Patent Application
20170306363
The instant application contains a Sequence Listing which has been submitted via EFS-web and is hereby incorporated by reference in its entirety. The ASCII copy, created on Apr. 24, 2017, is named 16-53_ST25.txt, and is 95,228 bytes in size.
Biocatalytic production of commodity and specialty chemicals from renewable resources offers a high-potential, sustainable route to establish a viable bio-based economy. Recent advances in metabolic engineering and fermentation optimization have enabled the establishment of bio-based routes for the production of an array of biochemicals, fuel precursors, and pharmaceuticals. Four-carbon dicarboxylic acids are particularly promising precursors for the development of commodity and specialty chemicals. One such di-acid, succinic acid (SA), possesses properties analogous to petrochemically-derived maleic anhydride, a primary precursor to 1,4-butanediol (BDO), unsaturated polyester resins (UPR), lubricating oil additives, and an array of pharmaceutical and nutraceutical products.
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.
The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods that are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.
Exemplary embodiments provide engineered cells with at least one exogenously added gene encoding an enzyme from the reductive branch of the tricarboxylic acid (TCA) cycle, where the cells are able to produce succinic acid from a carbon source.
In some embodiments, the exogenously added gene encodes malate dehydrogenase, PEP-carboxykinase, fumarase, or combinations thereof. In some embodiments, the cells contain an additional exogenously added gene encoding XylE or phosphoglucose dehydrogenase.
In certain embodiments, the cells are bacterial cells, such as bacterial cells from the genus Actinobacillus, from Actinobacillus succinogenes, or from Actinobacillus succinogenes strain 130Z.
In various embodiments, the cells produce a higher amount of succinic acid than the wild type cells.
In some embodiments, the carbon source is derived from lignocellulosic biomass and/or comprises glucose, xylose, galactose or arabinose.
In certain embodiments, the cells have an additional genetic modification that reduces the production of acetate or formate by the cell or reduces the expression of pyruvate formate lyase or acetate kinase.
Additional embodiments provide methods for producing succinic acid by culturing the engineered cells with a carbon source and recovering the succinic acid from the culture.
In some embodiments, the methods use carbon sources derived from lignocellulosic biomass.
In certain embodiments, the methods use bacterial cells from Actinobacillus succinogenes, cells that contain an exogenously added gene encoding malate dehydrogenase, or cells that have a genetic modification that reduces the expression of pyruvate formate lyase or acetate kinase.
In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions.
Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.
Presented herein are biocatalysts and methods for the production of succinic acid from carbon sources. The biocatalysts include microbial cells that have been engineered to overexpress exogenously added genes that encode enzymes active in the reductive branch of the tricarboxylic acid (TCA) cycle. The cells may also include genetic modifications that ablate the expression of genes that encode enzymes involved in formate or acetate biosynthesis. The modifications allow the engineered cells to produce higher amounts of succinic acid or higher purity succinic acid in comparison to wild type cells.
Succinic acid is a key chemical intermediate and potential platform alternative to petro-derived maleic anhydride for the synthesis of an array of high-value industrial products. Succinic acid is a specialty chemical and an important precursor for the synthesis of high-value products that can be applied across many industries, such as biopolymers, pharmaceutical products, and foods.
Microbial production of commodity and specialty chemicals from renewable resources offers a promising, sustainable route to establish a viable bioeconomy. Four-carbon dicarboxylic acids represent interesting precursors for bioproducts. Succinic acid (SA) in particular exhibits properties analogous to petrochemically-derived maleic anhydride, a primary precursor to 1,4-butanediol (BDO), unsaturated polyester resins, lubricating oil additives, and an array of other products. The similarity of SA to maleic anhydride, coupled with its natural occurrence as a byproduct in microbial fermentation, has made it an attractive target chemical. SA also presents potential for a series of unique product suites, including the biodegradable polyester polybutylene succinate (PBS). Elucidation of mechanisms governing SA biosynthesis in relevant industrial hosts offers a means to develop strain engineering strategies aimed at flux enhancement.
At present, bio-based production of succinic acid is not competitive with petroleum-derived analogs, largely due to technical hurdles associated with low cost fermentation and product recovery. Biosynthetic enhancements to succinic acid titers, productivity rates, and carbon yields via rational metabolic and fermentation engineering strategies offer a targeted approach to address these hurdles. Further, reduction or elimination of heterofermentative co-products via additional strain-engineering approaches offers an additional means to enhance carbon yield, while concurrently increasing product purity, which will in turn improve product recovery (estimated to account for upwards of 60-70% of the overall bioprocess cost). Thus, development of economically viable biobased routes to succinic acid will require rational strain-engineering approaches targeting increased flux to succinic acid and elimination of competitive carbon pathways.
Development and deployment of anaerobic strains with high native flux to SA, such as Actinobacillus succinogenes 130Z, offers an appealing foundation for effective biocatalysts. A. succinogenes is a gram-negative, capnophilic, facultatively anaerobic, biofilm-forming bacterium with the capacity to convert a broad range of carbon sources to SA as a primary fermentative product, achieving among the highest reported SA titers and yields to date. Because A. succinogenes is capnophilic, it can incorporate CO2 into SA, making this organism an ideal host for conversion of lignocellulosic sugars and CO2 to an emerging commodity bioproduct sourced from renewable feedstocks.
The core metabolic and SA biosynthetic pathways in A. succinogenes (and related organisms), including an incomplete TCA cycle which natively terminates at SA, are depicted in
Disclosed herein are genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in organisms such as Actinobacillus succinogenes. These metabolic engineering capabilities enable examination of SA flux determinants via knockout of the primary competing pathways—namely acetate and formate production—and overexpression of enzymes in the reductive branch of the TCA cycle leading to SA. The genetic modifications disclosed herein can lead to succinic acid production improvements, and also allow the identification of key flux determinants and new bottlenecks and energetic needs when removing by-product pathways in microbial metabolism. The overexpression of the SA biosynthetic machinery, for example the enzyme malate dehydrogenase, enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher purity SA. The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe.
The resulting strains are capable of fermentation on myriad carbon sources, including carbohydrate streams derived from lignocellulosic biomass. Examples include pentose-rich sugar streams from corn stover or culturing the cells in media with added sugars such as glucose, xylose, arabinose, galactose, or other sugars. Lignocellulose may be pretreated (e.g., with dilute acids) or contacted with enzymes such as cellulases to generate fermentable carbon sources.
Also disclosed are gene overexpression and/or marker-less gene knockout microbial strains, including strains of A. succinogenes. The knockout of competitive carbon pathway targets leads to higher-purity production of SA. Concurrently, up-regulation of the reductive branch of the TCA cycle enhances flux to SA, resulting in titer, rate, and yield enhancements. These modifications reveal a finely tuned energetic and redox system and previously unidentified mechanisms of secondary organic acid biosynthesis. Additionally, the resultant strains present promising metabolic engineering strategies for the economically viable, sustainable production of SA from pentose-rich sugar streams.
Disclosed herein are metabolic modeling techniques to identify strain-engineering targets and novel genetic tools to pursue rational metabolic engineering strategies coupled with fermentation optimization routes. Specific examples are provided below, but the content of this disclosure also includes overexpression and knockout strains (and combinations thereof) of microorganisms wherein the gene targets are part of the succinic acid pathway or alter the ability of a microorganism to produce succinic acid.
Gene knockout targets also include biosynthetic components governing acetate and formate biosynthesis, both in isolated and coupled background strains. Knockout of these targets leads to near homo-fermentative production of succinic acid. Concurrently, the reductive branch of the TCA cycle may be up-regulated via overexpression of PEP-carboxykinase (PEPCK), malate dehydrogenase (MDH), and fumarase (FUM). For example, overexpression of these three genes on an operon leads to significant enhancement of succinic acid titers, rates, and yields. Exemplary gene targets include PEP-carboxykinase, malate dehydrogenase, fumarase, pyruvate formate lyase, acetate kinase, malic enzyme, formate dehydrogenase, and others. Additional strain-engineering targets and combinations thereof are presented in Tables 2 and 3. Microorganisms may be engineered to overexpress any of the gene targets disclosed herein or sequences presented herein. Likewise, any of these genes or sequences may be ablated in a microorganism.
Actinobacillus
succinogenes
E. coli - A. succinogenes shuttle vector; Ampr;
Phosphoenolpyruvate carboxykinase (PEP-carboxykinase or PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and carbon dioxide. Nucleic acid and amino acid sequences for PEPCK from A. succinogenes are provided as SEQ ID NOS:1 and 2, respectively. Malate dehydrogenase (MDH) reversibly catalyzes the oxidation of malate to oxaloacetate using the reduction of NAD+ to NADH. Nucleic acid and amino acid sequences for two MDH isoforms from A. succinogenes are provided as SEQ ID NOS:3 and 4, respectively (MDH1) and SEQ ID NOS:5 and 6, respectively (MDH2). Fumarase (fumarate hydratase or FUM) catalyzes the reversible hydration/dehydration of fumarate to malate. Nucleic acid and amino acid sequences for FUM from A. succinogenes are provided as SEQ ID NOS:7 and 8, respectively.
Pyruvate formate lyase (PFL or pflB) catalyzes the reversible conversion of pyruvate and coenzyme-A into formic acid and acetyl-CoA. Nucleic acid and amino acid sequences for PFL from A. succinogenes are provided as SEQ ID NOS:9 and 10, respectively. Acetate kinase (AK or ackA) catalyzes conversion of acetyl-phosphate and ADP to acetate and ATP. Nucleic acid and amino acid sequences for AK from A. succinogenes are provided as SEQ ID NOS:11 and 12, respectively.
XylE is a transporter protein that facilitates cellular uptake and utilization of the sugar xylose. Nucleic acid and amino acid sequences for XylE from A. succinogenes are provided as SEQ ID NOS:13 and 14, respectively. Phosphogluconate dehydrogenase (GDH) is an enzyme in the pentose phosphate pathway that catalyzes the production of ribulose-5-phosphate from 6-phosphogluconate. Nucleic acid and amino acid sequences for GDH from A. succinogenes are provided as SEQ ID NOS:15 and 16, respectively.
Exemplary microorganisms suitable for genetic engineering as described herein include bacteria, including those from the genus Actinobacillus or strains of A. succinogenes such as A. succinogenes 130Z. Additional examples include the following Actinobacillus strains: A. actinomycetemcomitans, A. anseriformium, A. arthritidis, A. capsulatus, A. delphinicola, A. equuli, A. hominis, A. indolicus, A. hgnieresii, A. minor, A. muris, A. pleuropneumoniae, A. porcinus, A. rossii, A. scotiae, A. seminis, A. suis, and A. ureae. Bacteria may also include E. coli, Anaerobiospirillum succiniciproducens, Corynebacterium glutamicum or Mannheimia succiniciproducens.
Biocatalytic production of industrial platform chemicals from renewable substrates offers a promising means to generate petrochemical alternatives. Biological production of SA from lignocellulosic substrates in particular has the potential to displace a significant fraction of petroleum-derived maleic anhydride, serving as a potentially high-volume functional replacement in the production of an array of biopolymers and biomaterials. The development of biocatalysts with enhanced SA biosynthetic capacity via metabolic engineering strategies focused upon removal of alternative carbon sinks and/or enhanced biosynthetic pathway flux leads to strains that demonstrate a number of favorable characteristics, including enhanced titers, rates, and yields of SA, as well as reduction of alternative fermentative products, all of which will serve to enhance bioprocess economics.
Interplay exists between formic and acetic acid biosynthesis in redox and energetic balance, and biosynthesis of acetic acid may be a key contributor to SA productivity. Additionally, acetic acid biosynthetic capacity exists in A. succinogenes mutants lacking a canonical acetate kinase gene, indicating an alternative biosynthetic route. The genetic and/or flux modifications disclosed herein may also lead to increased co-production of lactic acid.
Modified microorganisms disclosed herein may be used in batch or continuous fermentation processes. Growth lags and delays in the onset of SA production may be mitigated by employing a continuous fermentation process. For example, a continuous process may benefit from deployment of the ΔpflB/pPMF combinatorial strain, wherein steady-state SA production is relatively unaffected compared to wild-type, yet heterofermentative products are significantly reduced, enabling more facile in-line separations.
Additional strain modifications, including components of the pentose phosphate pathway, may also be used to increase upstream flux enhancement. For example, overexpression of phosphoglucose dehydrogenase (GDH) in wild-type and pflB mutant backgrounds may result in SA biosynthetic production enhancement (
In certain embodiments, a nucleic acid may be identical to the sequence represented herein. In other embodiments, the nucleic acids may be least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence presented herein, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence presented herein. Sequence identity calculations can be performed using computer programs, hybridization methods, or calculations. Exemplary computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, BLASTN, BLASTX, TBLASTX, and FASTA. The BLAST programs are publicly available from NCBI and other sources. For example, nucleotide sequence identity can be determined by comparing query sequences to sequences in publicly available sequence databases (NCBI) using the BLASTN2 algorithm.
The nucleic acid molecules exemplified herein encode polypeptides with amino acid sequences represented herein. In certain embodiments, the polypeptides may be at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the reference amino acid sequence while possessing the enzymatic function. The present disclosure encompasses bacterial cells such as A. succinogenes cells that contain the nucleic acid molecules described herein, have genetic modifications to the nucleic acid molecules, or express the polypeptides described herein.
“Nucleic acid” or “polynucleotide” as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotide or mixed polyribo-polydeoxyribonucleotides. This includes single- and double-stranded molecules (i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids) as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases.
Nucleic acids referred to herein as “isolated” are nucleic acids that have been removed from their natural milieu or separated away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library), and may have undergone further processing. Isolated nucleic acids include nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acids that are isolated. In certain embodiments, the nucleic acids are complementary DNA (cDNA) molecules.
The nucleic acids described herein may be used in methods for production of organic acids (e.g., succinic acid) through incorporation into cells, tissues, or organisms. In some embodiments, a nucleic acid may be incorporated into a vector for expression in suitable host cells. Alternatively, gene-targeting or gene-deletion vectors may also be used to disrupt or ablate a gene. The vector may then be introduced into one or more host cells by any method known in the art. One method to produce an encoded protein includes transforming a host cell with one or more recombinant nucleic acids (such as expression vectors) to form a recombinant cell. The term “transformation” is generally used herein to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell, but can be used interchangeably with the term “transfection.”
Suitable vectors for gene expression may include (or may be derived from) plasmid vectors that are well known in the art, such as those commonly available from commercial sources. Vectors can contain one or more replication and inheritance systems for cloning or expression, one or more markers for selection in the host, and one or more expression cassettes. The inserted coding sequences can be synthesized by standard methods, isolated from natural sources, or prepared as hybrids. Ligation of the coding sequences to transcriptional regulatory elements or to other amino acid encoding sequences can be carried out using established methods. A large number of vectors, including algal, bacterial, yeast, and mammalian vectors, have been described for replication and/or expression in various host cells or cell-free systems, and may be used with genes encoding the enzymes described herein for simple cloning or protein expression.
Certain embodiments may employ promoters or regulatory operons. The efficiency of expression may be enhanced by the inclusion of enhancers that are appropriate for the particular cell system that is used, such as those described in the literature. Suitable promoters also include inducible promoters. Expression systems for constitutive expression in bacterial cells are available from commercial sources. Inducible expression systems are also suitable for use.
Host cells can be transformed, transfected, or infected as appropriate with gene-disrupting constructs or plasmids (e.g., an expression plasmid) by any suitable method including electroporation, calcium chloride-, lithium chloride-, lithium acetate/polyethylene glycol-, calcium phosphate-, DEAE-dextran-, liposome-mediated DNA uptake, spheroplasting, injection, microinjection, microprojectile bombardment, phage infection, viral infection, or other established methods. Alternatively, vectors containing a nucleic acid of interest can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, for example, by injection. Exemplary embodiments include a host cell or population of cells expressing one or more nucleic acid molecules or expression vectors described herein (for example, a genetically modified microorganism). The cells into which nucleic acids have been introduced as described above also include the progeny of such cells.
Vectors may be introduced into host cells by direct transformation, in which DNA is mixed with the cells and taken up without any additional manipulation, by conjugation, electroporation, or other means known in the art. Expression vectors may be expressed by host cells episomally or the gene of interest may be inserted into the chromosome of the host cell to produce cells that stably express the gene with or without the need for selective pressure. For example, expression cassettes may be targeted to neutral chromosomal sites by double recombination.
Host cells with targeted gene disruptions or carrying an expression vector (i.e., transformants or clones) may be selected using markers depending on the mode of the vector construction. The marker may be on the same or a different DNA molecule. In prokaryotic hosts, the transformant may be selected, for example, by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker.
In exemplary embodiments, the host cell may be a microbial cell, such as a bacterial cell, and may be from any genera or species of bacteria that is known to be genetically manipulable. Exemplary microorganisms include, but are not limited to, bacteria; fungi; archaea; protists; eukaryotes, such as algae; and animals such as plankton, planarian, and amoeba.
Host cells may be cultured in an appropriate fermentation medium. An appropriate, or effective, fermentation medium refers to any medium in which a host cell, including a genetically modified microorganism, when cultured, is capable of growing and/or expresing recombinant proteins. Such a medium is typically an aqueous medium comprising assimilable carbon, nitrogen and phosphate sources, but can also include appropriate salts, minerals, metals and other nutrients. Microorganisms and other cells can be cultured in conventional fermentation bioreactors or photobioreactors and by any fermentation process, including batch, fed-batch, cell recycle, and continuous fermentation. The pH of the fermentation medium is regulated to a pH suitable for growth of the particular organism. Culture media and conditions for various host cells are known in the art. A wide range of media for culturing bacterial cells, for example, are available from ATCC.
Isolation or extraction of succinic acid from the cells may be aided by mechanical processes such as crushing, for example, with an expeller or press, by supercritical fluid extraction, pH-induced precipitation, or the like. Once the succinic acid has been released from the cells, it can be recovered or separated from a slurry of debris material (such as cellular residue, by-products, etc.). This can be done, for example, using techniques such as sedimentation or centrifugation. Recovered succinic acid can be collected and directed to a conversion process if desired.
Processes for producing succinic acid may also comprise a removing step, wherein a portion of the succinic acid is removed from the mixture to form substantially pure succinic acid. In some embodiments, the removing step may comprise a two-step process, wherein the first step comprises an adsorption process wherein the succinic acid is not adsorbed, and other components are selectively adsorbed. In some embodiments of the present invention, a first step comprising adsorption will result in a substantially pure succinic acid stream.
The first step may be followed by a second polishing step. The second step may comprise crystallization. In still further embodiments of the present invention, the removing step is at least one of affinity chromatography, ion exchange chromatography, solvent extraction, liquid-liquid extraction, distillation, filtration, centrifugation, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, chromatofocusing, differential solubilization, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, or countercurrent distribution, or combinations thereof. In some embodiments of the present invention, the removing step may be performed either during the mixing step or subsequent to the mixing step or both.
Separation/purification operations may be used to generate succinic acid free of or substantially free of a wide variety of impurities that may be introduced during the biological production of the succinic acid. These impurities may include fermentation salts, nutrients and media to support growth, unconverted substrate, extracellular proteins and lysed cell contents, as well as the buildup of non-target metabolites. Accumulation of these constituents in culture broth may vary greatly depending on the microorganism, substrate used for conversion, biological growth conditions and bioreactor design, and broth pretreatment.
Cell removal from the broth may be achieved by a variety of solid removal unit operations. Some examples include filtration, centrifugation, and combinations thereof. Once the microorganism cell matter has been removed, further impurity removal operations may be utilized such as exposing the mixture to activated carbon.
Examples of biomass and other lignocellulosic-containing materials that may be degraded to sugars for use as carbon sources include bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood, forestry waste, corn grain, corn cobs, crop residues such as corn husks, corn stover, corn fiber, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood (e.g., poplar) chips, sawdust, shrubs and bushes, vegetables, fruits, flowers and animal manure.
Biomass or other lignocellulosic feedstocks may be subjected to pretreatment at an elevated temperature in the presence of a dilute acid, concentrated acid or dilute alkali solution for a time sufficient to at least partially hydrolyze the hemicellulose components before adding enzymes capable of producing carbohydrates such as sugars from the lignocellulose. Additional suitable pretreatment regimens include ammonia fiber expansion (AFEX), treatment with hot water or steam, or lime pretreatment. Following any pretreatment steps, lignocellulose may be subjected to saccharification by contacting the lignocellulose with enzymes such as cellulases, endoglucanases, β-glucosidases and other enzymes to hydrolyze the lignocellulose to sugars.
Core-Carbon Metabolic Model of A. succinogenes 130Z
A core-carbon metabolic model of A. succinogenes 130Z was created following the genome annotation provided in McKinlay et al., BMC Genomics 11:680 (2010). The model consists of 51 intracellular metabolites, 20 extracellular metabolites, and 89 mass-balanced reactions. Pathways for the metabolism of glucose, xylose, galactose, and arabinose were included based on annotated pathways and stoichiometry from the MetaCyc database. The model was used to predict metabolite fluxes, including NADH and CO2. By condensing the model to a number of balanced reactions that produce the observed fermentation products, the amount of CO2 and NADH absorbed and consumed by the creation of each product was found. This accounting allowed the overall mass balance (
Actinobacillus succinogenes 130Z (ATCC 55618) was anaerobically cultivated in sterile capped bottles (100 mL) containing 50 mL of Tryptic Soy Broth (TSB) supplemented with 10 g/L glucose (TSBG; Fluka Analytical, India) and incubated overnight at 37° C. and 120 rpm. Cells were harvested by centrifugation (Sorvall) then resuspended in 5 mL TSB and 5 mL glycerol, aliquoted in cryovials, and stored at −70° C. Prior to the inoculum preparation, bacteria were revived from the glycerol stock at the same conditions detailed above. Bacterial growth was followed by optical density measurements at 600 nm (OD600). Serum bottle cultures were inoculated at an OD600 of 0.1 with plate-harvested biomass.
A. succinogenes strains were anaerobically grown in 150 mL sterile capped bottles containing 50 mL of TSBG (TSB+glucose) and antibiotic (30 mg/L chloramphenicol or 100 mg/L ampicillin, as indicated in Table 2) (excluding the control strain, 130Z), and incubated overnight at 37° C. and 200 rpm. Cells were inoculated in the fermenter at an initial OD600 of 0.05. To ensure anaerobic fermentation, CO2 was sparged overnight before bacterial inoculation.
The media used for fermentations consisted of 6 g yeast extract, 10 g corn steep liquor (Sigma-Aldrich, USA) (prepared as described below), 0.3 g Na2HPO4, 1.4 g NaH2PO4, 1.5 g K2HPO4, 1 g NaCl, 0.2 g MgCl2×6H2O, and 0.2 g CaCl2×2H2O. Corn steep liquor was prepared at a concentration of 200 g/L (20×) and then boiled at 105° C. for 15 minutes. After cooling, solids were separated and the supernatant was autoclaved and used as nutrient source. As a carbon source, a mixture of sugars mimicking the concentration in real biomass hydrolysates (in particular deacetylated and diluted acid pretreated hydrolysate (DDAPH) was utilized. The final sugar concentration was 60 g/L and consisted of 6.5 g/L glucose, 44 g/L xylose, 3.5 g/L galactose, and 6.5 g/L arabinose. In addition, acetic acid (1.7 g/L) was supplemented to the media since DDAPH contains this acid. Appropriate antibiotic was added to the fermentor prior to inoculation.
Fermentations were performed in 0.5-L BioStat-Q Plus fermentors (Sartorious) with 300 mL of media. The pH was maintained at 6.8 by supplementing 4N NaOH. The temperature was controlled at 37° C. and the agitation at 300 rpm. During the fermentation, CO2 was sparged at 0.1 vvm. Samples (about 1 mL) from the fermentations were taken in aseptic conditions at various time points to follow bacterial growth, sugar consumption, and the production or uptake of acids (e.g., SA, formic acid, acetic acid, lactic acid, pyruvic acid) or ethanol. All the fermentations were performed at least in duplicate.
Bacterial growth was tracked by OD600 in a spectrophotometer, as a measurement of cells in suspension in the fermentation broth. Growth rates (μ−1) were calculated from the maximum growth slope before cell aggregation occurred. Samples were then filtered through a 0.2 μm syringe filter before placing them in high pressure liquid chromatography (HPLC) vials to analyze carbohydrates (glucose, xylose, arabinose, and galactose), organic acids (SA, formic acid, acetic acid, lactic acid, pyruvic acid), and ethanol. Carbohydrate HPLC analysis was performed by injecting 6.0 μL of 0.2 μm filtered culture supernatant onto an Agilent 1100 series system equipped with a Phenomenex Shodex SUGAR 7u SP0810 20A Column 300 mm×8 mm plus an anion guard column and cation guard column (Bio-Rad Laboratories) at 85° C. run using a mobile phase of Nanopure water at a flow rate of 0.6 mL/min and a refractive index detector for detection.
HPLC analyses for ethanol and all organic acids, except for pyruvic acid, were performed by injecting 6.0 μL of 0.2 μm filtered culture supernatant onto an Agilent 1100 series system equipped with a Bio-Rad HPLC Organic Acid Analysis Column, Aminex HPX-87H Ion Exclusion Column 300 mm×7.8 mm and a cation H+ guard column (Bio-Rad Laboratories) at 55° C. using a mobile phase of 0.01 N sulfuric acid at a flow rate of 0.6 mL/min and a refractive index detector for detection. Pyruvic acid HPLC analysis was performed by injecting 6.0 μL of 0.2 μm filtered culture supernatant onto an Agilent 1100 series system equipped with a Phenomenex Rezex RFQ-Fast Acid H+ (8%) Column 100 mm×7.8 mm and a cation guard cartridge (Bio-Rad Laboratories) at 85° C. using a mobile phase of 0.01 N sulfuric acid at a flow rate of 1.0 mL/min and a diode array detector at 315 nm for detection. Analytes were identified by comparing retention times and spectral profiles with pure standards.
Succinic acid (SA) yield is calculated as the ratio of SA (g) and total sugar consumption (g) at the end of the fermentation. Metabolic yield is calculated similarly to SA yield but correcting substrate and product concentrations with the dilution produced from base addition. Compensation was also made for the removal of substrate and products via sampling (considering 1 mL as sample volume). SA titer (g/L) is the SA concentration at the end of the fermentation and values are not corrected by the dilution factor. Overall productivity (g/L/h) is calculated as SA production (g/L) divided by the time (h) at the end of the fermentation. The end of the fermentation is considered when total sugar concentration is close to zero. Maximum instantaneous productivity (g/L/h) is the maximum productivity peak value observed during the fermentation course.
Q5 Hot Start High-Fidelity Polymerase and 2× Master Mix (New England Biolabs, MA) and primers were used in all PCR amplification for plasmid construction. Plasmids were constructed using restriction enzyme subcloning or Gibson Assembly Master Mix (New England Biolabs, MA) following the manufacturer's instructions. Plasmids were transformed into competent Zymo 5-alpha E. coli (Zymo Research, CA) following the manufacturer's instructions or electroporation into A. succinogenes. Transformants were selected on LB or TSB plates containing 10 g/L glucose, supplemented with either 50 μg/mL chloramphenicol or 50 μg/mL ampicillin and grown at 37° C. The sequences of all plasmid inserts were confirmed using Sanger sequencing.
Construction of Marker-Less Knockouts in A. succinogenes 130Z
To knock out pflB and ackA in the genome of 130Z via homologous recombination, a knockout cassette was constructed (
Construction of Gene Overexpression Strains of A. succinogenes 130Z
All plasmids were constructed based on an E. coli—A. succinogenes shuttle vector pLGZ920 (ATCC PTA-6140 and its derivative pL920Cm (
Electroporation of A. succinogenes
Plasmids and/or linear DNA cassettes were transformed into A. succinogenes via electroporation using Gene Pulser Xcell (Bio-Rad, CA). Electro-competent cells were prepared by harvesting the exponential growth phase cells (OD600=0.4-0.5) and centrifuging at 4° C., 3300×g for 10 minutes. Cells were washed twice in ½ volume of ice-cold 15% glycerol and concentrated 100× to 150× before electroporation. One hundred microliters of competent cells were mixed with 2-15 μL DNA in a 0.2-cm gap cuvette and electroporated at 2.5 kV, 25 μF and 600 ohms. One-milliliter TSBG was added to the cuvette after electroporation. Cells were transferred to a microtube and incubated at 37° C. for one hour before plating on TSBG agar plates supplemented with appropriate antibiotics. Plates were incubated at 37° C. for 1 to 2 days or until colonies were observed.
While prior metabolic engineering efforts in A. succinogenes have employed a positive selection strategy that leveraged the organism's glutamate auxotrophy, an alternative set of positive selection tools were developed via the conventional utilization of antibiotic resistance markers. Electroporation of A. succinogenes with linear PCR fragments containing genomic homology regions flanking an antibiotic resistance marker enabled homologous recombination-mediated chromosomal integration and gene disruption (
Wild-type A. succinogenes splits carbon flux between two main fermentative pathways: a SA producing, reductive C4 pathway, and a number of oxidative C3 pathways that produce acetic acid, formic acid, and ethanol as byproducts. As SA is a highly reduced product, biosynthesis of these additional compounds serves in part to generate cellular energy and reducing power. However, from a process perspective, these byproducts also serve as competitive carbon sinks, potentially reducing carbon yields and/or flux to SA. Additionally, the presence of organic acids other than SA reduces separation efficiency and product purity in downstream recovery of SA from fermentation broth, serving as a negative techno-economic cost driver.
To examine the effects of removal of competitive carbon pathways in A. succinogenes, the targeted knockout of genetic components encoding biosynthetic machinery of C3 pathway products acetic and formic acid was conducted. Pyruvate formate lyase (pflB), which catalyzes the reversible conversion of pyruvate and coenzyme-A into formic acid and acetyl-CoA, and acetate kinase (ackA), which catalyzes conversion of acetyl-phosphate and ADP to acetate and ATP (
Wild-type and mutant A. succinogenes strains were cultivated on mock biomass hydrolysate containing a 60 g/L total sugars stream rich in xylose as a carbon source over a 96-hour batch fermentation.
Both single and double mutants displayed a growth defect, demonstrating an additional 9-hour lag phase relative to wild-type (
Although overall productivity was similar between ΔpflB and wild-type strains, the maximum instantaneous productivity was lower in ΔpflB (
Despite delayed growth and onset of SA biosynthesis, effective reduction in acetate and formate was observed in all three mutants (
Additional pyruvate accumulation was observed in both ΔackA and ΔpflB single mutants, indicating a flux bottleneck upon removal of the acetate and formate carbon sinks, respectively (
As an alternative means to enhance flux to SA, overexpression of SA biosynthetic machinery was examined. Wild-type A. succinogenes channels carbon through the reductive branch of the TCA cycle (
Initially, three single gene overexpression strains were compared. The three strains, overexpressing PCK (pPCK,
In addition to enhanced SA titers, all three overexpression strains also exhibit higher final titers of acetic acid relative to wild-type (
Titer and yield enhancements were observed for all overexpression strains (
The effects of incorporating pPMF overexpression into three mutant backgrounds (ΔpflB, ΔackA, and ΔpflBΔackA) was investigated. pPMF was selected for further combinatorial engineering to ensure maximum flux through the reductive branch of the pathway in combination with knockouts in the oxidative branch.
As with the above described knockout mutant strains (
The acetic acid production rate lagged in the mutant backgrounds, as observed for mutants in the absence of MDH overexpression. Acetic acid accumulation was again observed in all ackA mutant backgrounds, further supporting an alternative route to acetic acid in A. succinogenes (
Final SA titers were enhanced (4%) in the ΔpflB/pPMF strain relative to wild-type, with minimal impact on yield (
Strains of A. succinogenes were also engineered to overexpress either phosphoenolpyruvate carboxykinase (pPCK) or fumarase (pFUM).
Strains of A. succinogenes were also engineered to overexpress phosphoglucose dehydrogenase in wild type strains (pGDH) or in strains where pyruvate formate lyase has also been knocked out (ΔpflBpGDH).
The Examples discussed above are provided for purposes of illustration and are not intended to be limiting. Still other embodiments and modifications are also contemplated.
While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.
This application claims priority to U.S. Provisional Application No. 62/326,895, filed Apr. 25, 2016, the contents of which are incorporated by reference in their entirety.
The United States Government has rights in this invention under Contract No. DE-AC36-08GO28308 between the United States Department of Energy and Alliance for Sustainable Energy, LLC, the Manager and Operator of the National Renewable Energy Laboratory.
| Number | Date | Country | |
|---|---|---|---|
| 62326895 | Apr 2016 | US |
