Patent Application
20210207108
From plastics to pharmaceuticals, synthetic organic compounds are predominately manufactured from petrochemical sources. Plastic production alone already consumes 10% of US oil and gas each year, and the market is continuing to grow at 15% a year (Criddle et al., 2014). Use of biodegradable plastic could mitigate the consumption of fossil byproducts and reduce the persistence of waste materials in the environment, yet its production typically requires the microbial conversion of plant-based carbohydrate feedstocks that could otherwise be used as a food source.
Bioplastic production has a significant carbon footprint and is vulnerable to the economics of volatile energy and food markets (Cheali et al., 2016; Ghatak, 2011; Tegtmeier and Duffy, 2004). Genetic engineering of a single species of cyanobacteria or algae may reroute cellular resources towards overproduction of a target renewable product (Leong et al., 2014; Reddy et al., 2013; Venkata Mohan and Venkateswar Reddy, 2013). However, such a monoculture approach to bioproduction requires extensive research and development to optimize each strain and is vulnerable to economic shifts during the development and application of such strains—such as exposure to volatile petroleum markets when creating a biofuel (Cheali et al., 2016). Furthermore, monocultures of algae and cyanobacteria are vulnerable to many inefficiencies when grown in scaled cultures, including contamination by foreign microbial species (Gavrilescu and Chisti, 2005; Vickers et al., 2012).
Described herein are cyanobacteria encapsulated in hydrogels. Encapsulation of cyanobacteria can block predation by grazer microbial species, infection by cyanobacterial viruses, and slows division. Such slower cell division can reduce loss of genetically engineered traits and reduce the incidence of genetic mutation. The encapsulated cyanobacteria can be modified to express a sucrose/proton symporter.
Also described herein are consortia that include encapsulated (e.g., autotrophic) cyanobacteria and heterotrophic microbes. The encapsulated cyanobacteria can provide nutrients (e.g., carbon-based nutrients) and the heterotrophic microbes can utilize the nutrients to grow and produce useful products. In some cases, the encapsulated (e.g., autotrophic) cyanobacteria can be modified to express a sugar transporter that they do not naturally express. In addition, the heterotrophic microbes can be modified to produce products that they do not naturally produce.
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Described herein are mixtures (e.g., co-cultures) of cyanobacteria that can supply nutrients (e.g., sugar) with at least one heterotrophic microorganism modified to produce useful products, and methods for making and using such mixtures. The cyanobacteria can therefore supply the heterotrophic microorganism(s) with nutrients to support their growth and production of useful products. Also as described herein, encapsulation of the cyanobacteria improves the stability of the cyanobacteria and the productivity of the co-cultures.
Cyanobacteria, also known as blue-green algae, blue-green bacteria, or Cyanophyta, is a phylum of bacteria that obtain their energy through photosynthesis. Cyanobacteria can produce metabolites, such as carbohydrates, proteins, lipids and nucleic acids, from CO2, water, inorganic salts and light. Any cyanobacteria may be used according to the present disclosure.
Cyanobacteria include both unicellular and colonial species. Colonies may form filaments, sheets or even hollow balls. Some filamentous colonies have the ability to differentiate into several different cell types, such as vegetative cells, the normal, photosynthetic cells that are formed under favorable growing conditions; akinetes, the climate-resistant spores that may form when environmental conditions become harsh; and thick-walled heterocysts, which contain the enzyme nitrogenase, vital for nitrogen fixation.
Heterocysts may also form under the appropriate environmental conditions (e.g., anoxic) whenever nitrogen is necessary. Heterocyst-forming species are specialized for nitrogen fixation and are able to fix nitrogen gas, which cannot be used by plants, into ammonia (NH3), nitrites (NO2−), or nitrates (NO3−), which can be absorbed by plants and converted to protein and nucleic acids.
Many cyanobacteria also form motile filaments, called hormogonia, which travel away from the main biomass to bud and form new colonies elsewhere. The cells in a hormogonium are often thinner than in the vegetative state, and the cells on either end of the motile chain may be tapered. In order to break away from the parent colony, a hormogonium often must tear apart a weaker cell in a filament, called a neridium.
Each individual cyanobacterial cell typically has a thick, gelatinous cell wall. Cyanobacteria differ from other gram-negative bacteria in that the quorum sensing molecules autoinducer-2 and acyl-homoserine lactones are absent. They lack flagella, but hormogonia and some unicellular species may move about by gliding along surfaces. In water columns, some cyanobacteria float by forming gas vesicles, like in archaea.
Cyanobacteria have an elaborate and highly organized system of internal membranes that function in photosynthesis. Photosynthesis in cyanobacteria generally uses water as an electron donor and produces oxygen as a by-product, though some cyanobacteria may also use hydrogen sulfide, similar to other photosynthetic bacteria. Carbon dioxide is reduced to form carbohydrates via the Calvin cycle. In most forms, the photosynthetic machinery is embedded into folds of the cell membrane, called thylakoids.
In some cases, the amount of oxygen produced by the cyanobacteria can inhibit the growth or functioning of heterotrophic organism that is co-cultured with the cyanobacteria. As illustrated herein, the atmosphere or the culture media in which a consortium of cyanobacteria and heterotrophic cells is cultured can be modulated to reduce the inhibitory effects of such oxygen production. For example, the consortium can be sparged with low-oxygen containing gases or anti-oxidants can be added to the culture medium.
In some cases, the cultures can be sparged with a gas having less than 20% oxygen, or less than 17% oxygen, or less than 15% oxygen, or less than 12% oxygen, or less than 10% oxygen, or less than 7% oxygen, or less than 5% oxygen, or less than 3% oxygen, or less than 1% oxygen. In some cases, the cultures can be sparged with a gas that is devoid of oxygen (e.g., 12:10:82 H2:CO2:N2).
In some cases, antioxidants such as DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), thiosulfate, or a combination thereof can be used in the culture medium. Concentrations of such antioxidants can vary from about 1 μM to 1000 mM, or about 10 μM to 200 mM.
Due to their ability to fix nitrogen in aerobic conditions, cyanobacteria are often found as symbionts with a number of other groups of microorganisms such as fungi (e.g., lichens), corals, pteridophytes (e.g., Azolla), and angiosperms (e.g., Gunnera), among others.
Cyanobacteria are the only group of microorganisms that are able to reduce nitrogen and carbon in aerobic conditions. The water-oxidizing photosynthesis is accomplished by coupling the activity of photosystem (PS) II and I (Z-scheme). In anaerobic conditions, cyanobacteria are also able to use only PS I (i.e., cyclic photophosphorylation) with electron donors other than water (e.g., hydrogen sulfide, thiosulphate, or molecular hydrogen), similar to purple photosynthetic bacteria. Furthermore, cyanobacteria share an archaeal property; the ability to reduce elemental sulfur by anaerobic respiration in the dark. The cyanobacterial photosynthetic electron transport system shares the same compartment as the components of respiratory electron transport. Typically, the plasma membrane contains only components of the respiratory chain, while the thylakoid membrane hosts both respiratory and photosynthetic electron transport.
Cyanobacteria of the present disclosure may be from any genera or species of cyanobacteria that is genetically manipulable, i.e., permissible to the introduction and expression of exogenous genetic material. Examples of cyanobacteria that can be employed include, but are not limited to, the genus Synechocystis, Synechococcus, Thermosynechococcus, Nostoc, Prochlorococcu, Microcystis, Anabaena, Spirulina, and Gloeobacter. In some cases, the cyanobacteria are Synechococcus elongatus. For example, the cyanobacteria can include Synechococcus elongatus PCC 7942.
In some cases, the cyanobacteria can be engineered to express a sucrose transporter. One example of such a sucrose transporter is the CscB transporter protein. An example of a CscB protein that is useful for sucrose export is an Escherichia coli H+/sucrose symporter CscB, for example having the following sequence (SEQ ID NO:1).
A nucleotide sequence that encodes the SEQ ID NO:1 Escherichia coli CscB protein is shown below as SEQ ID NO:2.
The NCBI database provides a variety of sucrose transporters (also called sucrose symporter or sucrose permeases). For example, an Escherichia coli CscB sucrose permease that has accession number CAA45274 can be employed and has the following sequence (SEQ ID NO:3).
Another sucrose transporter can be used such as the sucrose transport protein from uncultured bacterium that has accession number CCG34807.1, provided below as SEQ ID NO:5.
The sucrose transporter with SEQ ID NO:5 has only 39% sequence identity to SEQ ID NO:1 but is still a recognized sucrose transporter. Hence, the transporter employed can exhibit sequence variability compared to the sequences described herein.
For example, the sucrose transporter employed can have at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity to a sequence described herein.
In some cases, enzymes can have conservative changes such as one or more deletions, insertions, replacements, or substitutions that have no significant effect on the activities of the sucrose transporter.
The cyanobacteria are autotrophs that can thrive when cultured in a simple culture medium and exposed to light. Moreover, the cyanobacteria can produce organic nutrients (e.g., sugars) that can support the growth and functioning of organisms grown with the cyanobacteria. Organisms that are co-cultured with the cyanobacteria are referred to as heterotrophs because they cannot manufacture its own food and instead obtain nutrients and energy by taking in organic substances from the autotrophic cyanobacteria.
As illustrated herein, S. elongatus with a cscB+ transgene supports diverse heterotrophic microbes in co-culture, demonstrating that a flexible autotroph/heterotroph consortia platform can be employed for manufacture of useful products.
As described herein, the cyanobacteria can be encapsulated within a hydrogel matrix. There are benefits to embedding cyanobacteria within a matrix that includes the cyanobacteria and one or more types of hydrogels. As described herein, encapsulation of cyanobacteria greatly enhances stability, productivity, and modularity of co-cultures with heterotrophs, while also enabling selective recovery of heterotrophic biomass from the mixed cyanobacteria-heterotroph cultures. Encapsulation of cyanobacteria minimizes cell escape while still maintaining viability of trapped cells for more than five months. The hydrogel matrix did not degrade over more than five months of culture. Furthermore, encapsulation of cyanobacteria in a restrictive matrix can block predation by grazers, infection by cyanobacterial viruses, and slows division—greatly reducing the chance of loss of engineered pathways due to genetic mutation.
Examples of hydrogel materials that can be used include alginate, latex, silica, and combinations thereof. As illustrated herein, alginate forms an excellent encapsulation material that provides the benefits described in the foregoing paragraph. Latex is also a good encapsulation material. Latex is an easy-to-use, and inexpensive resin that has been used to embed bacteria. For example, one type of latex that can be used is Rhoplex SF012. Similarly, silica materials have superior properties for industrial use (e.g., thermostability). One example of a silica material that can be used is polyol silanes93.
The encapsulation procedure can include generation of beads or sheets of a diameter that allow diffusion of gases, nutrients, and other molecules to foster the stability and productivity of the cyanobacteria and the heterotroph cells. For example, the size and highly cross-linked network of encapsulation polymers may present a physical constraint on the diffusion of small molecules and can present a barrier to large molecules, such as proteins. The most salient molecules to consider are the permeability of CO2 into the matrix, and escape of O2 and sucrose. The cell density, percent alginate slurry, crosslinking efficiency, photosynthetic rates, and mixing rates are all variables that can influence the diffusion of substrates and products within the bead, so calculation of an optimal encapsulation diameter is non-trivial.
Optimal encapsulation diameters can vary and can include encapsulation diameters of about 0.05 mm to about 4 mm, or of about 0.075 mm to about 3.5 mm, or of about 0.1 mm to about 3 mm, or of about 0.15 mm to about 3 mm, or of about 0.15 mm to about 2.75 mm, or of about 0.25 mm to about 2.75 mm, or of about 0.2 mm to about 2 mm.
To evaluate or confirm optimal encapsulation diameters, titers of encapsulated cyanobacteria can be determined and compared to a control. The control can be non-encapsulated cyanobacteria or cyanobacteria encapsulated to a specified or desired diameter.
For example, the specific productivity of secreted sucrose and chlorophyll content of encapsulated cyanobacteria beads can be measured over time (see, e.g.,
One method for encapsulation of cyanobacteria is to drip a mixture of at least one hydrogel with cyanobacteria cells into a curing solution. For example, a cyanobacteria cell/alginate suspension can be dripped into a barium chloride (BaCl) curing solution (see, e.g.,
Much higher volumes of encapsulated material can be generated with customizable bead sizes through the use of various commercially available equipment. For example, the encapsulation methods can include use of a Nisco Encapsulation Unit VAR V1, which uses electrostatically assisted spraying for rapid generation of large encapsulated bead volumes of user-specified sizes, for example, over the range of about 0.2 mm to 2 mm.
A variety of heterotrophic microorganisms can be co-cultured with the cyanobacteria. Such heterotrophic microorganisms or heterotrophs can include bacteria, fungi, algae, and combinations thereof. For example, strains of Escherichia, Bacillus, Saccharormyces, Halomnonas, Pseudomnonas, or combinations are illustrated herein as examples of heterotrophs. Specific types of heterotrophs that can be employed include, for example, Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and combinations thereof.
Some synthetic communities have been constructed by cross-feeding of organisms [see, e.g., Song et al. Front Microbiol. 6:1298 (2015); Kim et al., Proc Natl Acad Sci. 105:18188-93 (2008); Wintermute & Silver, Genes Dev. 24:2603-14 (2010); Mee et al., Proc Natl Acad Sci USA 111:E2149-56 (2014); Song et al. Chem Soc Rev Chem Soc Rev. 6954:6954-81 (2014)]. However, sucrose is a metabolite that is naturally bioavailable to many microbes, and therefore the diversity of heterotrophic species with potential to be supported by cscB+ S. elongatus is broad.
For example, bacteria can be used as heterotrophic microorganisms that can be co-cultured with the cyanobacteria. Bacteria that naturally produce useful products, or bacteria that have been engineered to produce useful products are particularly useful as heterotrophs that can be co-cultured with the cyanobacteria.
In some cases, the heterotroph can be modified to reduce the function (activity) of a sucrose catabolism repressor cscR. As illustrated herein, E. coli with a deletion of the sucrose catabolism repressor cscR exhibit superior growth. One example of a CscR protein from the E. coli W strain has the following sequence (SEQ ID NO:6).
A nucleotide sequence encoding the CscR protein with SEQ ID NO:6 from the E. coli W strain, where the nucleotide sequence includes at least part of the natural promoter in the E. coli W strain is shown below at SEQ ID NO:7.
Other types of bacteria, fungi, algae, and combinations thereof can also be heterotrophs that can be cultured with cyanobacteria.
The heterotrophs can express a variety of useful products. Examples include drugs, enzymes, nutrients, proteins, oils, carbohydrates, alcohols, fatty acids, vitamins, pigments, pharmaceutical enzymes, biotechnological enzymes, hydrogen gas, polymer substrates/monomers, polymers, biofuels, metabolites, and combinations thereof. In some cases, the heterotrophs can perform useful functions such as metabolizing undesirable compounds or materials (e.g., pollutants), sequestering useful materials or compounds, and the like.
For example, as illustrated herein B. subtilis strain 168 naturally produces and secretes alpha-amylase (
The enzymes involved in synthesis of polyhydroxybutyrate (P1B) include those encoded by phbA, phbB, and phbC genes (see, e.g., Peoples et al., J. Biol. Chem. 264 (26): 15293-15297 (1989)). These genes can be present in a variety of bacterial species such as Alcaligenes eutrolropus, Burkholderia pseudoinallei, Cupravidus necator, Ralstonia eutropha, or Zoogloea ramigera.
The phbA gene encodes a beta-thiolase that can, for example, have the following sequence (SEQ ID NO:8 from Zoogloea ranigera).
The phbB gene encodes a NADP-specific acetoacetyl-CoA reductase that can, for example, have the following sequence (SEQ ID NO:9 from Ralstonia eutropha).
The phbC gene encodes a PHB polymerase that can, for example, have the following sequence (SEQ ID NO:10 from Cupriavidus necator).
In another example, an industrial pollutant that is of increasing concern to the U.S. Environmental Protection Agency (EPA) is the compound 2,4-dinitrotolulene (2,4-DNT; EPA. Emerging Contaminants—Dinitrotoluene (DNT)—EPA.; 2014. https://www.epa.gov/sites/production/files/2014-03/documents/ffrrofactsheet-contaminant-dnt_january2014_final.pdf). A byproduct of manufacturing of polyurethane polymers, explosives, and plasticizing agents, 2,4-DNT is classified as toxic to most organisms and is increasingly detected in ground water and soils at levels above those regarded as safe. Exposure to 2,4-DNT causes damage to the central nervous system, the circulatory system, and is associated with liver damage and oncogenes (Tchounwou et al. Rev Env Heal. 18(3):203-229 (2003)). Therefore, the EPA classifies 2,4-DNT as a priority pollutant.
One effective method for the remediation of environmental 2,4-DNT is through the use of microbial strains capable of degrading 2,4-DNT and other nitrotoluene derivatives (Han et al., Chemosphere. 85(5):848-853 (2011); Wang et al., J Appl Microbiol. 110(6):1476-1484 (2011); Aburto-Medina et al., Appraising the Role of Microorganisms. In: Enhancing Cleanup of Environmental Pollutants. Springer International Publishing; 2017:5-20 (2017)).
One such strain that has natural capacities to metabolize diverse aromatic compounds is Pseudomonas putida. While P. putida naturally exhibits some capacity to degrade recalcitrant aromatic compounds, it does not degrade 2,4-DNT. However, as described herein Pseudomonas putida can be engineered to express enhanced 2,4-DNT degradation capabilities such as the enzymes DntA, DntB, DntC, DntD, DntG, DntE, or combinations thereof. Such DntA, DntB, DntC, DntD, DntG, and DntE enzymes are expressed, for example, by Burkholderia (Pdrez-Pantoja et al., PLoS Genet. 9(8) (2013)). For example, 2,4-DNT can be degraded into non-toxic products (e.g., pyruvate and propionyl-CoA) by such enzymes as illustrated by the metabolic pathway shown below.
The DntA enzyme is a DNT dioxygenase that hydroxylates the aromatic ring in positions 4 and 5 to yield 4-methyl-5-nitrocatechol, releasing, at the same time, the first nitro substituent. The substituted catechol is subsequently mono-oxygenated by the DntB enzyme, which is a hydroxylase; this step eliminates the remaining nitro group in the structure, thereby producing 2-hydroxy-5-methylquinone. The rest of the pathway (executed by DntCDGE) includes a ring cleavage reaction and channeling of the products towards the central metabolism, in which they are finally metabolized.
The DntA gene encodes a 2,4-DNT dioxygenase that can, for example, have the following sequence (SEQ ID NO:11 from Burkholderia sp. DNT).
The DntB gene encodes a hydroxylase that can, for example, have the following sequence (SEQ ID NO:12 from Burkholderia).
DntCDGE provides ring cleavage and other functions and can, for example, have the following sequence (SEQ ID NO:13 from Burkholderia sp. BC1).
Species of bacteria such as Pseudomonas putida can be used as a heterotrophic organism, particularly after modification to include expression cassettes encoding enzymes such as the DntA, DntB, DntC, DntD, DntG, and/or DntE enzymes using recombinant methods.
In addition, Pseudomonas putida can be successfully co-cultivated with S. elongatus CscB+(Lowe et al. Biotechnol Biofuels. 10(1):190 (2017)). Hence, the co-culture mixtures and methods described herein can be used for light-driven bioproduction, and also for light-catalyzed bioremediation of toxic aromatics.
Therefore, encapsulated cyanobacterial cells can secrete sucrose, while allowing the P. putida strains to grow on the secreted sucrose. P. putida can grow in co-culture with S. elongatus CscB+. Optimized media are described herein that support growth of many heterotrophic species in co-culture with S. elongatus CscB+. Engineered strains of P. putida (e.g., derivatives of strain KT2440) having, for example, genomically integrated dntABCDEG genes from Burkholderia can breakdown 2,4-DNT while providing core carbon intermediates that can be utilized for cell growth. Furthermore, these strains also express cscAB genes that improve the utilization of sucrose.
In another example, the heterotroph can express enzymes that can facilitate manufacture of indole. Indole is the parent compound for the synthesis of a number of natural products and pharmaceuticals. Natural occurring indoles with physiological roles in humans include, for example, the neurotransmitter serotonin and the hormone melatonin. Some indole-derived compounds with pharmacological significance include sumatriptan, ondnsetron, alosetron, 2-aroylindole, 2-aryl-3arylcorbonylindole, indolyl-3-glycoxamide, ellipticine, mycotoxin gliotoxin. A number of indole-containing compounds have been utilized in drug therapies for humans including Vincristine, Roxindole, Atevirdine, Proamanullin, Reserpine, Pindolol, Oglufanide. Hence, by employing the co-culture and/or the encapsulation methods described herein, products such as indole can be made more efficiently and at lower cost.
For example, as illustrated herein, E. coli that can transgenically express tryptophanase and that are co-cultured with cyanobacteria can produce indole. One example of a sequence for a tryptophanase enzyme is shown below as SEQ ID NO:14.
The tryptophanase need not have the foregoing sequence (SEQ ID NO:14). Tryptophanases with a variety of different sequences can be employed. For example, the tryptophanase can have a sequence such as SEQ ID NO:15 from Sediminispirochaeta smaragdinae.
As shown below, the tryptophanases with SEQ ID NOs: 14 and 15 share about 46% sequence identity.
The heterologous functions employed herein by the cyanobacteria and the heterotrophic organism (e.g., any of the enzymes transgenes and/or expression cassettes) can exhibit sequence variability compared to the sequences described herein. For example, any of the enzymes, transgenes and/or expression cassettes employed can have at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence identity to a sequence described herein.
In some cases, enzymes can have conservative changes such as one or more deletions, insertions, replacements, or substitutions that have no significant effect on the activities of the enzymes. Examples of conservative substitutions are provided below.
In some cases, the heterotroph can be a fungal or yeast strain such as Saccharomyces cerevisiae, Saccharomyces uvarum (also known as Saccharomyces carlsbergensis), Saccharomyces pastorianus, or other strains. Such fungal or yeast strains can be modified to take up sugars and/or carbohydrates more efficiently. For example, S. cerevisiae W303Clump (originally called Recreated02 in Koschwanez et al. 2013) which contain mutations in genes CSE2, IRA1, MTH1, and UBR1 that enhance fitness in dilute sucrose.
In constructing these consortia, unforeseen interactions were observed that were shared across different heterotrophic species. For example, light-driven processes of cyanobacteria have negative impacts on all tested heterotrophic species while, conversely, growth of all heterotrophic species simulates cyanobacterial growth. However, as illustrated herein, the negative impact was photosynthetically produced oxygen by cyanobacteria when exposed to light. Such negative impacts on heterotroph growth are controlled by a variety of processes (e.g., sparging with low-oxygen containing gases or adding anti-oxidants). By taking measures to mitigate deleterious interactions, the inventors could stabilize consortia over time. Experiments described herein demonstrate that consortia persist in the face of fluctuations in light availability, population density, and composition. These consortia can be functionalized to produce target compounds, where the end product is dictated by the heterotrophic partner.
Also described herein are expression systems that include at least one expression cassette (e.g., expression vectors or transgenes) that encode one or more of the enzyme(s) described herein. These expression systems can be present in the cyanobacteria or in the heterotroph. The expression systems can also include one or more expression cassettes encoding an enzyme that can synthesize or degrade a product.
Cells containing such expression systems are further described herein. The cells containing such expression systems can be used to manufacture the enzymes (e.g., for in vitro use) or products produced by the enzymes. Methods of using the enzymes or cells containing expression cassettes encoding such enzymes to make products, degrade products, and combinations thereof are also described herein.
Nucleic acids encoding the enzymes can have sequence modifications. For example, nucleic acid sequences described herein can be modified to express enzymes that have modifications. Most amino acids can be encoded by more than one codon. When an amino acid is encoded by more than one codon, the codons are referred to as degenerate codons. A listing of degenerate codons is provided in the table below.
Different organisms may translate different codons more or less efficiently (e.g., because they have different ratios of tRNAs) than other organisms. Hence, when some amino acids can be encoded by several codons, a nucleic acid segment can be designed to optimize the efficiency of expression of an enzyme by using codons that are preferred by an organism of interest. For example, the nucleotide coding regions of the enzymes described herein can be codon optimized for expression in various cyanobacterial or heterotroph species.
An optimized nucleic acid can have less than 98%, less than 97%, less than 95%, or less than 94%, or less than 93%, or less than 92%, or less than 91%, or less than 90%, or less than 89%, or less than 88%, or less than 85%, or less than 83%, or less than 80%, or less than 75% nucleic acid sequence identity to a corresponding non-optimized (e.g., a non-optimized parental or wild type enzyme nucleic acid) sequence.
The enzymes described herein can be expressed from an expression cassette and/or an expression vector. Such an expression cassette can include a nucleic acid segment that encodes an enzyme operably linked to a promoter to drive expression of the enzyme. Convenient vectors, or expression systems can be used to express such enzymes. In some instances, the nucleic acid segment encoding an enzyme is operably linked to a promoter and/or a transcription termination sequence. The promoter and/or the termination sequence can be heterologous to the nucleic acid segment that encodes an enzyme. Expression cassettes can have a promoter operably linked to a heterologous open reading frame encoding an enzyme. The invention therefore provides expression cassettes or vectors useful for expressing one or more enzyme(s).
Constructs (e.g., expression cassettes) and vectors comprising the isolated nucleic acid molecule, e.g., with optimized nucleic acid sequence, as well as kits comprising the isolated nucleic acid molecule, construct or vector are also provided.
Nucleic acids encoding one or more enzyme(s) can have one or more nucleotide deletions, insertions, replacements, or substitutions. For example, the nucleic acids encoding one or more enzyme(s) can, for example, have less than 95%, or less than 94.8%, or less than 94.5%, or less than 94%, or less than 93.8%, or less than 94.50% nucleic acid sequence identity to a corresponding parental or wild-type sequence. In some cases, the nucleic acids encoding one or more enzyme(s) can have, for example, at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at 90% sequence identity to a corresponding parental or wild-type sequence. Examples of parental or wild type nucleic acid sequences for unmodified enzyme(s) with amino acid sequences SEQ ID NOs:1, 3, 5, 6, 8, 9, 10, 11, 12, or 13, and nucleic acid sequences SEQ ID NOs:2, 4, 7, 9, 11, 14, 16-24, or 25. Any of these nuclei acid or amino acid sequences can, for example, encode or have sequences with less than 99.5%, less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, less than 94.8%, less than 94.5%, less than 94%, less than 93.8%, less than 93.5%, less than 93%, less than 92%, less than 91%, or less than 90% sequence identity to a corresponding parental or wild-type sequence.
A variety of promoters can be included in the transgenes, expression cassettes and/or expression vectors. Promoter regions are typically found in the flanking DNA upstream from the coding sequence in both prokaryotic and eukaryotic cells. A promoter sequence provides for regulation of transcription of the downstream gene sequence and typically includes from about 50 to about 2,000 nucleotide base pairs. Promoter sequences can also contain regulatory sequences such as enhancer sequences that can influence the level of gene expression. Some isolated promoter sequences can provide for gene expression of heterologous DNAs, that is a DNA different from the native or homologous DNA.
Promoters can be strong or weak, or inducible. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression. An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus. For example, a bacterial promoter such as the Ptac promoter can be induced to vary levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed cells. Promoters can also provide for tissue specific or developmental regulation. A strong promoter for heterologous DNAs can be advantageous because it provides for a sufficient level of gene expression for easy detection and selection of transformed cells and provides for a high level of gene expression when desired. In some cases, the promoter within such expression cassettes/vectors can be functional during plant development or growth.
The promoter can be a promoter functional in a heterotrophic organism or a cyanobacteria such as a bacterial promoter, yeast promoter, viral promoter, or a mammalian promoter. The promoter can be a heterologous promoter.
As used herein, “heterologous” when used in reference to a gene or nucleic acid refers to a gene, nucleic acid, or enzyme that has been manipulated in some way. For example, a heterologous promoter is a promoter that contains sequences that are not naturally linked to an associated coding region. Thus, a heterologous promoter is not the same one as the natural promoter that drives expression of an operably linked coding region.
Examples of promoters that can be used include, but are not limited to, the T7 promoter (e.g., optionally with the lac operator), the CaMV 35S promoter (Odell et al., Nature. 313:810-812 (1985)), the CaMV 19S promoter (Lawton et al., Plant Molecular Biology. 9:315-324 (1987)), nos promoter (Ebert et al., Proc. Natl. Acad. Sci. USA. 84:5745-5749 (1987)), Adh1 promoter (Walker et al., Proc. Natl. Acad. Sci. USA. 84:6624-6628 (1987)), sucrose synthase promoter (Yang et al., Proc. Natl. Acad. Sci. USA. 87:4144-4148 (1990)), α-tubulin promoter, ubiquitin promoter, actin promoter (Wang et al., Mol. Cell. Biol. 12:3399 (1992)), cab (Sullivan et al., Mol. Gen. Genet. 215:431 (1989)), PEPCase promoter (Hudspeth et al., Plant Molecular Biology. 12:579-589 (1989)), the CCR promoter (cinnamoyl CoA:NADP oxidoreductase, EC 1.2.1.44) isolated from Lollium perenne, (or a perennial ryegrass) and/or those associated with the R gene complex (Chandler et al., The Plant Cell. 1:1175-1183 (1989)).
Other constitutive or inducible promoters can be used with or without associated enhancer elements. Examples include a baculovirus derived promoter, the p10 promoter. Although some heterotrophs are bacteria or yeast, cyanobacteria and heterotrophs may also employ plant promoters.
Expression cassettes that include a promoter operably linked to a nucleic acid segment encoding a polypeptide or peptide can include other elements such as a segment encoding 3′ nontranslated regulatory sequences, and restriction sites for insertion, removal and manipulation of segments of the expression cassettes. The 3′ nontranslated regulatory DNA sequences can act as a signal to terminate transcription and in some cases can allow for the polyadenylation of the resultant mRNA. The 3′ nontranslated regulatory DNA sequence preferably includes from about 300 to 1,000 nucleotide base pairs and contains prokaryotic or eukaryotic transcriptional and translational termination sequences. Various 3′ can be employed. For example, such 3′ nontranslated regulatory sequences can be obtained as described in An (Methods in Enzymology. 153:292 (1987)). Many such 3′ nontranslated regulatory sequences are also present in plasmids available from commercial sources such as Clontech, Palo Alto, Calif.
Cyanobacteria can be cultured in a variety of simple media in the presence of light. For example, cyanobacteria can be grown in sea water. In some cases, cyanobacteria can be cultured in a medium that contains a nitrate salt, a phosphate salt, a magnesium salt, a calcium salt, a carbonate salt, a chelator, citric acid, ferric ammonium, and combinations thereof. Cyanobacteria are often cultured at about neutral pH or in slightly alkaline culture media. One example of a cyanobacteria medium can contain the following components.
When cyanobacteria are co-cultured with heterotrophs, the cyanobacteria culture media can be supplemented with a source of nitrogen, a buffer, electrolytes, an alkali, an acid, or a combination thereof.
As described herein, co-culture media have been optimized for growth and maintenance of either prokaryotes (referred to as CoBBG-11 medium) or fungi such as yeast (referred to as CoYBG-11 medium). CoBBG-11 consists of BG-11 medium supplemented with 106 mM NaCl, 4 mM NH4Cl and 25 mM HEPPSO, pH 8.3-KOH. Indole (100 μM) can be added to the CoBBG-11 medium when co-culturing some types of heterotrophs such as B. subtilis 168.
The CoYBG-11 medium consists of BG-11 medium supplemented with 0.36 g/L Yeast Nitrogen Base without amino acids (Sigma Aldrich), 106 mM NaCl, 25 mM HEPPSO, pH 8.3-KOH and 1 mM KPO3.
Bacteria can be co-cultured on solid co-culture plates that can include CoBBG-11 media with 1% autoclaved agar.
As illustrated herein, the atmosphere or the culture media in which a consortium of cyanobacteria and heterotrophic cells is cultured can be modulated to reduce the inhibitory effects of such oxygen production. For example, the consortium can be sparged with low-oxygen containing gases or anti-oxidants can be added to the culture medium.
In some cases, the cultures can be sparged or incubated with a gas having less than 20% oxygen, or less than 17% oxygen, or less than 15% oxygen, or less than 12% oxygen, or less than 10% oxygen, or less than 7% oxygen, or less than 5% oxygen, or less than 3% oxygen, or less than 1% oxygen. In some cases, the cultures can be sparged with a gas that is devoid of oxygen (e.g., 12:10:82 H2:CO2:N2).
In some cases, antioxidants such as DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), thiosulfate, or a combination thereof can be used in the culture medium. Concentrations of such antioxidants can vary from about 1 μM to 1000 mM, or about 10 μM to 200 mM.
The following Examples describe some experimental work performed during development of the invention.
This Example describes some of the materials and methods used in the development of aspects of the invention.
S. elongatus PCC 7942 (obtained from ATCC #33912) was engineered to secrete sucrose through the expression of the sucrose/proton symporter cscB [Ducat et al., Appl Environ Microbiol. 78: 2660-82 (2012)]. E. coli W was obtained from ATCC (accession no. 9637) and the E. coli W ΔcscR strain is described in Arifin et al. (Biotechnol. 156:275-8 (2011)). B. subtilis 168 was obtained from ATCC (accession no. 23857) and B. subtilis 3610 ΔsinI is described by Kearns et al. (Mol Microbiol. 55:739-49 (2005)). The ΔsinI mutant strain of 3610 was used to minimize chained growth making CFU counts of the strain reproducible (Kearns et al. Mol Microbiol. 55:739-49 (2005)). S. cerevisiae strains, WT W303 and W303Clump (previously referred to as Ancestor and Recreated02 strains, respectively) are described by Koschwanez et al. (Elife. 2013:1-27 (2013)). All strains are listed in Table 1.
Synechococcus elongatus PCC7942
Synechococcus elongatus
Bacillus subtilis 168
Bacillus subtilis 3610 ΔsinI
Escherichia coli K-12 BW25113
Escherichia coli W
Escherichia coli W ΔcscR
Saccharomyces cerevisiae W303
Saccharomyces cerevisiae
S. elongatus was propagated in BG-11 media (Sigma-Aldrich) plus 1 g/L HEPES, pH 8 in constant light at 35° C. In some cases S. elongatus engineered to express an IPTG-inducible sucrose symporter CscB (sometimes referred to S. elongatus CscB) was propagated in BG11 medium with 1 g/L HEPES, pH 8.3-NaOH. BG11 medium was prepared from 50× stock solution (Sigma-Aldrich, St. Louis, Mo.). M1 and M2 media were modifications of standard BG11 and were prepared from laboratory-created stock solutions (Table 2). Briefly, base BG11 medium was additionally supplemented with 15 mM NaNO3, 4.5 mM K2HPO4 (phosphate buffering), 1.5 mM MgSO4, 5 mM Na2SO4, 30 μM FeCl3, 3 μM Na2MoO4, and 1× additional trace metals before the addition of KOH to pH 8.3. This mineral-enriched medium was designated “M1.” Second, a BG11 medium with reduced nitrate (only 2 mM NaNO3), but an additional 4.5 mM K2HPO4 and 10 mM MgSO4, was HCl corrected to pH 8.3. This nitrate-reduced medium was designated “M2.” Additional sodium chloride added to the three media recipes varied-depending on the intrinsic sodium content of each to maintain equivalent sodium contents.
H. boliviensis was routinely cultured in liquid 1097 medium (ATCC BAA-759; (Quillaguamán et al., 2004)) and on 1.5% agar (BD Biosciences, San Jose, Calif.) 1097 medium plates for propagation and colony counts.
E. coli W strains deleted for the sucrose catabolism repressor cscR—which exhibit superior growth in co-culture with S. elongatus CscB (Hays et al., 2017)—were routinely cultured in LB medium and on 1.5% agar plates. The E. coli W strain was used to make chemically competent cells by the standard CaCl method, then transformed with the pAET41 plasmid (containing a pabABC expression cassette (Peoples et al., Journal of Biological Chemistry, 264(26): 15298-15303 (1989).
Co-cultures of CscB and H. boliviensis or E. coli were grown in the indicated media. Heterotrophic contamination was assessed using TSB agar plates as needed. All media elements were purchased from Sigma-Aldrich unless otherwise stated.
Axenic cyanobacteria were checked for contamination via plating on rich media. B. subtilis and E. coli were propagated in LB-Miller (EMD Millipore) while S. cerevisiae was maintained in YEPD media (MP Biomedicals). E. coli, B. subtilis, and S. cerevisiae were struck from frozen stocks on rich media plates (LB for bacteria and YEPD for yeast).
Co-culture media were optimized for either prokaryotes (CoBBG-11) or S. cerevisiae (CoYBG-11). CoBBG-11 consists of BG-11 supplemented with 106 mM NaCl, 4 mM NH4Cl and 25 mM HEPPSO, pH 8.3-KOH. Indole (100 μM) was added to B. subtilis 168 co-cultures as indicated and in alpha-amylase experiments. CoYBG-11 consists of BG-11 supplemented with 0.36 g/L Yeast Nitrogen Base without amino acids (Sigma Aldrich), 106 mM NaCl, 25 mM HEPPSO, pH 8.3-KOH and 1 mM KPO3. Solid co-culture plates were composed of CoBBG-11 media with 1% autoclaved noble agar (BD Biosciences).
Where appropriate, media conditioned by S. elongatus was acquired by taking CoBBG-11 or CoYBG-11 and inoculating it with OD750 0.5 of cscB+ S. elongatus in baffled flasks, grown for 48 hours in constant light before filtration. Media conditioned by prokaryotic heterotrophs was made by inoculating CoBBG-11 supplemented with 0.2% sucrose with B. subtilis 3610 or W cscR E. coli at an OD600 of 0.01 and allowing growth for 48 hours in a baffled flask before filtration.
For characterization of S. elongatus growth and sucrose production, S. elongatus was cultured axenically in baffled flasks of CoBBG-11 or CoYBG-11 and allowed to acclimate for at least 12 hours. Then cultures were adjusted to 25 mL with a final density of 0.5 OD750. IPTG (1 mM) was added, as appropriate. This was the start of the experiment and is referred to as time 0. Cultures were monitored at 24-hour intervals by withdrawal of 1 mL culture. OD750 was measured via photospectrometer (ThermoScientific NonoDrop 2000c) and culture supernatant was analyzed for sucrose content via a colorimetric Glucose-Sucrose Assay (Megazyme).
To prepare heterotrophic strains, single colonies were picked into their respective rich media and grown until turbid at varying temperatures before co-culture (37° C. for E. coli and B. subtilis; 30° C. for S. cerevisiae). Cells were diluted into the appropriate co-culture media +2% sucrose to acclimate to co-culture media and maintained within log phase growth (OD600<0.70) before use in co-cultures. All acclimating cultures and co-cultures were grown at 35° C., 150 rpm, 2% CO2, in light (PAR=about 80 μmol m−2 s−1 with 15 W Gro-Lux Sylvania fluorescent bulbs) within a Multitron Infors HT incubator. Heterotrophic growth was measured by inoculating rinsed cells at 0.01 OD600 (bacteria) or 0.05 OD600 (yeast) into fresh co-culture media at the indicated sucrose concentration. Data for growth rate was collected from 25 mL flask cultures while 96-well plates with 1 mL culture volumes were used to assay growth in a gradient of sucrose concentrations (0.156 mg/mL to 10 mg/mL,
Flask co-cultures were completed in 25 mL volumes in baffled flasks. Cyanobacteria and heterotrophs were acclimated to CoBBG-11 or CoYBG-11 media prior to inoculation into co-cultures. All co-cultures were grown at 35° C., 150 rpm, 2% CO2, in light (15 W; Gro-Lux; Sylvania) within a Multitron Infors HT incubator. 1 mM IPTG was added when indicated. Growth in co-cultures was monitored every 12 hours: S. elongatus was measured by the count of gated red-fluorescent events on a quantitative flow cytometer (BD Accuri); heterotrophs were assayed by plating dilution series on rich media to count colony forming units (CFU). Estimates of W303Clump cell number were derived by counting CFUs, but numbers were adjusted for the 6.6 cells/clump, and as confirmed under our culture conditions. For dilution experiments, co-cultures containing E. coli or B. subtilis were grown for 24 hours before 10 or 100-fold dilutions.
In some cases, cultures and plates were grown at a constant 32-35° C. Some experiments were carried out in 125 mL baffled flasks in a Multitron HT Pro incubator (Infors, Bottmingen, Switzerland). Heterotrophs were grown in the dark on air with shaking at 250 rpm, while all phototrophic cultures and co-cultures were grown at 130 rpm, with 2% CO2 and ˜80 μE m−2 s−1 PAR (15W Gro-Lux Sylvania bulbs). Media volumes were often 30 mL. S. elongatus CscB were routinely passaged by back-dilution to OD750=0.3 and with 12.5 mg/L chloramphenicol. Chloramphenicol was not applied during co-culture experiments. CscB expression was induced with 1 mM IPTG (as previously reported (Ducat et al., 2012)) and also at greater than or equal to 12 h before experimentation or alginate encapsulation. H. boliviensis were maintained on 1097 plates and grown in 1097 liquid for 2 d before use in experiments.
S. elongatus CscB Alginate Encapsulation
3% sodium alginate (Sigma-Aldrich) was created by slow mixing over several hours, followed by vacuum degassing and autoclave sterilization. IPTG induced, S. elongatus CscB cells grown in BG11 medium for 24 h were harvested at OD750≈1.5 by centrifugation and resuspended in 1/24 volume sulfur-free (no MgSO4 added) BG11 medium. The resuspended cells were added 1:12 to 3% sodium alginate- and thoroughly mixed by gentle stirring to a final 2.75% sodium alginate content and roughly two-fold concentrated CscB cells (OD750=3.0). In a sterile hood, this solution was then added dropwise to a ≥20-fold larger volume of 20 mM BaCl2 using a vertically-oriented syringe pump (KD Scientific, Holliston, Mass.), 5 mL syringes (BD Biosciences), and 30G needles. The drops traveled ˜35 cm from needle to the slowly stirred BaCl2 solution and cured at least 15 min in BaCl2 before being twice rinsed with deionized water and further incubated in deionized water for 5 min. The beads were transferred into >2 μL of BG11 medium with 1 mM IPTG and indicated NaCl concentrations; the medium was exchanged at least 2× after >30 min incubations and the removal of excess residual barium was evaluated by precipitate formation after adding 1 M Na2SO4 to withdrawn supernatant. Equilibration was considered successful when residual barium no longer precipitated. Finally, beads were transferred into a baffled 4 μL Fernbach flask for 12 h, with the intended final medium and salt under constant light. Beads intended for use in M2 medium experiments were transferred into BG11 containing no nitrate (BG11-N) at a volume appropriate to diluting the 17.6 mM nitrate contained within the BG11 infused beads to the desired 2 mM. The completed beads were then apportioned into experimental flasks with fresh medium to begin experiments.
B. subtilis and E. coli were recovered from rich media as above, washed in CoBBG-11 and inoculated at an OD600 of 0.01 in CoBBG-11 media+2% sucrose with cyanobacteria at different densities (OD750 at 0, 0.5, 1, and 2). S. cerevisiae was treated identically except they were inoculated at about 3×105 cells/mL (OD750=0.03) and CoYBG-1 was used. These samples were split into two 36-well plates and incubated and exposed to either constant light or dark conditions while maintaining the other growth parameters. Additional cultures of B. subtilis strain 3610 were set up as described above before addition of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) in ethanol and (thiosulfate) in water to final concentrations of 40 μM and 190 mM, respectively. Vehicle was added to control cultures and further cultures were split and sealed with septa. One was kept with atmospheric gas, while the other was sparged for 5 minutes with gas devoid of oxygen (12:10:82 H2:CO2:N2). Heterotroph counts were determined by plating on rich media for colony counts as above after initial setup (time 0) and after 12 hours of culture. Ratios of the viable cell counts from the light vs. dark cultures or log 10 of these ratios after 12 hours are reported.
To test the ability of co-cultures to withstand environmental perturbation, flask co-cultures were inoculated and grown as previously described for 24 hours before plating of 100 μL on solid co-culture Petri dishes. After five days, uneven lawns of heterotrophs and cyanobacteria arose. Cells were picked from these plates into 96-well plates and allowed to grow for 2-5 additional days. Any well that demonstrated cyanobacterial growth (as judged visually by green appearance) at the end of 48 hours was spotted on rich media to determine the presence or absence of heterotrophic symbionts. Solid culture and 96-well plate growth was completed at 35° C., 0 rpm, 2% CO2, in constant light (15 W; Gro-Lux; Sylvania) within a Multitron Infors HT incubator.
Lawns of cscB+ cyanobacteria were achieved via spreading of 250 μL of cscB+ cyanobacteria (OD750 0.5) on solid co-culture plates with or without 1 mM IPTG. After the cyanobacteria had absorbed on to the plate (more than 3 hours in the dark), 3 μL drops of heterotrophs were spotted on to the lawns. Heterotrophs had been previously grown up in rich media and washed three times to remove any media components before spotting. Media blanks and boiled cells were spotted as negative controls. Plates were then grown at 35 C, 2% CO2,in constant light (15 W; Gro-Lux; Sylvania) within a Multitron Infors HT incubator.
Long-term co-cultures were incubated in Phenometrics Environmental Photo-Bioreactors with 150 mL liquid volumes of a mix of cscB+ S. elongatus with either S. cerevisiae W303Clump or E. coli W ΔcscR in the appropriate co-culture BG-11 media+1 mM IPTG. Reactors were seeded with about 1×108 cells/mL of S. elongalus (OD750=0.5) and a final concentration of heterotroph equivalent to about 1×106 cells/mL S. cerevisiae W303Clump (final OD600 about 0.1) or about 5×107 cells/mL E. coli W ΔcscR (OD600˜0.05). Light was provided by onboard white, high-power LEDs (400 μmol m−2 s−1) continuously for E. coli W ΔcscR cultures. and with a 16:8 light:dark photoperiod for S. cerevisiae W303Clump co-cultures. The total density of co-cultures was monitored by on-board infrared diodes, following a brief (3-12 hour) acclimation period where the time-averaged optical density was allowed to settle to a fixed point following culture initiation. This measurement was used to control attached peristaltic pumps that eject fresh media to maintain the set target OD as previously described [Lucker et al. Algal Res. 6(PB):242-9 (2014)]. Co-culture temperature was maintained at 30° C. by a heated jacket; cells were agitated continuously by a magnetic stirbar. Daily, about 2 mL of co-culture volume was withdrawn and cyanobacterial and heterotrophic cell counts determined by flow cytometry and plating, respectively (as described above).
For the production of alpha-amylase, co-cultures of cscB S. elongatus and B. subtilis strain 168 were completed in 8 mL volumes of CoBG-11 supplemented with 100 μM indole in 6 well dishes. When specified, cyanobacteria were present (OD750=0.5) with or without 1 mM IPTIG. Control cultures did not contain cyanobacteria. Alpha-amylase production was measured after 24 hours of culture at 35° C., 0 rpm, 2% CO2, in constant light (15 W; Gro-Lux; Sylvania) within a Multitron Infors HT incubator. Alpha-amylase activity in supernatants was measured immediately after pelleting of cultures with the EnzChek Ultra Amylase Assay Kit, Molecular Probes Life Technologies using the manufacturer's protocol. Western blots confirmed presence of alpha-amylase in supernatants after addition of NuPAGE LDS sample buffer (Invitrogen) followed by 10 minutes at 100° C. Protein (10 L) was run on NuPage 4-12% Bis-Tris gels (Life Technologies) for in MES SDS running buffer for 50 minutes at 185 V. The iBlot 2 Dry Blot System (ThermoScientific) was used to transfer protein to nitrocellulose membranes (iBlot 2 NC Regular Transfer Stacks). Anti-alpha amylase antibodies (polyclonal rabbit; LS-C147316; LifeSpan BioSciences; 1:3,000 dilution) were used as the primary antibody followed by peroxidase-conjugated donkey anti-rabbit antibodies (AffiniPure 711-035-152 lot 92319; Jackson ImmunoResearch; 1:5,000 dilution) as the secondary antibody. The western blot was visualized via Western Lightning Plus-ECL, Enhanced Chemiluminescence Substrate (PerkinElmer, ProteinSimple FluorChem M). Purified alpha-amylase (Sigma Aldrich) was used as a control in all assays.
E. coli strains were transformed with pAET41 (Table 1) before use in co-cultures for production [Peoples et al. J Biol Chem. 264:15298-303 (1989)]. Co-cultures were set up as previously described in 25 mL flasks. After one week of growth, the entire culture was spun down, frozen, and stored at −80° C. until PHB content was quantified. PHB content was quantified by methods described by Torella et al. (Proc Natl Acad Sci. 112:2337-42 (2015)) and Lui et al. (Nano Lett 15(5):3634-9 (2015)). Briefly: cell pellets were digested with concentrated H2SO4 at 90° C. for 60 min. The digestion solution was diluted 500 times (500×) with water and passed through 0.2 μm filter. The solutions were subsequently analyzed by a high-performance liquid chromatography (HPLC, Agilent HPLC 1200) equipped with Aminex HPX-87H column and UV absorption detector. The volume of each sample injection was 100 PL. The mobile phase was 2.5 mM H2SO4 aqueous solution, with a flow rate of 0.5 mL/min for 60 min. 5 mM sodium acetate (Sigma Aldrich) was added as an internal standard. The concentrations of PHB were determined by comparing the peak area with that in standard curves from 0.1 to 30 mM.
Cells were imaged on agarose pads using an inverted Observer D1 Microscope (Zeiss, Jena, Germany) with a Plan-Neofluar 100× oil objective (Zeiss), AxioCam ICC5 camera (Zeiss), X-Cite Series 120 mercury halide bulb (Lumen Dynamics, Mississauga, Canada), and using ZEN 2012 software (Zeiss).
Alginate encapsulated S. elongatus were fixed for 48 h at 4° C. in 2.5% paraformaldehyde/glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), while H. boliviensis cells were fixed for 24 h. All samples were washed three times with 0.1 M sodium cacodylate buffer and subsequently fixed in 1% osmium tetroxide/1.6% potassium ferricyanide solution in 0.1 M sodium cacodylate buffer utilizing a MS-9000 Laboratory Microwave Oven (Electron Microscopy Sciences, Hatfield, Pa.; 5 min at 30 C). Samples were washed with deionized water until clear from osmium and post-fixed with 1% aqueous uranyl acetate for 2 min at 30° C. Samples were washed with deionized water three times more and subsequently dehydrated with an increasing acetone series (2 min at 30° C.) and then embedded in Spurr's resin (20 min, 30° C., 25% increments). Blocks were left to polymerize at 60° C. for 3 d. Sections (45-nm thick) were cut on a MX ultramicrotome (RMC Boeckeler, Tucson, Ariz.). Prior to imaging on a JEM 100CX II transmission electron microscope (JEOL) equipped with a Prius SC200-830 CCD camera (Gatan, Pleasanton, Calif.), sections were stained with 1% uranyl acetate and Reynolds lead citrate (Reynolds, 1963) for 5 min each.
Culture optical densities were measured using >500 μL of culture in a cuvette with a Genesys 20 (Thermo Fisher Scientific, Waltham, Mass.) spectrophotometer. H. boliviensis were typically measured at OD600, S. elongatus CscB at OD750, and both for co-culture experiments, unless otherwise stated. Chlorophyll a was similarly measured at OD665 following extraction of an appropriate cell volume within 1 mL 95% methanol (Zavirel et al., 2015).
H. boliviensis or E. coli cell densities were quantified by 96-well plate serial dilution in BG11+175 mM NaCl medium and plating on agar solidified 1097 plates or LB plates, respectively. Colony forming unit (CFU) counts were made after 36 h of incubation at 30° C. for H. boliviensis, or overnight at 37° C. for E. coli. Heterotrophic contamination was routinely assessed from the same serial dilutions by spotting onto TSB plates.
Sucrose was measured using a Sucrose/D-Glucose Assay Kit (Megazyme, Bray, Ireland). Briefly, 200 μL of cell culture was pipetted into a 96-well plate, centrifuged at 3 krpm for 30 min at 4° C. (Sorvall 75006445; New Castle, Del.), and 100 μL of cell-free medium withdrawn. To assay sucrose, 20 μL of cell-free medium was mixed with 10 μL invertase solution, sealed, and incubated at 50° C. for >20 min, then incubated with 150 μL GOPOD reagent solution at 50° C. for 20 min. Absorbance at 510 nm after cooling was measured on a microplate reader (SpectraMax M2; Molecular Devices, Sunnyvale, Calif.). Sucrose concentrations were determined in comparison to a six-point standard curve containing 0 to 0.25 mg/mL glucose samples, which typically exhibited a R2≥0.98 linear fit.
Polyhydroxybutyrate (PHB) was measured using the crotonic acid method and high-performance liquid chromatography (HPLC; (Van-Thuoc et al., 2008)). Briefly, cells were centrifuged at 3,700×g for 30 min, decanted, and lyophilized before weighing of the resultant dry cell pellets. In a sealed glass vial, the dry cell pellets were dissolved in 1 mL of concentrated sulfuric acid, heated to 90° C. for 1 h, cooled to room temperature, and then diluted 100-fold with deionized water. The resulting solutions were filtered with GHP membranes (Pall Life Sciences, Port Washington, N.Y.) to remove particulates and 20 μL injected onto an Aminex 300-mm HPX-87H (Bio-Rad Laboratories, Hercules, Calif.) column. The mobile phase was 0.028 N H2SO4 and flowed at 1 mL/min, while the column was maintained at 60° C. UV-absorption was monitored at 210 nm. Two standards of commercial polyhydroxybutyric acid (Sigma-Aldrich) were similarly treated, injected at 6 volumes at the start and end of sampling each, and averaged to create a standard curve with R2=1 linear fit.
Free nitrate and nitrite were estimated by AquaChek (Hach) test strips during co-culture of S. elongatus CscB and H. boliviensis. This colorimetric assay is sensitive to free nitrate and nitrite levels ≥2 ppm (≥32 μM). In the nitrogen limited experiments described in
Contaminant 16s rRNA Sequencing
Contaminating bacterial colonies isolated from co-culture were grown for use in 16s rRNA PCR sequencing reactions with universal primers 8F, 27F, 1100R, and 1492R, as previously described (Lane, 1991; Turner et al., 1999). Sequence BLAST searches (https://blast.ncbi.nlm.nih.gov/Blast.cgi) returned only >97% identity and e-values >7e−126 results with Stenotrophomonas maltophilia the most numerous and consistently identified of named species.
All equations were modeled in Mathematica (Wolfram Research, Inc., Mathematica, Version 11.0). Statistics were completed in GraphPad Prism version 7 GraphPad Software. La Jolla Calif. USA (see website at graphpad.com).
Pairwise consortia were designed where cscB* S. elongatus secreted sucrose in response to osmotic pressure combined with induction of cscB expression by isopropyl β-D-1-thiogalactopyranoside (IPTG) [Ducat et al. Appl Environ Microbiol. 78: 2660-8 (2012)]. Carbon secreted by cyanobacteria promoted growth of co-cultured heterotrophs as schematically illustrated in
Media with optimized compositions of nitrogen, salt, and buffer were developed: termed CoBBG-11 for use in cyanobacteria/bacteria consortia and CoYBG-11 for cyanobacteria/yeast co-culture (see Example 1). Tests verified that S. elongatus grows and produces sucrose in both CoBBG-11 and CoYBG-11 (
CscB+ S. elongatus directly supported heterotroph growth in co-cultures that contain no external carbon sources (
B. subtilis growth in co-culture is dependent on IPTG-induced sucrose secretion from cscB+ S. elongatus (
S. cerevisiae growth in co-culture is dependent on genetic engineering to improve sucrose utilization. Wild type (WT) S. cerevisiae W303 did not grow in co-culture with or without IPTG induction (
The engineered strain, referred to as W303Clump, from directed evolution experiments of S. cerevisiae W303 in low sucrose media [Koschwanez et al. Elife 2013:1-2725 (2013)]. W303Clump (originally called Recreated02 in Koschwanez et al. 2013) contains mutations in genes CSE2, IRA1, MTH1, and UBR1 that enhance fitness in dilute sucrose, and also contains a nonsense mutation in ACE2 that compromises the full septation of budding daughter cells from the mother, resulting in small clonal cell aggregates (about 6.6 cells per clump on average). These aggregates grow in low sucrose due to increased local cell concentration and increased hexose availability after extracellular cleavage of sucrose by an invertase [Koschwanez et al., PLoS Biol. 9(8):e1001122 (2011)]. Unlike the parental strain, axenic cultures of W303Clump exhibited some growth at all tested sucrose concentrations greater than or equal to 0.156 g/L (
E. coli W grows in co-culture independently of induced sucrose secretion from cscB+ S. elongatus (
Monocultures of ΔcscR E. coli exhibit the capacity to grow on lower concentrations of sucrose (as low as 1.25 g sucrose/L;
These data indicate that in the first days of co-culture, while exported sucrose concentrations are low (less than or equal to I g/L), E. coli strains cannot utilize sucrose effectively and dominantly depend on other metabolites from S. elongatus; perhaps extracellular polymeric substances.
As illustrated in
Experiments were performed to determine the source of the heterotroph growth inhibition. To focus on products other than sucrose concentrations that could influence heterotrophic viability and eliminate the confounding factor that cyanobacteria only generate sucrose in the light [Ducat et al., Appl Environ Microbiol. 78: 2660-8 (2012)], co-cultures were supplemented with exogenous sucrose (2%) and cultivated in the light or dark. After 12 hours of co-cultivation, heterotroph viability of each of the three species (S. elongatus, B. subtilis and S. cerevisiae) was determined.
However, the inhibition of B. subtilis growth while in co-culture with S. elongalus was mitigated when cells were incubated with DCMU, an inhibitor of oxygen evolution from Photosystem 11, or thiosulfate, a potent antioxidant (
These data indicate that the factor that inhibited heterotroph growth when high concentrations of cyanobacteria were present, was oxygen. Oxygen levels can readily be regulated (e.g., reduced) by sparging with low oxygen-containing gases or addition of antioxidants, as illustrated in
Unlike the effects on heterotroph viability, co-culture with heterotrophs can stimulate growth of S. elongatus (
The growth-promoting effect of heterotrophs on cyanobacteria is also observed when co-cultivated on solid media. Dilutions of B. subtilis or E. coli cultures were spotted on a lawn of dilute cyanobacteria with or without IPTG (
The growth-promoting effect of B. subtilis on cscB+ S. elongatus was dependent upon induction of sucrose export. Without IPTG, spots of B. subtilis inhibited cyanobacterial growth (
Collectively, these experiments indicate that all three evolutionarily unrelated heterotrophs can significantly increase cyanobacterial growth under a range of growth conditions.
The inhibitory effects of cyanobacteria on the viability of heterotrophs was only observed at relatively high density and in the light (
Phenometrics environmental Photo-bioreactors (ePBRs) were first employed. Such ePBRs have turbidostat capabilities in addition to control of light, temperature, and culture stirring [Lucker et al. Algal Res. 6(PB):242-9 (2014)].
When cultivated at a constant density, E. coli/cscB+ S. elongatus co-cultures persist over time and heterotrophic viability is maintained. Co-cultures of induced cscB+ S. elongatus and strain W ΔcsR E. coli were grown continuously in ePBRs under constant light (
Similarly, when cultured in ePBRs, S. cerevisiae W303Clump maintained viability in co-cultures with cscB+ S. elongatus over time and also persisted through variable light conditions. The cscB+ S. elongatus was induced to secrete sucrose and inoculated with S. cerevisiae into culture media containing ePBRs. To examine the co-culture capacity to persist through light perturbations, these cultures were programmed with an alternating diurnal illumination regime (16 hours light: 8 hours dark,
Prokaryotic co-cultures with cyanobacteria persist through population bottlenecks and changes in environmental structure, B. subtilis/cscB+ S. elongatus and W ΔcscR E. coli/cscB+ S. elongatus co-cultures were subjected to large dilutions (1 to 10 or 1 to 100) to determine viability following the introduction of a population bottleneck. Cyanobacterial growth was monitored via flow cytometry following dilution, while heterotroph growth was measured via CFU, In perturbed cultures, heterotrophs can return to pre-dilution levels within three days (
As multiple species can be co-cultured with cscB+ S. elongatus, it is possible to exchange heterotrophs to functionalize consortia for a desired activity. In this design, the heterotrophic species of the consortia acts as a “conversion module” to metabolize the products of photosynthesis into target bioproducts in a “one-pot reaction” (
Alpha-amylase was produced in co-cultures of B. subtilis strain 168 and cscB+ S. elongatus (
Engineered E. coli/S. elongatus communities produced PHB. E. coli strains harboring a previously described PHB production plasmid, pAET41 [Peoples et al. J Biol Chem. 264:15298-303 (1989)] were co-cultured with cscB+ S. elongatus for one week in constant light and measured PHB in the total biomass by liquid chromatography (
Taken together, these results demonstrate that consortia can be flexibly programmed for photoproduction of different bioproducts by employing different heterotrophic organisms.
Halomonas boliviensis was selected as a potential co-culture partner with cyanobacteria partially because H. boliviensis can produce useful compounds such as hydroxylated alkyl acids. H. boliviensis also grows well under limited nutritional conditions, similar to those routinely used as cyanobacterial minimal media (see, e.g., Guzmán et al. Appl. Microbiol. Biotechnol. 84, 1069-1077 (2009); Quillaguamán et al., Appl. Microbiol. Biotechnol. 74, 981-986 (2007); Quillaguamán et al., J. Appl. Microbiol. 99, 151-157 (2005); Rivera-Terceros et al., J. Biol. Res. 22: 8 (2015).
Indeed, H. boliviensis grew readily in standard BG11 medium as well as a modified BG11 medium (M1 medium; see Table 2) when supplemented with sucrose and NaCl (
S. elongatus CscB grew in both BG11 and M1 media and produced sucrose when induced to express cscB via the addition of IPTG (
If cyanobacteria are encapsulated, they could facilitate selective harvesting of H. boliviensis biomass from mixed co-cultures. To test this, sodium alginate-suspended S. elongatus CscB were dripped into a barium chloride gelling solution, generating spherical, barium-alginate hydrogel beads of about 2.6 mm in diameter that contained entrapped cyanobacterial cells (
The sucrose productivity of encapsulated S. elongatus CscB was examined relative to planktonic (i.e., “free floating”) suspensions by culturing equal numbers of free and encapsulated cyanobacteria into separate cultures. As shown in
Cyanobacterial chlorophyll a concentrations are influenced by growth conditions (Muramatsu & Hihara, J. Plant Res. 125: 11-39 (2012)). Attempts were made to estimate sucrose specific productivity on a per cell basis, but unfortunately, a treatment was not identified that could both sufficiently dissolve the resilient Ba-alginate and yield viable, planktonic cells. Cyanobacterial growth was therefore examined within the alginate matrix by transmission electron microscopy (TEM) of bead cross-sections over time. Immediately following encapsulation, approximately 1 cell/pocket was observed within the alginate matrix, and this was unchanged after 7 days of cultivation (
Assuming that the cell number did not significantly change within the beads after 66 hours of encapsulation (
The capacity of sucrose secreted from encapsulated S. elongatus CscB was examined to support the growth of H. boliviensis in direct co-culture. Ba-alginate beads containing S. elongatus CscB were prepared as described in the Examples above and added to liquid cultures of planktonic H. boliviensis (see
The cellular biomass was evaluated by withdrawal of media from the liquid phase after each week, and PHB levels were determined to be between 2.5-5.5 mg PHB L−1 (
Alginate-encapsulated S. elongatus CscB in M2 medium supported robust growth of co-cultivated H. boliviensis for more than five months using only CO2 as the input carbon source (
These data indicate that alginate encapsulation delayed the onset of nitrogen stress responses relative to planktonic cells, perhaps because alginate encapsulation restricts growth and thereby reduces cellular nitrogen requirements (as seen in other encapsulated cells, see e.g., Kaya & Picard, Biotechnol. Bioeng. 46, 459-464 (1995). Indeed, intact S. elongatus CscB cells were observed by TEM at greater than 150 days-post encapsulation, although nitrogen was limited for the majority of the cultivation period (see
Co-cultivated H. boliviensis produced high titers of PHB and productivity during five months of co-culture, as monitored by brightfield microcopy, Nile red staining, electron microcopy, and chromatography. Following each back-dilution, large, light-diffracting bodies accumulated in H. boliviensis, and these inclusions stained with the hydrophobic fluorescent dye, Nile red (
In addition to productivity, S. elongatus CscB/H. boliviensis co-cultures also demonstrated resistance to both perturbation and contamination. Encapsulated cyanobacterial cells remained viable through repeated cycles of nitrogen availability and also were stable through a deliberate extended period of nitrogen deprivation (
To further evaluate the promising stability and productivity observed in synthetic co-cultures (
To compare the effectiveness of H. boliviensis for PHB production relative to the co-culture work illustrated in Examples 2-7, the co-culture conditions represented in
This Example illustrates that encapsulated S. elongatus CscB+ cells are more tolerant of the toxin 2,4-dinitrotoluene (2,4-DNT) than non-encapsulated S. elongatus CscB+ cells, and that co-cultures of these cyanobacteria with dnt-transgenic Pseudomonas putida can degrade dinitrotoluenes (e.g., 2,4-DNT). Such dinitrotoluenes can be environmental pollutants from industrial activities such as from the manufacture of the explosive trinitrotoluene (TNT).
The transgenic Pseudononas putida have the dnt the operon (expression cassette; pSEVA221-cscRABY) that was integrated in order to confer the capacity to degrade 2,4-DNT. This operon is derived from Burkholderia cepacia sp. R34 This dnt expression cassette includes nucleic acid segments encoding the DntA, DntB, DntD, DntE, DntG and DntR enzymes. Nucleic acid segments in the pSEVA221-cscRABY have the following sequences.
The nucleic acid segment encoding DntR (LysR-type regulatory protein) has the following sequence (SEQ ID NO:16).
The nucleic acid segment encoding DntAa (2,4-dinitrotoluene dioxygenase system ferredoxin—NAD(+), reductase component) has the following sequence (SEQ ID NO: 17).
The nucleic acid segment encoding DntAb (2,4-dinitrotoluene dioxygenase system, ferredoxin component) has the following sequence (SEQ ID NO:18).
The nucleic acid segment encoding DntAc (2,4-dinitrotoluene dioxygenase system, large oxygenase component) has the following sequence (SEQ ID NO:19).
The nucleic acid segment encoding DntAd (2,4-dinitrotoluene dioxygenase system, small oxygenase component) has the following sequence (SEQ ID NO:20).
The nucleic acid segment encoding DntB (4-methyl-5-nitrocatechol 5-monooxygenase) has the following sequence (SEQ ID NO:21).
The nucleic acid segment encoding DntD (trihydroxytoluene oxygenase) has the following sequence (SEQ ID NO:22).
The nucleic acid segment encoding DntE (methylmalonate-semialdehyde dehydrogenase) has the following sequence (SEQ ID NO:23).
The nucleic acid segment encoding DntG has the following sequence (SEQ ID NO:24).
A primary consideration when expanding the range of functionalities for consortia into the capacity to degrade harmful chemical compounds is the sensitivity of S. elongatus CscB+ to the toxic and bacteriostatic properties of the harmful chemicals such as dinitrotoluenes (e.g., 2,4-DNT).
Preliminary toxicity assays of planktonic (i.e., free-floating) cultures of S. elongatus cyanobacteria were monitored in the presence of increasing 2,4-DNT. Such S. elongatus cyanobacteria did not contain the enzymatic machinery to breakdown 2,4-DNT. As illustrated in
S. elongatus CscB+ cyanobacterial cells were encapsulated in alginate hydrogels as illustrated in
Significantly, encapsulation also improved the resilience of cyanobacterial cells to the toxic effects of 2,4-DNT. Encapsulated S. elongatus CscB+ cells were exposed to increasing concentrations of 2,4-DNT.
This Example illustrates that E. coli strains engineered to overexpress the tryptophanase gene (tnaA) can grow in co-culture with sucrose secreting S. elongatus to produce and secrete the product indole.
A nucleic acid encoding the TnaA tryptophanase enzyme that was used in E. coli is shown below as SEQ ID NO:25.
The tryptophanase with SEQ ID NO:14, is encoded by this nucleic acid segment with SEQ ID NO:25, and was expressed in the E. coli-S. elongatus co-culture.
As shown in
All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.
The following statements are intended to describe and summarize various embodiments of the invention according to the foregoing description in the specification.
The specific products, consortia, methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential.
The specific products, consortia, methods and compositions illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a microbe,” “a compound,” “a nucleic acid” or “a promoter” includes a plurality of such microbes, compounds, nucleic acids or promoters (for example, a solution of microbes, compounds or nucleic acids, or a series of promoters), and so forth.
The term “about” can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value, or of a stated limit of a range. When a range or a list of sequential values is given, unless otherwise specified any value within the range or any value between the given sequential values is also disclosed.
Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.
The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention.
This application is a continuation of U.S. patent application Ser. No. 16/250,672, filed Jan. 17, 2019, which claims benefit of priority to the filing date of U.S. Provisional Application Ser. No. 62/618,859, filed Jan. 18, 2018, the contents of which are specifically incorporated herein by reference in their entirety.
This invention was made with government support under 1437657 and 1144152 awarded by National Science Foundation, and under DE-SC0012658 awarded by the U.S. Department of Energy. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62618859 | Jan 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16250672 | Jan 2019 | US |
| Child | 17143045 | US |
