U.S. patent application number 16/363052 was filed with the patent office on 2021-10-14 for bio-manufacturing process.
This patent application is currently assigned to Ecovative Design LLC. The applicant listed for this patent is Ecovative Design LLC. Invention is credited to Damen Schaak.
Application Number | 20210317433 16/363052 |
Document ID | / |
Family ID | 1000005866140 |
Filed Date | 2021-10-14 |
United States Patent
Application |
20210317433 |
Kind Code |
A9 |
Schaak; Damen |
October 14, 2021 |
Bio-Manufacturing Process
Abstract
The process of making a biocomposite material utilize a
bacterial species and a fungal species in an agricultural feedstock
composed of a substrate of non-nutrient discrete particles and a
nutrient material wherein the bacterial species imparts mechanical
properties to the biocomposite material and the fungal species
binds the biocomposite material. Both bacterium and fungus can be
genetically engineered to produce desired properties within the
microbial communities.
Inventors: |
Schaak; Damen; (Clifton
Park, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ecovative Design LLC |
Green Island |
NY |
US |
|
|
Assignee: |
Ecovative Design LLC
Green Island
NY
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20190322997 A1 |
October 24, 2019 |
|
|
Family ID: |
1000005866140 |
Appl. No.: |
16/363052 |
Filed: |
March 25, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62649175 |
Mar 28, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/80 20130101;
A01G 18/10 20180201; C12Y 305/01041 20130101; C12N 15/746 20130101;
C12N 11/14 20130101; C12N 9/88 20130101; A23K 40/30 20160501; A23K
10/18 20160501; C12N 11/02 20130101; C12N 15/52 20130101; C12N 9/80
20130101; C12N 1/14 20130101; C12Y 402/01001 20130101 |
International
Class: |
C12N 11/02 20060101
C12N011/02; A01G 18/10 20060101 A01G018/10; C12N 1/14 20060101
C12N001/14; C12N 11/14 20060101 C12N011/14; A23K 10/18 20060101
A23K010/18; A23K 40/30 20060101 A23K040/30; C12N 15/74 20060101
C12N015/74; C12N 15/80 20060101 C12N015/80; C12N 9/88 20060101
C12N009/88; C12N 9/80 20060101 C12N009/80; C12N 15/52 20060101
C12N015/52 |
Claims
1. A process of making a biocomposite material comprising the steps
of forming a substrate of non-nutrient discrete particles and a
nutrient material; adding a filamentous fungus to said substrate;
adding a Bacillus subtilis strain characterized in producing a
bio-film with poly-gamma-glutamic acid (PGA) to said substrate;
co-cultivating said fungus and said Bacillus subtilis strain in
said substrate and allowing said fungus to digest said nutrient
material in said substrate over a period sufficient to grow hyphae
and to allow said hyphae to form a network of interconnected
mycelia cells through and around said non-nutrient discrete
particles thereby bonding said discrete particles together to form
a self-supporting composite material.
2. A biocomposite material comprising a substrate of non-nutrient
discrete particles; a bio-film containing poly-gamma-glutamic acid
(PGA) dispersed within said substrate; and a network of
interconnected mycelia cells extending through and around said
discrete particles and bonding said discrete particles
together.
3. A process of making a biocomposite material comprising the steps
of forming a substrate of non-nutrient discrete particles and a
nutrient material; adding a filamentous fungus to said substrate;
adding a Bacillus subtilis strain characterized in producing
melanin to said substrate; co-cultivating said fungus and said
Bacillus subtilis strain in said substrate and allowing said fungus
to digest said nutrient material in said substrate over a period
sufficient to grow hyphae and to allow said hyphae to form a
network of interconnected mycelia cells through and around said
non-nutrient discrete particles thereby bonding said discrete
particles together to form a self-supporting composite
material.
4. A biocomposite material comprising a substrate of non-nutrient
discrete particles; an amount of melanin dispersed within said
substrate; and a network of interconnected mycelia cells extending
through and around said discrete particles and bonding said
discrete particles together, said biocomposite material being
characterized in being radiation hard.
5. A process of making a biocomposite material comprising the steps
of forming a substrate of non-nutrient discrete particles and a
nutrient material; adding a selected fungus to said substrate;
adding Streptomyces natalensis characterized in producing natamycin
to said substrate; co-cultivating said fungus and said Streptomyces
natalensis in said substrate and allowing said fungus to digest
said nutrient material in said substrate over a period sufficient
to grow hyphae and to allow said hyphae to form a network of
interconnected mycelia cells through and around said non-nutrient
discrete particles thereby bonding said discrete particles together
to form a self-supporting composite material while allowing said
Streptomyces natalensis to produce natamycin as a fungicide in said
self-supporting composite material.
6. A biocomposite material comprising a substrate of non-nutrient
discrete particles; an amount of natamycin dispersed within said
substrate; and a network of interconnected mycelia cells extending
through and around said discrete particles and bonding said
discrete particles together, said biocomposite material being
characterized in having fungicidal properties.
7. A biocomposite material as set forth in claim 6 wherein said
biocomposite material is resistant to Trichoderma.
8. A process of making a biocomposite material comprising the steps
of forming a substrate of non-nutrient discrete particles and a
nutrient material; adding a filamentous fungus to said substrate;
adding a Bacillus subtilis strain characterized in producing an
antifungal protein (AFP1) native to Streptomyces tendae to said
substrate; co-cultivating said fungus and said Bacillus subtilis
strain in said substrate and allowing said fungus to digest said
nutrient material in said substrate over a period sufficient to
grow hyphae and to allow said hyphae to form a network of
interconnected mycelia cells through and around said non-nutrient
discrete particles thereby bonding said discrete particles together
to form a self-supporting composite material.
9. A biocomposite material comprising a substrate of non-nutrient
discrete particles; an amount of an antifungal protein (AFP1)
native to Streptomyces tendae dispersed within said substrate; and
a network of interconnected mycelia cells extending through and
around said discrete particles and bonding said discrete particles
together, said biocomposite material being characterized in having
fungicidal properties.
10. A biocomposite material as set forth in claim 9 wherein said
biocomposite material is resistant to Trichoderma.
11. A process comprising the steps of obtaining a feedstock
including non-nutrient discrete particles, a nutrient material and
at least one native microbial species; isolating said at least one
native microbial species from said feedstock; subjecting said
isolated native microbial species to genetic processing to
transform said native microbial species into a genetically
engineered microbial species having predetermined characteristics;
and thereafter returning said genetically engineered microbial
species into said feedstock.
12. A process as set forth in claim 11 wherein said native
microbial species is one of Bacillus spp., Streptomyces alboniger,
Streptomyces natalensis, and Streptomyces tendae.
13. A process as set forth in claim 12 wherein said characteristics
are one of producing a bio-film containing poly-gamma-glutamic acid
(PGA) in said feedstock, producing melanin in said feedstock,
producing natamycin in said feedstock and producing an antifungal
protein (AFP1) native to Streptomyces tendae in said feedstock.
14. A process of making a biocomposite material comprising the
steps of forming a substrate of non-nutrient discrete particles and
a nutrient material; adding a filamentous fungus genetically
engineered to have predetermined characteristics to said substrate;
adding a bacterial species to said substrate; and co-cultivating
said fungus and said bacterial species in said substrate and
allowing said fungus to digest said nutrient material in said
substrate over a period sufficient to grow hyphae and to allow said
hyphae to form a network of interconnected mycelia cells through
and around said non-nutrient discrete particles thereby bonding
said discrete particles together to form a self-supporting
composite material.
15. A process as set forth in claim 14 wherein said filamentous
fungus is genetically engineered to express carbonic anhydrase (CA)
and said step of co-cultivating is performed in an environment
without regulation of carbon dioxide (CO.sub.2) through external
inputs.
16. A process as set forth in claim 14 wherein said filamentous
fungus is of the genus Trametes genetically engineered to
overexpress chlamydospore production.
17. A process as set forth in claim 14 wherein said filamentous
fungus is genetically engineered to overexpress a chitin
deacetylase (DCA) gene to increase material strength in the formed
self-supporting composite material.
18. A process as set forth in claim 14 wherein said filamentous
fungus is genetically engineered to overexpress the production of
hydrophobins to decrease water absorption of the formed
self-supporting composite material.
19. A process as set forth in claim 14 wherein said filamentous
fungus is of the genus Ganoderma genetically engineered to
overexpress the genes BGS1 and BGS2 that encode the two
.beta.-1,3-glucan synthases therein to increase glucans in said
cells of the formed self-supporting composite material.
Description
[0001] This is a Non-Provisional patent Application and claims the
benefit of Provisional Patent Application 62/659,175, filed Mar.
28, 2018.
[0002] This invention relates to method a bio-manufacturing
process. More particularly, this invention relates to method a
bio-manufacturing process involving the development of a
cohabitation platform incorporating reprogrammed (genetically
engineered) bacterial and fungal components in order to improve
existing processes of producing myceliated material.
[0003] As is known from U.S. Pat. No. 9,485,917, a composite
material comprised of discrete particles and a network of
interconnected mycelia cells bonding the discrete particles
together can be made by inoculating a substrate of the discrete
particles and a nutrient material with a preselected fungus. As
described, the fungus digests the nutrient material over a period
of time sufficient to grow hyphae and to allow the hyphae to form a
network of interconnected mycelia cells through and around the
discrete particles thereby bonding the discrete particles together
to form a self-supporting composite material.
[0004] It has also been known to employ a bio-manufacturing process
to make a composite material as described in U.S. Pat. No.
9,485,917 by leveraging domestic agricultural waste products, e.g.
corn stalks, and inoculating these with various fungal species. The
fungi utilize the agricultural substrate as the sole energy source,
growing new cells (mycelia) that ramify throughout the
material.
[0005] It has also been known from U.S. Pat. No. 10,125,347 to make
a composite biomaterial that employs a binding organism, such as a
filamentous fungi that produce mycelium, based on the material
physical properties required for the composite biomaterial and a
modulating organism, such as a bacteria, fungus or yeast, based on
a desired effect of the modulating organism on the binding
organism. As described in U.S. Pat. No. 10,125,347 a method is
provided for stimulating the expression of specific tissue
morphologies in filamentous fungi via interactions with competing
microorganisms.
[0006] It is an object of the invention to incorporate reprogrammed
(genetically engineered) bacterial and fungal components in a
process of producing myceliated material.
[0007] It is another object of the invention to cohabitate both
bacterial and fungal species together in a substrate of discrete
particles and a nutrient material to improve existing processes of
producing myceliated material and produce a new class of composite
materials.
[0008] Briefly, the invention provides a process of making a
biocomposite material utilizing a bacterial species and a fungal
species in an agricultural feedstock composed of a substrate of
non-nutrient discrete particles and a nutrient material wherein the
bacterial species imparts mechanical properties to the biocomposite
material and the fungal species binds the biocomposite
material.
[0009] In accordance with the invention, both bacterium and fungus
can be genetically engineered to produce desired properties within
the microbial communities. This provides the ability to tightly
regulate excreted compounds and fungal morphologies related to the
production of antimicrobials, and final mechanical properties. This
has also results in unique materials with a myriad of
applications.
Bacterium Processing
[0010] In one embodiment, the process comprises the steps of
forming a substrate of non-nutrient discrete particles and a
nutrient material (i.e. a feedstock); adding a filamentous fungus
to the substrate; adding a Bacillus subtilis strain characterized
in producing a bio-film with poly-gamma-glutamic acid (PGA) to the
substrate; and co-cultivating the fungus and the Bacillus subtilis
strain in the substrate and allowing the fungus to digest the
nutrient material in the substrate over a period of time sufficient
to grow hyphae and to allow the hyphae to form a network of
interconnected mycelia cells through and around all the
non-nutrient discrete particles thereby bonding all the discrete
particles together to form a self-supporting composite
material.
[0011] This embodiment of the process produces a self-supporting
biocomposite material comprising a substrate of non-nutrient
discrete particles; a bio-film containing poly-gamma-glutamic acid
(PGA) dispersed within the substrate; and a network of
interconnected mycelia cells extending through and around the
discrete particles and bonding the discrete particles together.
[0012] The bio-film containing poly-gamma-glutamic acid (PGA)
dispersed within the substrate enhances the mechanical properties
of the biocomposite material. For example, when feedstocks were
co-cultivated with both fungus and PGA producing Bacillus, there
was a demonstrated two-fold increase in the elastic modulus of the
final material when compared to materials cultivated with only
fungus.
[0013] In another embodiment, the process comprises the steps of
forming a substrate of non-nutrient discrete particles and a
nutrient material; adding a filamentous fungus to the substrate;
adding a Bacillus subtilis strain characterized in producing
melanin to the substrate; and co-cultivating the fungus and the
Bacillus subtilis strain in the substrate and allowing the fungus
to digest the nutrient material in the substrate over a period of
time sufficient to grow hyphae and to allow the hyphae to form a
network of interconnected mycelia cells through and around the
non-nutrient discrete particles thereby bonding the discrete
particles together to form a self-supporting composite
material.
[0014] This embodiment of the process produces a self-supporting
biocomposite material comprising a substrate of non-nutrient
discrete particles; an amount of melanin dispersed within the
substrate; and a network of interconnected mycelia cells extending
through and around the discrete particles and bonding the discrete
particles together.
[0015] The melanin dispersed within biocomposite material renders
the biocomposite material radiation hard. Melanin is a complex
molecule that is difficult to synthesize in vitro, and possesses
energy absorption properties. By co-culturing a melanin producing
bacteria within the composite material, one is able to manufacture
melanin in situ. Once the composite is imbedded with melanin, the
composite material is capable of absorbing UV and other types of
radiation the material is exposed to, thus rendering the material
radiation hard or resistant. The melanin imbedded material now has
the capability to protect itself from dangerous radiation exposure,
as well as protecting other living organisms i.e., humans, if the
composite materials are used to build shelters or barriers were
radiation is present i.e., laboratories or Mars.
[0016] In another embodiment, the process comprises the steps of
forming a substrate of non-nutrient discrete particles and a
nutrient material; adding a filamentous fungus to substrate; adding
Streptomyces natalensis characterized in being able to produce
natamycin during cultivation to the substrate; and co-cultivating
the fungus and the Streptomyces natalensis in the substrate and
allowing the fungus to digest the nutrient material in the
substrate over a period of time sufficient to grow hyphae and to
allow the hyphae to form a network of interconnected mycelia cells
through and around the non-nutrient discrete particles thereby
bonding the discrete particles together to form a self-supporting
composite material while allowing the Streptomyces natalensis to
produce natamycin in the self-supporting composite material.
[0017] This embodiment of the process produces a self-supporting
biocomposite material comprising a substrate of non-nutrient
discrete particles; an amount of natamycin dispersed within the
substrate; and a network of interconnected mycelia cells extending
through and around the discrete particles and bonding the discrete
particles together.
[0018] The natamycin dispersed within biocomposite material imparts
fungicidal properties to the biocomposite material and, in
particular, renders a biocomposite material made with a filamentous
fungus from the genus Ganoderma resistant to Trichoderma. Natamycin
primarily targets fungi from the Ascomycota phylum. Ganoderma is
from the Basidiomycota phylum.
[0019] In particular, the Streptomyces spp. is genetically
engineered to produce a fungicidal agent that prevents Trichoderma
spp. contamination when the material is bioactive i.e. during the
manufacturing process, and throughout a living materials usable
life span. This technique provides several advantages, namely, the
technique [0020] a. enables non-sterile in-field cultivation
practices through the reduction of contaminating microbes [0021] b.
reduces bio-control infrastructure and sterile processes during
manufacturing [0022] c. provides protection against contaminants
such as Trichoderma during the usable life of "living
materials".
[0023] In another embodiment, the process comprises the steps of
forming a substrate of non-nutrient discrete particles and a
nutrient material; adding a filamentous fungus to the substrate;
adding a Bacillus subtilis strain characterized in producing
Antifungal Protein (AFP1) native to Streptomyces tendae (previously
characterized by Bormann, C., Baier, D., Horr, I., Raps, C.,
Berger, J., Jung, G., & Schwarz, H. (1999, Sep. 27,
Characterization of a Novel, Antifungal, Chitin-Binding Protein
from Streptomyces tendae Tu901 That Interferes with Growth
Polarity. Journal of Bacteriology, 181(24), 7421-7429) to the
substrate; and co-cultivating the fungus and the Bacillus subtilis
strain in the substrate and allowing the fungus to digest the
nutrient material in the substrate over a period of time sufficient
to grow hyphae and to allow the hyphae to form a network of
interconnected mycelia cells through and around the non-nutrient
discrete particles thereby bonding the discrete particles together
to form a self-supporting composite material.
[0024] This embodiment of the process produces a self-supporting
biocomposite material comprising a substrate of non-nutrient
discrete particles; an amount of an antifungal protein (AFP1)
native to Streptomyces tendae dispersed within the substrate; and a
network of interconnected mycelia cells extending through and
around the discrete particles and bonding the discrete particles
together.
[0025] The antifungal protein (AFP1) dispersed within biocomposite
material impacts fungicidal properties to the biocomposite material
and, in particular, renders the biocomposite material resistant to
Trichoderma as is the case with natamycin dispersed within the
biocomposite material.
[0026] In each of the above described embodiments, the bacterial
strain (i.e. microbial species) may be obtained from various
sources. However, in accordance with the invention, the bacterial
strain is obtained from the feedstock for making a biocomposite
material. To this end, the invention provides a process of
isolating a microbial species from a feedstock; genetically
processing the microbial species and then returning the microbial
species to the feedstock.
[0027] In accordance with the invention, this process comprises the
steps of obtaining a feedstock including non-nutrient discrete
particles, a nutrient material and at least one native microbial
species; isolating the native microbial species from the feedstock;
subjecting the isolated native microbial species to genetic
processing to transform the native microbial species into a
genetically engineered microbial species having predetermined
characteristics; and thereafter returning the genetically
engineered microbial species into the feedstock.
[0028] The native microbial species in the feedstock of interest
would be one of Bacillus spp., Streptomyces alboniger, Streptomyces
natalensis, and Streptomyces tendae and the characteristics of
interest are one of producing a bio-film containing poly-gamma
glutamic acid (PGA) in the feedstock, producing melanin in the
feedstock, producing natamycin in the feedstock and producing an
antifungal protein (AFP1) native to Streptomyces tendae in the
feedstock.
[0029] Of note, B. subtilis has the ability to produce durable
endospores which allows the B. subtilis to be used in
co-inoculation. In this respect, the spore inoculum provides a
robust precursor that can be prepared in advance to material
cultivation, stored, transported, and more easily introduced into
the feedstock during manufacturing particularly during infield
deployment applications.
Fungal Processing
[0030] In accordance with the invention, the fungus for making a
biocomposite material is genetically engineered to have
predetermined characteristics.
[0031] In one embodiment, the filamentous fungus is genetically
engineered to express carbonic anhydrase (CA) and the step of
co-cultivating in the process of making a biocomposite material is
performed in an environment without regulation of carbon dioxide
(CO.sub.2) through external inputs, such as, by using incubation
chambers to regulate the carbon dioxide in the growth
environment.
[0032] In another embodiment, the filamentous fungus is of the
genus Trametes and is genetically engineered to overexpress
chlamydospore production to increase the ability of the fungus to
disperse through the growth substrate.
[0033] In another embodiment, the filamentous fungus is genetically
engineered to overexpress a chitin deacetylase (DCA) gene to
increase material strength in the formed self-supporting composite
material. Overexpressing a chitin deacetylase gene in the fungal
genome alters important structural components in the fungal cell
wall i.e., chitin and chitosan. Modulating these ratios changes the
mechanical properties of the cell wall and the final performance of
the resultant biocomposite material when cultivated with these
mutant strains.
[0034] In another embodiment, the filamentous fungus is of the
genus Ganoderma and is genetically engineered to overexpress the
production of hydrophobins to enhance the mycelium skin on the
cells of the formed self-supporting composite material.
Overexpressing hydrophobins (i.e. increasing the levels of
hydrophobins) enhances the aesthetics of the resultant biocomposite
material, produces a water-proofing skin encapsulating the
material, which prevents water from entering the composite
material, and resists swelling from humidity.
[0035] In another embodiment, the filamentous fungus is of the
genus Ganoderma and is genetically engineered to overexpress the
ortholog Ganoderma genes BGS1 and BGS2 that encode the two
.beta.-1,3-glucan synthases therein to increase glucans in the
cells of the formed self-supporting composite material.
[0036] These and other objects of the invention will become more
apparent from the following more detailed description.
DETAILED DESCRIPTION
[0037] The two current main production strains of filamentous
fungus used in manufacturing processes to make a composite material
according to U.S. Pat. No. 9,485,917, are members of the genus
Ganoderma and Trametes, respectively.
[0038] Initially, two types of ATMT vectors were constructed, one
type for use in Ganoderma, and another for use in Trametes. Each
utilized the pOSCAR plasmid backbone developed for gene deletion in
fungi. See Paz, Z., Garcia-Pedrajas, M. D., Andrews, D. L.,
Klosterman, S. J., Baeza-Montanez, L., Gold, S. E. (2011), One Step
Construction of Agrobacterium-Recombination-ready-plasmids (OSCAR),
an efficient and robust tool for ATMT based gene deletion
construction in fungi. Fungal Genet. Biol. 48(7): 677-84.
[0039] Each vector had a hygromycin-resistance cassette regulated
by the glyceraldehyde-3-phosphate dehydrogenase (GPD) controlling
sequences native to that particular fungus. GPD has been used
extensively to drive the expression of selectable markers in
filamentous fungi, and in particular mushrooms (Kim et al., 2015,
Current technologies and related issues for mushroom
transformation. Mycobiology, 43(1): 1-8).
[0040] Use was made of two Agrobacterium-based transformation
protocols for filamentous fungi found in the literature. One
procedure was adapted from a protocol described by Kemppainen and
Pardo (2011, Transformation of the mycorrhizal fungus Laccaria
bicolor by using Agrobacterium tumefaciens. Bioengineered Bugs 2:1,
38-44) for the mushroom Laccaria bicolor and another used by
Michielse et al. (2008 Agrobacterium-mediated transformation of the
filamentous fungus Aspergillus awamori. Nature Protocols 3(10):
1671-1678) for the ascomycete fungus Aspergillus awamori.
[0041] Both fungal mycelia and fungal protoplasts (cell wall-less
derivatives of mycelia) were used as the target tissue in the
initial transformation experiments. Binary vector (pOSCAR)
recombinant DNA plasmids were cloned in E. coli, and then
transformed into Agrobacterium tumefaciens strain AGL-1, which
already contained the Ti plasmid. The phenolic compound
acetosyringone (AS) was used during the pre-cultivation of A.
tumefaciens and also during co-cultivation with the fungus. Three
different ratios of bacterium to fungus were used during
co-cultivation, as well as three different temperatures (20.degree.
C., 22.5.degree. C., 25.degree. C.). After 4 days of
co-cultivation, mycelia were transferred to selection plates,
containing hygromycin at 50 .mu.M to select for fungal
transformants, and cefotaxin at 200 .mu.M to select against
Agrobacterium.
Work Flow of Agrobacterium Tumefaciens Mediated Transformation
(ATMT)
[0042] Membranes were seeded with fungal mycelium, then infected
with A. tumefaciens (AGL-1) harboring the recombinant DNA plasmid.
Membranes with co-cultured Mycelium/AGL-1 were then transferred
onto drug selection agar to remove AGL-1 and select for putative
transformants. The mycelium was then sub-cultured for further
isolation and PCR screening.
[0043] Transformation efficiencies between 15-30% were achieved
using the ATMT platform with fungal mycelium.
Bacterium Processing
[0044] Identification and assembling of plasmids, controlling
sequences, and drug marker cassettes for four primary bacterium
strains were conducted
[0045] Transformation protocols were also developed and optimized
to allow for the efficient DNA transfer of these constructs (DNA
sequences on the engineered plasmids) into each of our bacterium
strains.
[0046] Engineering toolkits were developed for wild type isolates
of Bacillus spp., Streptomyces alboniger, Streptomyces natalensis,
and Streptomyces tendae. The "toolkits" involved the procedures for
1) cultivation in the lab i.e., temperatures, culture conditions 2)
refined DNA transformation methods and 3) Gene controlling
sequences used to engineer DNA plasmids which are then transformed
into the bacteria.
[0047] Optimized transformation protocols for Bacillus spp. were
based on inducing DNA uptake by nutrient starvation to increase the
competency of recipient cells. These approaches were then tuned to
overcome the more recalcitrant nature of non-domesticated strains
through increased cell numbers and optimized competent cell
preparations and media components.
[0048] Strong constitutive promoters were also identified, and used
to drive expression in the overexpression plasmids. These technical
achievements were then successfully used to engineer the Bacillus
strains to produce melanin, PGA, antifungals, and confer drug
resistance when co-cultivated with the fungus in agricultural
feedstocks.
[0049] All three Streptomyces strains were cultivated at various
temperatures to establish optimal growth conditions, in addition to
best incubation conditions related to DNA conjugation
protocols.
Bacterium Strain Engineering and Co-Cultivation
[0050] Using the toolkits for the bacterium community, these
strains were engineered for enhanced material features by
co-cultivation in feedstocks with the fungus.
[0051] One of the goals was to develop a novel microbial community
that incorporates other soil microbes such as Streptomyces spp.,
and Bacillus spp. in farm waste inoculum. This more diversified
community allowed the introduction of more complex, and novel
properties into the resultant materials than were achieved using
fungal species alone.
Co-Inoculating PGA Producing Bacillus Strains
[0052] Poly-gamma-glutamic acid (PGA) is a polypeptide, which
consists of D-and L-glutamic acid units linked between amino and
carboxyl groups.
[0053] Due to PGA's viscous, water soluble, and biodegradable
properties, PGA has gained momentum in the fields of food science,
agriculture, and biomedical devices. PGA is the primary constituent
in the biofilm produced by some Bacillus subtilis strains.
[0054] The biofilm-producing B. subtilis strains were cohabited
within our assembled microbiome.
[0055] Co-cultivating PGA producing Bacillus strains with the
production fungus, leveraged the sticky viscus biofilm produced by
the Bacillus to enhance the grown-in place bio-resin to
significantly increase the flexure strength of the co-cultivated
material by two-fold.
Co-Inoculating Melanin Producing Bacillus Strains
[0056] A melanin producing Bacillus strain was co-cultured with the
fungus to produce a radiation hard material.
[0057] In this regard, a melanin expression pathway was engineered,
then transformed into a Bacillus strain (ECO-ISO) isolated from a
production feedstock.
[0058] The engineered Bacillus strain was co-cultivated with
pre-myceliated feedstocks to produce a novel material with light
and energy absorption properties. Bacillus was isolated from
feedstocks, engineered to produce melanin, then introduced back
into the community, and co-cultivated to add enhanced material
properties.
Streptomyces spp., as a Co-Cultivation Chassis
[0059] In this particular case, the production fungus was
co-inoculated with a strain of Streptomyces natalensis that
provides substrates with fungicidal properties.
[0060] The goal in this case was to develop a cultivation paradigm
that can utilize raw non-sterilized feedstocks. These biological
controls could, in turn, reduce or eliminate the need for
sterilization of raw agricultural substrates prior to inoculation
and provide relief for sterile controls throughout the material
growth cycle and manufacturing process.
[0061] Eliminating the need for sterilization provides significant
energy savings during manufacture. Also, these biomaterials could
be grown and produced outside of a manufacturing facility, thus
reducing infrastructure, and enabling "in field" production using
various low-quality agriculture substrates specific to the
region.
[0062] Streptomyces natalensis naturally produces low levels of
pimaricin, also known as natamycin, which is a fungicide with the
ability to bind to sterols found in fungal cell membranes, thus
making the cell wall permeable and lysing the cell. This fungicide
has greater activity against Ascomycete contaminants, versus
production fungi Ganoderma or Trametes. Natamycin's affinity for
non-basidiomycetes species make this particular antifungal an
attractive target for expression during cultivated
manufacturing.
[0063] The overexpression of the pimM protein was engineered. This
pimM protein has been characterized as a positive regulator for the
natamycin biosynthesis gene cluster. See Anton, A.,
Santos-Aberturas, J., Mendes, M. V., Guerra, S. M., Martin, J. F.,
Aparicio, J. F. (2007). PimM, a PAS domain positive regulator of
pimaricin biosynthesis in Streptomyces natalensis. Microbiology
153, 3174-3183.
[0064] By up-regulating the expression of pimM, the total yield of
natamycin produced by our Streptomyces natalensis strains was
increased. In this respect, natamycin production in wild type
Streptomyces natalensis is within the effective range of inhibition
(5 ug/mL) when cultured in vitro (liquid media). However, the
production of natamycin must be increased to produce a more potent
drug titer in co-cultivated feedstocks. Engineering a bacterium
strain with increased natamycin potency helps to reduce the
bacterial loading needed for substrate bioburden mitigation, thus
further reducing process and material cost.
[0065] A pimM overexpression construct was designed and cloned
using Streptomyces specific constitutive promoter (ermE) and a
second expression construct was designed and cloned utilizing the
native pimM promoter sequence.
[0066] The constructs were constructed using Gibson Assembly and
subcloned into DH5a, and then transformed into a DNA donor
non-methylation E. coli strain. A conjugation protocol was
optimized to transform the DNA constructs into the unique
Streptomyces strain.
[0067] Due to the complexities and diversification of Streptomyces
species, there is no universal standard protocol that works
efficiently with all Streptomyces spp. Accordingly, a spore
harvesting technique (determined spores are best DNA recipients)
was developed and optimized and a conjugation method was developed
and optimized for non-domesticated Streptomyces strains.
[0068] The conjugation protocol was implemented to transform the
pimM constructs from the E. coli donor strain into S.
natalensis.
[0069] The efficacy of inhibitory properties was tested in
feedstocks without the addition of the fungus. Feedstocks were
loaded with Trichoderma spores, a common mold contaminate.
Engineered Streptomyces was then co-cultured in these contaminated
feedstocks, and the inhibitory effects were recorded. A strong
linear inhibitory effect of Trichoderma in feedstocks was
demonstrated as a function of natamycin expression.
[0070] A Bacillus strain was engineered to express an antifungal
protein (AFP1) native to Streptomyces tendae. AFP1 is a more
attractive protein for expression in a non-native host than
natamycin based on its simple expression pathway. AFP1 is produced
by the expression of a single protein (87 amino acids). This
antifungal protein is of particular interest because of its
resistance to degradation in harsh environments. AFP1 is stable
over a pH range of 1.5-12, is highly resistant to digestion via
peptidases, and can retain 50% of the antifungal activity after 60
min heat treatments of 70-100.degree. C.
[0071] Bacillus AFP1 overexpression plasmids were designed by
cloning our Streptomyces AFP1-Mature sequence into our Bacillus
backbone p1664. The mature version of the AFP1 sequence has been
truncated (42 AA cleavage) to eliminate the need for
post-translational modifications. A strong constitutive promoter
Pveg with an optimized ribosomal binding site was cloned in front
of the AFP1 ORF to drive expression. The full expression sequence
was cloned between a 5' and 3' flanking region for the thrC locus
native to Bacillus spp. Taking advantage of homologous
recombination, we integrated our expression sequence into the thrC
gene of the Bacillus genome.
[0072] Once the plasmid was assembled using Gibson Assembly, the
plasmid was transformed into 10-Beta E. coli cells for propagation.
The plasmid was then isolated and verified by PCR, digestion, and
sanger sequencing. The sequence verified plasmid was then
transformed into two different Bacillus strains; Bacillus
subtilis_168 (B.s_168), and Bacillus subtilis_KO7 (B.s_KO7).
B.s_168 is a common lab strain that we have already been able to
co-cultivate with our fungus in feedstocks.
[0073] Putative transformants were recovered by drug selection
plating, and PCR verified.
[0074] To determine if our constructs were successfully expressing
AFP1 in Bacillus, we performed Sq-RT-PCR to test the AFP1
transcription levels in our engineered strains. Both engineered
strains had robust AFP1 transcription levels, and the
non-engineered wild type strains had no detectable expression as
expected.
[0075] To establish the effectiveness of Bacillus KO7_AFP1 as an
antifungal agent when co-cultivated in our agricultural feedstocks,
we performed experiments where we co-cultured both our Bacillus
KO7_AFP1 strain (1.times.10.sup.8 bac/1 g dry feedstock) and
Trichoderma spores in standard feedstock blends of Ecovative
Design, LLC of Green Island, N.Y., without the addition of a
production fungus. The exclusion of fungus was intentionally done
to eliminate any possible community interactions between the native
fungal defense mechanisms (i.e., the secretion of antimicrobial
compounds) which could interfere with the interpretation of the
Bacillus KO7_AFP1 inhibitory effects.
[0076] Three experimental sets were prepared for each experiment;
1) Trichoderma spores only, 2) Trichoderma spores co-cultured with
the empty vector Bacillus strain KO7, and 3) Trichoderma spores
co-cultured with Bacillus KO7_AFP1.
[0077] The Trichoderma spore load remained constant across sets,
and was orders of magnitude higher than typical ambient
contamination titers. Once feedstocks were inoculated, the material
was allowed to incubate at standard incubation parameters of
Ecovative Design, LLC.
[0078] Samples were visually inspected for Trichoderma sporulation
over the course of ten days. We observed complete Trichoderma
inhibition in the feedstocks through the first six days when
co-cultured with Bacillus KO7_AFP1, after which point, Trichoderma
began to sporulate throughout the co-cultured feedstocks at reduced
rates when compared to the Trichoderma only control sets. This six
day window of mold inhibition provides ample time to allow a
co-cultivated fungus to fully colonize the feedstocks and out
compete any bioburden molds that may reside within the feedstocks
at inoculation.
[0079] In addition to determining the inhibitory properties of
Bacillus KO7_AFP1 as it relates to bioburden (Trichoderma spp.), we
needed to assess the potential for any significant inhibition of
the production fungus when co-cultured in feedstocks with Bacillus
KO7_AFP1.
[0080] Standard Ecovative feedstock materials (blends) were
co-inoculated with production fungus (Ganoderma spp.) and cultured
Bacillus KO7 AFP1 at 1.times.10.sup.8 bac/1 g dry feedstock. The
material was cultivated using Ecovative's manufacturing guidelines,
and grown into 2''.times.6''.times.6'' testing plaques. The
co-cultivated material was then tested for mechanical properties;
ASTM D1621-16. Material co-cultured with Bacillus KO7 (chassis
control) and Bacillus KO7_APF1 (AFP1 expression) yielded no
significant detrimental effects on compressive modulus (composite
strength). The high inoculation titers of chassis bacteria did not
reduce fungal colonization even in the presence AFP1
expression.
[0081] We successfully demonstrated the construction of a Bacillus
KO7_AFP1 strain capable of expressing high titers of AFP1 protein.
We further demonstrated the ability of our engineered strain to
inhibit known bioburden molds (i.e., Trichoderma spp., and C.
sitophila) isolated in the facilities and manufacturing process of
Ecovative Design using plate based assays, and competition assays
in our production feedstock blends.
[0082] Bacillus KO7 has been successfully co-cultured with the
Ecovative production fungus on feedstocks without any negative
effects on fungal colonization and final material mechanical
properties while maintaining high viable bacterium survivability in
the microbial community. Additionally, when AFP1 is expressed in
situ by this new Bacillus chassis, we observed no detrimental
effects on fungal growth and material performance.
Fungal Strain Engineering
[0083] A fungal network was engineered to bind the feedstock
matrix.
[0084] Leveraging the co-cultivation platforms and fungal strain
engineering provides for increased growth and enhanced
proliferation of a mycelium network throughout the inoculated
feedstocks, and increased resistance to competing microbes native
to the feedstocks or contaminants encountered during scaled
cultivation.
[0085] Several fungal strains were genetically engineered to
increase performance in the bio-manufacturing process as described
in U.S. Pat. No. 9,485,917 through the increased distribution of
inoculum spores in feedstocks and the upregulation of carbonic
anhydrase (CA) to help reduce environmental cultivation
infrastructure.
[0086] The upregulation of cell surface hydrophobins, B-glucans,
and chitosan was performed to increase fungal resin properties and
enhance material performance i.e., strength, and water
resistance.
Enhanced Manufacturing Process--nrg1 and efg1 Regulation
(Chlamydospore)
[0087] Chlamydospores are produced by some fungi as a survival
strategy when exposed to harsh environmental conditions (nutrient
depletion, temperature variations). From a practical point of view,
these spores enable production of an inoculum using Trametes sp.
that is more resistant to contamination, with the increased ability
to disperse throughout the substrate providing better acquisition
of available resources.
[0088] The spore induction pathway was engineered by overexpressing
a positive transcriptional regulator egf1, and knocking out a
negative transcriptional regulator gene sequence nrg1. Both efg1
overexpression, and nrg1 knockout strains were made.
[0089] Using an alternative approach to upregulating chlamydospore
production in Trametes by overexpression of a positive regulator, a
plasmid was designed and cloned with the potential to knockout the
ortholog of nrg1,characterized as a negative transcriptional
regulator in the chlamydospore induction pathway.
[0090] The knockout strategy was designed around homologous
recombination. A hygromycin drug cassette was cloned between a 5'
and 3' flanking region of the nrg1 gene. This setup allowed
recombination between the homologous flanking regions thus flipping
the drug cassette into the nrg1 ORF effectively interrupting or
knocking out the gene.
[0091] Microscopy was performed to characterize the nrg1_KO mutant.
The nrg1_KO was grown on ME agar for three days, then sampled for
characterization on bright field microscopy to evaluate asexual
spore formation. Knocking out nrg1 significantly upregulated the
production of asexual anthrospores. Anthrospores are asexual spores
that form in monokaryon basidiomycete fungus. When compared, the
relative numbers of anthrospores between the wild type and nrg1_KO
showed a 42% increase in asexual spore formation in the mutant.
[0092] The growth rate of nrg1_KO was also measured and compared to
the wild type. Both the nrg1_KO and wild type were inoculated onto
the center of ME agar plates in triplicate, and incubated at
24.degree. C. The total area of growth was measured over a period
of five days. A significant increase was observed in the growth
rate of the nrg1_KO when compared to the wild type. Here, a more
robust, faster growing strain was observed from the deletion of
nrg1.
[0093] The macro-morphology of the nrg1_KO is also distinctly
different from the wild type. The wild type maintains uniform and
tight leading-edge growth, while the mutant displays a more
reaching non-uniform morphology. This phenotype is typically
associated with a nutritional "searching" behavior.
Enhanced Material Performance-Chitin Deacetylase (CDA)
Expression
[0094] Fungi cell walls consist in part of glycoproteins,
hydrophobins, chitin, and chitosan. The ratio of all these
constituents contributes to cell wall structure (i.e., mechanical
properties, and permeability). Chitosan is derived through the
deacetylation of nascent chitin by various chitin deacetylase (CDA)
proteins which hydrolyze the acetamido group in the
N-acetylglucosamine units of chitin, thus generating glucosamine
units and acetic acid.
[0095] A chitin deacetylase overexpression plasmid was engineered
and cloned.
[0096] The plasmid contained the CDA1 ORF cloned from Saccharomyces
cerevisiae S288c, and the expression driven by the
glyceraldehyde-3-phosphate dehydrogenase GPD promoter.
[0097] The S. cerevisiae CDA1 gene was the first chitin deacetylase
gene identified, and its function has been characterized using both
in vitro and in vivo models. Assembled constructs were transformed
into a production fungus and verified by PCR.
[0098] For each of the two mutants used, the insertion of the CDA1
expression construct was positioned at different sites within the
fungal genome. In fungal genomes, it is not uncommon for expression
levels of the same genes to be different when expressed from
different locations on the genome. Because of this, both
transformants generated from the same plasmid were screened. Each
strain was characterized for micro-morphology, growth rates,
antifungal properties, and the mechanical strength of material
generated by these strains.
[0099] Feedstocks were inoculated with each strain, and cultivated
in bags and then in plastic molds (tools) to set the geometry for
mechanical testing plaques. On visual observation, all strains
colonized the feedstocks well and in similar fashion.
[0100] Materials cultivated from the engineered CDA strains were
tested for their mechanical properties (compressive modulus) and
compared to a wild type strain. Compressive modulus is a function
of material stiffness and a standard metric used to assess material
strength.
[0101] Material cultivated with the CDA1.18-3 strain, had
significantly higher compressive modulus when compared to both wild
type and CDA1.14-2. No significant differences were observed
between CDA1.14-2 and the wild type.
[0102] Material cultivated with the CDA1.18-4 engineered strain was
significantly stronger (compressive modulus) than material grown
with the wild type strain.
Enhanced Material Performance--frt1 Regulation (Hydrophobins)
[0103] Hydrophobins are cysteine rich proteins that are anchored on
the outer surface of fungal cell walls. These proteins give the
outside surface of the cell its hydrophobic properties.
Hydrophobins are unique to fungi, and are linked to growth
morphology and cell signaling.
[0104] Two separate publicly available materials from Ecovative
Design, LLC of Green Island, N.Y., were cultivated using unique
cultivation paradigms and agricultural feedstock blends; 1)
Ecovative's standard MycoComposite.TM. mycelium bound agricultural
byproduct sold as "Protective Packaging" used as protective
packaging or molded shapes, 2) Ecovative's MycoComposite.TM. 584
structural material used for construction building material.
[0105] Each of the two materials were cultivated and processed to
either induce or reduce the amount of the mycelium skin that
encapsulates the final forms. By reducing or inducing the amount of
mycelium skin, we can quantitate the effects of the hydrophobin
layer as a function of water absorption.
[0106] For standard MycoComposite.TM. Protective Packaging
materials, environmental conditions such as temperature, CO2, and
relative humidity (RH) were tuned to drive mycelial growth to the
outer surface of the material. As such, Ecovative's structural
materials were grown in conditions which enabled optimal internal
colonization to add overall mechanical strength properties, thus
reducing the external myceliation and reducing the hydrophobin skin
on the surface of the material.
[0107] Once testing plaques were grown and processed to either
reduce or induce the quantity of mycelium skin on the surface of
the parts, they were subjected to water submersion testing (ASTM
C1134). Parts were measured (physical dimensions and weight) and
then submerged into a water tank. The plaques were held to complete
submersion for 24 hours, then removed and re-measured. The percent
of water mass absorbed was calculated and plotted for each of the
two materials with variable mycelium skins. Water absorption of the
standard Structural material was measured at 35% water mass
absorption, but when the hydrophobic skin was reduced, there was a
61% water mass absorption for the material. This is a 43% increase
in water absorption.
[0108] Standard MycoComposite.TM. Protective Packaging material was
measured at a 15% water mass absorption, but significantly
increased to 55% when the hydrophobic skin was removed
demonstrating a staggering 77% increase in water absorption.
[0109] The amount of hydrophobic mycelium skin coating the part is
proportional to the percent of water absorption. Both materials
have similar absorption performance at reduced skin formats, but
the MycoComposite.TM. Protective Packaging material performs better
than the structural material in the induced hydrophobin skin sets.
One explanation for this difference would be the paradigm which
MycoComposite.TM. Protective Packaging is grown when compared to
the Structural materials. MycoComposite.TM. Protective Packaging is
cultivated to "force" myceliation to the outer surfaces of the
material to aid in aesthetics (softer feel), and to promote
"cushioning" properties gained with reduced internal
colonization.
[0110] As for MycoComposite.TM. 584 structural materials, they are
primarily cultivated to increase internal colonization with limited
surface flush, thus enhancing mechanical properties such as
internal bond and modulus. Engineering the fungus to increase
hydrophobin production while still driving internal part
colonization enables the growth of strong structural materials
while retaining some of the critical mycelium hydrophobins on the
surface of the material to protect the grown material from the
elements.
Enhanced Material Performance--Regulation of .beta.-Glucans
[0111] Glucans are the major structural polysaccharides of the
fungal cell wall, constituting approximately 50-60% of the wall by
dry weight. These polysaccharides are of particular interest with
regards to increasing the internal bond strength of composites
through enhanced fungal resin properties. The most abundant glucan
in the fungal cell wall is .beta.-1,3-glucan, which makes up
between 65% and 90% of the whole .beta.-glucan content.
[0112] Recombinant DNA constructs were made with the goal of
over-expressing the genes (BGS1 and BGS2) that encode the two
.beta.-1,3-glucan synthases found in the Ganoderma genome. Use was
made of the controlling sequences from the constitutively-expressed
gene encoding glyceraldehyde-3-phosphate dehydrogenase (GPD) to
drive expression of BGS1 and BGS2.
[0113] These constructs were used in classical PEG-mediated
co-transformation experiments using a second plasmid containing a
resistance gene to the fungicide carboxin. Three carboxin-resistant
co-transformants were verified via PCR to have the integrated BGS
overexpression constructs: two with BGS1 (BGS1-1, 1-7), and a third
with BGS2 (BGS2-1). Assays for glucan content suggested a
significant increase in the .beta.-glucan fraction in each of the
three co-transformants (i.e. 165%, 135%, and 147%,
respectively).
[0114] The engineered Ganoderma_BGS strains had about a two-fold
increase in .beta.-1,3-glucan levels in the cell wall fractions
when compared to the unmodified wild type Gandoerma.
[0115] The invention thus provides unique techniques for
incorporating reprogrammed (genetically engineered) bacterial and
fungal components in a process of producing myceliated
material.
[0116] The invention also provides a process of cohabitating both
bacterial and fungal species together in a substrate of discrete
particles and a nutrient material to improve existing processes of
producing myceliated material and produce a new class of composite
materials.
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