U.S. patent application number 16/793873 was filed with the patent office on 2020-07-02 for production of beta-phellandrene using genetically engineered photosynthetic microorganisms.
The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Fiona K. Davies, Anastasios MELIS, Hsu-Ching Chen Wintz, Andreas Zurbriggen.
Application Number | 20200208178 16/793873 |
Document ID | / |
Family ID | 48947953 |
Filed Date | 2020-07-02 |
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United States Patent
Application |
20200208178 |
Kind Code |
A1 |
MELIS; Anastasios ; et
al. |
July 2, 2020 |
PRODUCTION OF BETA-PHELLANDRENE USING GENETICALLY ENGINEERED
PHOTOSYNTHETIC MICROORGANISMS
Abstract
The present invention provides methods and compositions for
producing .beta.-phellandrene hydrocarbons from a photosynthetic
microorganism such as cyanobacteria.
Inventors: |
MELIS; Anastasios; (El
Cerrito, CA) ; Davies; Fiona K.; (Berkeley, CA)
; Wintz; Hsu-Ching Chen; (El Cerrito, CA) ;
Zurbriggen; Andreas; (Berkeley, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA |
Oakland |
CA |
US |
|
|
Family ID: |
48947953 |
Appl. No.: |
16/793873 |
Filed: |
February 18, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15888939 |
Feb 5, 2018 |
10563228 |
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16793873 |
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14376392 |
Aug 1, 2014 |
9951354 |
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PCT/US2013/024908 |
Feb 6, 2013 |
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15888939 |
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61595610 |
Feb 6, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 5/007 20130101;
C12N 9/88 20130101; C12N 15/52 20130101; C12N 1/20 20130101; C12P
5/002 20130101; C12Y 402/03052 20130101 |
International
Class: |
C12P 5/00 20060101
C12P005/00; C12N 9/88 20060101 C12N009/88; C12N 15/52 20060101
C12N015/52; C12N 1/20 20060101 C12N001/20 |
Claims
1. A method of obtaining .beta.-phellandrene from photosynthetic
microorganisms, the method comprising: culturing a strain of a
photosynthetic microorganism that has been genetically modified to
express a heterologous .beta.-phellandrene synthase under
conditions in which the .beta.-phellandrene synthase is expressed;
and collecting from the surface of the culture medium
.beta.-phellandrene hydrocarbons that have spontaneously diffused
into the medium from the photosynthetic microorganisms across the
cell wall, wherein the culture is in a continuous growth phase and
the .beta.-phellandrene hydrocarbons are continuously
generated.
2. The method of claim 1, wherein the strain of the photosynthetic
microorganism is a cyanobacteria strain or a microalgae strain.
3. The method of claim 1, wherein the strain of the photosynthetic
microorganism is a cyanobacteria strain.
4. The method of claim 3, wherein the cyanobacteria strain is from
a genus selected from the group consisting of Synechocystis,
Synechococcus, Arthrospira, Nostoc, and Anabaena.
5. The method of claim 3, where the .beta.-phellandrene synthase is
encoded by a nucleic acid having at least 80% nucleic acid sequence
identity to SEQ ID NO:3.
6. The method of claim 1, wherein the strain of the photosynthetic
microorganism is a microalgae strain.
7. The method of claim 1, wherein the .beta.-phellandrene synthase
has at least 70% identity to SEQ ID NO:1.
8. The method of claim 7, wherein the .beta.-phellandrene synthase
has at least 90% identity to SEQ ID NO:1.
9. The method of claim 1, wherein collecting .beta.-phellandrene
hydrocarbons comprises siphoning or skimming the
.beta.-phellandrene hydrocarbons from the surface of the culture
medium.
10. The method of claim 1, wherein collecting .beta.-phellandrene
hydrocarbons comprises overlaying a solvent onto the surface of the
culture medium.
11. The method of claim 10, wherein the solvent is selected from
the group consisting of heptane, decane, and dodecane.
12. A cell culture comprising a photosynthetic microorganism,
wherein the photosynthetic microorganism is genetically modified to
express a heterologous .beta.-phellandrene synthase; and cell
culture media comprises .beta.-phellandrene produced by the
photosynthetic microorganism that has diffused into the cell
culture media from the photosynthetic microorganism and floats on
the surface of the culture medium, wherein the cell culture is in a
continuous growth phase and the .beta.-phellandrene hydrocarbons
are continuously generated.
13. The cell culture of claim 12, wherein the photosynthetic
microorganism strain is a cyanobacteria strain or a microalgae
strain.
14. The cell culture of claim 12, wherein the photosynthetic
microorganism strain is a cyanobacteria strain.
15. The cell culture of claim 14, wherein the cyanobacteria strain
is of a genus selected from the group consisting of the genera
Synechocystis, Synechococcus, Arthrospira, Nostoc, and Anabaena.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 15/888,939, filed Feb. 5, 2018, which is a divisional of U.S.
application Ser. No. 14/376,392, filed Aug. 1, 2014, which is a
National Stage of International Application No. PCT/US2013/024908,
filed Feb. 6, 2013, and which claims the benefit to U.S.
Provisional Application No. 61/595,610, filed Feb. 6, 2012, each of
which is herein incorporated by reference for all purposes.
REFERENCE TO SEQUENCE LISTING SUBMITTED AS AN ASCII TEXT FILE
[0002] This application includes a Sequence Listing submitted as a
text file named "086540-1178882-SEQ.txt" created Feb. 18, 2020, and
containing 50,607 bytes. The material contained in this text file
is incorporated by reference in its entirety for all purposes.
BACKGROUND OF THE INVENTION
[0003] There is a need to develop renewable biofuels and chemicals
that will help meet global demands for energy and synthetic
chemistry feedstock, but without contributing to climate change or
other environmental degradation.
[0004] Terpenoids represent the largest and most diverse group of
naturally occurring organic compounds, and are all derived from the
monomeric isoprene five-carbon building block. More than 25,000
different naturally occurring terpenoids have been identified, and
many have plant origin. Terpenoids are classified into groups based
on the number of five-carbon isoprene units they comprise;
monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20),
triterpenes (C30), tetraterpenes (C40) and polyterpenes (greater
than C40). .beta.-Phellandrene (C.sub.10H.sub.16) offers example of
such monoterpenes as a constituent of the essential oils
synthesized by many plant species. It has significant commercial
potential for use in the cosmetics and personal care industries, in
cleaning products for household and industrial use, and medicinal
use. There is also potential for .beta.-phellandrene and other
monoterpenes to be developed as feedstock in the synthetic
chemistry and pharmaceutical industries, and as a renewable
biofuel, where .beta.-phellandrene itself may serve as supplement
to gasoline or oligomerization of such monoterpene units may
generate second order fuel molecules, suitable for use as
supplements to jet fuel and diesel.
[0005] A number of plant species naturally produce
.beta.-phellandrene as a constituent of their essential oils,
including lavender and grand fir. Essential oils are produced and
stored in specialized organs called glandular trichomes, which form
on the surface of leaves and flowers. Essential oils are mainly
composed of monoterpenes and function in chemical defence against
potential herbivores. The harvesting of essential oils from
glandular trichomes, and subsequent purification of individual
monoterpenes, such as .beta.-phellandrene, is labour intensive and
costly with relatively limited yields. The use of microorganisms,
both photosynthetic and non-photosynthetic, for the production of
such commercially useful and valuable chemicals is an attractive
alternative to harvesting the product from plants.
[0006] All terpenoids are produced by two biosynthetic pathways: 1)
the mevalonic acid (MVA) pathway, which operates in the cytosol of
eukaryotes and archaea; and 2) the methyl-erythritol-4-phosphate
(MEP) pathway, which is of prokaryotic bacterial origin and present
in cyanobacteria, as well as in plant and algal plastids (see, FIG.
1). Synthesis of .beta.-phellandrene in plants is due to the
presence of a .beta.-phellandrene synthase (.beta.-PHLS) gene. This
is a nuclear gene encoding a chloroplast-localized protein that
catalyzes the conversion of geranyl diphosphate (GPP) to
.beta.-phellandrene. Plant .beta.-phellandrene synthases, encoded
by the gene .beta.-PHLS, have been cloned and characterized from
lavender, grand fir, tomato, and spruce (see, e.g., Demissie et
al., Planta, 233:685-696 (2011); Bohlmann et al., Arch. Biochem.
Biophys., 368:232-243 (1994); Schilmiller et al., Proc. Nat. Acad.
Sci. U.S.A., 106:10865-10870 (2009); and Keeling et al., BMC Plant
Biol. 11:43-57 (2011)).
[0007] Although photosynthetic microorganisms, such as microalgae
and cyanobacteria utilize the MEP pathway, which generates GPP
precursors, these microorganisms do not natively possess a
.beta.-phellandrene synthase gene or enzyme and thus, do not
natively catalyze the conversion of GPP to (3-phellandrene.
However, they do express the MEP pathway and utilize the
corresponding isoprenoid pathway enzymes for the biosynthesis of a
great variety of needed terpenoid-type molecules like carotenoids,
tocopherols, phytol, sterols, hormones, among many others) (see,
FIG. 1). The MEP isoprenoid biosynthetic pathway (Lindberg et al.,
Metab Eng., 12:70-79 (2010)) consumes pyruvate and
glyceraldehyde-3-phosphate (G3P) as substrates, which are combined
to form deoxyxylulose-5-phosphate (DXP), as first described for
Escherichia coli (Rohmer et al., Biochem. J., 295:517-524 (1993)).
DXP is then converted into methyl-erythitol phosphate (MEP), which
is subsequently modified to form
hydroxy-2-methyl-2-butenyl-4-diphosphate (HMBPP). HMBPP is the
substrate required for the formation of isopentenyl pyrophosphate
(IPP) and dimethylallyl pyrophosphate (DMAPP), which are terpenoid
precursors. Cyanobacteria also contain an IPP isomerase that
catalyzes the inter-conversion of IPP and DMAPP. In addition to
reactants G3P and pyruvate, the MEP pathway consumes reducing
equivalents and cellular energy in the form of NADPH, reduced
ferredoxin, CTP and ATP, ultimately derived from photosynthesis.
For reviews, see also (Ershov et al., J. Bacteriol.
184(18):5045-51; Sharkey et al., Ann. Bot. 101(1):5-18 (2002)).
[0008] Evidence in the literature shows that 15-carbon hydrophobic
terpenoid hydrocarbons can be transgenically expressed in
photosynthetic and fermentative microorganisms, but are trapped
within the cell, where they are synthesized, requiring dewatering
of the culture, drying of the biomass, followed by product
extraction from within the cells. For example, the sesquiterpene
.beta.-caryophyllene was produced in a transgenic strain of the
cyanobacterium Synechocystis. However, isolation of the product
required an extensive protocol that included treating the isolated
cellular biomass with an application of a chloroform:methanol:water
solvent mixture to solubilize lipid bilayers, releasing all
intracellular compounds, and extracting the lipophilic components
(Reinsvold et al., Plant 168: 848-852 (2011)).
[0009] Ten-carbon monoterpene hydrocarbon products occur in
different distinct configurations, such as acyclic (e.g., myrcene),
monocyclic (e.g., limonene and .beta.-phellandrene), and bicyclic
molecules (e.g., pinene). Spontaneous emission of monoterpene
hydrocarbons from single-celled microorganisms to the extracellular
space depends on the chemical nature of the monoterpene, and also
depends on the lipid bilayer configuration and cell wall
hydrophobic barriers imposed by the microorganism. For example,
yield of limonene production increased substantially in transgenic
E. coli upon the additional heterologous expression of an efflux
pump from Alcanivorax borkumensis (AcrB/AcrD/AcrFa gene product;
GenBank Accession No. YP692684) in the cell, suggesting limonene
product feedback inhibition and/or toxicity to the cell.
[0010] The Lavandula angustifolia .beta.-phellandrene synthase
protein has been over-expressed in E. coli upon transformant cell
induction with isopropyl .beta.-D-1-thiogalactopyranoside, IPTG
(Demissie et al., Planta, 233:685-696, 2011). However, IPTG
induction in E. coli can be toxic to the cell, causing loss of cell
fitness, thereby hindering a continuous and large scale production
of .beta.-phellandrene synthase by this method. Host cell toxicity
could be due to accumulation of the recombinant protein itself
and/or due to synthesis and intracellular accumulation of the
transgenic product. The latter is one of the most common barriers
in the commercial application of synthetic biology approaches for
product generation.
[0011] This invention in based, in part, on the discovery of
nucleic acids and expression systems that can be introduced and
expressed in cyanobacteria and enable these microorganisms to
produce .beta.-phellandrene. Such genetically modified
cyanobacteria can be used commercially in an enclosed mass culture
system to provide a source of .beta.-phellandrene which can be
potentially developed as feedstock in the synthetic chemistry and
pharmaceutical industries. For instance, .beta.-phellandrene may
serve as supplement to gasoline or oligomerization of such
monoterpene units may generate second order fuel molecules,
suitable for use as supplements to jet fuel and diesel.
BRIEF SUMMARY OF THE INVENTION
[0012] The current invention addresses the need of generating
monoterpene hydrocarbons by providing methods and composition for
the generation of .beta.-phellandrene hydrocarbons in
photosynthetic microorganisms, e.g. cyanobacteria and microalgae.
.beta.-Phellandrene, derived entirely via photosynthesis, i.e.,
from sunlight, carbon dioxide (CO.sub.2) and water (H.sub.2O),
could serve as renewable biofuels or feedstock in the synthetic
chemistry and pharmaceutical industries.
[0013] The invention is based, in part, on the discovery of
improvements to the engineering of cyanobacteria which, upon
suitable modification, produce 10-carbon monoterpenes, such as
.beta.-phellandrene. In one aspect, the invention therefore
provides methods and compositions for producing and harvesting
.beta.-phellandrene from cyanobacteria. Such genetically modified
organisms can be used commercially in an enclosed mass culture
system, e.g., a photobioreactor, to provide a source of renewable
fuel for internal combustion engines or, upon on-board reformation,
in fuel-cell operated engines; or to provide a source of
.beta.-phellandrene for use in chemical processes such as chemical
synthesis, pharmaceuticals and perfume cosmetics.
[0014] Photosynthetic microorganisms, such as microalgae and
cyanobacteria do not possess a .beta.-phellandrene synthase gene or
enzyme by which to catalyze the formation of .beta.-phellandrene
from GPP. However, they do express the
methyl-erythritol-4-phosphate (MEP) pathway and utilize the
corresponding isoprenoid pathway enzymes for the biosynthesis of a
variety of terpenoid-type molecules. This invention provides
methods and compositions to genetically modify microorganisms to
express a P-phellandrene synthase gene, e.g., a codon-optimized
Lavandular angustifolia .beta.-phellandrene synthase gene, in order
to produce .beta.-phellandrene in cyanobacteria.
[0015] In one aspect, the invention provides a method of producing
.beta.-phellandrene hydrocarbons in cyanobacteria, the method
comprising: introducing an expression cassette that comprises a
nucleic acid encoding .beta.-phellandrene synthase into the
cyanobacteria, wherein the nucleic acid encoding
.beta.-phellandrene synthase is operatively linked to a PsbA2
promoter, or other suitable promoter; and culturing the
cyanobacteria under conditions in which the nucleic acid encoding
.beta.-phellandrene synthase is expressed. In some embodiments, the
expression cassette is introduced into the PsbA2 gene locus and the
PsbA2 promoter is the native cyanobacteria promoter. In some
embodiments, the cyanobacteria are unicellular cyanobacteria, e.g.,
a Synechocystis sp or a Synechococcus sp. In alternative
embodiments, the cyanobacteria are multicellular, e.g., a
Gloeocapsa sp. The multicellular cyanobacteria may be a filamentous
cyanobacteria sp. such as a Nostoc sp, an Anabaena sp, or an
Arthrospira sp. In some embodiments, the nucleic acid encodes a
.beta.-phellandrene synthase that has at least 55%, 60%, 70%, 75%,
or 80% sequence identity, often at least 85%, 90%, 95%, or 100%
sequence identity, to SEQ ID NO:3. In some embodiments, the nucleic
acid encodes a .beta.-phellandrene synthase that comprises amino
acid SEQ ID NO:1. In typical embodiments, the nucleic acid that
encodes the .beta.-phellandrene synthase is codon-adjusted for
expression in cyanobacteria, e.g., in some embodiments, the nucleic
acid is a codon-modified variant of SEQ ID NO:2. In some
embodiments, the .beta.-phellandrene synthase nucleic acid
comprises SEQ ID NO:3, or a sequence having at least 80% identity,
typically at least 85% identity or 90%, 95%, 96%, 97%, 98%, 99%, or
100% identity to the nucleic acid sequence of SEQ ID NO:3.
[0016] In other aspects, the invention provides a cyanobacteria
cell, wherein the cyanobacteria cell comprises a heterologous
nucleic acid that encodes .beta.-phellandrene synthase and is
operably linked to a promoter such as a PsbA2 promoter. In some
embodiments, the PsbA2 promoter is an endogenous promoter. In some
embodiments, the cyanobacteria are unicellular cyanobacteria, e.g.,
a Synechocystis sp or a Synechococcus sp. In alternative
embodiments, the cyanobacteria are multicellular cyanobacteria,
e.g., a Gloeocapsa sp. In some embodiments, the multicellular
cyanobacteria sp is a filamentous cyanobacteria sp. such as a
Nostoc sp, an Anabaena sp, or an Arthrospira sp. In some
embodiments, the heterologous nucleic acid encodes a P-phellandrene
synthase and has at least 55%, 60%, 70%, 75%, or 80% sequence
identity, often at least 85%, 90%, 95%, or 100% sequence identity,
to the nucleic acid sequence of SEQ ID NO:3. In some embodiments,
the cyanobacteria cell comprises a heterologous nucleic acid that
comprises the nucleic acid sequence of SEQ ID NO:3. Preferably, the
heterologous nucleic acid present in the cyanobacterial cell that
encodes the .beta.-phellandrene synthase is codon-optimized for
expression in cyanobacteria, e.g., in some embodiments, the nucleic
acid is a codon-optimized variant of SEQ ID NO:2. In some
embodiments, the .beta.-phellandrene synthase nucleic acid
comprises SEQ ID NO:3, or a sequence having at least 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:3.
The invention additionally provides vectors comprising the nucleic
acid and cyanobacterial host cells into which the nucleic acid has
been introduced.
[0017] In a further aspect, the invention provides a nucleic acid
encoding a .beta.-phellandrene synthase that comprises amino acid
SEQ ID NO:1, where the nucleic acid is a codon-optimized variant of
SEQ ID NO:2 where codons used with an average frequency of less
than 12% by Synechocystis are replaced by more frequently used
codons. In some embodiments, the nucleic acid comprises the
sequence set forth in SEQ ID NO:3, or a sequence having at least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID
NO:3. The invention additionally provides vectors comprising the
nucleic acid and cyanobacterial host cells into which the nucleic
acid has been introduced.
[0018] In another aspect, the invention provides a method of
obtaining .beta.-phellandrene hydrocarbons in cyanobacteria as
described herein that express a heterologous .beta.-phellandrene
synthase gene, where the method comprises mass-culturing
cyanobacteria as described herein under conditions in which the
.beta.-phellandrene synthase gene is expressed.
[0019] In another aspect, the invention provides a method of
obtaining .beta.-phellandrene in cell culture comprising
genetically modified cyanobacteria, wherein the photosynthetically
generated .beta.-phellandrene accumulates as a non-miscible product
floating on the top of the liquid culture.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1. Terpenoid biosynthesis via the MEP
(methylerythritol-4-phosphate) pathway in photosynthetic
microorganisms, e.g. Synechocystis sp. Abbreviations used:
G3P=glyceraldehyde 3-phosphate; DXP=deoxyxylulose 5-phosphate;
HMBPP=hydroxymethylbutenyl diphosphate; IPP=isopentenyl
diphosphate; DMAPP=dimethylallyl diphosphate; GPP=geranyl
diphosphate; FPP=farnesyl diphosphate; GGPP=geranylgeranyl
diphosphate; .beta.-PHLS=/.beta.-phellandrene synthase. Solid lines
represent reactions catalyzed by endogenous Synechocystis enzymes,
whereas the dashed line show the reaction catalyzed by the
heterologously expressed S-/.beta.-PHLS construct.
[0021] FIG. 2. Amino acid sequence alignment of .beta.-phellandrene
synthase protein from lavender (Lavandula angustifolia), SEQ ID
NO:4; tomato (Solanum lycopersicum), SEQ ID NO:5; grand fir (Abies
grandis), SEQ ID NO:6, and spruce (Picea sitchensis 2, 3, 1, and
4), SEQ ID NOS:7, 8, 9, and 10, respectively. The Clustal 2.1
software application (University College, Dublin, Ireland) was used
to perform the multiple sequence alignment analysis.
[0022] FIG. 3. The .beta.-phellandrene synthase nucleotide and
protein sequences employed in the present invention. (Part A) Amino
acid sequence of S-.beta.-PHLS protein (SEQ ID NO:1) catalyzing the
conversion of GPP to .beta.-PHL. (Part B) The L. angustifolia
.beta.-PHLS (La-.beta.-PHLS) cDNA nucleotide sequence (SEQ ID NO:2;
GenBank Accession No. HQ404305). The chloroplast transit peptide is
indicated in bold, and start and stop codons are underlined. (Part
C) Codon-optimized version of Lavandula angustifolia .beta.-PHLS
cDNA nucleotide sequence minus the chloroplast transit peptide (SEQ
ID NO:3) for expression in microorganisms, e.g. Synechocystis sp.
PCC 6803 and E. coli. This codon-optimized sequence was termed
S-.beta.-PHLS. Start and stop codons are indicated. Restriction
sites incorporated into the synthesized sequence for cloning
purposes are underlined; PacI and NdeI sites at the start of the
sequence, and BglII and NotI sites after the stop codon.
[0023] FIG. 4. Plasmid construct for expression of S-.beta.-PHLS in
cyanobacteria, e.g. Synechocystis. The Synechcystis codon-optimized
.beta.-phellandrene synthase gene (S-.beta.-PHLS) and a
chloramphenicol resistance cassette (CamR), were cloned into a
vector containing upstream and downstream regions of the
Synechocystis PsbA2 gene. Restriction sites used for cloning
purposes are indicated. This plasmid was used for the
transformation of wild-type Synechocystis cells, and facilitated
the integration of the S-.beta.-PHLS-CamR cassette within the
Synechocystis genome at the PsbA2 locus via double homologous
recombination.
[0024] FIG. 5. Double homologous recombination and Synechocystis
DNA copy segregation. Panel A shows maps of the PsbA2 gene locus in
wild-type Synechocystis and in the S-.beta.-PHLS transformants upon
integration of the S-.beta.-PHLS-CamR gene construct into the
Synechocystis genome via double homologous recombination upon
transformation with plasmid pBA2Syn.beta.PHLSCamRA2. Genomic PCR
primers (arrows) were designed to flanking regions of the upstream
and downstream regions of the PsbA2 gene (PsbA2 us, PsbA2 ds) that
were used for homologous recombination. These amplify a 3.6 kb
product in the S-.beta.-PHLS transformant compared to a 2.3 kb
product in the wild type. Panel B shows complete DNA copy
segregation in 12 transformant lines following the replacement of
PsbA2 with the heterologous S-.beta.-PHLS transgene construct using
the above-mentioned primers. A PCR product of .about.2.3 kb was
amplified in the wild type (WT) containing the endogenous PsbA2,
whereas larger products of .about.3.6 kb were amplified in twelve
different S-.beta.-PHLS transformant lines (1-12). Absence of the
2.3 kb product from the latter indicates homoplasmy for the
introduced transgene. M, 1 kb plus marker.
[0025] FIG. 6. Western blot analysis of the S-.beta.-PHLS protein
in transformant Synechocystis cells. (A) Western blot analysis of
wild type (WT) and S-.beta.-PHLS transformant cells probed with
.beta.-PHLS specific polyclonal antibodies. Lanes were loaded with
a total cell extract (TCE) sample, or the soluble fraction of
Synechocystis cells (SP) as obtained by collection of the
supernatant following cell disruption and centrifugation to pellet
insoluble material. (B) Coomassie-stained SDS-PAGE gel
corresponding to the protein profile of the Western blot in panel
A, shown as a control for protein loading.
[0026] FIG. 7. GC-MS analyses of gases from the headspace of wild
type culture. Accumulated headspace gases in sealed cultures were
analyzed by GC-MS following 48 h of photoautotrophic growth in the
presence of CO.sub.2 in gaseous/aqueous two-phase bioreactors. GC
profile of gasses from wild-type culture.
[0027] FIG. 8. GC-MS analyses of gases from the headspace of
S-.beta.-PHLS transformant culture. Accumulated headspace gases in
sealed cultures were analyzed by GC-MS following 48 h of
photoautotrophic growth in the presence of CO.sub.2 in
gaseous/aqueous two-phase bioreactors. GC profile of gasses from
S-.beta.-PHLS transformant culture. The .beta.-phellandrene peak is
labeled with asterisks and has a retention time of around 4.6
min.
[0028] FIG. 9. GC profile of gasses from a vaporized
.alpha.-phellandrene standard (containing .beta.-phellandrene as a
contaminant). The .beta.-phellandrene peak is labeled with
asterisks and has a retention time of around 4.6 min.
[0029] FIG. 10. GC-MS analyses of gases from the headspace of an
S-.beta.-PHLS culture. Accumulated headspace gases in sealed
cultures were analyzed by GC-MS following 48 h of photoautotrophic
growth in the presence of CO.sub.2 in gaseous/aqueous two-phase
bioreactors. MS analysis of the products eluted at 4.6 min in the
S-.beta.-PHLS transformant culture showing the signature [77, 91,
93 and 136] MS lines of .beta.-phellandrene.
[0030] FIG. 11. MS analysis of the products eluted at 4.6 min with
a contaminating .beta.-phellandrene peak in the standard
solution.
[0031] FIG. 12. Absorbance spectra of phellandrene hydrocarbons in
heptane. (Panel A) Absorbance spectra of heptane-extracted samples
from the surface of wild type (black) and S-.beta.-PHLS
transformant (S-.beta.-PHLS) liquid cultures. The
.beta.-phellandrene absorbance peak is observed at 230 nm,
exclusively in the heptane extracts from the S-.beta.-PHLS
cultures. (Panel B) Absorbance spectra of the .alpha.-phellandrene
standard diluted in heptane. The .alpha.-phellandrene absorbance
peak is observed at 260 nm.
[0032] FIG. 13. Comparative photoautotrophic growth measurements of
wild type and S-.beta.-PHLS transformants in liquid culture.
Photoautotrophic growth kinetics of wild type (open squares) and
four different S-.beta.-PHLS transformant lines (closed squares,
circles, diamonds and triangles), as measured by optical density of
the culture at 730 nm. Cultures were grown under conditions of
continuous aeration and illumination at 20 .mu.mol photons m.sup.-2
s.sup.-1.
[0033] FIG. 14. Quantum yields of photosynthesis as measured by
oxygen evolution in wild type and S-.beta.-PHLS transformants in
liquid culture. Light saturation curves of photosynthesis for wild
type and S-.beta.-PHLS transformant cells, as measured by the
oxygen-evolution activity of an aliquot of the cultures incubated
in the presence of 15 mM NaHCO.sub.3, pH 7.4 under a range of
actinic light intensities.
[0034] FIG. 15. Absence of .beta.-phellandrene hydrocarbons in
heptane extracts from the surface of Escherichia coli cultures
induced by isopropyl .beta.-D-1-thiogalactopyranoside (IPTG) and
over-expressing the .beta.-phellandrene protein. Absorbance spectra
of heptane-extracted samples from the surface of E. coli liquid
cultures, measured in the wavelength region between 200 and 400 nm.
Extraction time of cultures, i.e., application of the heptane
solvent on the surface of the liquid phase of IPTG-induced
cultures, was either 1 h or 48 h. No distinctive
.beta.-phellandrene absorbance peak could be observed at 230 nm
from these .beta.-PHLS cultures, as compared to that of FIG.
12.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0035] A ".beta.-phellandrene hydrocarbon", ".beta.-phellandrene"
or ".beta.-PHL" in the context of this invention refers to a
monoterpene with a chemical formula C.sub.10H.sub.16. The IUPAC
name is 3-Methylene-6-(1-methylethyl)cyclohexene or
3-methylidene-6-propan-2-ylcyclohexene. .beta.-phellandrene is also
referred to as 3-isopropyl-6-methylene-1-cyclohexene or
p-mentha-1(7),2-diene. The CAS number is 555-10-2 and Pubchem CID
number is 11142. .beta.-Phellandrene is a water-insoluble cyclic
monoterpene with an endocyclic and an exocyclic double bond.
[0036] A ".beta.-PHLS gene" or ".beta.-PHLS polynucleotide" in the
context of this invention refers to a nucleic acid that encodes a
.beta.-PHLS protein, or fragment thereof In some embodiments, the
gene is a cDNA sequence that encodes .beta.-PHLS. In other
embodiments, a .beta.-PHLS gene may include sequences, such as
introns, that are not present in a cDNA. In some embodiments, a
".beta.-PHLS gene" refers to a nucleic acid sequence that encodes a
.beta.-PHLS polypeptide, e.g., a .beta.-PHLS polypeptide shown in
FIG. 2, or a homolog, fragment, or variant of a .beta.-PHLS
polypeptide shown in FIG. 2. In some embodiments, a ".beta.-PHLS
gene" encodes a .beta.-PHLS polypeptide having a sequence set forth
in SEQ ID NO:1 or encodes a homolog, fragment, or variant of the
polypeptide of SEQ ID NO:1. In some embodiments, a ".beta.-PHLS
gene" comprises the coding region of SEQ ID NO:2 or SEQ ID NO:3; or
comprises a nucleic acid sequence that is substantially similar to
the .beta.-PHLS protein coding region of SEQ ID NO:2 or SEQ ID
NO:3. Thus, in some embodiments, a .beta.-PHLS polynucleotide: 1)
comprises a region of about 15 to about 50, 100, 150, 200, 300,
500, 1,000, 1500, or 1700 or more nucleotides, sometimes from about
20, or about 50, to about 1800 nucleotides and sometimes from about
200 to about 600 or about 1700 nucleotides of SEQ ID NO:2 or SEQ ID
NO:3; or 2) hybridizes to SEQ ID NO:2 or SEQ ID NO:3, or the
complements thereof, under stringent conditions, or 3) encodes a
.beta.-PHLS polypeptide or fragment of at least 50 contiguous amino
acids, typically of at least 100, 150, 200, 250, 300, 350, 400,
450, 500, or 550, or more contiguous residues of a .beta.-PHLS
polypeptide shown in FIG. 2, such as the lavender .beta.-PHLS
sequence SEQ ID NO:1; or 4) encodes a .beta.-PHLS polypeptide or
fragment that has at least 25%, 30%, 35%, 40%, 45%, 45%, 50%, or
55%, and often at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
greater, identity to SEQ ID NO:1, or over a comparison window of at
least 100, 200, 300, 400, 500, or 550 amino acid residues of SEQ ID
NO:1; or 5) has a nucleic acid sequence that has greater than about
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
higher nucleotide sequence identity to SEQ ID NO:2 or SEQ ID NO:3,
at least 80%, 85%, 90%, or at least 95%, 96%, 97%, 98%, 99% or
greater identity over a comparison window of at least about 50,
100, 200, 500, 1000, 1500, 2000, or more nucleotides of SEQ ID NO:2
or SEQ ID NO:3; or 6) is amplified by primers to SEQ ID NO:2 or SEQ
ID NO:3. The term ".beta.-PHLS polynucleotide" refers to double
stranded or singled stranded nucleic acids. The .beta.-PHLS nucleic
acids for use in the invention encode an active .beta.-PHLS that
catalyzes the conversion of geranyl diphosphate (GPP) or
neryl-diphosphate (NPP), which is the cis isomer of GPP, to
.beta.-phellandrene.
[0037] A "codon-optimized variant of a .beta.-PHLS nucleic acid",
e.g., a codon-optimized variant of SEQ ID NO:2 in the context of
this invention, refers to a variant that encodes the same protein,
e.g., SEQ ID NO:1, but contains nucleotide substitutions based on
frequency of codon occurrence in cyanobacteria. For instance, SEQ
ID NO:3 represents a codon-optimized variant of SEQ ID NO:2 for
expression in the glucose-tolerant cyanobacterial strain
Synechocystis sp. PCC 6803. The method of generating a
codon-optimized variant includes modifying one or more codons of a
gene to eliminate codons that are rarely used in the host cell, and
adjusting the AT/GC ratio to that of the host cell. Rare codons can
be defined, e.g., by using a codon usage table derived from the
sequenced genome of the host cell.
[0038] A ".beta.-PHLS polypeptide" as herein refers to a
.beta.-PHLS polypeptide a protein that catalyzes the conversion of
geranyl diphosphate (GPP) or neryl-diphosphate (NPP) to
.beta.-phellandrene. A ".beta.-PHLS polypeptide" thus refers to a
polypeptide having the amino acid sequence of a .beta.-PHLS shown
in FIG. 2, or a fragment or variant thereof. In some embodiments, a
.beta.-PHLS polypeptide has the amino acid sequence of SEQ ID NO:1,
or a fragment or variant thereof. Thus, a .beta.-PHLS polypeptide
can: 1) have at least 25%, 30%, 35%, 40%, 45%, 45%, 50%, or 55%,
and typically at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or
greater identity to SEQ ID NO:1, or over a comparison window of at
least 100, 200, 250, 300, 250, 400, 450, 500, or 550 amino acids of
SEQ ID NO:1, or has at least 70%, 75%, 80%, 85%, 90%, 95% or
greater identity to a .beta.-PHLS polypeptide of FIG. 2; or a
subfragment comprising at least 100, 200, 250, 300, 250, 400, 450,
500, or 550 amino acids of a .beta.-PHLS polypeptide of FIG. 2; or
2) comprise at least 100, typically at least 200, 250, 300, 350,
400, 450, 500, 550, or more contiguous amino acids of a .beta.-PHLS
shown in FIG. 2, or comprise at least 100, typically at least 200,
250, 300, 350, 400, 450, 500, 550, or more contiguous amino acids
of SEQ ID NO:1; or 3) specifically binds to antibodies raised
against an immunogen comprising an amino acid sequence of a
.beta.-PHLS of FIG. 2, e.g., SEQ ID NO:1.
[0039] As used herein, a homolog or ortholog of a particular
.beta.-PHLS gene (e.g., SEQ ID NO:2) is a second gene in the same
plant type or in a different plant type that is substantially
identical (determined as described below) to a sequence in the
first gene.
[0040] In the case of expression of transgenes one of skill will
recognize that the inserted polynucleotide sequence need not be
identical and may be "substantially identical" to a sequence of the
gene from which it was derived. As explained below, these variants
are specifically covered by this term.
[0041] In the case where the inserted polynucleotide sequence is
transcribed and translated to produce a functional polypeptide, one
of skill will recognize that because of codon degeneracy a number
of polynucleotide sequences will encode the same polypeptide. These
variants are specifically covered by the term ".beta.-PHLS
polynucleotide sequence" or ".beta.-PHLS gene".
[0042] Two nucleic acid sequences or polypeptides are said to be
"identical" if the sequence of nucleotides or amino acid residues,
respectively, in the two sequences is the same when aligned for
maximum correspondence as described below. The term "complementary
to" is used herein to mean that the sequence is complementary to
all or a portion of a reference polynucleotide sequence.
[0043] Optimal alignment of sequences for comparison may be
conducted by the local homology algorithm of Smith and Waterman
Add. APL. Math. 2:482 (1981), by the homology alignment algorithm
of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search
for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci.
(U.S.A.) 85: 2444 (1988), by computerized implementations of these
algorithms (CLUSTAL, GAP, BESTFIT, BLAST, FASTA, and TFASTA), or by
inspection.
[0044] "Percentage of sequence identity" is determined by comparing
two optimally aligned sequences over a comparison window, wherein
the portion of the polynucleotide sequence in the comparison window
may comprise additions or deletions (i.e., gaps) as compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison and multiplying the result by 100 to yield the
percentage of sequence identity. A "comparison window", as used
herein, includes reference to a segment of any one of the number of
contiguous positions, e.g., 20 to 600, usually about 50 to about
200, more usually about 100 to about 150 in which a sequence may be
compared to a reference sequence of the same number of contiguous
positions after the two sequences are optimally aligned.
[0045] The term "substantial identity" in the context of
polynucleotide or amino acid sequences means that a polynucleotide
or polypeptide comprises a sequence that has at least 50% sequence
identity to a reference sequence. Alternatively, percent identity
can be any integer from 50% to 100%. Exemplary embodiments include
at least: at least 25%, 30%, 35%, 40%, 45%, 50%55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 99% or 100% identity compared to a
reference sequence using the programs described herein; preferably
BLAST using standard default parameters, as described below.
Accordingly, .beta.-PHLS sequences of the invention include nucleic
acid sequences that have substantial identity to the
codon-optimized version of the L. angustifolia .beta.-PHLS coding
region (SEQ ID NO:3) or to the L. angustifolia .beta.-PHLS coding
region (SEQ ID NO:2). As noted above, .beta.-PHLS polypeptide
sequences of the invention include polypeptide sequences having
substantial identify to SEQ ID NO:1.
[0046] The terms "nucleic acid" and "polynucleotide" are used
synonymously and refer to a single or double-stranded polymer of
deoxyribonucleotide or ribonucleotide bases read from the 5' to the
3' end. A nucleic acid of the present invention will generally
contain phosphodiester bonds, although in some cases, nucleic acid
analogs may be used that may have alternate backbones, comprising,
e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or
O-methylphophoroamidite linkages (see, e.g., Eckstein, F.,
Oligonucleotides and Analogues: A Practical Approach, Oxford
University Press, 1991); and peptide nucleic acid backbones and
linkages. Other analog nucleic acids include those with positive
backbones; non-ionic backbones, and non-ribose backbones. Thus,
nucleic acids or polynucleotides may also include modified
nucleotides, that permit correct read through by a polymerase.
"Polynucleotide sequence" or "nucleic acid sequence" includes both
the sense and antisense strands of a nucleic acid as either
individual single strands or in a duplex. As will be appreciated by
those in the art, the depiction of a single strand also defines the
sequence of the complementary strand; thus the sequences described
herein also provide the complement of the sequence. Unless
otherwise indicated, a particular nucleic acid sequence also
implicitly encompasses variants thereof (e.g., degenerate codon
substitutions) and complementary sequences, as well as the sequence
explicitly indicated. The nucleic acid may be DNA, both genomic and
cDNA, RNA or a hybrid, where the nucleic acid may contain
combinations of deoxyribo- and ribo-nucleotides, and combinations
of bases, including uracil, adenine, thymine, cytosine, guanine,
inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc
[0047] The phrase "a nucleic acid sequence encoding" refers to a
nucleic acid which contains sequence information for a structural
RNA, or the primary amino acid sequence of a specific protein or
peptide, or a binding site for a trans-acting regulatory agent.
This phrase specifically encompasses degenerate codons (i.e.,
different codons which encode a single amino acid) of the native
sequence or sequences that may be introduced to conform with codon
preference in a specific host cell. In the context of this
invention, the term ".beta.-PHLS coding region" when used with
reference to a nucleic acid reference sequence such as SEQ ID NO:2
or 3 refers to the region of the nucleic acid that encodes
.beta.-PHLS protein.
[0048] The term "promoter" or "regulatory element" refers to a
region or sequence determinants located upstream or downstream from
the start of transcription that direct transcription. 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 elements, which can be located as much
as several thousand base pairs from the start site of
transcription. A "constitutive" promoter is a promoter that is
active under most environmental and developmental conditions. An
"inducible" promoter is a promoter that is active under
environmental or developmental regulation. The term "operably
linked" refers to a functional linkage between a nucleic acid
expression control sequence (such as a promoter) and a second
nucleic acid sequence, such as a .beta.-PHLS gene, wherein the
expression control sequence directs transcription of the nucleic
acid corresponding to the second sequence. A "cyanobacteria
promoter" is a promoter capable of initiating transcription in
cyanobacterial cells, respectively. Such a promoter is therefore
active in a cyanobacteria cell, but need not originate from that
organism. It is understood that limited modifications can be made
without destroying the biological function of a regulatory element
and that such limited modifications can result in cyanobacteria
regulatory elements that have substantially equivalent or enhanced
function as compared to a wild type cyanobacteria regulatory
element. These modifications can be deliberate, as through
site-directed mutagenesis, or can be accidental such as through
mutation in hosts harboring the cyanobacteria regulatory element as
long as the ability to confer expression in unicellular and
multicellular cyanobacteria is substantially retained.
[0049] An "expression construct" in the context of this invention
refers to a nucleic acid encoding a .beta.-PHLS protein operably
linked to a promoter. The nucleic acid encoding the .beta.-PHLS
protein is considered to be heterologous to a cyanobacterial host
cell, as cyanobacteria do not have a .beta.-PHLS. An expression
construct includes embodiments in which the .beta.-PHLS nucleic
acid is linked to an endogenous promoter, e.g., the .beta.-PHLS
nucleic acid may be integrated into cyanobacterial DNA such that
expression is controlled by the native promoter. In further
embodiments, the .beta.-PHLS nucleic acid is operably linked to a
promoter that is introduced into the cyanbacterial host cell with
the .beta.-PHLS.
[0050] A "PsbA2 promoter" refers to a promoter region that
regulates expression of psbA2. The promoter region the psbA2 gene
has been well characterized (Eriksson et al., Mol Cell Biol Res
Commun 3: 292-298 (2000); Mohamed et al., Mol Gen Genet 238:
161-168 1(993); Mohamed and Jansson, Plant Mol Biol 13: 693-700
(1989)). Often, the PsbA2 promoter that is operably linked to the
.beta.-PHLS gene of this invention is the endogenous cyanobacteria
promoter, but a heterologous PsbA2 promoter may also be employed.
Such promoter sequences typically include High Light Regulatory 1
(HLR1) sequences that are involved in photoregulation as well as
minimal promoter sequences (see, e.g., Eriksson et al., Mol. Cell
Biol Res. Commun. 3: 292-298 (2000)).
[0051] "Expression" of a .beta.-PHLS gene in the context of this
invention typically refers introducing a .beta.-PHLS gene into
cyanobacteria cells, in which it is not normally expressed.
Accordingly, an "increase" in .beta.-PHLS activity or expression is
generally determined relative to wild-type cyanobacteria that have
no .beta.-PHLS activity.
[0052] A polynucleotide sequence is "heterologous to" a second
polynucleotide sequence if it originates from a foreign species,
or, if from the same species, is modified by human action from its
original form. For example, a promoter operably linked to a
heterologous coding sequence refers to a coding sequence from a
species different from that from which the promoter was derived,
or, if from the same species, a coding sequence which is different
from any naturally occurring allelic variants. A "heterologous"
promoter is a promoter that is not native to the host cell or that
has been modified by human action.
[0053] Polypeptides that are "substantially similar" share
sequences as noted above except that residue positions that are not
identical may differ by conservative amino acid changes.
Conservative amino acid substitutions refer to the
interchangeability of residues having similar side chains. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulfur-containing
side chains is cysteine and methionine. Exemplary conservative
amino acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic
acid-glutamic acid, and asparagine-glutamine.
[0054] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each
other, or a third nucleic acid, under stringent conditions. The
phrase "stringent hybridization conditions" refers to conditions
under which a probe will hybridize to its target subsequence,
typically in a complex mixture of nucleic acid, but to no other
sequences. Stringent conditions are sequence-dependent and will be
different in different circumstances. Longer sequences hybridize
specifically at higher temperatures. An extensive guide to the
hybridization of nucleic acids is found in Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic
Probes. (Tijssen, P., ed.), Elsevier, N.Y. (1993). Generally,
stringent conditions are selected to be about 5-10.degree. C. lower
than the thermal melting point (Tm) for the specific sequence at a
defined ionic strength pH. The Tm is the temperature (under defined
ionic strength, pH, and nucleic concentration) at which 50% of the
probes complementary to the target hybridize to the target sequence
at equilibrium (as the target sequences are present in excess, at
Tm, 50% of the probes are occupied at equilibrium). Stringent
conditions will be those in which the salt concentration is less
than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium
ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. For selective or specific hybridization, a positive
signal is at least two times background, optionally 10 times
background hybridization. Exemplary stringent hybridization
conditions can be as following: 50% formamide, 5.times.SSC, and 1%
SDS, incubating at 42.degree. C., or 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2X SSC, and 0.1% SDS at
55.degree. C., 60.degree. C., or 65.degree. C. Such washes can be
performed for 5, 15, 30, 60, 120, or more minutes.
[0055] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides that they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. For example, P-phellandrene
synthase polynucleotides, can also be identified by their ability
to hybridize under stringent conditions (e.g., Tm .about.40.degree.
C.) to nucleic acid probes having the sequence of SEQ ID NO:2 or by
their ability to hybridize under stringent conditions (e.g., Tm
.about.40.degree. C.) to nucleic acid probes having the sequence of
SEQ ID NO:3. Such a P-phellandrene synthase nucleic acid sequence
can have, e.g., about 25-30% base pair mismatches or less relative
to the selected nucleic acid probe. SEQ ID NOS:2 and 3 are examples
of nucleic acids that encode a L. angustifolia .beta.-phellandrene
synthase polypeptide. Exemplary "moderately stringent hybridization
conditions" include a hybridization in a buffer of 40% formamide, 1
M NaCl, 1% SDS at 37.degree. C., and a wash in 1.times.SSC at
45.degree. C. Such washes can be performed for 5, 15, 30, 60, 120,
or more minutes. A positive hybridization is at least twice
background. Those of ordinary skill will readily recognize that
alternative hybridization and wash conditions can be utilized to
provide conditions of similar stringency.
[0056] The term "isolated", when applied to a nucleic acid or
protein, denotes that the nucleic acid or protein is essentially
free of other cellular components with which it is associated in
the natural state. It is preferably in a homogeneous state and may
be in either a dry or aqueous solution. Purity and homogeneity are
typically determined using analytical chemistry techniques such as
polyacrylamide gel electrophoresis or high performance liquid
chromatography. A protein that is the predominant species present
in a preparation is substantially purified. In particular, an
isolated gene is separated from open reading frames that flank the
gene and encode a protein other than the gene of interest.
[0057] As used herein, "mass-culturing" refers to growing large
quantities of cyanobacteria, that have been modified to express a
.beta.-phellandrene synthase gene. A "large quantity" is generally
in the range of about 100 liters to about 1,500,000 liters, or
more. In some embodiments, the organisms are cultured in large
quantities in modular bioreactors, each having a capacity of about
1,000 to about 1,000,000 liters.
[0058] A "bioreactor" in the context of this invention is any
enclosed large-capacity vessel in which cyanobacteria are grown. A
"large-capacity vessel" in the context of this invention can hold
about 100 liters, often about 500 liters, or about 1,000 liters to
about 1,000,000 liters, or more.
[0059] As used herein, "harvesting" or "isolating"
.beta.-phellandrene hydrocarbons refers to collecting the
.beta.-phellandrene that has diffused into culture medium from the
culture medium.
Introduction
[0060] The invention employs various routine recombinant nucleic
acid techniques. Generally, the nomenclature and the laboratory
procedures in recombinant DNA technology described below are those
well-known and commonly employed in the art. Many manuals that
provide direction for performing recombinant DNA manipulations are
available, e.g., Molecular Cloning, A Laboratory Manual. (Sambrook,
J. and Russell, D., eds.), CSHL Press, New York (3rd Ed, 2001); and
Current Protocols in Molecular Biology. (Ausubel et al., eds.), New
Jersey (1994-1999).
[0061] In one aspect, the invention is based, in part, on the
discovery that in cyanobacteria, .beta.-phellandrene diffuses into
the culture media, unlike other long-chain hydrocarbons of the
terpenoid and fatty acid biosynthetic pathways. Accordingly, the
invention provides methods and compositions for producing
.beta.-phellandrene by expressing .beta.-phellandrene synthase in
cyanobacteria.
[0062] .beta.-Phellandrene Synthase Nucleic Acids
[0063] .beta.-phellandrene synthase nucleic acid and polypeptide
sequences are known in the art. .beta.-phellandrene synthase genes
have been isolated, sequenced and characterized from lavender
(Lavandular angustifolia), grand fir (Abies grandis), tomato
(Solanum lycopersicum) and spruce (Picea abies, Picea sitchensis).
See, e.g., Demissie et al., Planta, 233:685-696 (2011); Bohlmann et
al., Arch. Biochem. Biophys., 368:232-243 (1994); Schilmiller et
al., Proc. Nat. Acad. Sci. U.S.A., 106:10865-10870 (2009); and
Keeling et al., BMC Plant Biol. 11:43-57 (2011). Illustrative
accession numbers are: lavender (Lavandula angustifolia cultivar
Lady), Accession: HQ404305; tomato (Solanum lycopersicum),
Accession: FJ797957; grand fir (Abies grandis), Accession:
AF139205; spruce (Picea sitchensis) (4 genes identified, Accession
Nos: Q426162 (PsTPS-Phel-1), HQ426169 (PsTPS-Phel-2), HQ426163
(PsTPS-Phel-3), HQ426159 (PsTPS-Phel-4). FIG. 2 illustrates an
amino acid alignment of .beta.-phellandrene synthases from
lavender, grand fir, tomato and spruce. The conserved motifs are
underlined.
[0064] Amino acid sequence comparison of lavender (Lavandular
angustifolia) .beta.-phellandrene synthase with those of grand fir
(Abies grandis) and tomato (Solanum lycopersicum) showed 29% and
15% identity, respectively. There is a 26% amino acid sequence
identity between Lavandular angustifolia (lavender) and Picea
sitchensis (spruce) .beta.-phellandrene synthases. In terms of
similarity, amino acid sequence comparison of lavender (Lavandular
angustifolia) .beta.-phellandrene synthase with those of grand fir
(Abies grandis) and tomato (Solanum lycopersicum) showed 75% and
61% similarity, respectively. There is 73% amino acid sequence
similarity between Lavandular angustifolia (lavender) and Picea
sitchensis (spruce) .beta.-phellandrene synthases. Although amino
acid sequence comparison of all known .beta.-phellandrene
synthases, shown in FIG. 2, revealed a low amino acid identity over
the length of all the sequences, there are regions that are
conserved.
[0065] .beta.-Phellandrene synthases (and other monoterpene
synthases such as linalool synthase) share several conserved motifs
(see, e.g., Demissie et al., Planta 233:685-696, (2011)). FIG. 2
illustrates an amino acid alignment of .beta.-phellandrene synthase
proteins from Lavandular angustifolia, Abies grandis, Solanum
lycopersicum and Picea sitchensis. The monoterpene synthase
signature arginine-rich N-terminal RR(x8)W motif (underlined in
FIG. 2) is required for cyclization of geranyl-diphosphate (see,
e.g., Williams J G K. Methods Enzymol., 167:766-778 (1988)). The
arginine rich motif is located near the N-terminus of the mature
protein (Demissie et al., Planta 233:685-696, (2011)). The highly
conserved aspartate-rich DDxxD motif (underlined in FIG. 2) is
required for substrate binding, a process usually assisted by
divalent cations, e.g. Mg.sup.2+ (see, e.g., Nieuwenhuizen et al.,
J. Exp. Bot. 60(11):3203-3219 (2009)). The partially conserved
amino acid sequences, LQLYEASFLL (SEQ ID NO:11) (underlined in FIG.
2) and (N,D)D(L,I,V)x(S,T)xxxE (underlined in FIG. 2) play roles in
catalysis and second metal ion binding, respectively (see, e.g.,
Wise et al., J. Biol. Chem., 273:14891-14899 (1998); Degenhardt et
al, Phytochemistry, 70 (15-16):1621-1637 (2009).; Roeder et al.,
Plant Mol Biol 65(1):107-124 (2007)). A .beta.-phellandrene
synthase gene for use in the invention encodes a protein retaining
the motifs. Further, one of skill can employ an alignment of the
protein sequences to select residues that may be varied, e.g., by
conservative substitution, that retain function.
[0066] Differences between monoterpene synthases have been
identified in several plant organisms. For instance, monoterpene
synthases (e.g., .beta.-phellandrene synthase and linalool
synthase) from a given organism have greater homology to each
other, compared to monoterpene synthase orthologs from different
species. Substrates of monoterpene synthases can also vary between
plant species. For example, tomato .beta.-phellandrene synthase
uses neryl diphosphate (NPP), which is the cis-isomer of GPP, as a
substrate, rather than geranyl-diphosphate, a common substrate for
other known monoterpene synthases (Schilmiller et al., Proc. Natl.
Acad. Sci. USA 106:10865-10870, 2009).
[0067] The methods of the invention comprise expressing a nucleic
acid sequence that encodes a .beta.-phellandrene synthase
polypeptide, e.g., a polypeptide having a sequence at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, or at least
95%, or greater, identity to a .beta.-PHLS polypeptide set forth in
FIG. 2, e.g., a SEQ ID NO:1, in cyanobacteria. A .beta.-PHLS
polypeptide encoded by a nucleic acid employed in the methods of
the invention have the catalytic activity of converting GPP or its
cis-isomer to .beta.-phellandrene. In some embodiments, the
invention provides a .beta.-PHLS gene that encodes a modified
version of a .beta.-PHLS polypeptide from a plant, such as
lavender, grand fir, tomato, or spruce. A .beta.-PHLS polypeptide
variant suitable for use in the present invention possesses the
ability to convert GPP or NPP to .beta.-phellandrene when
heterologously expressed in cyanobacteria. In some embodiments, the
.beta.-PHLS polypeptide variant employs GPP. In some embodiments, a
.beta.-PHLS for use in the invention has at least 70%, typically at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
or greater, identity to a .beta.-PHLS polypeptide from lavender,
grand fir or spruce set forth in FIG. 2. Typically, the level of
activity is equivalent to the activity exhibited by a natural
.beta.-phellandrene synthase polypeptide (e.g., SEQ ID NO:1) to
produce .beta.-phellandrene. A .beta.-phellandrene synthase
polypeptide suitable for producing .beta.-phellandrene has at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at least 90% or at least 95%, or greater,
of the activity of an endogenous .beta.-PHLS polypeptide from a
plant, such as lavender, grand fir, tomato, and spruce, e.g., a
.beta.-PHLS having a sequence set forth in SEQ ID NO:2.
[0068] Activity of a heterologous .beta.-phellandrene synthase of
the present invention can be assayed by methods known to those
skilled in the art. Non-limiting examples of assays that measure
the function of .beta.-phellandrene synthase to produce
.beta.-phellandrene from the substrate GPP or NPP include in vitro
enzymatic assays using purified recombinant .beta.-phellandrene
synthase protein, assays that determine the enzyme saturation
kinetics, GC and GC-MS analysis to measure .beta.-phellandrene
production (detailed description in Example), spectrophotometric
analysis for .beta.-phellandrene quantification (detailed
description in Example).
.beta.-Phellandrene Synthase Expression Constructs
[0069] .beta.-PHLS nucleic acid sequences of the invention are
expressed recombinantly in cyanobacteria. Expression constructs can
be designed taking into account such properties as codon usage
frequencies of the organism in which the .beta.-PHLS nucleic acid
is to be expressed. Codon usage frequencies can be tabulated using
known methods (see, e.g., Nakamura et al. Nucl. Acids Res. 28:292
(2000)). Codon usage frequency tables, including those for
cyanobacteria, are also available in the art (e.g., in codon usage
databases of the Department of Plant Genome Research, Kazusa DNA
Research Institute, Japan).
[0070] In certain embodiments, the invention provides a .beta.-PHLS
gene that encodes a L. angustifolia .beta.-PHLS protein, where the
gene is a codon-optimized variant of a lavender .beta.-PHLS gene,
e.g., a codon-modified variant of SEQ ID NO:2.
[0071] Isolation or generation of .beta.-PHLS polynucleotide
sequences can be accomplished by well-known techniques, including
amplification techniques and/or library screening.
[0072] Appropriate primers and probes for generating a .beta.-PHLS
gene can be designed based on known principles using, e.g., the
.beta.-PHLS sequences provided herein. For a general overview of
PCR see PCR Protocols: A Guide to Methods and Applications. (Innis,
M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press,
San Diego (1990). An illustrative PCR for amplifying a .beta.-PHLS
nucleic acid sequence is provided in the examples.
[0073] .beta.-PHLS nucleic acid sequences for use in the invention
include genes and gene products identified and characterized by
techniques such as hybridization and/or sequence analysis using an
exemplary nucleic acid sequence, e.g., SEQ ID NO:3. In some
embodiments, a .beta.-PHLS nucleic acid sequence for use in the
invention has at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99% to SEQ ID NO:3. In some embodiments the
.beta.-PHLS nucleic acid sequence comprises SEQ ID NO:3.
[0074] To use isolated sequences in the above techniques,
recombinant DNA vectors suitable for transformation of
cyanobacteria are prepared. Techniques for transformation are well
known and described in the technical and scientific literature. For
example, a DNA sequence encoding a .beta.-PHLS gene (described in
further detail below), can be combined with transcriptional and
other regulatory sequences which will direct the transcription of
the sequence from the gene in the intended cells of the transformed
cyanobacteria. In some embodiments, an expression vector that
comprises an expression cassette that comprises the .beta.-PHLS
gene further comprises a promoter operably linked to the
.beta.-PHLS gene. In other embodiments, a promoter and/or other
regulatory elements that direct transcription of the .beta.-PHLS
gene are endogenous to the cyanobacteria and the expression
cassette comprising the .beta.-PHLS gene is introduced, e.g., by
homologous recombination, such that the heterologous .beta.-PHLS
gene is operably linked to an endogenous promoter and is expression
driven by the endogenous promoter.
[0075] Regulatory sequences include promoters, which may be either
constitutive or inducible. In some embodiments, a promoter can be
used to direct expression of .beta.-PHLS nucleic acids under the
influence of changing environmental conditions. Examples of
environmental conditions that may affect transcription by inducible
promoters include anaerobic conditions, elevated temperature, or
the presence of light. Promoters that are inducible upon exposure
to chemicals reagents are also used to express .beta.-PHLS nucleic
acids. Other useful inducible regulatory elements include
copper-inducible regulatory elements (Mett et al., Proc. Natl.
Acad. Sci. USA 90:4567-4571 (1993); Furst et al., Cell 55:705-717
(1988)); tetracycline and chlor-tetracycline-inducible regulatory
elements (Gatz et al., Plant J. 2:397-404 (1992); Roder et al.,
Mol. Gen. Genet. 243:32-38 (1994); Gatz, Meth. Cell Biol.
50:411-424 (1995)); ecdysone inducible regulatory elements
(Christopherson et al., Proc. Natl. Acad. Sci. USA 89:6314-6318
(1992); Kreutzweiser et al., Ecotoxicol. Environ. Safety 28:14-24
(1994)); heat shock inducible promoters, such as those of the
hsp70/dnaK genes (Takahashi et al., Plant Physiol. 99:383-390
(1992); Yabe et al., Plant Cell Physiol. 35:1207-1219 (1994); Ueda
et al., Mol. Gen. Genet. 250:533-539 (1996)); and lac operon
elements, which are used in combination with a constitutively
expressed lac repressor to confer, for example, IPTG-inducible
expression (Wilde et al., EMBO J. 11:1251-1259 (1992)). An
inducible regulatory element also can be, for example, a
nitrate-inducible promoter, e.g., derived from the spinach nitrite
reductase gene (Back et al., Plant Mol. Biol. 17:9 (1991)), or a
light-inducible promoter, such as that associated with the small
subunit of RuBP carboxylase or the LHCP gene families (Feinbaum et
al., Mol. Gen. Genet. 226:449 (1991); Lam and Chua, Science 248:471
(1990)), or a light.
[0076] In some embodiments, the promoter may be from a gene
associated with photosynthesis in the species to be transformed or
another species. For example such a promoter from one species may
be used to direct expression of a protein in transformed
cyanobacteria cells. Suitable promoters may be isolated from or
synthesized based on known sequences from other photosynthetic
organisms. Preferred promoters are those for genes from other
photosynthetic species, or other photosynthetic organism where the
promoter is active in cyanobacteria.
[0077] In some embodiments, a promoter used to drive expression of
a heterologous .beta.-PHLS gene is a constitutive promoter.
Examples of constitutive strong promoters for use in cyanobacteria
include, for example, the psbDI gene or the basal promoter of the
psbDII gene. Various other promoters that are active in
cyanobacteria are also known. These include the light inducible
promoters of the psbA and psbA3 genes in cyanobacteria and
promoters such as those set forth in U.S. Patent Application
Publication No. 20020164706, which is incorporated by reference.
Other promoters that are operative in plants, e.g., promoters
derived from plant viruses, such as the CaMV35S promoters, can also
be employed in cyanobacteria. For a description of strong and
regulated promoters, e.g., active in the cyanobacterium Anabaena
sp. strain PCC 7120, see e.g., Elhai, FEMS Microbiol Lett
114:179-184, (1993)). In other embodiments, other locus in the
cyanobacterial chloroplast genome can be used to drive expression
of the heterologous .beta.-PHLS gene, provided that the locus
permits relatively high expression levels of the heterologous gene.
In particular embodiments,
[0078] In some embodiments, promoters are identified by analyzing
the 5' sequences of a genomic clone corresponding to a .beta.-PHLS
gene. Sequences characteristic of promoter sequences can be used to
identify the promoter.
[0079] A promoter can be evaluated, e.g., by testing the ability of
the promoter to drive expression in cyanobacteria in which it is
desirable to introduce a .beta.-PHLS expression construct.
[0080] A vector comprising .beta.-PHLS nucleic acid sequences will
typically comprise a marker gene that confers a selectable
phenotype on cyanobacteria transformed with the vector. Such
markers are known. For example, the marker may encode antibiotic
resistance, such as resistance to chloramphenicol, kanamycin, G418,
bleomycin, hygromycin, and the like.
Heterologous Expression of .beta.-phellandrene Synthase Gene in
Cyanobacteria
[0081] Cell transformation methods and selectable markers for
cyanobacteria are well known in the art (Wirth, Mol. Gen. Genet.,
216(1):175-7 (1989); Koksharova, Appl. Microbiol. Biotechnol.,
58(2): 123-37 (2002); Thelwell et al., Proc. Natl. Acad. Sci.
U.S.A., 95:10728-10733 (1998)). Transformation methods and
selectable markers for are also well known (see, e.g., Sambrook et
al., supra).
[0082] The codon-optimized .beta.-phellandrene synthase gene of the
present invention can be expressed in any number of cyanobacteria
where it is desirable to produce .beta.-phellandrene. Suitable
unicellular cyanobacteria include Synechocystis sp., such as strain
Synechocystis PCC 6803; and Synechococcus sp., e.g., the
thermophilic Synechococcus lividus; the mesophilic Synechococcus
elongatus or Synechococcus 6301. Multicellular, including
filamentous cyanobacteria, may also be engineered to express
.beta.-PHLS in accordance with this invention. Multicellularr
cyanobacteria that can be used include, e.g., Gloeocapsa, as well
as filamentous cyanobacteria such as Nostoc sp., e.g., Nostoc sp.
PCC 7120, Nostoc sphaeroides); Anabaena sp., e.g., Anabaena
variabilis; and Arthrospira sp. ("Spirulina"), such as Arthrospira
platensis and Arthrospira maxima. Cyanobacteria that are
genetically modified in accordance with the invention to express a
.beta.-PHLS gene may also contain other genetic modifications,
e.g., modifications to the terpenoid pathway, to enhance production
of .beta.-phellandrene.
[0083] In some embodiments, an expression construct is generated to
allow the heterologous expression of the .beta.-phellandrene
synthase gene in Synechocystis through the replacement of the
Synechocystis PsbA2 gene with the codon-optimized .beta.-PHLS gene
via double homologous recombination. In some embodiments, the
expression construct comprises a codon-optimized
.beta.-phellandrene synthase gene operably linked to an endogenous
cyanobacteria promoter. In some aspects, the promoter is the PsbA2
promoter.
[0084] In some embodiments, cyanobacteria are transformed with an
expression vector comprising a .beta.-PHLS gene and an antibiotic
resistance gene. A detailed description is set forth in PCT
Application No. PCT/US2007/71465, which is incorporated by
reference. Transformants are cultured in selective media containing
an antibiotic to which an untransformed host cell is sensitive.
Cyanobacteria normally have up to 100 copies of identical circular
DNA chromosomes in each cell. Successful transformation with an
expression vector comprising a .beta.-PHLS gene and an antibiotic
resistance gene normally occurs in only one, or just a few, of the
many cyanobacterial DNA copies. Hence, presence of the antibiotic
is necessary to encourage expression of the transgenic copy(ies) of
the DNA for .beta.-phellandrene production. In the absence of the
selectable marker (antibiotic), the transgenic copy(ies) of the DNA
would be lost and replaced by wild-type copies of the DNA.
[0085] In some embodiments, cyanobacterial transformants are
cultured under continuous selective pressure conditions (presence
of antibiotic over many generations) to achieve DNA homoplasmy in
the transformed host organism. One of skill in the art understands
that the number of generations and length of time of culture varies
depending on the particular culture conditions employed. Homoplasmy
can be determined, e.g., by monitoring the DNA composition in the
cells to determine the presence of wild-type copies of the
cyanobacterial DNA.
[0086] "Achieving homoplasmy" refers to a quantitative replacement
of most, e.g., 70% or greater, or typically all, wild-type copies
of the cyanobacterial DNA in the cell with the transformant DNA
copy that carries the .beta.-PHLS transgene. This is normally
attained over time, under the continuous selective pressure
(antibiotic) conditions applied, and entails the gradual during
growth replacement of the wild-type copies of the DNA with the
transgenic copies, until no wild-type copy of the cyanobacterial
DNA is left in any of the transformant cells. Achieving homoplasmy
is typically verified by quantitative amplification methods such as
genomic-DNA PCR using primers and/or probes specific for the
wild-type copy of the cyanobacterial DNA. In some embodiments, the
presence of wild-type cyanobacterial DNA can be detected by using
primers specific for the wild-type cyanobacterial DNA and detecting
the presence of the PsbA2 gene. Transgenic DNA is typically stable
under homoplasmy conditions and present in all copies of the
cyanobacterial DNA.
[0087] In some embodiments, cyanobacterial cultures can be cultured
under conditions in which the light intensity is varied. Thus, for
example, when a psbA2 promoter is used as a promoter to drive
.beta.-phellandrene synthase expression, transformed cyanobacterial
cultures can be grown at low light intensity conditions (e.g.,
10-50 .mu.mol photons m.sup.-2 s.sup.-1), then shifted to higher
light intensity conditions (e.g., 500 .mu.mol photons m.sup.-2
s.sup.-1). The psbA2 promoter responds to the shift in light
intensity by up-regulating the expression of the .beta.-PHLS gene
in Synechocystis, typically at least about 10-fold. In other
embodiments, cyanobacterial cultures can be exposed to increasing
light intensity conditions (e.g., from 50 .mu.mol photons m.sup.-2
s.sup.-1 to 2,500 .mu.mol photons m.sup.-2 s.sup.-1) corresponding
to a diurnal increase in light intensity up to full sunlight. The
psbA2 promoter responds to the gradual increase in light intensity
by up-regulating the expression of the .beta.-PHLS gene in
Synechocystis in parallel with the increase in light intensity.
Production of .beta.-phellandrene in Cyanobacteria
[0088] Transformed cyanobacteria (transformant cyanobacteria) are
grown under conditions in which the heterologous .beta.-PHLS gene
is expressed. Methods of mass culturing cyanobacteria are known to
one skilled in the art. For example, cyanobacteria can be grown to
high cell density in photobioreactors (see, e.g., Lee et al.,
Biotech. Bioengineering 44:1161-1167, 1994; Chaumont, J Appl.
Phycology 5:593-604, 1990). Examples of photobioreactors include
cylindrical or tubular bioreactors, see, e.g., U.S. Pat. Nos.
5,958,761, 6,083,740, US Patent Application Publication No.
2007/0048859; WO 2007/011343, and WO2007/098150. High density
photobioreactors are described in, for example, Lee, et al.,
Biotech. Bioengineering 44: 1 161-1 167, 1994. Other
photobioreactors suitable for use in the invention are described,
e.g., in WO/2011/034567 and references cited in the background
section. Photobioreactor parameters that can be optimized,
automated and regulated for production of photosynthetic organisms
are further described in (Puiz (2001) Appl Microbiol Biotechnol
57:287-293). Such parameters include, but are not limited to,
materials of construction, efficient light incidence into reactor
lumen, light path, layer thickness, oxygen released, salinity and
nutrients, pH, temperature, turbulence, optical density, and the
like.
[0089] Transformed cyanobacteria that express a heterologous
.beta.-PHLS gene are grown under mass culture conditions for the
production of .beta.-phellandrene. In typical embodiments, the
transformed organisms are growth in bioreactors or fermentors that
provide an enclosed environment. For example, in some embodiments
for mass culture, the cyanobacteria are grown in enclosed reactors
in quantities of at least about 500 liters, often of at least about
1000 liters or greater, and in some embodiments in quantities of
about 1,000,000 liters or more. One of skill understands that
large-scale culture of transformed cyanobacteria that comprise a
.beta.-phellandrene synthase gene where expression is driven by a
light sensitive promoter, such as a PsbA2 promoter, is typically
carried out in conditions where the culture is exposed to natural
light. Accordingly, in such embodiments appropriate enclosed
reactors are used that allow light to reach the cyanobacteria
culture.
[0090] Growth media for culturing cyanobacteria transformants are
well known in the art. For example, cyanobacteria may be grown on
solid media such as BG-11 media (see, e.g., Rippka et al., J Gen
Microbiol. 111:1-61, 1979). Alternatively, they may be grown in
liquid media (see, e.g., Bentley & Melis, Biotechnol. Bioeng.
109:100-109, 2012). In typical embodiments for production of
.beta.-phellandrene, liquid cultures are employed. For example,
such a liquid culture may be maintained at about 25.degree. C.
under a slow stream of constant aeration and illumination, e.g., at
20 .mu.mol photons m.sup.-2 s.sup.-1. In certain embodiments, an
antibiotic, e.g., chloramphenical, is added to the liquid culture.
For example, chloramphenicol may be used at a concentration of 15
.mu.g/ml.
[0091] In some embodiments, cyanobacteria transformants are grown
photoautotrophically in a gaseous/aqueous two-phase photobioreactor
(see, e.g., Bentley & Melis, 2012, supra, and U.S. patent
application no. 61/477,896). In certain embodiments, the methods of
the present invention comprise obtaining .beta.-phellandrene using
a diffusion-based method for spontaneous gas exchange in a
gaseous/aqueous two-phase photobioreactor. In particular aspects of
the method, carbon dioxide is used as a feedstock for the
photosynthetic generation of .beta.-phellandrene in cell culture
and the headspace of the bioreactor is filled with 100% CO.sub.2
and sealed. This allows diffusion-based CO.sub.2 uptake and
assimilation by the cells via photosynthesis, and concomitant
replacement of the CO.sub.2 in the headspace with
.beta.-phellandrene vapour and O.sub.2. Typically, the
photosynthetically generated .beta.-phellandrene accumulates as a
non-miscible product floating on the top of the liquid culture.
[0092] In particular embodiments, a gaseous/aqueous two-phase
photo-bioreactor is seeded with a culture of cyanobacterial cells
and grown under continuous illumination, e.g., at 75 .mu.mol
photons m.sup.-2 s.sup.-1, and continuous bubbling with air.
Inorganic carbon is delivered to the culture in the form of
aliquots of 100% CO.sub.2 gas, which is slowly bubbled through the
bottom of the liquid culture to fill the bioreactor headspace. Once
atmospheric gases is replaced with 100% CO.sub.2, the headspace of
the reactor is sealed and the culture is incubated, e.g., at about
25.degree. C. to 37.degree. C. under continuous illumination, e.g.,
of 150 .mu.mol photons m.sup.-2 s.sup.-1. Slow continuous
mechanical mixing is also employed to keep cells in suspension and
to promote balanced cell illumination and nutrient mixing into the
liquid culture in support of photosynthesis and biomass
accumulation. Uptake and assimilation of headspace CO.sub.2 by
cells is concomitantly exchanged for O.sub.2 during
photoautotrophic growth. The sealed bioreactor headspace allows for
the trapping, accumulation and concentration of photosynthetically
produced .beta.-phellandrene.
[0093] In some embodiments, the photoautotrophic cell growth
kinetics of the cyanobacteria transformants are similar to those of
wild type cyanobacteria cells. In some embodiments, the rates of
oxygen consumption during dark respiration are about the same in
wild type cyanobacteria cells. In other embodiments, the rates of
oxygen evolution and the initial slopes of photosynthesis as a
function of light intensity re comparable in wild-type
Synechocystis cells and Synechocystis transformants, when both are
at sub-saturating light intensities between 0 and 250 .mu.mol
photons m.sup.-2 s.sup.31 1.
[0094] Conditions for growing .beta.-PHLS -expressing cyanobacteria
for the purposes illustrated above are known in the art (see, e.g.,
the illustrative references cited herein). .beta.-phellandrene
hydrocarbons produced by the modified cyanobacteria can be
harvested using known techniques. .beta.-phellandrene hydrocarbons
are not miscible in water and they rise to and float at the surface
of the microorganism growth medium. In typical embodiments, they
are siphoned off from the surface and sequestered in suitable
containers. In addition, and depending on the prevailing
temperature during the mass cultivation of the cyanobacteria,
.beta.-phellandrene can exist in vapor form above the water medium
in the bioreactor container (monoterpene hydrocarbons have a
relatively high boiling temperature T=170-175.degree. C.). In some
embodiments, .beta.-phellandrene vapor is piped off the bioreactor
container and condensed into liquid 1 form upon cooling or
low-level compression.
[0095] In typical embodiments, the photosynthetically produced
.beta.-phellandrene is in liquid form and floating on the aqueous
phase of the liquid culture. In some embodiments, extraction of the
.beta.-phellandrene produced in accordance with the invention is
performed by skimming the floating .beta.-phellandrene from the
surface of the liquid phase of the culture that is producing the
.beta.-phellandrene and isolating .beta.-phellandrene in pure form.
In certain embodiments, photosynthetically produced non-miscible
.beta.-phellandrene in liquid form is extracted from the liquid
phase by a method comprising overlaying a solvent such as heptane,
decane, or dodecane, on top of the liquid culture in the
bioreactor, incubating for at room temperature, e.g. 30 minutes or
longer; and removing the solvent, e.g., heptane, layer containing
.beta.-phellandrene. In some embodiments, photosynthetically
produced .beta.-phellandrene is a volatile product accumulating in
the headspace of the bioreactor used for .beta.-phellandrene
production.
EXAMPLES
[0096] The examples described herein are provided by way of
illustration only and not by way of limitation. Those of skill in
the art will readily recognize a variety of non-critical parameters
that could be changed or modified to yield essentially similar
results.
Example 1. .beta.-Phellandrene Production Using Genetically
Engineered Cyanobacteria
[0097] The invention provides method and compositions for the
genetic modification of cyanobacteria to confer upon these
microorganisms the ability to produce .beta.-phellandrene
(C.sub.10H.sub.16) upon heterologous expression of a
.beta.-phellandrene synthase gene, e.g., a .beta.-phellnadrene
syntahse gene from lavender (Lavandular angustifolia), grand fir
(Abies grandis), tomato (Solanum lycopersicum) or spruce (Picea
abies, Picea sitchensis), or a variant thereof In some embodiments,
the invention provides for production of .beta.-phellandrene
hydrocarbons in gaseous-aqueous two-phase photobioreactors and
results in the renewable generation of a hydrocarbon bio-product,
which can be used, e.g., for generating fuel, chemical synthesis,
or pharmaceutical and cosmetics applications. This example
illustrates expression of a .beta.-phellandrene synthase gene from
lavender in cyanobacteria to produce .beta.-phellandrene.
[0098] This example further illustrates that .beta.-phellandrene
can be continuously-generated in cyanobacteria transformants that
express a .beta.-phellandrene synthase gene. Further, this example
demonstrates that .beta.-phellandrene can spontaneously diffuse out
of cyanobacteria transformants and into the extracellular water
phase, and be collected from the surface of the liquid culture as a
water-floating product. This example also demonstrates that this
strategy for production of .beta.-phellandrene alleviates product
feedback inhibition, product toxicity to the cell, and the need for
labor-intensive extraction protocols.
[0099] In the present example, photosynthetic microorganisms, with
the cyanobacterium Synechocystis sp. PCC6803 as the model organism,
were genetically engineered to express a .beta.-phellandrene
synthase gene from lavender (Lavandular angustifolia), thereby
endowing upon them with the property of photosynthetic
.beta.-phellandrene production (FIG. 1). Genetically modified
strains were used in an enclosed mass culture system to provide a
renewable hydrocarbon in the form of .beta.-phellandrene that is
suitable as biofuel or feedstock in chemical synthesis.
.beta.-Phellandrene hydrocarbon products were spontaneously emitted
by the cells into the extracellular space, followed by floating to
the surface of the liquid phase, where they were easily be
collected without imposing any disruption to the
growth/productivity of the cells. The genetically modified
cyanobacteria remained in a continuous growth phase, constitutively
generating and emitting .beta.-phellandrene. The example further
provides an example of a codon-optimized .beta.-phellandrene
synthase gene for improved yield of .beta.-phellandrene in
photosynthetic cyanobacteria, e.g., Synechocystis.
Materials and Methods
Strains and Growth Conditions
[0100] The E. coli strain DH5.alpha. was used for routine
subcloning and plasmid propagation, and grown in LB media with
appropriate antibiotics as selectable markers at 37.degree. C.,
according to standard protocols. The glucose-tolerant
cyanobacterial strain Synechocystis sp. PCC 6803 (Williams, J G K.
Methods Enzymol., 167:766-768 (1988)) was used as the recipient
strain in this study, and is referred to as the wild type. Wild
type and transformant strains were maintained on solid BG-11 media
supplemented with 10 mM TES-NaOH (pH 8.2), 0.3% sodium thiosulfate,
and 5 mM glucose. Where appropriate, chloramphenicol was used at a
concentration of 15 .mu.g/mL. Liquid cultures were grown in BG-11
containing 25 mM sodium phosphate buffer, pH 7.5. Liquid cultures
for inoculum purposes and for photoautotrophic growth experiments
and SDS-PAGE analyses were maintained at 25.degree. C. under a slow
stream of constant aeration and illumination at 20 .mu.mol photons
m.sup.-2 s.sup.-1. Growth conditions employed, when measuring the
production of .beta.-phellandrene from Synechocystis cultures, are
described below in the .beta.-phellandrene production assays
section.
Codon-Use Optimization of the .beta.-phellandrene Synthase Gene for
Expression in Synechocystis sp. PCC 6803 and Escherichia coli
[0101] The nucleotide and translated protein sequences of the
.beta.-phellandrene synthase gene from Lavandula angustifolia
cultivar Lady (GenBank Accession Number HQ404305) were obtained
from the NCBI GenBank database (National Center for Biotechnology
Information; see, e.g.,
http:/lwww.ncbi.nlm.nih.gov/nuccore/HQ404305). The protein sequence
of the .beta.-phellandrene synthase gene was firstly analyzed by
TargetP software (see, e.g.,
http://www.cbs.dtu.dk/services/TargetP/) for the prediction of the
subcellular localization of the protein and for identification of
the presence and length of any targeting/transit amino acid
sequence. Based on this analysis, the .beta.-phellandrene synthase
from Lavandula angustifolia cultivar Lady was predicted to be a
chloroplast localized protein with the first 42 amino acids of the
protein serving as a chloroplast transit peptide. This analysis
indicated that the first 42 amino acids are not part of the mature
protein that functions in the catalysis of GPP conversion to
.beta.-phellandrene in the chloroplast. Based on this information,
we designed a protein sequence from the original sequence of
Lavandula .beta.-phellandrene synthase (La-.beta.-PHLS) gene by
replacing the first 42 amino acids with a methionine. The codon-use
of the resulting cDNA was then optimized for expression in
Synechocystis sp. PCC 6803 and E. coli. The protein sequence of the
.beta.-phellandrene synthase that was employed in this work is
composed of 540 amino acids of which the sequence is shown in FIG.
3A. To maximize the expression of .beta.-phellandrene synthase in
Synechocystis sp. PCC 6803 and E. coli, this protein sequence was
back-translated and codon-optimized according to the frequency of
the codon usage in Synechocystis sp. PCC 6803. The
codon-optimization process was performed based on the codon usage
table obtained from Kazusa DNA Research Institute, Japan (see,
e.g., http://www.kazusa.or.jp/codon/), and using the "Gene Designer
2.0" software from DNA 2.0 (see, e.g., https://www.dna20.com/) at a
cut-off thread of 15%. The codon-optimized gene was designed with
appropriate restriction sites flanking the S-.beta.-PHLS sequence
to aid subsequent cloning steps. The nucleotide sequences of the
original .beta.-phellandrene synthase gene from Lavandula
angustifolia (La-.beta.-PHLS) is shown in FIG. 3B, while the
codon-optimized sequence for expression in Synechocystis sp. PCC
6803 and E. coli (S-.beta.-PHLS) is shown in FIG. 3C.
Plasmid Construction and Generation of Synechocystis Transformants
with Heterologous Expression of the S-.beta.-PHLS Gene
[0102] A plasmid construct was generated to allow the heterologous
expression of the .beta.-phellandrene synthase gene in
Synechocystis through the replacement of the Synechocystis PsbA2
gene with the Syn-.beta.-PHLS gene via double homologous
recombination. The synthesized Syn-.beta.-PHLS was PCR amplified
using the following primers: PHLS_F, 5'-CCTGGGCGGTTCTGATAACG-3'
(SEQ ID NO:12), and PHLS_BamHI_R,
5'-CGCGGATCCTTTTGACGGCGGCCGCAGAT-3' (SEQ ID NO:13). A BamHI site
was incorporated into the PHLS_BamHI_R primer to allow the cloning
of S-.beta.-PHLS PCR product into the NdeI and BamHI sites of the
plasmid pBA2A2, which contains 500 bp of the upstream and
downstream sequences of the PsbA2 gene (Lindberg et al., Metab.
Eng., 12:70-79 (2010)), generating plasmid pBA2Synf.beta.PHLSA2.
Finally, a chloramphenicol resistance cassette from plasmid
pACYC184 was PCR amplified using primers with strategically
incorporated restriction sites: CamR_NotI_F,
5'-AAGGAAAAAAGCGGCCGCGTTGATCGGCACGTAAGAGGTTC-3' (SEQ ID NO:14), and
CamR_BamHI_R, 5'-CGCGGATCCCCAGGCGTTTAAGGGCACCAATAAC-3' (SEQ ID
NO:15), and cloned into the NotI and BamHI sites of plasmid
pBA2Synf.beta.PHLSA2, to generate plasmid pBA2Synf.beta.PHLSCamRA2.
This plasmid was used to transform wild-type Synechocystis sp. PCC
6803 according to established procedures (Williams J G K. Methods
Enzymol., 167:766-778 (1988); Eaton-Rye J J. Methods Mol. Biol.,
684:295-312 (2011)). Chloramphenicol was used for selection and
maintenance of transformant strains on agar plate. The heterologous
transformed Synechocystis PCC 6803 cyanobacteria are referred to as
S-.beta.-PHLS transformants. Successful transgene incorporation and
complete DNA cyanobacterial copy segregation for the S-.beta.-PHLS
gene was verified by genomic DNA PCR, using primers designed to
genomic DNA regions just outside of the upstream and downstream
regions of the PsbA2 gene that were used for homologous
recombination:
TABLE-US-00001 (SEQ ID NO: 16) A2us_F,
5'-TATCAGAATCCTTGCCCAGATG-3', and (SEQ ID NO: 17) A2ds_R,
5'-GGTAGAGTTGCGAGGGCAAT-3'.
Antibody Generation and Western Blot Analysis
[0103] For expression in E. coli, the Synechocystis codon optimized
.beta.-PHLS gene (S-.beta.-PHLS) was PCR amplified using the
forward primer 5'-GGAATTCCATATGTGTAGTTTGCAAGTTTCTGAT-3'(SEQ ID
NO:18) and reverse primer 5'-ACAGGATCCTCACTCATAGCGCTCAATCAGCGT-3'
(SEQ ID NO:19), and subcloned into the pET28a(+) vector (Novagen).
Expression of the S-.beta.-PHLS construct was induced by IPTG in E.
coli BL21 (DE3) cells (Novagen), and the 6xHis-tagged S-.beta.-PHLS
protein was purified under native conditions through a
nickel-nitrilotriacetic acid agarose column (NTA, Qiagen) according
to the manufacturer's instructions. Specific polyclonal antibodies
were generated in rabbit against the full length mature
.beta.-phellandrene synthase recombinant protein as the antigen,
following the instructions of ProSci Inc, USA.
[0104] Samples for SDS-PAGE analyses were prepared from
Synechocystis cells resuspended in phosphate buffer pH 7.4 at a
concentration of 0.12 mg/ml chlorophyll. The suspension was
supplemented with 0.05% w/v lysozyme (Thermo Scientific) and
incubated with shaking at 37.degree. for 45 min. Cells were then
pelleted at 4,000 g, washed twice with fresh phosphate buffer and
disrupted with a French Pressure chamber (Aminco, USA) at 1500 psi
in the presence of 1 mM PMSF. Soluble protein was separated from
the total cell extract by centrifugation at 21,000 g and removed as
the supernatant fraction. Samples for SDS-PAGE analysis were
solubilized with 1 volume of 2.times. denaturing protein
solubilization buffer (0.2 M Tris, pH 6.8, 4% SDS, 2 M urea, 1 mM
EDTA and 20% glycerol). In addition, all samples in denaturing
solutions were supplemented with a 5% (v/v) of
.beta.-mercaptoethanol and centrifuged at 17,900 g for 5 min prior
to gel loading. For Western blot analyses, Any kD.TM. (BIO-RAD)
precast SDS-PAGE gels were utilized to resolve proteins, which were
then transferred to PVDF membrane (Immobilon-FL 0.45 .mu.m,
Millipore,USA) for immunodetection using the rabbit immune serum
containing specific polyclonal antibodies against the S-.beta.-PHLS
protein. Cross-reactions were visualized by Supersignal West Pico
Chemiluminiscent substrate detection system (Thermo Scientific,
USA).
Chlorophyll Determination, Photosynthetic Productivity and Biomass
Quantitation
[0105] Chlorophyll a concentrations in cultures were determined
spectrophotometrically in 90% methanol extracts of the cells
according to Meeks and Castenholz (Arch. Mikrobiol., 78:25-41
(1971)). Photosynthetic productivity of the cultures was tested
polarographically with a Clark-type oxygen electrode (Rank
Brothers, Cambridge, England). Cells were harvested at
mid-exponential growth phase, and maintained at 25.degree. C. in
BG11 containing 25 mM HEPES-NaOH, pH 7.5, at a chlorophyll a
concentration of 10 .mu.g/mL. Oxygen evolution was measured at
25.degree. C. in the electrode upon yellow actinic illumination,
which was defined by a CS 3-69 long wavelength pass cutoff filter
(Corning, Corning, N.Y.). Photosynthetic activity of a 5 mL aliquot
of culture was measured at varying actinic light intensities in the
presence of 15 mM NaHCO.sub.3 pH 7.4, to generate the light
saturation curve of photosynthesis. Culture biomass accumulation
was measured gravimetrically as dry cell weight, where 5 mL samples
of culture were filtered through 0.22 .mu.m Millipore filters and
the immobilized cells dried at 90.degree. C. for 6 h prior to
weighing the dry cell weight.
.beta.-Phellandrene Production and Quantification Assays
[0106] Synechocystis cultures for 13-phellandrene production assays
were grown photoautotrophically in 1 L gaseous/aqueous two-phase
photobioreactors, described in detail by Bentley and Melis
(Biotechnol Bioeng., 109:100-109 (2012)). Bioreactors were seeded
with a 700 ml culture of Synechocystis cells at an OD730 nm of 0.05
in BG11 medium containing 25 mM sodium phosphate buffer, pH 7.5,
and grown under continuous illumination at 75 .mu.mol photons
m.sup.-2 s.sup.-1, and continuous bubbling with air, until an OD730
nm of approximately 0.5 was reached. Inorganic carbon was delivered
to the culture in the form of 500 mL aliquots of 100% CO.sub.2 gas,
which was slowly bubbled though the bottom of the liquid culture to
fill the bioreactor headspace. Once atmospheric gases were replaced
with 100% CO.sub.2, the headspace of the reactor was sealed and the
culture was incubated under continuous illumination of 150 .mu.mol
photons m.sup.-2 s.sup.-1 at 35.degree. C. Slow continuous
mechanical mixing was employed to keep cells in suspension and to
promote balanced cell illumination and nutrient mixing into the
liquid culture in support of photosynthesis and biomass
accumulation. Uptake and assimilation of headspace CO.sub.2 by
cells was concomitantly exchanged for O.sub.2 during
photoautotrophic growth. The sealed bioreactor headspace allowed
for the trapping, accumulation and concentration of
photosynthetically produced .beta.-phellandrene, as either a
volatile product in the headspace, or in liquid form floating on
the aqueous phase.
[0107] Gas from the headspace of sealed bioreactors was sampled and
analyzed by gas chromatographymass spectrometry (GC-MS) in an
effort to detect volatilized, photosynthetically produced
monoterpene hydrocarbons (.beta.-phellandrene). Comparison of
retention time and mass spectrum with a vaporized mixture of
.alpha.-phellandrene and .beta.-phellandrene standard (MP
Biomedicals) allowed for positive identification of
.beta.-phellandrene in the headspace. Photosynthetically produced
non-miscible .beta.-phellandrene in liquid form was extracted from
the liquid phase upon overlaying 20 mL heptane on top of the liquid
culture in the bioreactor, and upon incubating for 30 min, or
longer, at room temperature. The heptane layer was subsequently
removed and analysed by GC-MS for the detection of
.beta.-phellandrene by comparison with the liquid a-phellandrene
and .beta.-phellandrene standard also dissolved in heptane. GC-MS
analyses were performed with an Agilent 6890GC/5973 MSD equipped
with a DB-XLB column (0.25 mm i.d..times.0.25 .mu.m.times.30 m, J
&W Scientific). Oven temperature was initially maintained at
40.degree. C. for 4 min, followed by a temperature increase of
5.degree. C./min to 80.degree. C., and a carrier gas (helium) flow
rate of 1.2 ml per minute.
[0108] Accumulation of .beta.-phellandrene in the liquid phase was
quantified spectrophotometrically according to known absorbance
spectra and extinction coefficients of .beta.-phellandrene in
organic solvents (Macbeth et al., J Chem. Soc. 119-123 (1938);
Booker et al., J. Chem. Soc. 1453-1463 (1940); Gross K P, Schnepp
O., J. Chem. Phys. 68:2647-2657 (1978)). The majority of
photosynthetically produced .beta.-phellandrene accumulated as a
liquid floating over the aqueous phase of the bioreactor.
Therefore, the non-miscible, heptane-extracted .beta.-phellandrene
was used to generate the absorption spectra of .beta.-phellandrene
in heptane for quantification purposes.
Results
[0109] The native L. angustifolia cDNA sequence has a codon usage
different from that preferred by photosynthetic microorganisms,
e.g., cyanobacteria and microalgae. The unicellular cyanobacteria
Synechocystis sp. were used as a model organism in the development
of the present invention. A de novo codon-optimized .beta.-PHLS
gene was designed and synthesized. In the optimized version of the
gene, termed S-.beta.-PHLS, the codon usage was adapted to
eliminate codons rarely used in Synechocystis, and to adjust the
AT/GC ratio to that of the host. Rare codons were defined using a
codon usage table derived from the sequenced genome of
Synechocystis. The .beta.-phellandrene synthase sequences used in
this example were: the .beta.-PHLS protein sequence for expression
in Synechocystis and E. coli (S-.beta.-PHLS), the native L.
angustifolia .beta.-PHLS cDNA sequence including the predicted
chloroplast transit peptide (GenBank Accession No. HQ404305), and
the L. angustifolia .beta.-PHLS cDNA sequence minus the chloroplast
transit peptide, with codon usage optimized for Synechocystis
(S-.beta.-PHLS). In the native L. angustifolia .beta.-PHLS sequence
a substantial number of codons are present that are used with a
frequency of less than 15% by Synechocystis. In the codon-optimized
gene, such low-frequency codons were not allowed.
[0110] SDS-PAGE analyses and immuno-detection of the
.beta.-phellandrene synthase enzyme, using specific polyclonal
antibodies raised against the E. coli-expressed recombinant
protein, confirmed the presence of the S-.beta.-PHLS protein in
Synechocystis (FIG. 6). The S-.beta.-PHLS protein was localized in
the soluble fraction of Synechocystis cell extracts, consistent
with the notion of a soluble protein. FIG. 6 (top panel) shows the
absence of cross-reaction between the anti-S-.beta.-PHLS polyclonal
antibodies and any protein of the Synechocysis wild type (WT) in
the total cell extract (TCE) or supernatant (SP) fractions.
However, a specific cross-reaction was observed between the
anti-S-.beta.-PHLS polyclonal antibodies and a protein band at
about 65 kD in both of the total cell extract (TCE) and supernatant
fractions (SP) of the S-.beta.-PHLS transformant. These results
clearly show that the recombinant S-.beta.-PHLS protein was
expressed in Synechocystis transformants, and that it accumulated
as a soluble protein in the cell.
[0111] The above results demonstrated that Synechocystis can be
used for heterologous transformation using a .beta.-PHLS gene, and
that such transformants expressed and accumulated the S-.beta.-PHLS
protein in their cytosol. To determine whether the expressed
S-.beta.-PHLS protein is metabolically competent, wild type and
S-.beta.-PHLS transformants were cultivated under the conditions of
the gaseous/aqueous two-phase bioreactor (Bentley F K and Melis A.,
Biotechnol Bioeng., 109:100-109 (2012)), with 100% CO.sub.2 gas
occupying the headspace prior to sealing the reactor to allow
autotrophic biomass accumulation. Samples were obtained from both
the headspace of sealed cultures (to detect vaporized
.beta.-phellandrene) and from the surface of liquid cultures (to
detect non-miscible liquid .beta.-phellandrene floating on top of
the aqueous phase) and analyzed by GC-MS.
[0112] Analysis of the accumulated reactor headspace gases in the
wild type after 48 h incubation showed no evidence of
.beta.-phellandrene hydrocarbons (FIG. 7). The headspace of the
S-.beta.-PHLS transformant, however, showed .beta.-phellandrene
accumulation, as evidenced by the GC peak with a 4.6 min retention
time (FIG. 8, asterisk), which is comparable to the retention time
of the .beta.-phellandrene peak observed in the phellandrene
standard (FIG. 9, asterisk). A supply of commercially available
pure .beta.-phellandrene standard was difficult to source,
therefore we opted to use a commercially-available
.alpha.-phellandrene standard. Upon GC-MS analysis of the standard,
a major peak was identified as .alpha.-phellandrene, however, the
MS-analysis indicated presence of a number of other monoterpene
impurities, including .beta.-myrcene, 2-carene, benzene, eucalyptol
and .beta.-phellandrene (FIG. 9). Accordingly, we employed the
.beta.-phellandrene peak in the standard as a reference for
identification of .beta.-phellandrene produced by the Synechocystis
transformant cultures. The height of the .beta.-phellandrene peak
from the gas sample of the S-.beta.-PHLS transformant clearly
showed that .beta.-phellandrene was the major volatile hydrocarbon
generated by photosynthesis in the transformant (FIG. 8). This peak
was positively identified as .beta.-phellandrene by comparison of
its mass spectrum with the .beta.-phellandrene peak in the standard
sample, showing distinct mass spectral lines [77, 91, 93 and 136]
that signify .beta.-phellandrene hydrocarbons (FIGS. 10 and 11).
These results provided evidence that the S-.beta.-PHLS transgene
and its encoded .beta.-phellandrene synthase enzyme were
responsible for the catalysis of .beta.-phellandrene production in
the transformant Synechocystis strains.
[0113] Unlike molecular hydrogen (H.sub.2) and isoprene
hydrocarbons (C.sub.5H.sub.8), which are small and easily escape
from the cells that produce them (Melis A., Energy Environ. Sci.,
5(2): 5531-5539; (2012)), monoterpene hydrocarbons, including
.beta.-phellandrene, are large enough to be trapped in the
hydrophobic domain of the cell's lipid bilayers. Such outcome would
most likely have very adverse consequences for cell growth and
fitness. Accordingly, our efforts have focused to investigate
whether the cells can freely emit .beta.-phellandrene, and whether
cell growth and properties of photosynthesis are adversely affected
in the S-.beta.-PHLS transformants.
[0114] Monoterpene hydrocarbons have a relatively high boiling
point (170-175.degree. C.) and are non-miscible in aqueous
solution. If freely emitted by the transformant photosynthetic
microorganisms, one would expect that monoterpene molecules,
including .beta.-phellandrene, would float on the aqueous phase of
the reactor. Surprisingly, this was indeed observed in the case of
.beta.-phellandrene, produced by the transformed cyanobacteria. A
small volume of heptane was layered on top of the liquid culture to
trap non-miscible liquid hydrocarbons, such as .beta.-phellandrene,
floating on the surface of the culture, and spectrophotometic
analyses were performed as the method for .beta.-phellandrene
quantification. FIG. 12A shows representative absorbance spectra of
heptane-extracted samples from wild type (WT) and S-.beta.-PHLS
transformant cultures. The absorbance maximum of
.beta.-phellandrene occurs at 230 nm (Macbeth et al., J. Chem.
Soc., 119-123 (1938); Booker et al., J. Chem. Soc., 1453-1463
(1940); Gross K P and Schnepp O., J. Chem. Phys., 68:2647-2657
(1978)). A well-defined band peaking at 230 nm was observed in the
heptane extracts of S-.beta.-PHLS-transformants, while absent in
wild-type samples. Importantly, .alpha.-phellandrene has an
absorbance maximum of 260 nm (Macbeth et al., J. Chem. Soc.,
119-123 (1938); Booker et al., J. Chem. Soc., 1453-1463 (1940);
Gross K P and Schnepp O., J. Chem. Phys., 68:2647-2657 (1978)),
which allows .beta.-phellandrene to be easily distinguished from
.alpha.-phellandrene using the spectrophotometric method. FIG. 12B
shows the absorbance spectrum of the liquid .alpha.-phellandrene
standard (used for the GC-MS analyses, e.g. FIG. 9) diluted in
heptane with its absorbance maximum of 260 nm. The absence of
absorbance peaks at 260 nm in the S-.beta.-PHLS transformants (FIG.
12A) indicated that little, if any, .alpha.-phellandrene
accumulated as a non-miscible product of photosynthesis in
transformant lines, and that .beta.-phellandrene exclusively
accumulated in more substantial quantities.
[0115] Quantification of .beta.-phellandrene in the
heptane-extracted samples from S-.beta.-PHLS transformants was
determined according to the Beer-Lambert Law, using the absorbance
values measured at 230 nm and the known molar extinction
coefficient of .beta.-phellandrene. During 48 h of active
photoautotrophic growth in the presence of CO.sub.2 in a sealed
gaseous/aqueous two-phase bioreactor, a 700 ml culture of
S-.beta.-PHLS transformant produced .beta.-phellandrene in the form
of a non-miscible product floating on the surface of the
culture.
[0116] The photoautotrophic cell growth kinetics of the
S-.beta.-PHLS transformants were similar to those of the wild type,
with a cell doubling time of 16 h under a light intensity of 20
.mu.mol photons m.sup.-2 s.sup.-1 under continuous bubbling with
air (FIG. 13). The light saturation curves of photosynthesis of
wild type and the S-.beta.-PHLS transformants were also similar to
one another (FIG. 14), where oxygen evolution saturated at about
500 .mu.mol photons m.sup.-2 s.sup.-1, with an average P.sub.max of
216 .mu.mol O.sub.2 (mg Chl).sup.-1h.sup.-1 in wild type and 263
.mu.mol O.sub.2 (mg Chl).sup.-1 11.sup.-1 in the S-.beta.-PHLS
transformant (FIG. 14). Similarly, rates of oxygen consumption
during dark respiration were about the same in the wild type and
S-.beta.-PHLS transformants and equal to about -14 .mu.mol O.sub.2
(mg Chl).sup.-1h.sup.-1. Importantly, at sub-saturating light
intensities between 0 and 250 .mu.mol photons m.sup.-2 s.sup.-1,
rates of oxygen evolution and the initial slopes of photosynthesis
as a function of light intensity were comparable in wild-type and
S-.beta.-PHLS-transformant cells (FIG. 14), suggesting similar
quantum yields of photosynthesis for the two strains (Melis A.,
Plant Science, 177:272-280 (2009)). These results demonstrated that
deletion of the endogenous PsbA2 coding region from the
Synechocystis genome, with the attendant replacement/integration
and expression of the S-.beta.-PHLS transgene in the cell, as well
as the subsequent generation and accumulation of
.beta.-phellandrene, had no adverse effects on the photoautotrophic
growth parameters of the transformants.
[0117] The .beta.-phellandrene synthase protein has been
successfully over-expressed in E. coli (Demissie et al., Planta,
233:685-696 (2011)). However, only in vitro enzymatic assays were
performed with the .beta.-phellandrene synthase recombinant
protein. This suggests that there was little .beta.-phellandrene in
E. coli and/or limited or no efflux of .beta.-phellandrene from E.
coli and/or adverse effects of the .beta.-phellandrene synthase
protein or of its product on the E. coli host cells. Absence of
.beta.-phellandrene hydrocarbons in heptane extracts from the
surface of IPTG-induced .beta.-PHLS-transformed Escherichia coli
cultures was also observed in the illustrative experiments
described herein (FIG. 15). In this study, transformant E. coli
cells were induced by isopropyl .beta.-D-1-thiogalactopyranoside
(IPTG), resulting in the over-expression of the .beta.-phellandrene
protein. Absorbance spectra of heptane-extracted samples from the
surface of such E. coli liquid cultures failed to show the presence
of the .beta.-phellandrene molecule. No distinctive
.beta.-phellandrene absorbance peak could be observed at 230 nm
from the .beta.-PHLS E. coli cultures (compare with the results of
FIG. 12A).
Discussion
[0118] "Photosynthetic biofuels", as defined in the present
invention, are produced in a system where the same organism serves
both as photo-catalyst and producer of ready-made fuel or chemical.
A number of guiding principles have been applied in the endeavor of
photosynthetic biofuels, as they pertain to the selection of
organisms and, independently, to the selection of potential
biofuels. Criteria for the selection of organisms include,
foremost, the solar-to-biofuel energy conversion efficiency, which
must be as high as possible. This important criterion is better
satisfied with photosynthetic microorganisms than with crop plants
(Melis A., Plant Science, 177:272-280 (2009)). Criteria for the
selection of potential biofuels include (i) the relative energy
content and potential utility of the molecule. Pure hydrocarbons
are preferred over sugars or alcohols because of the greater
relative energy stored in hydrocarbon molecules (Schakel et al., J.
Food Comp. Anal., 10:102-114 (1997); Berg J, Tymoczko J L, Stryer
L. (2002) Biochemistry (5th ed.). W. H. Freeman, San Francisco,
Calif. p.603.); and (ii) the question of product separation from
the biomass, which enters prominently in the economics of the
process and is a most important aspect in commercial application.
This example demonstrates that .beta.-phellandrene is suitable in
this respect, as it is not miscible in water, spontaneously
separating from the biomass and end-up floating on the aqueous
phase of the reactor and culture that produced them. Such
spontaneous product separation from the liquid culture alleviates
the requirement of time-consuming, expensive, and technologically
complex biomass dewatering (Danquah et al., J Chem Tech. Biotech.,
84:1078-1083 (2009); Saveyn et al., J. Res. Sci Tech., 6:51-56
(2009)) and product excision from the cells that otherwise would be
needed for product isolation.
[0119] In the pursuit of renewable biofuels, photosynthesis,
cyanobacteria or microalgae and .beta.-phellandrene meet the
above-enumerated criteria for "process", "organism" and "product",
respectively. This example shows that .beta.-phellandrene can be
heterologously produced via photosynthesis in microorganisms, e.g.,
cyanobacteria, genetically engineered to express a plant
.beta.-phellandrene synthase. In this example, the Lavandular
angustifolia .beta.-PHLS gene was employed via heterologous
expression in Synechocystis. The DNA sequence of the Lavandular
.beta.-PHLS gene was optimized for Synechocystis codon-usage. A
diffusion-based method for spontaneous gas exchange in
gaseous/aqueous two-phase photobioreactors was employed, using
carbon dioxide as a feedstock for the photosynthetic generation of
.beta.-phellandrene. The headspace of the bioreactor was filled
with 100% CO.sub.2 and sealed, allowing the diffusion-based
CO.sub.2 uptake and assimilation by the cells via photosynthesis,
and the concomitant replacement of the CO.sub.2 in the headspace
with .beta.-phellandrene vapour and O.sub.2. A considerable amount
of photosynthetically generated .beta.-phellandrene accumulated as
a non-miscible product floating on the top of the liquid culture,
which is explained as .beta.-phellandrene has a boiling point of
171.degree. C.
[0120] In the plasmid constructs employed for the expression of the
.beta.-phellandrene synthase in Synechocystis, we used the PsbA2
gene locus for insertion of the transgenes. Upon transformation of
Synechocystis with these constructs, the .beta.-PHLS gene replaced
the coding sequence of the PsbA2 gene, and the PsbA2 promoter was
used to drive expression of .beta.-PHLS. The PsbA2 gene is one of
three homologous genes in cyanobacteria, the other two being PsbA1
and PsbA3, that encode the 32 kD/D1 reaction center protein of
photosystem-II. The promoter region and regulation of expression of
the PsbA2 gene has been characterized (Eriksson et al., Mol. Cell
Biol. Res. Commun., 3:292-8 (2000); Mohamedet al., Mol Gen Genet.,
238:161-8 (1993); Mohamed A, Jansson C., Plant Mol. Biol.
13:693-700 (1989)). It has also been shown that a knock-out mutant
of either PsbA2 or PsbA3 is able to grow photoautotrophically,
provided that the other PsbA genes are still active, while PsbA1 on
its own was not able to compensate for the loss of both PsbA2 and
PsbA3 (Mohamed A, Jansson C., Plant Mol. Biol. 13:693-700 (1989)).
Inactivation of PsbA2 resulted in a strong up-regulation of PsbA3
(Mohamedet al., Mol Gen Genet., 238:161-8 (1993)). This example
illustrates that replacement of PsbA2 by the codon-optimized
.beta.-PHLS gene does not significantly alter normal
photoautotrophic growth of the transformants.
[0121] The monoterpene .beta.-phellandrene is an energy rich
10-carbon hydrocarbon molecule, useful industrially as in cosmetics
industry, cleaning products for household and industrial use, and
medicinal use. Currently, .beta.-phellandrene for use in commercial
industry is extracted from plants, such as lavender, which contain
.beta.-phellandrene in their glandular trichome essential oils.
However, this example shows that .beta.-phellandrene can be
produced by photosynthetic microorganisms, e.g., cyanobacteria and
microalgae, through heterologous expression of the gene encoding
for the .beta.-phellandrene synthase (.beta.-PHLS), in a reaction
of the MEP pathway, driven by the process of cellular
photosynthesis. Since the carbon atoms used to generate
.beta.-phellandrene in such a system originate from CO.sub.2, this
would make cyanobacterial and microalgal .beta.-phellandrene
production a carbon-neutral source of synthetic chemistry and
biofuel feedstock. .beta.-Phellandrene would also be suitable as a
building block for the production of longer chain hydrocarbons, to
be used as longer chain renewable and carbon-neutral biofuels,
pharmaceuticals, and cosmetics.
[0122] All publications, accession numbers, and patent applications
cited in this specification are herein incorporated by reference as
if each individual publication or patent application were
specifically and individually indicated to be incorporated by
reference.
Illustrative Sequences
TABLE-US-00002 [0123] Amino acid sequence of the mature
S.beta.-PHLS protein SEQ ID NO: 1
MCSLQVSDPIPTGRRSGGYPPALWDFDTIQSLNTEYKGERHMRREEDLIGQVREMLV
HEVEDPTPQLEFIDDLHKLGISCHFENEILQILKSIYLNQNYKRDLYSTSLAFRLLRQY
GFILPQEVEDCFKNEEGTDFKPSFGRDIKGLLQLYEASFLSRKGEETLQLAREFATKIL
QKEVDEREFATKMEFPSHWTVQMPNARPFIDAYRRRPDMNPVVLELAILDTNIVQA
QFQEELKETSRWWESTGIVQELPFVRDRIVEGYFWTIGVTQRREHGYERIMTAKVIA
LVTCLDDIYDVYGTIEELQLFTSTIQRWDLESMKQLPTYMQVSFLALHNEVTEVAYD
TLKKKGYNSTPYLRKTWVDLVESYIKEATWYYNGYKPSMQEYLNNAWISVGSMAI
LNHLFFRFTNERMHKYRDMNRVSSNIVRLADDMGTSLAEVERGDVPKAIQCYMNET
NASEEEAREYVRRVIQEEWEKLNTELMRDDDDDDDFTLSKYYCEVVANLTRMAQFI
YQDGSDGFGMKDSKVNRLLKETLIERYE The native La-.beta.-PHLS cDNA
nucleotide sequence SEQ ID NO: 2
ATGTCTACCATTATTGCGATACAAGTGTTGCTTCCTATTCCAACTACTAAAACATA
CCCTAGTCATGACTTGGAGAAGTCCTCTTCGCGGTGTCGCTCCTCCTCCACTCCTC
GCCCTAGACTGTGTTGCTCGTTGCAGGTGAGTGATCCGATCCCAACGGGCCGGCG
ATCCGGAGGCTACCCGCCCGCCCTATGGGATTTCGACACTATTCAATCGCTCAAC
ACCGAGTATAAGGGAGAGAGGCACATGAGAAGGGAAGAAGACCTAATTGGGCA
AGTTAGAGAGATGCTGGTGCATGAAGTAGAGGATCCCACTCCACAGCTGGAGTT
CATTGATGATTTGCATAAGCTTGGCATATCTTGCCATTTTGAGAATGAAATCCTCC
AAATCTTGAAATCCATATATCTTAATCAAAACTACAAAAGGGATTTGTACTCAAC
ATCTCTAGCATTCAGACTCCTCAGACAATATGGCTTCATCCTTCCACAAGAAGTA
TTTGATTGTTTCAAGAATGAGGAGGGTACGGATTTCAAGCCAAGCTTCGGCCGTG
ATATCAAAGGCTTGTTACAATTGTATGAAGCTTCTTTCCTATCAAGAAAAGGAGA
AGAAACTTTACAACTAGCAAGAGAGTTTGCAACAAAGATTCTGCAAAAAGAAGT
TGATGAGAGAGAGTTTGCAACCAAGATGGAGTTCCCTTCTCATTGGACGGTTCAA
ATGCCGAATGCAAGACCTTTCATCGATGCTTACCGTAGGAGGCCGGATATGAATC
CAGTTGTGCTCGAGCTAGCCATACTTGATACAAATATAGTTCAAGCACAATTTCA
AGAAGAACTCAAAGAGACCTCAAGGTGGTGGGAGAGTACAGGCATTGTCCAAGA
GCTTCCATTTGTGAGGGATAGGATTGTGGAAGGCTACTTTTGGACGATTGGAGTG
ACTCAGAGACGCGAGCATGGATACGAAAGAATCATGACCGCAAAGGTTATTGCC
TTAGTAACATGTTTAGACGACATATACGATGTTTATGGCACGATAGAAGAGCTTC
AACTTTTCACAAGCACAATCCAAAGATGGGATTTGGAATCAATGAAGCAACTCC
CTACCTACATGCAAGTAAGCTTTCTTGCACTACACAACTTTGTAACCGAGGTGGC
TTACGATACTCTCAAGAAAAAGGGCTACAACTCCACACCATATTTAAGAAAAAC
GTGGGTGGATCTTGTTGAATCATATATCAAAGAGGCAACTTGGTACTACAACGGT
TATAAACCTAGTATGCAAGAATACCTTAACAATGCATGGATATCAGTCGGAAGT
ATGGCTATACTCAACCACCTCTTCTTCCGGTTCACAAACGAGAGAATGCATAAAT
ACCGCGATATGAACCGTGTCTCGTCCAACATTGTGAGGCTTGCTGATGATATGGG
AACATCATTGGCTGAGGTGGAGAGAGGGGACGTGCCGAAAGCAATTCAATGCTA
CATGAATGAGACGAATGCTTCTGAAGAAGAAGCAAGAGAATATGTAAGAAGAGT
CATACAGGAAGAATGGGAAAAGTTGAACACAGAATTGATGCGGGATGATGATGA
TGATGATGATTTTACACTATCCAAATATTACTGTGAGGTGGTTGCTAATCTTACA
AGAATGGCACAGTTTATATACCAAGATGGATCGGATGGCTTCGGCATGAAAGAT
TCCAAGGTTAATAGACTGCTAAAAGAGACGTTGATCGAGCGCTACGAATAA Codon-optimized
version of the L. angustifolia (La-S-.beta.-PHLS) cDNA nucleotide
sequence for expression in cyanobacteria, e.g., Synechocystis ("S"
in gene designation). SEQ ID NO: 3
TTAATTAACATATGTGTAGTTTGCAAGTTTCTGATCCTATTCCTACCGGACGCCGT
TCCGGTGGTTATCCCCCGGCCTTATGGGATTTCGATACTATTCAATCCCTGAATAC
CGAATATAAGGGCGAACGTCACATGCGTCGGGAAGAAGACTTAATTGGTCAAGT
TCGGGAAATGTTGGTGCACGAAGTAGAAGATCCCACTCCCCAGTTGGAATTCATT
GACGATCTGCATAAATTGGGCATTTCCTGCCATTTTGAAAACGAGATTCTGCAAA
TTCTCAAATCCATTTATCTCAACCAAAACTATAAACGGGACCTCTATTCTACCAG
TTTAGCCTTCCGTCTCTTGCGTCAATACGGGTTTATCTTGCCGCAGGAAGTTTTTG
ACTGCTTTAAAAACGAAGAAGGTACGGATTTTAAACCCAGCTTCGGCCGGGATA
TTAAGGGTCTGTTACAGTTGTACGAAGCCTCCTTTTTGTCCCGGAAGGGGGAAGA
AACTTTACAACTCGCCCGCGAATTTGCTACCAAAATCTTGCAAAAGGAAGTCGAT
GAACGGGAATTTGCTACTAAAATGGAATTTCCCAGTCACTGGACCGTACAAATGC
CTAACGCTCGGCCTTTTATCGATGCCTATCGTCGGCGTCCCGACATGAACCCCGT
GGTTCTGGAACTCGCCATTCTCGATACCAATATCGTGCAAGCTCAGTTTCAAGAA
GAATTGAAGGAGACCTCCCGTTGGTGGGAAAGCACGGGGATTGTTCAAGAACTG
CCGTTTGTTCGGGACCGGATTGTGGAAGGTTATTTTTGGACCATTGGTGTTACTCA
ACGCCGTGAACACGGTTACGAACGTATTATGACGGCCAAAGTCATCGCTTTGGTG
ACCTGTTTGGATGATATTTATGACGTATATGGCACTATTGAAGAATTGCAACTCT
TCACCTCTACGATTCAGCGTTGGGATTTGGAGTCTATGAAGCAGTTACCGACTTA
TATGCAGGTAAGCTTCCTGGCCTTGCACAATTTTGTAACCGAAGTGGCCTATGAT
ACGCTGAAGAAAAAGGGCTACAACTCTACCCCCTATTTGCGGAAGACTTGGGTG
GATTTGGTCGAAAGTTACATTAAGGAAGCCACTTGGTACTATAATGGGTACAAAC
CCTCTATGCAGGAATACCTCAACAACGCCTGGATCTCTGTGGGCAGCATGGCTAT
TTTGAATCATTTGTTTTTTCGCTTTACTAATGAACGCATGCATAAGTACCGGGACA
TGAATCGTGTATCCTCTAATATTGTGCGGTTAGCCGACGATATGGGAACCTCTTT
GGCCGAAGTTGAACGCGGTGACGTGCCCAAAGCTATCCAATGTTACATGAATGA
AACGAACGCCTCTGAGGAGGAGGCCCGCGAATATGTGCGGCGCGTTATCCAGGA
AGAATGGGAAAAACTGAACACTGAACTGATGCGCGACGACGACGATGACGATG
ATTTCACCTTAAGTAAATACTACTGCGAAGTCGTTGCTAACCTGACCCGGATGGC
TCAGTTCATTTACCAAGATGGTTCCGATGGGTTTGGGATGAAAGATTCCAAAGTA
AATCGTTTACTGAAAGAAACGCTGATTGAGCGCTATGAGTGAAGATCTGCGGCC GC
Sequence CWU 1
1
201540PRTLavandula angustifolialavender cultivar Lady mature beta-
phellandrene synthase (beta-PHLS, S-beta-PHLS) 1Met Cys Ser Leu Gln
Val Ser Asp Pro Ile Pro Thr Gly Arg Arg Ser1 5 10 15Gly Gly Tyr Pro
Pro Ala Leu Trp Asp Phe Asp Thr Ile Gln Ser Leu 20 25 30Asn Thr Glu
Tyr Lys Gly Glu Arg His Met Arg Arg Glu Glu Asp Leu 35 40 45Ile Gly
Gln Val Arg Glu Met Leu Val His Glu Val Glu Asp Pro Thr 50 55 60Pro
Gln Leu Glu Phe Ile Asp Asp Leu His Lys Leu Gly Ile Ser Cys65 70 75
80His Phe Glu Asn Glu Ile Leu Gln Ile Leu Lys Ser Ile Tyr Leu Asn
85 90 95Gln Asn Tyr Lys Arg Asp Leu Tyr Ser Thr Ser Leu Ala Phe Arg
Leu 100 105 110Leu Arg Gln Tyr Gly Phe Ile Leu Pro Gln Glu Val Phe
Asp Cys Phe 115 120 125Lys Asn Glu Glu Gly Thr Asp Phe Lys Pro Ser
Phe Gly Arg Asp Ile 130 135 140Lys Gly Leu Leu Gln Leu Tyr Glu Ala
Ser Phe Leu Ser Arg Lys Gly145 150 155 160Glu Glu Thr Leu Gln Leu
Ala Arg Glu Phe Ala Thr Lys Ile Leu Gln 165 170 175Lys Glu Val Asp
Glu Arg Glu Phe Ala Thr Lys Met Glu Phe Pro Ser 180 185 190His Trp
Thr Val Gln Met Pro Asn Ala Arg Pro Phe Ile Asp Ala Tyr 195 200
205Arg Arg Arg Pro Asp Met Asn Pro Val Val Leu Glu Leu Ala Ile Leu
210 215 220Asp Thr Asn Ile Val Gln Ala Gln Phe Gln Glu Glu Leu Lys
Glu Thr225 230 235 240Ser Arg Trp Trp Glu Ser Thr Gly Ile Val Gln
Glu Leu Pro Phe Val 245 250 255Arg Asp Arg Ile Val Glu Gly Tyr Phe
Trp Thr Ile Gly Val Thr Gln 260 265 270Arg Arg Glu His Gly Tyr Glu
Arg Ile Met Thr Ala Lys Val Ile Ala 275 280 285Leu Val Thr Cys Leu
Asp Asp Ile Tyr Asp Val Tyr Gly Thr Ile Glu 290 295 300Glu Leu Gln
Leu Phe Thr Ser Thr Ile Gln Arg Trp Asp Leu Glu Ser305 310 315
320Met Lys Gln Leu Pro Thr Tyr Met Gln Val Ser Phe Leu Ala Leu His
325 330 335Asn Phe Val Thr Glu Val Ala Tyr Asp Thr Leu Lys Lys Lys
Gly Tyr 340 345 350Asn Ser Thr Pro Tyr Leu Arg Lys Thr Trp Val Asp
Leu Val Glu Ser 355 360 365Tyr Ile Lys Glu Ala Thr Trp Tyr Tyr Asn
Gly Tyr Lys Pro Ser Met 370 375 380Gln Glu Tyr Leu Asn Asn Ala Trp
Ile Ser Val Gly Ser Met Ala Ile385 390 395 400Leu Asn His Leu Phe
Phe Arg Phe Thr Asn Glu Arg Met His Lys Tyr 405 410 415Arg Asp Met
Asn Arg Val Ser Ser Asn Ile Val Arg Leu Ala Asp Asp 420 425 430Met
Gly Thr Ser Leu Ala Glu Val Glu Arg Gly Asp Val Pro Lys Ala 435 440
445Ile Gln Cys Tyr Met Asn Glu Thr Asn Ala Ser Glu Glu Glu Ala Arg
450 455 460Glu Tyr Val Arg Arg Val Ile Gln Glu Glu Trp Glu Lys Leu
Asn Thr465 470 475 480Glu Leu Met Arg Asp Asp Asp Asp Asp Asp Asp
Phe Thr Leu Ser Lys 485 490 495Tyr Tyr Cys Glu Val Val Ala Asn Leu
Thr Arg Met Ala Gln Phe Ile 500 505 510Tyr Gln Asp Gly Ser Asp Gly
Phe Gly Met Lys Asp Ser Lys Val Asn 515 520 525Arg Leu Leu Lys Glu
Thr Leu Ile Glu Arg Tyr Glu 530 535 54021746DNALavandula
angustifolianative lavender cultivar Lady beta- phellandrene
synthase (La-beta-PHLS) cDNACDS(1)...(1746)native lavender cultivar
Lady beta- phellandrene synthase (La-beta-PHLS) 2atgtctacca
ttattgcgat acaagtgttg cttcctattc caactactaa aacataccct 60agtcatgact
tggagaagtc ctcttcgcgg tgtcgctcct cctccactcc tcgccctaga
120ctgtgttgct cgttgcaggt gagtgatccg atcccaacgg gccggcgatc
cggaggctac 180ccgcccgccc tatgggattt cgacactatt caatcgctca
acaccgagta taagggagag 240aggcacatga gaagggaaga agacctaatt
gggcaagtta gagagatgct ggtgcatgaa 300gtagaggatc ccactccaca
gctggagttc attgatgatt tgcataagct tggcatatct 360tgccattttg
agaatgaaat cctccaaatc ttgaaatcca tatatcttaa tcaaaactac
420aaaagggatt tgtactcaac atctctagca ttcagactcc tcagacaata
tggcttcatc 480cttccacaag aagtatttga ttgtttcaag aatgaggagg
gtacggattt caagccaagc 540ttcggccgtg atatcaaagg cttgttacaa
ttgtatgaag cttctttcct atcaagaaaa 600ggagaagaaa ctttacaact
agcaagagag tttgcaacaa agattctgca aaaagaagtt 660gatgagagag
agtttgcaac caagatggag ttcccttctc attggacggt tcaaatgccg
720aatgcaagac ctttcatcga tgcttaccgt aggaggccgg atatgaatcc
agttgtgctc 780gagctagcca tacttgatac aaatatagtt caagcacaat
ttcaagaaga actcaaagag 840acctcaaggt ggtgggagag tacaggcatt
gtccaagagc ttccatttgt gagggatagg 900attgtggaag gctacttttg
gacgattgga gtgactcaga gacgcgagca tggatacgaa 960agaatcatga
ccgcaaaggt tattgcctta gtaacatgtt tagacgacat atacgatgtt
1020tatggcacga tagaagagct tcaacttttc acaagcacaa tccaaagatg
ggatttggaa 1080tcaatgaagc aactccctac ctacatgcaa gtaagctttc
ttgcactaca caactttgta 1140accgaggtgg cttacgatac tctcaagaaa
aagggctaca actccacacc atatttaaga 1200aaaacgtggg tggatcttgt
tgaatcatat atcaaagagg caacttggta ctacaacggt 1260tataaaccta
gtatgcaaga ataccttaac aatgcatgga tatcagtcgg aagtatggct
1320atactcaacc acctcttctt ccggttcaca aacgagagaa tgcataaata
ccgcgatatg 1380aaccgtgtct cgtccaacat tgtgaggctt gctgatgata
tgggaacatc attggctgag 1440gtggagagag gggacgtgcc gaaagcaatt
caatgctaca tgaatgagac gaatgcttct 1500gaagaagaag caagagaata
tgtaagaaga gtcatacagg aagaatggga aaagttgaac 1560acagaattga
tgcgggatga tgatgatgat gatgatttta cactatccaa atattactgt
1620gaggtggttg ctaatcttac aagaatggca cagtttatat accaagatgg
atcggatggc 1680ttcggcatga aagattccaa ggttaataga ctgctaaaag
agacgttgat cgagcgctac 1740gaataa 174631648DNAArtificial
Sequencesynthetic codon-optimized variant of lavender Lavandula
angustifolia beta-phellandrine synthase (beta-PHLS) cDNA minus
chloroplast transit peptide for expression in glucose- tolerant
cyanobacterial strain Synechocystis sp. PCC 6803 or E. coli
(S-beta-PHLS, La-S-beta-PHLS)CDS(12)...(1634)synthetic
codon-optimized variant of lavender Lavandula angustifolia
beta-phellandrine synthase (beta-PHLS) minus chloroplast transit
peptide for expression in glucose- tolerant cyanobacterial strain
Synechocystis sp. PCC 6803 or E. coli (S-beta-PHLS, La-S-beta-PHLS)
3ttaattaaca tatgtgtagt ttgcaagttt ctgatcctat tcctaccgga cgccgttccg
60gtggttatcc cccggcctta tgggatttcg atactattca atccctgaat accgaatata
120agggcgaacg tcacatgcgt cgggaagaag acttaattgg tcaagttcgg
gaaatgttgg 180tgcacgaagt agaagatccc actccccagt tggaattcat
tgacgatctg cataaattgg 240gcatttcctg ccattttgaa aacgagattc
tgcaaattct caaatccatt tatctcaacc 300aaaactataa acgggacctc
tattctacca gtttagcctt ccgtctcttg cgtcaatacg 360ggtttatctt
gccgcaggaa gtttttgact gctttaaaaa cgaagaaggt acggatttta
420aacccagctt cggccgggat attaagggtc tgttacagtt gtacgaagcc
tcctttttgt 480cccggaaggg ggaagaaact ttacaactcg cccgcgaatt
tgctaccaaa atcttgcaaa 540aggaagtcga tgaacgggaa tttgctacta
aaatggaatt tcccagtcac tggaccgtac 600aaatgcctaa cgctcggcct
tttatcgatg cctatcgtcg gcgtcccgac atgaaccccg 660tggttctgga
actcgccatt ctcgatacca atatcgtgca agctcagttt caagaagaat
720tgaaggagac ctcccgttgg tgggaaagca cggggattgt tcaagaactg
ccgtttgttc 780gggaccggat tgtggaaggt tatttttgga ccattggtgt
tactcaacgc cgtgaacacg 840gttacgaacg tattatgacg gccaaagtca
tcgctttggt gacctgtttg gatgatattt 900atgacgtata tggcactatt
gaagaattgc aactcttcac ctctacgatt cagcgttggg 960atttggagtc
tatgaagcag ttaccgactt atatgcaggt aagcttcctg gccttgcaca
1020attttgtaac cgaagtggcc tatgatacgc tgaagaaaaa gggctacaac
tctaccccct 1080atttgcggaa gacttgggtg gatttggtcg aaagttacat
taaggaagcc acttggtact 1140ataatgggta caaaccctct atgcaggaat
acctcaacaa cgcctggatc tctgtgggca 1200gcatggctat tttgaatcat
ttgttttttc gctttactaa tgaacgcatg cataagtacc 1260gggacatgaa
tcgtgtatcc tctaatattg tgcggttagc cgacgatatg ggaacctctt
1320tggccgaagt tgaacgcggt gacgtgccca aagctatcca atgttacatg
aatgaaacga 1380acgcctctga ggaggaggcc cgcgaatatg tgcggcgcgt
tatccaggaa gaatgggaaa 1440aactgaacac tgaactgatg cgcgacgacg
acgatgacga tgatttcacc ttaagtaaat 1500actactgcga agtcgttgct
aacctgaccc ggatggctca gttcatttac caagatggtt 1560ccgatgggtt
tgggatgaaa gattccaaag taaatcgttt actgaaagaa acgctgattg
1620agcgctatga gtgaagatct gcggccgc 16484581PRTLavandula
angustifolialavender cultivar Lady beta-phellandrene synthase
(La-beta-PHLS)PEPTIDE(1)...(42)chloroplast transit peptide 4Met Ser
Thr Ile Ile Ala Ile Gln Val Leu Leu Pro Ile Pro Thr Thr1 5 10 15Lys
Thr Tyr Pro Ser His Asp Leu Glu Lys Ser Ser Ser Arg Cys Arg 20 25
30Ser Ser Ser Thr Pro Arg Pro Arg Leu Cys Cys Ser Leu Gln Val Ser
35 40 45Asp Pro Ile Pro Thr Gly Arg Arg Ser Gly Gly Tyr Pro Pro Ala
Leu 50 55 60Trp Asp Phe Asp Thr Ile Gln Ser Leu Asn Thr Glu Tyr Lys
Gly Glu65 70 75 80Arg His Met Arg Arg Glu Glu Asp Leu Ile Gly Gln
Val Arg Glu Met 85 90 95Leu Val His Glu Val Glu Asp Pro Thr Pro Gln
Leu Glu Phe Ile Asp 100 105 110Asp Leu His Lys Leu Gly Ile Ser Cys
His Phe Glu Asn Glu Ile Leu 115 120 125Gln Ile Leu Lys Ser Ile Tyr
Leu Asn Gln Asn Tyr Lys Arg Asp Leu 130 135 140Tyr Ser Thr Ser Leu
Ala Phe Arg Leu Leu Arg Gln Tyr Gly Phe Ile145 150 155 160Leu Pro
Gln Glu Val Phe Asp Cys Phe Lys Asn Glu Glu Gly Thr Asp 165 170
175Phe Lys Pro Ser Phe Gly Arg Asp Ile Lys Gly Leu Leu Gln Leu Tyr
180 185 190Glu Ala Ser Phe Leu Ser Arg Lys Gly Glu Glu Thr Leu Gln
Leu Ala 195 200 205Arg Glu Phe Ala Thr Lys Ile Leu Gln Lys Glu Val
Asp Glu Arg Glu 210 215 220Phe Ala Thr Lys Met Glu Phe Pro Ser His
Trp Thr Val Gln Met Pro225 230 235 240Asn Ala Arg Pro Phe Ile Asp
Ala Tyr Arg Arg Arg Pro Asp Met Asn 245 250 255Pro Val Val Leu Glu
Leu Ala Ile Leu Asp Thr Asn Ile Val Gln Ala 260 265 270Gln Phe Gln
Glu Glu Leu Lys Glu Thr Ser Arg Trp Trp Glu Ser Thr 275 280 285Gly
Ile Val Gln Glu Leu Pro Phe Val Arg Asp Arg Ile Val Glu Gly 290 295
300Tyr Phe Trp Thr Ile Gly Val Thr Gln Arg Arg Glu His Gly Tyr
Glu305 310 315 320Arg Ile Met Thr Ala Lys Val Ile Ala Leu Val Thr
Cys Leu Asp Asp 325 330 335Ile Tyr Asp Val Tyr Gly Thr Ile Glu Glu
Leu Gln Leu Phe Thr Ser 340 345 350Thr Ile Gln Arg Trp Asp Leu Glu
Ser Met Lys Gln Leu Pro Thr Tyr 355 360 365Met Gln Val Ser Phe Leu
Ala Leu His Asn Phe Val Thr Glu Val Ala 370 375 380Tyr Asp Thr Leu
Lys Lys Lys Gly Tyr Asn Ser Thr Pro Tyr Leu Arg385 390 395 400Lys
Thr Trp Val Asp Leu Val Glu Ser Tyr Ile Lys Glu Ala Thr Trp 405 410
415Tyr Tyr Asn Gly Tyr Lys Pro Ser Met Gln Glu Tyr Leu Asn Asn Ala
420 425 430Trp Ile Ser Val Gly Ser Met Ala Ile Leu Asn His Leu Phe
Phe Arg 435 440 445Phe Thr Asn Glu Arg Met His Lys Tyr Arg Asp Met
Asn Arg Val Ser 450 455 460Ser Asn Ile Val Arg Leu Ala Asp Asp Met
Gly Thr Ser Leu Ala Glu465 470 475 480Val Glu Arg Gly Asp Val Pro
Lys Ala Ile Gln Cys Tyr Met Asn Glu 485 490 495Thr Asn Ala Ser Glu
Glu Glu Ala Arg Glu Tyr Val Arg Arg Val Ile 500 505 510Gln Glu Glu
Trp Glu Lys Leu Asn Thr Glu Leu Met Arg Asp Asp Asp 515 520 525Asp
Asp Asp Asp Phe Thr Leu Ser Lys Tyr Tyr Cys Glu Val Val Ala 530 535
540Asn Leu Thr Arg Met Ala Gln Phe Ile Tyr Gln Asp Gly Ser Asp
Gly545 550 555 560Phe Gly Met Lys Asp Ser Lys Val Asn Arg Leu Leu
Lys Glu Thr Leu 565 570 575Ile Glu Arg Tyr Glu 5805778PRTSolanum
lycopersicumtomato beta-phellandrene synthase (beta-PHLS) 5Met Ile
Val Gly Tyr Arg Ser Thr Ile Ile Thr Leu Ser His Pro Lys1 5 10 15Leu
Gly Asn Gly Lys Thr Ile Ser Ser Asn Ala Ile Phe Gln Arg Ser 20 25
30Cys Arg Val Arg Cys Ser His Ser Thr Thr Ser Ser Met Asn Gly Phe
35 40 45Glu Asp Ala Arg Asp Arg Ile Arg Glu Ser Phe Gly Lys Leu Glu
Leu 50 55 60Ser Pro Ser Ser Tyr Asp Thr Ala Trp Val Ala Met Val Pro
Ser Arg65 70 75 80His Ser Leu Asn Glu Pro Cys Phe Pro Gln Cys Leu
Asp Trp Ile Ile 85 90 95Glu Asn Gln Arg Glu Asp Gly Ser Trp Gly Leu
Asn Pro Thr His Pro 100 105 110Leu Leu Leu Lys Asp Ser Leu Ser Ser
Thr Leu Ala Cys Leu Leu Ala 115 120 125Leu Thr Lys Trp Arg Val Gly
Asp Glu Gln Ile Lys Arg Gly Leu Gly 130 135 140Phe Ile Glu Thr Tyr
Gly Trp Ala Val Asp Asn Lys Asp Gln Ile Ser145 150 155 160Pro Leu
Gly Phe Glu Val Ile Phe Ser Ser Met Ile Lys Ser Ala Glu 165 170
175Lys Leu Asp Leu Asn Leu Pro Leu Asn Leu His Leu Val Asn Leu Val
180 185 190Lys Cys Lys Arg Asp Ser Thr Ile Lys Arg Asn Val Glu Tyr
Met Gly 195 200 205Glu Gly Val Gly Glu Leu Cys Asp Trp Lys Glu Met
Ile Lys Leu His 210 215 220Gln Arg Gln Asn Gly Ser Leu Phe Asp Ser
Pro Ala Thr Thr Ala Ala225 230 235 240Ala Leu Ile Tyr His Gln His
Asp Gln Lys Cys Tyr Gln Tyr Leu Asn 245 250 255Ser Ile Phe Gln Gln
His Lys Asn Trp Val Pro Thr Met Tyr Pro Thr 260 265 270Lys Val His
Ser Leu Leu Cys Leu Val Asp Thr Leu Gln Asn Leu Gly 275 280 285Val
His Arg His Phe Lys Ser Glu Ile Lys Lys Ala Leu Asp Glu Ile 290 295
300Tyr Arg Leu Trp Gln Gln Lys Asn Glu Gln Ile Phe Ser Asn Val
Thr305 310 315 320His Cys Ala Met Ala Phe Arg Leu Leu Arg Met Ser
Tyr Tyr Asp Val 325 330 335Ser Ser Asp Glu Leu Ala Glu Phe Val Asp
Glu Glu His Phe Phe Ala 340 345 350Thr Asn Gly Lys Tyr Lys Ser His
Val Glu Ile Leu Glu Leu His Lys 355 360 365Ala Ser Gln Leu Ala Ile
Asp His Glu Lys Asp Asp Ile Leu Asp Lys 370 375 380Ile Asn Asn Trp
Thr Arg Ala Phe Met Glu Gln Lys Leu Leu Asn Asn385 390 395 400Gly
Phe Ile Asp Arg Met Ser Lys Lys Glu Val Glu Leu Ala Leu Arg 405 410
415Lys Phe Tyr Thr Thr Ser His Leu Ala Glu Asn Arg Arg Tyr Ile Lys
420 425 430Ser Tyr Glu Glu Asn Asn Phe Lys Ile Leu Lys Ala Ala Tyr
Arg Ser 435 440 445Pro Asn Ile Asn Asn Lys Asp Leu Leu Ala Phe Ser
Ile His Asp Phe 450 455 460Glu Leu Cys Gln Ala Gln His Arg Glu Glu
Leu Gln Gln Leu Lys Arg465 470 475 480Trp Phe Glu Asp Tyr Arg Leu
Asp Gln Leu Gly Leu Ala Glu Arg Tyr 485 490 495Ile His Ala Ser Tyr
Leu Phe Gly Val Thr Val Ile Pro Glu Pro Glu 500 505 510Leu Ser Asp
Ala Arg Leu Met Tyr Ala Lys Tyr Val Met Leu Leu Thr 515 520 525Ile
Val Asp Asp His Phe Glu Ser Phe Ala Ser Lys Asp Glu Cys Phe 530 535
540Asn Ile Ile Glu Leu Val Glu Arg Trp Asp Asp Tyr Ala Ser Val
Gly545 550 555 560Tyr Lys Ser Glu Lys Val Lys Val Phe Phe Ser Val
Phe Tyr Lys Ser 565 570 575Ile Glu Glu Leu Ala Thr Ile Ala Glu Ile
Lys Gln Gly Arg Ser Val 580 585 590Lys Asn His Leu Ile Asn Leu Trp
Leu Glu Leu Met Lys Leu Met Leu 595 600 605Met Glu Arg Val Glu Trp
Cys Ser Gly Lys Thr Ile
Pro Ser Ile Glu 610 615 620Glu Tyr Leu Tyr Val Thr Ser Ile Thr Phe
Cys Ala Lys Leu Ile Pro625 630 635 640Leu Ser Thr Gln Tyr Phe Leu
Gly Ile Lys Ile Ser Lys Asp Leu Leu 645 650 655Glu Ser Asp Glu Ile
Cys Gly Leu Trp Asn Cys Ser Gly Arg Val Met 660 665 670Arg Ile Leu
Asn Asp Leu Gln Asp Ser Lys Arg Glu Gln Lys Glu Val 675 680 685Ser
Ile Asn Leu Val Thr Leu Leu Met Lys Ser Met Ser Glu Glu Glu 690 695
700Ala Ile Met Lys Ile Lys Glu Ile Leu Glu Met Asn Arg Arg Glu
Leu705 710 715 720Leu Lys Met Val Leu Val Gln Lys Lys Gly Ser Gln
Leu Pro Gln Leu 725 730 735Cys Lys Asp Ile Phe Trp Arg Thr Ser Lys
Trp Ala His Phe Thr Tyr 740 745 750Ser Gln Thr Asp Gly Tyr Arg Ile
Ala Glu Glu Met Lys Asn His Ile 755 760 765Asp Glu Val Phe Tyr Lys
Pro Leu Asn His 770 7756630PRTAbies grandisgrand fir
beta-phellandrene synthase (beta- PHLS) 6Met Ala Leu Val Ser Ser
Ala Pro Lys Ser Cys Leu His Lys Ser Leu1 5 10 15Ile Arg Ser Thr His
His Glu Leu Lys Pro Leu Arg Arg Thr Ile Pro 20 25 30Thr Leu Gly Met
Cys Arg Arg Gly Lys Ser Phe Thr Pro Ser Val Ser 35 40 45Met Ser Leu
Thr Thr Ala Val Ser Asp Asp Gly Leu Gln Arg Arg Ile 50 55 60Gly Asp
Tyr His Ser Asn Leu Trp Asp Asp Asp Phe Ile Gln Ser Leu65 70 75
80Ser Thr Pro Tyr Gly Glu Pro Ser Tyr Arg Glu Arg Ala Glu Lys Leu
85 90 95Ile Gly Glu Val Lys Glu Met Phe Asn Ser Met Pro Ser Glu Asp
Gly 100 105 110Glu Ser Met Ser Pro Leu Asn Asp Leu Ile Glu Arg Leu
Trp Met Val 115 120 125Asp Ser Val Glu Arg Leu Gly Ile Asp Arg His
Phe Lys Lys Glu Ile 130 135 140Lys Ser Ala Leu Asp Tyr Val Tyr Ser
Tyr Trp Asn Glu Lys Gly Ile145 150 155 160Gly Cys Gly Arg Asp Ser
Val Phe Pro Asp Val Asn Ser Thr Ala Ser 165 170 175Gly Phe Arg Thr
Leu Arg Leu His Gly Tyr Ser Val Ser Ser Glu Val 180 185 190Leu Lys
Val Phe Gln Asp Gln Asn Gly Gln Phe Ala Phe Ser Pro Ser 195 200
205Thr Lys Glu Arg Asp Ile Arg Thr Val Leu Asn Leu Tyr Arg Ala Ser
210 215 220Phe Ile Ala Phe Pro Gly Glu Lys Val Met Glu Glu Ala Glu
Ile Phe225 230 235 240Ser Ser Arg Tyr Leu Lys Glu Ala Val Gln Lys
Ile Pro Val Ser Ser 245 250 255Leu Ser Gln Glu Ile Asp Tyr Thr Leu
Glu Tyr Gly Trp His Thr Asn 260 265 270Met Pro Arg Leu Glu Thr Arg
Asn Tyr Leu Asp Val Phe Gly His Pro 275 280 285Thr Ser Pro Trp Leu
Lys Lys Lys Arg Thr Gln Tyr Leu Asp Ser Glu 290 295 300Lys Leu Leu
Glu Leu Ala Lys Leu Glu Phe Asn Ile Phe His Ser Leu305 310 315
320Gln Gln Lys Glu Leu Gln Tyr Leu Ser Arg Trp Trp Ile His Ser Gly
325 330 335Leu Pro Glu Leu Thr Phe Gly Arg His Arg His Val Glu Tyr
Tyr Thr 340 345 350Leu Ser Ser Cys Ile Ala Thr Glu Pro Lys His Ser
Ala Phe Arg Leu 355 360 365Gly Phe Ala Lys Thr Cys His Leu Ile Thr
Val Leu Asp Asp Ile Tyr 370 375 380Asp Thr Phe Gly Thr Met Asp Glu
Ile Glu Leu Phe Asn Glu Ala Val385 390 395 400Arg Arg Trp Asn Pro
Ser Glu Lys Glu Arg Leu Pro Glu Tyr Met Lys 405 410 415Glu Ile Tyr
Met Ala Leu Tyr Glu Ala Leu Thr Asp Met Ala Arg Glu 420 425 430Ala
Glu Lys Thr Gln Gly Arg Asp Thr Leu Asn Tyr Ala Arg Lys Ala 435 440
445Trp Glu Val Tyr Leu Asp Ser Tyr Thr Gln Glu Ala Lys Trp Ile Ala
450 455 460Ser Gly Tyr Leu Pro Thr Phe Glu Glu Tyr Leu Glu Asn Ala
Lys Val465 470 475 480Ser Ser Gly His Arg Ala Ala Ala Leu Thr Pro
Leu Leu Thr Leu Asp 485 490 495Val Pro Leu Pro Asp Asp Val Leu Lys
Gly Ile Asp Phe Pro Ser Arg 500 505 510Phe Asn Asp Leu Ala Ser Ser
Phe Leu Arg Leu Arg Gly Asp Thr Arg 515 520 525Cys Tyr Lys Ala Asp
Arg Asp Arg Gly Glu Glu Ala Ser Ser Ile Ser 530 535 540Cys Tyr Met
Lys Asp Asn Pro Gly Leu Thr Glu Glu Asp Ala Leu Asn545 550 555
560His Ile Asn Ala Met Ile Asn Asp Ile Ile Lys Glu Leu Asn Trp Glu
565 570 575Leu Leu Lys Pro Asp Ser Asn Ile Pro Met Thr Ala Arg Lys
His Ala 580 585 590Tyr Glu Ile Thr Arg Ala Phe His Gln Leu Tyr Lys
Tyr Arg Asp Gly 595 600 605Phe Ser Val Ala Thr Gln Glu Thr Lys Ser
Leu Val Arg Arg Thr Val 610 615 620Leu Glu Pro Val Pro Leu625
6307623PRTPicea sitchensisspruce beta-phellandrene synthase
(beta-PHLS, PsTPS-Phel-2, P.sitchensis2) 7Met Ala Ile Val Ser Ser
Val Pro Leu Ala Ser Lys Ser Cys Leu His1 5 10 15Lys Ser Leu Ile Ser
Ser Ile His Lys Leu Lys Pro Phe Cys Arg Thr 20 25 30Ile Pro Thr Leu
Gly Met Ser Arg Pro Gly Lys Tyr Val Met Pro Ser 35 40 45Met Ser Met
Ser Ser Pro Val Ser Asp Asp Gly Val Gln Arg Arg Thr 50 55 60Gly Gly
Tyr His Ser Asn Leu Trp Asn Asp Asp Ile Ile Gln Phe Leu65 70 75
80Ser Thr Thr Tyr Gly Glu Pro Ala Tyr Arg Glu Arg Gly Glu Arg Leu
85 90 95Ile Asp Glu Val Lys Asn Met Phe Asn Ser Ile Ser Met Glu Asp
Val 100 105 110Glu Phe Ser Pro Leu Asn Asp Leu Ile Gln Arg Leu Trp
Ile Val Asp 115 120 125Ser Val Glu Arg Leu Gly Ile Asp Arg His Phe
Lys Asn Glu Ile Lys 130 135 140Ser Thr Leu Asp Tyr Val Tyr Ser Tyr
Trp Thr Gln Lys Gly Ile Gly145 150 155 160Cys Gly Ile Glu Ser Val
Val Pro Asp Leu Asn Ser Thr Ala Leu Gly 165 170 175Leu Arg Thr Leu
Arg Leu His Gly Tyr Pro Val Ser Ala Glu Val Leu 180 185 190Lys His
Phe Gln Asn Gln Asn Gly Gln Phe Ala Cys Ser Pro Ser Glu 195 200
205Thr Glu Gly Glu Met Arg Ser Ile Val Asn Leu Tyr Arg Ala Ser Leu
210 215 220Ile Ala Phe Pro Gly Glu Lys Val Met Glu Glu Ala Glu Ile
Phe Ser225 230 235 240Thr Lys Tyr Leu Lys Glu Ala Leu Gln Lys Ile
Pro Val Ser Ser Leu 245 250 255Ser Arg Glu Ile Gly Asp Val Leu Glu
Gln Asp Trp His Thr Asn Leu 260 265 270Pro Arg Leu Glu Ala Arg Asn
Tyr Ile Asp Val Phe Gly Gln Asp Thr 275 280 285Lys Asp Thr Lys Leu
Tyr Met Lys Thr Glu Lys Leu Leu Glu Leu Ala 290 295 300Lys Leu Glu
Phe Asn Ile Phe Gln Ser Leu Gln Lys Thr Glu Leu Asp305 310 315
320Ser Leu Leu Arg Trp Trp Lys Asp Ser Gly Phe His His Ile Thr Phe
325 330 335Ser Arg His Leu His Val Glu Tyr Tyr Thr Leu Ala Ser Cys
Ile Ala 340 345 350Ile Glu Pro Gln His Ser Arg Phe Arg Leu Gly Phe
Ala Lys Ala Cys 355 360 365His Val Ile Thr Ile Leu Asp Asp Met Tyr
Asp Val Phe Gly Thr Ile 370 375 380Asp Glu Leu Glu Leu Phe Thr Ala
Gln Ile Lys Arg Trp Asp Pro Ser385 390 395 400Ala Thr Asp Cys Leu
Pro Lys Tyr Met Lys Arg Met Tyr Met Ile Leu 405 410 415Tyr Asp Met
Val Asn Glu Met Ser Arg Glu Ala Glu Thr Ala Gln Gly 420 425 430Arg
Asp Thr Leu Asn Tyr Ala Arg Gln Ala Trp Glu Asp Phe Ile Asp 435 440
445Ser Tyr Met Gln Glu Ala Lys Trp Ile Ala Thr Gly Tyr Leu Pro Thr
450 455 460Phe Asp Glu Tyr Phe Glu Asn Gly Lys Val Ser Ser Gly His
Arg Val465 470 475 480Ala Ala Leu Gln Pro Ile Leu Thr Met Asp Ile
Pro Phe Pro His Asp 485 490 495Ile Leu Lys Glu Val Asp Phe Pro Ser
Lys Leu Asn Asp Leu Ala Ser 500 505 510Ala Ile Leu Arg Leu Arg Gly
Asp Thr Arg Cys Tyr Lys Ala Asp Arg 515 520 525Ala Arg Gly Glu Glu
Ala Ser Cys Ile Ser Cys Tyr Met Lys Asp Asn 530 535 540Pro Gly Ala
Thr Glu Glu Asp Ala Leu Ser His Ile Asn Ala Val Ile545 550 555
560Ser Asp Val Ile Lys Gly Leu Asn Trp Glu Leu Leu Asn Pro Asn Ser
565 570 575Ser Val Pro Ile Ser Ser Lys Lys His Val Phe Asp Val Ser
Arg Ala 580 585 590Leu His Tyr Gly Tyr Lys Tyr Arg Asp Gly Tyr Ser
Val Ser Asn Ile 595 600 605Glu Thr Lys Ser Leu Val Met Arg Thr Leu
Leu Glu Ser Val Pro 610 615 6208624PRTPicea sitchensisspruce
beta-phellandrene synthase (beta-PHLS, PsTPS-Phel-3, P.sitchensis3)
8Met Ala Ile Val Ser Ser Val Pro Leu Ala Ser Lys Ser Cys Leu His1 5
10 15Lys Ser Leu Ile Ser Ser Ile His Lys Leu Lys Pro Phe Cys Arg
Thr 20 25 30Ile Pro Thr Leu Gly Met Ser Arg Pro Gly Lys Tyr Val Met
Pro Ser 35 40 45Met Ser Met Ser Ser Pro Val Ser Asp Asp Gly Val Gln
Arg Arg Thr 50 55 60Gly Gly Tyr His Ser Asn Leu Trp Asn Asp Asp Ile
Ile Gln Phe Leu65 70 75 80Ser Thr Pro Tyr Gly Glu Pro Ala Tyr Arg
Glu Arg Gly Glu Arg Leu 85 90 95Ile Asp Glu Val Lys Asn Met Phe Asn
Ser Ile Ser Met Glu Asp Val 100 105 110Glu Phe Ser Pro Leu Asn Asp
Leu Ile Gln Arg Leu Trp Ile Val Asp 115 120 125Ser Val Glu Arg Leu
Gly Ile Asp Arg His Phe Lys Asn Glu Ile Lys 130 135 140Ser Thr Leu
Asp Tyr Val Tyr Ser Tyr Trp Thr Gln Lys Gly Ile Gly145 150 155
160Cys Gly Ile Glu Ser Val Asp Pro Asp Leu Asn Ser Thr Ala Leu Gly
165 170 175Leu Arg Thr Leu Arg Leu His Gly Tyr Pro Val Ser Ala Glu
Val Leu 180 185 190Lys His Phe Gln Asn Gln Asn Gly Gln Phe Ala Cys
Ser Pro Ser Glu 195 200 205Thr Glu Gly Glu Met Arg Ser Ile Val Asn
Leu Tyr Arg Ala Ser Leu 210 215 220Ile Ala Phe Pro Gly Glu Lys Val
Met Glu Glu Ala Glu Ile Phe Ser225 230 235 240Thr Lys Tyr Leu Lys
Glu Ala Leu Gln Lys Ile Pro Val Ser Ser Leu 245 250 255Ser Arg Glu
Ile Gly Asp Val Leu Glu Gln Asp Trp His Thr Asn Leu 260 265 270Pro
Arg Leu Glu Ala Arg Asn Tyr Ile Asp Val Phe Gly Gln Asp Thr 275 280
285Lys Asp Thr Lys Leu Tyr Met Lys Thr Glu Lys Leu Leu Glu Leu Ala
290 295 300Lys Leu Glu Phe Asn Ile Phe Gln Ser Leu Gln Lys Thr Glu
Leu Asp305 310 315 320Ser Leu Leu Arg Trp Trp Lys Asp Ser Gly Phe
His His Ile Thr Phe 325 330 335Ser Arg His Leu His Val Glu Tyr Tyr
Thr Leu Ala Ser Cys Ile Ala 340 345 350Ile Glu Pro Gln His Ser Arg
Phe Arg Leu Gly Phe Ala Lys Ala Cys 355 360 365His Val Ile Thr Ile
Leu Asp Asp Met Tyr Asp Val Phe Gly Thr Ile 370 375 380Asp Glu Leu
Glu Leu Phe Thr Ala Gln Ile Lys Arg Trp Asp Pro Ser385 390 395
400Ala Thr Asp Cys Leu Pro Lys Tyr Met Lys Arg Met Tyr Met Ile Leu
405 410 415Tyr Asp Met Val Asn Glu Met Ser Arg Glu Ala Glu Thr Ala
Gln Gly 420 425 430Arg Asp Thr Leu Asn Tyr Ala Arg Gln Ala Trp Glu
Asp Phe Ile Asp 435 440 445Ser Tyr Met Gln Glu Ala Lys Trp Ile Ala
Thr Gly Tyr Leu Pro Thr 450 455 460Phe Asp Glu Tyr Phe Glu Asn Gly
Lys Val Ser Ser Gly His Arg Val465 470 475 480Ala Ala Leu Gln Pro
Ile Leu Thr Met Asp Ile Pro Phe Pro His Asp 485 490 495Ile Leu Lys
Glu Val Asp Phe Pro Ser Lys Leu Asn Asp Leu Ala Ser 500 505 510Ala
Ile Leu Arg Leu Arg Gly Asp Thr Arg Cys Tyr Lys Ala Asp Arg 515 520
525Ala Arg Gly Glu Glu Ala Ser Cys Ile Ser Cys Tyr Met Lys Asp Asn
530 535 540Pro Gly Ala Thr Glu Glu Asp Ala Leu Ser His Ile Asn Ala
Val Ile545 550 555 560Ser Asp Val Ile Lys Gly Leu Asn Trp Glu Leu
Leu Asn Pro Asn Ser 565 570 575Ser Val Pro Ile Ser Ser Lys Lys His
Val Phe Asp Val Ser Arg Ala 580 585 590Leu His Tyr Gly Tyr Lys Tyr
Arg Asp Gly Tyr Ser Val Ser Asn Ile 595 600 605Glu Thr Lys Ser Leu
Val Met Arg Thr Leu Leu Glu Ser Val Pro Phe 610 615 6209624PRTPicea
sitchensisspruce beta-phellandrene synthase (beta-PHLS,
PsTPS-Phel-1, P.sitchensis1) 9Met Ala Ile Val Ser Ser Val Pro Leu
Ala Ser Lys Ser Cys Leu His1 5 10 15Lys Ser Leu Ile Ser Ser Ile His
Lys Leu Lys Pro Phe Cys Arg Thr 20 25 30Ile Pro Thr Leu Gly Met Ser
Arg Pro Gly Lys Tyr Val Met Pro Ser 35 40 45Met Ser Met Ser Ser Pro
Val Ser Asp Asp Gly Val Gln Arg Arg Thr 50 55 60Gly Gly Tyr His Ser
Asn Leu Trp Asn Asp Asp Ile Ile Gln Phe Leu65 70 75 80Ser Thr Pro
Tyr Gly Glu Pro Ala Tyr Arg Glu Arg Gly Glu Arg Leu 85 90 95Ile Asp
Glu Val Lys Asn Met Phe Asn Ser Ile Ser Met Glu Asp Val 100 105
110Glu Phe Ser Pro Leu Asn Asp Leu Ile Gln Arg Leu Trp Ile Val Asp
115 120 125Ser Val Glu Arg Leu Gly Ile Asp Arg His Phe Lys Asn Glu
Ile Lys 130 135 140Ser Thr Leu Asp Tyr Val Tyr Ser Tyr Trp Thr Gln
Lys Gly Ile Gly145 150 155 160Cys Gly Ile Glu Ser Val Val Pro Asp
Leu Asn Ser Thr Ala Leu Gly 165 170 175Leu Arg Thr Leu Arg Leu His
Gly Tyr Pro Val Ser Ala Glu Val Leu 180 185 190Lys His Phe Gln Asn
Gln Asn Gly Gln Phe Ala Cys Ser Pro Ser Glu 195 200 205Thr Glu Gly
Glu Met Arg Ser Ile Val Asn Leu Tyr Arg Ala Ser Leu 210 215 220Ile
Ala Phe Pro Gly Glu Lys Val Met Glu Glu Ala Glu Ile Phe Ser225 230
235 240Thr Lys Tyr Leu Lys Glu Ala Leu Gln Lys Ile Pro Val Ser Ser
Leu 245 250 255Ser Arg Glu Ile Gly Asp Val Leu Glu Gln Asp Trp His
Thr Asn Leu 260 265 270Pro Arg Leu Glu Ala Arg Asn Tyr Ile Asp Val
Phe Gly Gln Asp Thr 275 280 285Lys Asp Thr Lys Leu Tyr Met Lys Thr
Glu Lys Leu Leu Glu Leu Ala 290 295 300Lys Leu Glu Phe Asn Ile Phe
Gln Ser Leu Gln Lys Thr Glu Leu Asp305 310 315 320Ser Leu Leu Arg
Trp Trp Lys Asp Ser Gly Phe Pro His Ile Thr Phe 325 330 335Ser Arg
His Leu His Val Glu Tyr Tyr Thr Leu Ala Ser Cys Ile Ala 340 345
350Phe Glu Pro Gln His Ser Arg Phe Arg Leu Gly Phe Ala Lys Ala Cys
355 360 365His Val Ile Thr Ile Leu Asp Asp Met Tyr Asp Val Phe Gly
Thr Ile 370 375 380Asp Glu Leu Glu Leu
Phe Thr Ala Gln Ile Lys Arg Trp Asp Pro Ser385 390 395 400Ala Thr
Asp Cys Leu Pro Lys Tyr Met Lys Arg Met Tyr Met Ile Leu 405 410
415Tyr Asp Met Val Asn Glu Met Ser Arg Glu Ala Glu Thr Ala Gln Gly
420 425 430Arg Asp Thr Leu Asn Tyr Ala Arg Gln Ala Trp Glu Asp Phe
Ile Asp 435 440 445Ser Tyr Met Gln Glu Ala Lys Trp Ile Ala Thr Gly
Tyr Leu Pro Thr 450 455 460Phe Asp Glu Tyr Phe Glu Asn Gly Lys Val
Ser Ser Gly His Arg Val465 470 475 480Ala Ala Leu Gln Pro Ile Leu
Thr Met Asp Ile Pro Phe Pro His Asp 485 490 495Ile Leu Lys Glu Val
Asp Phe Pro Ser Lys Leu Asn Asp Leu Ala Ser 500 505 510Ala Ile Leu
Arg Leu Arg Gly Asp Thr Arg Cys Tyr Lys Ala Asp Arg 515 520 525Ala
Arg Gly Glu Glu Ala Ser Cys Ile Ser Cys Tyr Met Lys Asp Asn 530 535
540Pro Gly Ala Thr Glu Glu Asp Ala Leu Ser His Ile Asn Ala Val
Ile545 550 555 560Ser Asp Val Ile Lys Gly Leu Asn Trp Glu Leu Leu
Asn Pro Asn Ser 565 570 575Ser Val Pro Ile Ser Ser Lys Lys His Val
Phe Asp Val Ser Arg Ala 580 585 590Leu His Tyr Gly Tyr Lys Tyr Arg
Asp Gly Tyr Ser Val Ser Asn Ile 595 600 605Glu Thr Lys Ser Leu Val
Met Arg Thr Leu Leu Glu Ser Val Pro Phe 610 615 62010624PRTPicea
sitchensisspruce beta-phellandrene synthase (beta-PHLS,
PsTPS-Phel-4, P.sitchensis4) 10Met Ala Ile Val Ser Ser Val Pro Leu
Ala Ser Lys Ser Cys Leu His1 5 10 15Lys Ser Leu Ile Ser Ser Ile His
Lys Leu Lys Pro Phe Cys Arg Thr 20 25 30Ile Pro Thr Leu Gly Met Ser
Arg Pro Gly Lys Ser Val Met Pro Ser 35 40 45Met Ser Met Ser Ser Pro
Val Ser Asp Asp Gly Val Gln Arg Arg Thr 50 55 60Gly Gly Tyr His Ser
Asn Leu Trp Asn Asp Asp Ile Ile Gln Phe Leu65 70 75 80Ser Thr Pro
Tyr Gly Glu Pro Ala Tyr Arg Glu Arg Gly Glu Arg Leu 85 90 95Ile Asp
Glu Val Lys Asn Met Phe Asn Ser Ile Ser Met Glu Asp Val 100 105
110Glu Phe Ser Pro Leu Asn Asp Leu Ile Gln Arg Leu Trp Ile Val Asp
115 120 125Ser Val Glu Arg Leu Gly Ile Asp Arg His Phe Lys Asn Glu
Ile Lys 130 135 140Ser Thr Leu Asp Tyr Val Tyr Ser Tyr Trp Thr Gln
Lys Gly Ile Gly145 150 155 160Cys Gly Ile Glu Ser Val Val Pro Asp
Leu Asn Ser Thr Ala Leu Gly 165 170 175Leu Arg Thr Leu Arg Leu His
Gly Tyr Pro Val Ser Ala Glu Val Leu 180 185 190Lys His Phe Gln Asn
Gln Asn Gly Gln Phe Ala Cys Ser Pro Ser Glu 195 200 205Thr Glu Gly
Glu Met Arg Ser Ile Val Asn Leu Tyr Arg Ala Ser Leu 210 215 220Ile
Ala Phe Pro Gly Glu Lys Val Met Glu Glu Ala Glu Ile Phe Ser225 230
235 240Thr Lys Tyr Leu Lys Glu Ala Leu Gln Lys Ile Pro Val Ser Ser
Leu 245 250 255Ser Arg Glu Ile Gly Asp Val Leu Glu Gln Asp Trp His
Thr Asn Leu 260 265 270Pro Arg Leu Glu Ala Arg Asn Tyr Ile Asp Val
Phe Gly Gln Asp Thr 275 280 285Lys Asp Thr Lys Leu Tyr Met Lys Thr
Glu Lys Leu Leu Glu Leu Ala 290 295 300Lys Leu Glu Phe Asn Ile Phe
Gln Ser Leu Gln Lys Thr Glu Leu Asp305 310 315 320Ser Leu Leu Arg
Trp Trp Lys Asp Ser Gly Phe His His Ile Thr Phe 325 330 335Ser Arg
His Leu His Val Glu Tyr Tyr Thr Leu Ala Ser Cys Ile Ala 340 345
350Phe Glu Pro Gln His Ser Arg Phe Arg Leu Gly Phe Ala Lys Ala Cys
355 360 365His Val Ile Thr Ile Leu Asp Asp Met Tyr Asp Val Phe Gly
Thr Ile 370 375 380Asp Glu Leu Glu Leu Phe Thr Ala Gln Ile Lys Arg
Trp Asp Pro Ser385 390 395 400Ala Thr Asp Cys Leu Pro Lys Tyr Met
Lys Arg Met Tyr Met Ile Leu 405 410 415Tyr Asp Met Val Asn Glu Met
Ser Arg Glu Ala Glu Thr Ala Gln Gly 420 425 430Arg Asp Thr Leu Asn
Tyr Ala Arg Gln Ala Trp Glu Asp Phe Ile Asp 435 440 445Ser Tyr Met
Gln Glu Ala Lys Trp Ile Ala Thr Gly Tyr Leu Pro Thr 450 455 460Phe
Asp Glu Tyr Phe Glu Asn Gly Lys Val Ser Ser Gly His Arg Val465 470
475 480Ala Ala Leu Gln Pro Ile Leu Thr Met Asp Ile Pro Phe Pro His
Asp 485 490 495Ile Leu Lys Glu Val Asp Phe Pro Ser Lys Leu Asn Asp
Leu Ala Ser 500 505 510Ala Ile Leu Arg Leu Arg Gly Asp Thr Arg Cys
Tyr Lys Ala Asp Arg 515 520 525Ala Arg Gly Glu Glu Ala Ser Cys Ile
Ser Cys Tyr Met Lys Asp Asn 530 535 540Pro Gly Ala Thr Glu Glu Asp
Ala Leu Ser His Ile Asn Ala Val Ile545 550 555 560Asn Asp Val Ile
Lys Gly Leu Asn Trp Glu Leu Leu Asn Pro Asn Ser 565 570 575Ser Val
Pro Ile Ser Ser Lys Lys His Val Phe Asp Val Ser Arg Ala 580 585
590Leu His Tyr Gly Tyr Lys Tyr Arg Asp Gly Tyr Ser Val Ser Asn Ile
595 600 605Glu Thr Lys Ser Leu Val Met Arg Thr Leu Leu Glu Ser Val
Pro Phe 610 615 6201110PRTArtificial Sequencesynthetic
beta-phellandrine synthase (beta- PHLS) partially conserved amino
acid sequence with role in catalysis 11Leu Gln Leu Tyr Glu Ala Ser
Phe Leu Leu1 5 101220DNAArtificial Sequencesynthetic PCR
amplification primer PHLS_F for Syn-beta-PHLS 12cctgggcggt
tctgataacg 201329DNAArtificial Sequencesynthetic PCR amplification
primer PHLS_BamHI_R for Syn-beta-PHLS 13cgcggatcct tttgacggcg
gccgcagat 291441DNAArtificial Sequencesynthetic PCR amplification
primer CamR_NotI_F for chloramphenicol resistance cassette from
plasmid pACYC184 14aaggaaaaaa gcggccgcgt tgatcggcac gtaagaggtt c
411534DNAArtificial Sequencesynthetic PCR amplification primer
CamR_BamHI_R for chloramphenicol resistance cassette from plasmid
pACYC184 15cgcggatccc caggcgttta agggcaccaa taac
341622DNAArtificial Sequencesynthetic genomic DNA PCR primer A2us_F
for PsbA2 homologous recombination 16tatcagaatc cttgcccaga tg
221720DNAArtificial Sequencesynthetic genomic DNA PCR primer A2ds_R
for PsbA2 homologous recombination 17ggtagagttg cgagggcaat
201834DNAArtificial Sequencesynthetic PCR amplification forward
primer for codon-optimized beta-PHLS gene (S-beta-PHLS)
18ggaattccat atgtgtagtt tgcaagtttc tgat 341933DNAArtificial
Sequencesynthetic PCR amplification reverse primer for
codon-optimized beta-PHLS gene (S-beta-PHLS) 19acaggatcct
cactcatagc gctcaatcag cgt 33206PRTArtificial Sequencesynthetic
6xHis tag 20His His His His His His1 5
* * * * *
References