ttgacaagca
agcccctgtg 180caagcccatt cacggggtac cagtggtgct gaggtagatg
ggtttgaaaa ggattgctcg 240gtcgattgct gctcatggaa ttggcatgtg
catgcatgtt cacaatatgc caccaggctt 300tggagcaaga gagcatgaat
gccttcaggc aggttgaaag ttcctggggg tgaagaggca 360gggccgagga
ttggaggagg aaagcatcaa gtcgtcgctc atgctcatgt tttcagtcag
420agtttgccaa gctcacagga gcagagacaa gactggctgc tcaggtgttg
catcgtgtgt 480gtggtggggg ggggggggtt aatacggtac gaaatgcact
tggaattccc acctcatgcc 540agcggaccca catgcttgaa ttcgaggcct
gtggggtgag aaatgctcac tctgccctcg 600ttgctgaggt acttcaggcc
gctgagctca aagtcgatgc cctgctcgtc tatcagggcc 660tgcacctctg
ggctgaccgg ctcagcctcc ttcgcgggca tggagtaggc gccggcagcg
720ttcatgtccg ggcccagggc agcggtggtg ccataaatgt cggtgatggt
ggggaggggg 780gccgtcgcca caccattgcc gttgctggct gacgcatgca
catgtggcct ggctggcacc 840ggcagcactg gtctccagcc agccagcaag
tggctgttca ggaaagcggc catgttgttg 900gtccctgcgc atgtaattcc
ccagatcaaa ggagggaaca gcttggattt gatgtagtgc 960ccaaccggac
tgaatgtgcg atggcaggtc cctttgagtc tcccgaatta ctagcagggc
1020actgtgacct aacgcagcat gccaaccgca aaaaaatgat tgacagaaaa
tgaagcggtg 1080tgtcaatatt tgctgtattt attcgtttta atcagcaacc
aagttcgaaa cgcaactatc 1140gtggtgatca agtgaacctc atcagactta
cctcgttcgg caaggaaacg gaggcaccaa 1200attccaattt gatattatcg
cttgccaagc tagagctgat ctttgggaaa ccaactgcca 1260gacagtggac
tgtgatggag tgccccgagt ggtggagcct cttcgattcg gttagtcatt
1320actaacgtga accctcagtg aagggaccat cagaccagaa agaccagatc
tcctcctcga 1380caccgagaga gtgttgcggc agtaggacga caag
141431113DNAArtificialSynthetic B. braunii malate dehydrogenase
5'UTR 3aattggaaac cccgcgcaag accgggttgt ttggccgcct gaccggaaag
ggggggcctg 60tcccgaaggg ggtctatctc ttgggggatg tcgggcgcgg aaagtcgatg
ttgatggacc 120tcttcttcga ccatgtcggg gtcgaggcca agagccgcgt
ccatttcgcc gagttcatga 180tggaggtgaa tgaccgcatc gccaccgaac
gcgccaagaa gcgggcgacc gatcgccccc 240gtcgctgcag cccttgccga
ggaagtccgg ctgctggcgt tcgacgagat gatggtgacg 300aacagcccgg
acgcgatgat cctgtcgcgg ctgttcaccg cgctgatcga ggcgggggtg
360acgatcgtca ccacctccaa ccggccgccc agggatctct ataagaacgg
gctcaaccgc 420gagcatttcc tgcccttcat cgcgctgatc gaggcgcggc
tggacgtgct ggcgctgaac 480ggcccgaccg actatcggcg cgaccggctg
gggcggctgg acacgtggtt ggtgcccaat 540ggccccaagg cgacgattac
cttgtcggcg gcgttcttcc gcctgaccga ctatccggtc 600gaggatgccg
cgcatgtgcc ctctgaggac ctgaaggtgg gcgggcgcgt gctgaatgtc
660cccaaggcgc tgaagggcgt cgcggtcttc tcgttcaagc ggttgtgcgg
cgaagcgcgg 720ggggcggcgg actatctggc ggtcgcgcgg ggcttccaca
ccgtcatcct ggtcggaatc 780cccaagctgg gggcggagaa ccgcaacgag
gcggggcgct tcgtccagct gatcgacgcg 840ctctacgaac ataaggtcaa
gctgctcgcc gcagccgatg ccagcccgcc gaactctatg 900aaaccggcga
cggccggttc gagtttgagc gcagatcagc cggttggaag agatgcgctc
960cgaggattat ctggcccaag gccatggctc ggaggggcct tgatcaggcc
ttaatgcact 1020tcgcaaccat tatcgtttaa aatcttaaac tctgtggaat
aacggttccc cgacgccgca 1080atacacgtac gtccactacg gagtaggatt gga
11134253DNAArtificialSynthetic Chlamydomonas RBCS2 promoter
4cgcttagaag atttcgataa ggcgccagaa ggagcgcagc caaaccagga tgatgtttga
60tggggtattt gagcacttgc aacccttatc cggaagcccc ctggcccaca aaggctaggc
120gccaatgcaa gcagttcgca tgcagcccct ggagcggtgc cctcctgata
aaccggccag 180ggggcctatg ttctttactt ttttacaaga gaagtcactc
aacatcttaa acggtcttaa 240gaagtctatc cgg
25352435DNAArtificialSynthetic CMV-Hyg-CMV BamHI-SacII cassette
from pCAMBIA 5ggatccccgg gaattcggcg cgccgggccc aacatggtgg
agcacgacac tctcgtctac 60tccaagaata tcaaagatac agtctcagaa gaccaaaggg
ctattgagac ttttcaacaa 120agggtaatat cgggaaacct cctcggattc
cattgcccag ctatctgtca cttcatcaaa 180aggacagtag aaaaggaagg
tggcacctac aaatgccatc attgcgataa aggaaaggct 240atcgttcaag
atgcctctgc cgacagtggt cccaaagatg gacccccacc cacgaggagc
300atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc aagtggattg
atgtgataac 360atggtggagc acgacactct cgtctactcc aagaatatca
aagatacagt ctcagaagac 420caaagggcta ttgagacttt tcaacaaagg
gtaatatcgg gaaacctcct cggattccat 480tgcccagcta tctgtcactt
catcaaaagg acagtagaaa aggaaggtgg cacctacaaa 540tgccatcatt
gcgataaagg aaaggctatc gttcaagatg cctctgccga cagtggtccc
600aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc
aaccacgtct 660tcaaagcaag tggattgatg tgatatctcc actgacgtaa
gggatgacgc acaatcccac 720tatccttcgc aagaccttcc tctatataag
gaagttcatt tcatttggag aggacacgct 780gaaatcacca gtctctctct
acaaatctat ctctctcgag ctttcgcaga tcccgggggg 840caatgagata
tgaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
900aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc
tcgtgctttc 960agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
atagctgcgc cgatggtttc 1020tacaaagatc gttatgttta tcggcacttt
gcatcggccg cgctcccgat tccggaagtg 1080cttgacattg gggagtttag
cgagagcctg acctattgca tctcccgccg tgcacagggt 1140gtcacgttgc
aagacctgcc tgaaaccgaa ctgcccgctg ttctacaacc ggtcgcggag
1200gctatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg
cccattcgga 1260ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
tatgcgcgat tgctgatccc 1320catgtgtatc actggcaaac tgtgatggac
gacaccgtca gtgcgtccgt cgcgcaggct 1380ctcgatgagc tgatgctttg
ggccgaggac tgccccgaag tccggcacct cgtgcacgcg 1440gatttcggct
ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
1500agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt
ctggaggccg 1560tggttggctt gtatggagca gcagacgcgc tacttcgagc
ggaggcatcc ggagcttgca 1620ggatcgccac gactccgggc gtatatgctc
cgcattggtc ttgaccaact ctatcagagc 1680ttggttgacg gcaatttcga
tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc 1740cgatccggag
ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
1800accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag
cactcgtccg 1860agggcaaaga aatagagtag atgccgaccg gatctgtcga
tcgacaagct cgagtttctc 1920cataataatg tgtgagtagt tcccagataa
gggaattagg gttcctatag ggtttcgctc 1980atgtgttgag catataagaa
acccttagta tgtatttgta tttgtaaaat acttctatca 2040ataaaatttc
taattcctaa aaccaaaatc cagtactaaa atccagatcc cccgaattaa
2100ttcggcgtta attcagtaca ttaaaaacgt ccgcaatgtg ttattaagtt
gtctaagcgt 2160caatttgttt acaccacaat atatcctgcc accagccagc
caacagctcc ccgaccggca 2220gctcggcaca aaatcaccac tcgatacagg
cagcccatca gtccgggacg gcgtcagcgg 2280gagagccgtt gtaaggcggc
agactttgct catgttaccg atgctattcg gaagaacggc 2340aactaagctg
ccgggtttga aacacggatg atctcgcgga gggtagcatg ttgattgtaa
2400cgatgacaga gcgttgctgc ctgtgatcac cgcgg
2435620DNAArtificialSynthetic Chlorella virus promoter - CMV
construct forward primer 6caaccacgtc ttcaaagcaa
20720DNAArtificialSynthetic Chlorella virus promoter 7agcaatcgcg
catatgaaat 2081599DNAArtificialSynthetic sucrose invertase gene
8atgcttcttc aggcctttct ttttcttctt gctggttttg ctgccaagat cagcgcctct
60atgacgaacg aaacctcgga tagaccactt gtgcacttta caccaaacaa gggctggatg
120aatgacccca atggactgtg gtacgacgaa aaagatgcca agtggcatct
gtactttcaa 180tacaacccga acgatactgt ctgggggacg ccattgtttt
ggggccacgc cacgtccgac 240gacctgacca attgggagga ccaaccaata
gctatcgctc cgaagaggaa cgactccgga 300gcattctcgg gttccatggt
ggttgactac aacaatactt ccggcttttt caacgatacc 360attgacccga
gacaacgctg cgtggccata tggacttaca acacaccgga gtccgaggag
420cagtacatct cgtatagcct ggacggtgga tacactttta cagagtatca
gaagaaccct 480gtgcttgctg caaattcgac tcagttccga gatccgaagg
tcttttggta cgagccctcg 540cagaagtgga tcatgacagc ggcaaagtca
caggactaca agatcgaaat ttactcgtct 600gacgacctta aatcctggaa
gctcgaatcc gcgttcgcaa acgagggctt tctcggctac 660caatacgaat
gcccaggcct gatagaggtc ccaacagagc aagatcccag caagtcctac
720tgggtgatgt ttatttccat taatccagga gcaccggcag gaggttcttt
taatcagtac 780ttcgtcggaa gctttaacgg aactcatttc gaggcatttg
ataaccaatc aagagtagtt 840gattttggaa aggactacta tgccctgcag
actttcttca atactgaccc gacctatggg 900agcgctcttg gcattgcgtg
ggcttctaac tgggagtatt ccgcattcgt tcctacaaac 960ccttggaggt
cctccatgtc gctcgtgagg aaattctctc tcaacactga gtaccaggcc
1020aacccggaaa ccgaactcat aaacctgaaa gccgaaccga tcctgaacat
tagcaacgct 1080ggcccctgga gccggtttgc aaccaacacc acgttgacga
aagccaacag ctacaacgtc 1140gatctttcga atagcaccgg tacacttgaa
tttgaactgg tgtatgccgt caataccacc 1200caaacgatct cgaagtcggt
gttcgcggac ctctccctct ggtttaaagg cctggaagac 1260cccgaggagt
acctcagaat gggtttcgag gtttctgcgt cctccttctt ccttgatcgc
1320gggaacagca aagtaaaatt tgttaaggag aacccatatt ttaccaacag
gatgagcgtt 1380aacaaccaac cattcaagag cgaaaacgac ctgtcgtact
acaaagtgta tggtttgctt 1440gatcaaaata tcctggaact ctacttcaac
gatggtgatg tcgtgtccac caacacatac 1500ttcatgacaa ccgggaacgc
actgggctcc gtgaacatga cgacgggtgt ggataacctg 1560ttctacatcg
acaaattcca ggtgagggaa gtcaagtga 1599920DNAArtificialSynthetic CMV
construct reverse primer 9tccggtgtgt tgtaagtcca
201020DNAArtificialSynthetic CV construct forward primer
10ttgtcggaat gtcatatcaa 201120DNAArtificialSynthetic CV construct
reverse primer 11tccggtgtgt tgtaagtcca 201220DNAArtificialSynthetic
HUP1 construct forward primer 12aacgcctttg tacaactgca
201320DNAArtificialSynthetic HUP1 construct reverse primer
13tccggtgtgt tgtaagtcca 2014512PRTArtificialSynthetic sucrose
invertase 14Met Thr Asn Glu Thr Ser Asp Arg Pro Leu Val His Phe Thr
Pro Asn 1 5 10 15 Lys Gly Trp Met Asn Asp Pro Asn Gly Leu Trp Tyr
Asp Glu Lys Asp 20 25 30 Ala Lys Trp His Leu Tyr Phe Gln Tyr Asn
Pro Asn Asp Thr Val Trp 35 40 45 Gly Thr Pro Leu Phe Trp Gly His
Ala Thr Ser Asp Asp Leu Thr Asn 50 55 60 Trp Glu Asp Gln Pro Ile
Ala Ile Ala Pro Lys Arg Asn Asp Ser Gly 65 70 75 80 Ala Phe Ser Gly
Ser Met Val Val Asp Tyr Asn Asn Thr Ser Gly Phe 85 90 95 Phe Asn
Asp Thr Ile Asp Pro Arg Gln Arg Cys Val Ala Ile Trp Thr 100 105 110
Tyr Asn Thr Pro Glu Ser Glu Glu Gln Tyr Ile Ser Tyr Ser Leu Asp 115
120 125 Gly Gly Tyr Thr Phe Thr Glu Tyr Gln Lys Asn Pro Val Leu Ala
Ala 130 135 140 Asn Ser Thr Gln Phe Arg Asp Pro Lys Val Phe Trp Tyr
Glu Pro Ser 145 150 155 160 Gln Lys Trp Ile Met Thr Ala Ala Lys Ser
Gln Asp Tyr Lys Ile Glu 165 170 175 Ile Tyr Ser Ser Asp Asp Leu Lys
Ser Trp Lys Leu Glu Ser Ala Phe 180 185 190 Ala Asn Glu Gly Phe Leu
Gly Tyr Gln Tyr Glu Cys Pro Gly Leu Ile 195 200 205 Glu Val Pro Thr
Glu Gln Asp Pro Ser Lys Ser Tyr Trp Val Met Phe 210 215 220 Ile Ser
Ile Asn Pro Gly Ala Pro Ala Gly Gly Ser Phe Asn Gln Tyr 225 230 235
240 Phe Val Gly Ser Phe Asn Gly Thr His Phe Glu Ala Phe Asp Asn Gln
245 250 255 Ser Arg Val Val Asp Phe Gly Lys Asp Tyr Tyr Ala Leu Gln
Thr Phe 260 265 270 Phe Asn Thr Asp Pro Thr Tyr Gly Ser Ala Leu Gly
Ile Ala Trp Ala 275 280 285 Ser Asn Trp Glu Tyr Ser Ala Phe Val Pro
Thr Asn Pro Trp Arg Ser 290 295 300 Ser Met Ser Leu Val Arg Lys Phe
Ser Leu Asn Thr Glu Tyr Gln Ala 305 310 315 320 Asn Pro Glu Thr Glu
Leu Ile Asn Leu Lys Ala Glu Pro Ile Leu Asn 325 330 335 Ile Ser Asn
Ala Gly Pro Trp Ser Arg Phe Ala Thr Asn Thr Thr Leu 340 345 350 Thr
Lys Ala Asn Ser Tyr Asn Val Asp Leu Ser Asn Ser Thr Gly Thr 355 360
365 Leu Glu Phe Glu Leu Val Tyr Ala Val Asn Thr Thr Gln Thr Ile Ser
370 375 380 Lys Ser Val Phe Ala Asp Leu Ser Leu Trp Phe Lys Gly Leu
Glu Asp 385 390 395 400 Pro Glu Glu Tyr Leu Arg Met Gly Phe Glu Val
Ser Ala Ser Ser Phe 405 410 415 Phe Leu Asp Arg Gly Asn Ser Lys Val
Lys Phe Val Lys Glu Asn Pro 420 425 430 Tyr Phe Thr Asn Arg Met Ser
Val Asn Asn Gln Pro Phe Lys Ser Glu 435 440 445 Asn Asp Leu Ser Tyr
Tyr Lys Val Tyr Gly Leu Leu Asp Gln Asn Ile 450 455 460 Leu Glu Leu
Tyr Phe Asn Asp Gly Asp Val Val Ser Thr Asn Thr Tyr 465 470 475 480
Phe Met Thr Thr Gly Asn Ala Leu Gly Ser Val Asn Met Thr Thr Gly 485
490 495 Val Asp Asn Leu Phe Tyr Ile Asp Lys Phe Gln Val Arg Glu Val
Lys 500 505 510 1520PRTArtificialSynthetic secretion signal from
yeast 15Met Leu Leu Gln Ala Phe Leu Phe Leu Leu Ala Gly Phe Ala Ala
Lys 1 5 10 15 Ile Ser Ala Ser 20 1618PRTArtificialSynthetic
secretion signal from higher plants 16Met Ala Asn Lys Ser Leu Leu
Leu Leu Leu Leu Leu Gly Ser Leu Ala 1 5 10 15 Ser Gly
1710PRTArtificialSynthetic eukaryotic consensus secretion signal
17Met Ala Arg Leu Pro Leu Ala Ala Leu Gly 1 5 10
1820PRTArtificialSynthetic combination of signal sequence from
higher plants and eukaryotic consensus 18Met Ala Asn Lys Leu Leu
Leu Leu Leu Leu Leu Leu Leu Leu Pro Leu 1 5 10 15 Ala Ala Ser Gly
20 19532PRTArtificialSynthetic sucrose invertase gene 19Met Leu Leu
Gln Ala Phe Leu Phe Leu Leu Ala Gly Phe Ala Ala Lys 1 5 10 15 Ile
Ser Ala Ser Met Thr Asn Glu Thr Ser Asp Arg Pro Leu Val His 20 25
30 Phe Thr Pro Asn Lys Gly Trp Met Asn Asp Pro Asn Gly Leu Trp Tyr
35 40 45 Asp Glu Lys Asp Ala Lys Trp His Leu Tyr Phe Gln Tyr Asn
Pro Asn 50 55 60 Asp Thr Val Trp Gly Thr Pro Leu Phe Trp Gly His
Ala Thr Ser Asp 65 70 75 80 Asp Leu Thr Asn Trp Glu Asp Gln Pro Ile
Ala Ile Ala Pro Lys Arg 85 90 95 Asn Asp Ser Gly Ala Phe Ser Gly
Ser Met Val Val Asp Tyr Asn Asn 100 105 110 Thr Ser Gly Phe Phe Asn
Asp Thr Ile Asp Pro Arg Gln Arg Cys Val 115 120 125 Ala Ile Trp Thr
Tyr Asn Thr Pro Glu Ser Glu Glu Gln Tyr Ile Ser 130 135 140 Tyr Ser
Leu Asp Gly Gly Tyr Thr Phe Thr Glu Tyr Gln Lys Asn Pro 145 150 155
160 Val Leu Ala Ala Asn Ser Thr Gln Phe Arg Asp Pro Lys Val Phe Trp
165 170 175 Tyr Glu Pro Ser Gln Lys Trp Ile Met Thr Ala Ala Lys Ser
Gln Asp 180 185 190 Tyr Lys Ile Glu Ile Tyr Ser Ser Asp Asp Leu Lys
Ser Trp Lys Leu 195 200 205 Glu Ser Ala Phe Ala Asn Glu Gly Phe Leu
Gly Tyr Gln Tyr Glu Cys 210 215 220
Pro Gly Leu Ile Glu Val Pro Thr Glu Gln Asp Pro Ser Lys Ser Tyr 225
230 235 240 Trp Val Met Phe Ile Ser Ile Asn Pro Gly Ala Pro Ala Gly
Gly Ser 245 250 255 Phe Asn Gln Tyr Phe Val Gly Ser Phe Asn Gly Thr
His Phe Glu Ala 260 265 270 Phe Asp Asn Gln Ser Arg Val Val Asp Phe
Gly Lys Asp Tyr Tyr Ala 275 280 285 Leu Gln Thr Phe Phe Asn Thr Asp
Pro Thr Tyr Gly Ser Ala Leu Gly 290 295 300 Ile Ala Trp Ala Ser Asn
Trp Glu Tyr Ser Ala Phe Val Pro Thr Asn 305 310 315 320 Pro Trp Arg
Ser Ser Met Ser Leu Val Arg Lys Phe Ser Leu Asn Thr 325 330 335 Glu
Tyr Gln Ala Asn Pro Glu Thr Glu Leu Ile Asn Leu Lys Ala Glu 340 345
350 Pro Ile Leu Asn Ile Ser Asn Ala Gly Pro Trp Ser Arg Phe Ala Thr
355 360 365 Asn Thr Thr Leu Thr Lys Ala Asn Ser Tyr Asn Val Asp Leu
Ser Asn 370 375 380 Ser Thr Gly Thr Leu Glu Phe Glu Leu Val Tyr Ala
Val Asn Thr Thr 385 390 395 400 Gln Thr Ile Ser Lys Ser Val Phe Ala
Asp Leu Ser Leu Trp Phe Lys 405 410 415 Gly Leu Glu Asp Pro Glu Glu
Tyr Leu Arg Met Gly Phe Glu Val Ser 420 425 430 Ala Ser Ser Phe Phe
Leu Asp Arg Gly Asn Ser Lys Val Lys Phe Val 435 440 445 Lys Glu Asn
Pro Tyr Phe Thr Asn Arg Met Ser Val Asn Asn Gln Pro 450 455 460 Phe
Lys Ser Glu Asn Asp Leu Ser Tyr Tyr Lys Val Tyr Gly Leu Leu 465 470
475 480 Asp Gln Asn Ile Leu Glu Leu Tyr Phe Asn Asp Gly Asp Val Val
Ser 485 490 495 Thr Asn Thr Tyr Phe Met Thr Thr Gly Asn Ala Leu Gly
Ser Val Asn 500 505 510 Met Thr Thr Gly Val Asp Asn Leu Phe Tyr Ile
Asp Lys Phe Gln Val 515 520 525 Arg Glu Val Lys 530
2029DNAArtificialSynthetic primer 2383 20ctgacccgac ctatgggagc
gctcttggc 292128DNAArtificialSynthetic primer 2279 21cttgacttcc
ctcacctgga atttgtcg 282220DNAArtificialSynthetic primer 2336
22gtggccatat ggacttacaa 202341DNAArtificialSynthetic primer 2465
23caagggctgg atgaatgacc ccaatggact gtggtacgac g
412432DNAArtificialSynthetic primer 2470 24cacccgtcgt catgttcacg
gagcccagtg cg 32252615DNAArtificialSynthetic nucleotide sequence of
the construct used for transformation of multiple Chlorella species
25gaattcccca acatggtgga gcacgacact ctcgtctact ccaagaatat caaagataca
60gtctcagaag accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc
120ctcggattcc attgcccagc tatctgtcac ttcatcaaaa ggacagtaga
aaaggaaggt 180ggcacctaca aatgccatca ttgcgataaa ggaaaggcta
tcgttcaaga tgcctctgcc 240gacagtggtc ccaaagatgg acccccaccc
acgaggagca tcgtggaaaa agaagacgtt 300ccaaccacgt cttcaaagca
agtggattga tgtgaacatg gtggagcacg acactctcgt 360ctactccaag
aatatcaaag atacagtctc agaagaccaa agggctattg agacttttca
420acaaagggta atatcgggaa acctcctcgg attccattgc ccagctatct
gtcacttcat 480caaaaggaca gtagaaaagg aaggtggcac ctacaaatgc
catcattgcg ataaaggaaa 540ggctatcgtt caagatgcct ctgccgacag
tggtcccaaa gatggacccc cacccacgag 600gagcatcgtg gaaaaagaag
acgttccaac cacgtcttca aagcaagtgg attgatgtga 660tatctccact
gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc
720tatataagga agttcatttc atttggagag gacacgctga aatcaccagt
ctctctctac 780aaatctatct ctggcgcgcc atatcaatgc ttcttcaggc
ctttcttttt cttcttgctg 840gttttgctgc caagatcagc gcctctatga
cgaacgaaac ctcggataga ccacttgtgc 900actttacacc aaacaagggc
tggatgaatg accccaatgg actgtggtac gacgaaaaag 960atgccaagtg
gcatctgtac tttcaataca acccgaacga tactgtctgg gggacgccat
1020tgttttgggg ccacgccacg tccgacgacc tgaccaattg ggaggaccaa
ccaatagcta 1080tcgctccgaa gaggaacgac tccggagcat tctcgggttc
catggtggtt gactacaaca 1140atacttccgg ctttttcaac gataccattg
acccgagaca acgctgcgtg gccatatgga 1200cttacaacac accggagtcc
gaggagcagt acatctcgta tagcctggac ggtggataca 1260cttttacaga
gtatcagaag aaccctgtgc ttgctgcaaa ttcgactcag ttccgagatc
1320cgaaggtctt ttggtacgag ccctcgcaga agtggatcat gacagcggca
aagtcacagg 1380actacaagat cgaaatttac tcgtctgacg accttaaatc
ctggaagctc gaatccgcgt 1440tcgcaaacga gggctttctc ggctaccaat
acgaatgccc aggcctgata gaggtcccaa 1500cagagcaaga tcccagcaag
tcctactggg tgatgtttat ttccattaat ccaggagcac 1560cggcaggagg
ttcttttaat cagtacttcg tcggaagctt taacggaact catttcgagg
1620catttgataa ccaatcaaga gtagttgatt ttggaaagga ctactatgcc
ctgcagactt 1680tcttcaatac tgacccgacc tatgggagcg ctcttggcat
tgcgtgggct tctaactggg 1740agtattccgc attcgttcct acaaaccctt
ggaggtcctc catgtcgctc gtgaggaaat 1800tctctctcaa cactgagtac
caggccaacc cggaaaccga actcataaac ctgaaagccg 1860aaccgatcct
gaacattagc aacgctggcc cctggagccg gtttgcaacc aacaccacgt
1920tgacgaaagc caacagctac aacgtcgatc tttcgaatag caccggtaca
cttgaatttg 1980aactggtgta tgccgtcaat accacccaaa cgatctcgaa
gtcggtgttc gcggacctct 2040ccctctggtt taaaggcctg gaagaccccg
aggagtacct cagaatgggt ttcgaggttt 2100ctgcgtcctc cttcttcctt
gatcgcggga acagcaaagt aaaatttgtt aaggagaacc 2160catattttac
caacaggatg agcgttaaca accaaccatt caagagcgaa aacgacctgt
2220cgtactacaa agtgtatggt ttgcttgatc aaaatatcct ggaactctac
ttcaacgatg 2280gtgatgtcgt gtccaccaac acatacttca tgacaaccgg
gaacgcactg ggctccgtga 2340acatgacgac gggtgtggat aacctgttct
acatcgacaa attccaggtg agggaagtca 2400agtgagatct gtcgatcgac
aagctcgagt ttctccataa taatgtgtga gtagttccca 2460gataagggaa
ttagggttcc tatagggttt cgctcatgtg ttgagcatat aagaaaccct
2520tagtatgtat ttgtatttgt aaaatacttc tatcaataaa atttctaatt
cctaaaacca 2580aaatccagta ctaaaatcca gatcccccga attaa 2615
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References