Patent Application
20230123501
The present invention relates to methods of metabolic engineering a microbial host to synthesize chemicals from oil palm lignin. In particular, the invention relates to the production of adipic acid and levulinic acid from lignocellulose biomass in engineered Escherichia coli, and also provides recombinant cells made using such methods.
Lignocellulosic biomass is the most abundant renewable resource [Vardon et al., Energy & Environmental Science 8: 617-628 (2015); Deng et al., Biochemical Engineering Journal 105: 16-26 (2016)]. In particular, oil palm empty fruit bunches (OPEFBs), by-products products of palm oil production, are abundant lignocellulosic biomass largely burned for energy and regarded as waste. It is estimated that 1.1 tons of OPEFBs are generated per ton of oil palm produced, which amounts to 57 million tons annually [Murphy, Journal of Oil Palm Research 26: 1-24 (2014); Coral Medina et al., Bioresource Technology 194: 172-178 (2015)]. Given their availability in large quantities, OPEFBs are an attractive renewable lignocellulosic source that can serve as feedstock in biorefineries to produce value-added products. OPEFB can be converted to fermentable sugars [Li et al., Biotechnology and Applied Biochemistry 61: 426-431 (2014)] and lignin extract [Mohamad Ibrahim et al., CLEAN—Soil, Air, Water 36: 287-291 (2008)] through simple, cost-effective pre-treatments using chemicals and heat.
The use of OPEFB-derived fermentable sugars to support cell growth was explored in 2014 by Li et al., [Li et al., Biotechnology and Applied Biochemistry 61: 426-431 (2014)] who combined the use of dilute acid and whole fungal cell culture-catalyzed hydrolysis to extract fermentable sugars from OPEFBs. Hemicellulose was first stripped off from OPEFBs using acid hydrolysis, and the remaining cellulose-lignin complex was converted to glucose by the cellulases in the whole fungal cell culture. The OPEFB-derived sugars were subsequently used as a carbon source to cultivate Escherichia coli (E. coli) as a proof of concept. The use of OPEFB-derived lignin was explored in 2008 by Mohamad Ibrahim et al., where lignin was extracted from OPEFBs using 20% sulfuric acid, followed by nitrobenzene oxidation to break down the lignin. This extraction process releases a plethora of depolymerized lignin compounds, notably vanillin, p-coumaric acid, p-hydroxybenzaldehyde, vanillic acid, p-hydroxybenzoic acid and ferulic acid [Xu et al., ChemSusChem 5: 667-675 (2012)]. Vanillin and p-coumaric acid were the predominant degradation products, with concentrations of ˜1800 ppm (1.8 g/L) and ˜1000 ppm (1.0 g/L), respectively, [Mohamad Ibrahim et al., CLEAN—Soil, Air, Water 36: 287-291 (2008)]. These compounds are useful substrates for the production of commercially important organic acids. Taken together, the reported studies suggest that OPEFB derivatives can be potentially exploited for biorefinery processes, while microbial cells can be engineered to convert aromatic compounds into commodity chemicals while utilizing fermentable sugars for cell growth (
Adipic acid is a high-demand commodity chemical that is used as a lubricant and as a precursor for nylon 6,6, polyester polyols and plasticizers [Vardon et al., Energy & Envronmental Science 8: 617-628 (2015)]. It has a market volume of 2.6 million tons per year [Polen et al., J Biotechnol 167: 75-84 (2013)] and was valued at USD 5.56 billion in 2016 [Research, Adipic Acid Market Size, Share & Trends Analysis Report by Application (Nylon 66 Fiber, Nylon 66 Resin, Polyurethane, Adipate Ester), By Region (APAC, North America, Europe, MEA, CSA), and Segment Forecasts, 2018-2024 (2018), worldwidewebdotgrandviewresearchdotcom/industry-analysis/adipic-acid-market]. On the other hand, levulinic acid had a market value of USD 164 million in 2020 [MarketWatch, Levulinic Acid Market Size 2020: Top Countries Data, Definition, Detailed Analysis of Current Industry Figures with Forecasts Growth By 2026 (2020), worldwidewebdotmarketwatchdotcom/press-release/levulinic-acid-market-size-2020-top-countries-data-definition-detailed-analysis-of-current-industry-figures-with-forecasts-growth-by-2026-2020-07-13] and is a versatile chemical having roles in industrial products such as resins, plasticizers, textiles, animal feed, coatings, and as an antifreeze [Ghorpade and Hanna, Cereals: Novel Uses and Processes, eds. G. M. Campbell, C. Webb & S. L. Mckee. (Boston, Mass.: Springer 433 US), 49-55 (1997)].
There is a need for improved economics of exploiting OPEFB and its under-utilized lignin fraction to produce commodity chemicals.
Herein, a microbial-based bioprocess was devised that directly utilized unfractionated depolymerized OPEFB lignin as a substrate for commodity chemical production (
Herein, the inventors have identified and constructed a metabolic pathway consisting of 9 enzymes (11 genes) that will enable E. coli to utilize all 6 constituents of depolymerized lignin to produce a versatile precursor molecule, β-ketoadipate, via the β-ketoadipic acid pathway [Wells and Ragauskas, Trends Biotechnol 30: 627-637 (2012)], and subsequently convert this intermediate into various commercially important derivatives such as adipic acid (reduction) and levulinic acid (decarboxylation).
To further improve the bioconversion and simplify the bioprocess, E. coli cells were engineered to have a regulatory element that functions as a dynamic sensor-based genetic controller. In engineered cells, the expression of enzymatic pathway genes is commonly controlled by an induction system, where an artificial inducer is used to activate enzyme expression. The use of such inducers, although effective, is less favorable due to their high cost and toxicity and the corresponding increased bioprocess complexity. To this end, a two-layer genetic controller [Lo et al., Cell Syst 3: 133-143 (2016)] was employed that regulated the enzyme expression and hence bioconversion based on the availability of nutrient and OPEFB lignin derivatives. This enabled the engineered E. coli to autonomously activate the bioconversion process when the substrates are available without the need for additional inducer.
Biosynthesis of commodity chemicals using this intermediate was also demonstrated, where up to 9.5 mg/L adipic acid and 455.57 mg/L levulinic acid were produced from the reconstituted OPEFB lignin cocktail under fermenter-controlled conditions.
The microbial host, E. coli MG 1655 was also subjected to strain optimization, where native E. coli genes that are involved in competing metabolic pathways were systematically deleted to improve bioproduction yield. Deletion of sucCD and atoDA resulted in the greatest improvement for adipic acid and levulinic acid production, respectively.
The disclosure relates to a platform using E. coli for bioproduction of commodity chemicals from OPEFB lignin.
Broadly, the platform may use E. coli MG 1655 and comprise;
According to a first aspect, the present invention provides an isolated genetically engineered microorganism for producing β-ketoadipic acid from depolymerized lignin, wherein the microorganism has been transformed by at least one polynucleotide molecule; the at least one polynucleotide molecule comprising heterologous β-ketoadipic acid pathway genes, feruloyl-CoA synthetase (fcs), enoyl-CoA hydratase (ech), vanillin dehydrogenase (vdh), vanillate O-demethylase (vanAB; vanA and vanB), p-hydroxybenzoate hydroxylase (pobA), protocatechuate 3,4-dioxygenase (pcaGH; pcaG and pcaH), 3-carboxy-cis,cis-muconate cycloisomerase (pcaB), 4-carboxymuconolactone decarboxylase (pcaC) and p-ketoadipate enol-lactone hydrolase (pcaD) operably linked to at least one promoter, wherein said genetically engineered microorganism can convert depolymerized lignin to β-ketoadipic acid.
In some embodiments, the isolated genetically engineered microorganism further comprises:
In some embodiments:
An asterisk at the C-terminal end of the sequence denotes a stop or termination codon.
In some embodiments:
It would be understood that specific pathway genes described herein may be substituted by related genes encoding enzymes with equivalent catalytic function. It would also be understood that the nucleic acid sequence of genes used in the pathways of the invention may be codon optimized for the particular engineered host cell.
In some embodiments, the at least one promoter is modulated by a heterologous genetic controller. In some embodiments the at least one promoter is a constitutive promoter, such as T7. It would be understood that there are other promoters which may be suitable for the instant invention.
In some embodiments, the heterologous genetic controller is pBAD or hydroxycinnamic acid (HA). In some embodiments the pBAD controller comprises the nucleotide sequence set forth in SEQ ID NO: 41. In some embodiments the HA controller comprises the nucleotide sequence set forth in SEQ ID NO: 42.
In some embodiments, the isolated genetically engineered microorganism according to any aspect of the invention, further comprises an inactivated endogenous succinyl CoA synthetase gene, such as sucCD and/or an inactivated β-ketoadipyl-CoA thiolase gene, such as paaJ. In some embodiments the sucCD gene encodes an amino acid sequence set forth in SEQ ID NO: 54 and SEQ ID NO: 56 (SucC and SucD, respectively). In some embodiments the sucCD gene comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence set forth in SEQ ID NO: 53 and SEQ ID NO: 55 (sucC and sucD, respectively). In some embodiments the paaJ gene encodes an amino acid sequence set forth in SEQ ID NO: 52. In some embodiments the paaJ gene comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence set forth in SEQ ID NO: 51.
In some embodiments, the isolated genetically engineered microorganism according to any aspect of the invention, further comprises an inactivated endogenous Acyl CoA:acetate/3-ketoacid CoA transferase gene, such as atoDA. In some embodiments the atoDA gene encodes an amino acid sequence set forth in SEQ ID NO: 48 and SEQ ID NO: 50 (AtoD and AtoA, respectively). In some embodiments the atoDA gene comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95% sequence identity or 100% sequence identity to the polynucleotide sequence set forth in SEQ ID NO: 47 and SEQ ID NO: 49 (atoD and atoA, respectively).
In some embodiments, the isolated genetically engineered microorganism comprises a bacteria or yeast, preferably a bacteria such as Escherichia coli. In some embodiments the bacteria is Escherichia coli MG1655.
In some embodiments, the depolymerized lignin is from fibrous oil palm empty fruit bunches.
According to another aspect, the present invention provides the use of the isolated genetically engineered microorganism according to any aspect of the invention for the production of adipic acid, or for the production of levulinic acid.
According to another aspect, the present invention provides a recombinant vector comprising heterologous β-ketoadipic acid pathway genes, fcs, ech, vdh, vanAB (vanA and vanB), pobA, pcaGH (pcaG and pcaH), pcaB, pcaC and pcaD operably linked to at least one promoter, and/or
In some embodiments:
In some embodiments:
According to another aspect, the present invention provides a kit comprising an isolated genetically engineered microorganism according to any aspect of the invention, or a recombinant vector of any aspect of the invention.
According to another aspect, the present invention provides a method of producing β-ketoadipic acid from depolymerized lignin, comprising the step of culturing a plurality of genetically engineered microorganisms of any aspect of the invention under conditions for production of said β-ketoadipic acid.
According to another aspect, the present invention provides a method of producing adipic acid from depolymerized lignin, comprising the step of culturing a plurality of genetically engineered microorganisms of any aspect of the invention under conditions for production of said adipic acid.
According to another aspect, the present invention provides a method of producing levulinic acid from depolymerized lignin, comprising the step of culturing a plurality of genetically engineered microorganisms of any aspect of the invention under conditions for production of said levulinic acid.
In some embodiments, the method of any aspect of the invention further comprises isolating said product produced by the genetically engineered microorganisms.
In some embodiments, the microorganism comprises a bacterium, such as Escherichia coli, preferably Escherichia coli MG1655.
In some embodiments of the production methods of the invention, the depolymerized lignin is from fibrous oil palm empty fruit bunches.
The platform E. coli strain enables direct utilization of depolymerized lignin cocktail without fractionating into individual constituents. Moreover, in some embodiments the use of a genetic controller allows autonomous induction of gene expression, which reduces the cost of artificial inducers such as IPTG that is commonly used. An advantage of the platform is that it is customizable, where other pathways that can convert the precursor, β-ketoadipate, can be easily implemented for a chemical of interest.
Certain terms employed in the specification, examples and appended claims are collected here for convenience.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise.
As used herein, the term “comprising” or “including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. However, in context with the present disclosure, the term “comprising” or “including” also includes “consisting of”. The variations of the word “comprising”, such as “comprise” and “comprises”, and “including”, such as “include” and “includes”, have correspondingly varied meanings.
The term “isolated” is herein defined as a biological component (such as a nucleic acid, peptide or protein) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins which have been isolated thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
The terms “nucleotide”, “nucleic acid” or “nucleic acid sequence”, as used herein, refer to an oligonucleotide, polynucleotide, or any fragment thereof, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
Several of the pathway enzymes comprise 2 subunits that are encoded by 2 separate genes. As used herein, the two subunit genes may be referred to as, for example, sucCD or alternatively separately as sucC and sucD. Similarly, the pathway enzymes may be referred to by their name, such as succinyl CoA synthetase or SucCD. In the present disclosure it would be understood that if an enzyme is to be inactivated, the inactivation may be achieved by various means including, for example, deletion of one or more genes encoding the enzyme subunits, or mutation of the gene coding sequence to produce inactive truncated or nonsense peptides.
As used herein, the term “operably linked” means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions. For example, a control sequence which is “operably linked” to a protein coding sequence is ligated thereto, so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences. By way of an example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
The terms “amino acid” or “amino acid sequence,” as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
As used herein, the terms “polypeptide”, “peptide” or “protein” refer to one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or peptide can comprise a plurality of chains noncovalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. A “polypeptide”, “peptide” or “protein” can comprise one (termed “a monomer”) or a plurality (termed “a multimer”) of amino acid chains.
Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference. Any discussion about prior art is not an admission that the prior art is part of the common general knowledge in the field of the invention.
A vector can include one or more catalytic enzyme nucleic acid(s) in a form suitable for expression of the nucleic acid(s) in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence(s) to be expressed. The term “regulatory sequence” includes promoters, enhancers, ribosome binding sites and/or IRES elements, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence such as the T7 promoter disclosed in the Examples herein. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., catalytic enzyme proteins).
The recombinant expression vectors of the invention can be designed for expression of catalytic enzyme proteins in prokaryotic or eukaryotic cells, more particularly prokaryotic cells. For example, polypeptides of the invention can be expressed in bacteria (e.g., cyanobacteria) or yeast cells. Suitable host cells are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif.
The methods described hereinbefore make use of enzymes to catalyse a sequence of reactions. While these reactions may be performed individually or, more particularly, two or more of them in combination, it is particularly preferred that all of the reactions are combined into a cascade reaction sequence that provides the product from the initial starting material in one pot, thereby eliminating the need for isolation of the intermediates and, potentially, increasing the overall yield of the reaction sequence.
The engineered cells of the invention further comprise inactivated genes to limit the utilization by the host cell of intermediate compounds in other biosynthetic pathways and reducing yield of the target final products. The engineered cells may comprise an inactivated endogenous succinyl CoA synthetase gene, such as sucCD (sucC, SEQ ID NO: 53 and sucD, SEQ ID NO: 55) and/or an inactivated β-ketoadipyl-CoA thiolase gene, such as paaJ, (SEQ ID NO: 51) if adipic acid is the intended product, or an inactivated endogenous Acyl CoA:acetate/3-ketoacid CoA transferase gene, such as atoDA (atoD, SEQ ID NO: 47 and atoA, SEQ ID NO: 49), if levulinic acid is the intended target product.
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.
A person skilled in the art will appreciate that the present invention may be practiced without undue experimentation according to the methods given herein. The methods, techniques and chemicals are as described in the references given or from protocols in standard biotechnology and molecular biology textbooks.
The plasmid backbones used in this study were the BgIBrick vectors [Lee et al., Journal of Biological Engineering 5: 12 (2011)] pBbE8k and pBbE8a from the Joint BioEnergy Institute, U.S.A. Cloning and modification of DNA parts such as promoters, genes, and terminators required the use of the splicing overlap extension (SOE) technique [Heckman and Pease, Nature Protocols 2: 924-932 (2007)]. Biological parts were either PCR-cloned from genomic templates of P. putida KT2440 and E. coli K-12 MG1655 or SOE-assembled using gene fragments (gBlocks) from Integrated DNA Technologies, U.S.A. They were converted into the BgIBrick standard, which was comprised of universal linkers such as EcoRI, BgIII, BamHI and Xhol restriction sites for assembly. The standard BgIBrick assembly method, described by Anderson et al. (2010) [Anderson et al., Journal of Biological Engineering 4: 1 (2010)], was used to assemble the genetic constructs listed in
E. coli K-12
E. coli BL21(DE3) was transformed with each plasmid bearing one of the PcaI-, PcaJ-, PaaH-, Ech-, egTer-, tdTer-, Ptb-, or Buk1-encoding genes in either pBbE8k or pACYCDuet-1 (Novagen, Germany). Each gene was from P. putida KT2440 (PcaI and PcaJ), Ralstonia eutropha (PaaH1), R. eutropha H16 (Ech), Euglena gracilis (egTer), Treponema denticola (TdTer) or Clostridium acetobutylicum (Ptb and Buk1). The seed cultures for the transformants were prepared by overnight cultivation in LB medium supplemented with the appropriate antibiotic (30 μg/L kanamycin or 50 μg/L ampicillin) at 37° C. and 225 rpm. The seed cultures were diluted 1:100 (v/v) into Terrific Broth medium supplemented with appropriate antibiotics (30 μg/L kanamycin or 50 μg/L ampicillin) and cultivated at 37° C. and 225 rpm. The diluted E. coli cultures were induced with 0.1 mM IPTG at OD600 0.5-1.0 and cultivated at 16° C. and 225 rpm for 24 h. The cultures were harvested and resuspended with 0.5 mL of lysis buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM DTT, and 10% (v/v) glycerol, pH 7.5, final concentrations) and incubated at 25° C. and 150 rpm for 1 h with 1.5 mg/mL lysozyme. After the addition of 0.1% Triton X-100 and 1×protease inhibitor (Promega), the soluble fraction of crude cell extract was prepared by using FastPrep-24™ 5G (MP Biomedicals) and acid-washed beads (≤106 μm) (Sigma-Aldrich) at 6.5 m/s and 45 s, followed by centrifugation at 4° C. and 13,000 rpm for 10 min. Total proteins in the soluble extracts were manually quantified by using Bradford reagent (Sigma-Aldrich). The overexpression of each gene was verified by SDS-PAGE.
The activities of β-ketoadipic acid succinyl-CoA transferase (subunit, PcaI and subunit, PcaJ) were determined as described previously [MacLean et al., Appl Environ Microbiol 72: 5403-5413 (2006)], incorporated herein by reference, with slight modifications. Briefly, the reaction was started with the addition of a reaction mixture (200 mM Tris-HCl, 0.4 mM succinyl-CoA, 40 mM MgSO4, and 1 g/L β-ketoadipic acid, pH 8.0, final concentrations) to aliquots of cell extracts to a final volume of 0.1 mL. The formation of β-ketoadipyl-CoA:Mg2+ was monitored at 305 nm and 30° C. for 4 min by using a Biotek Synergy H1m microplate reader.
In vitro adipate production was performed as described previously [Yu et al., Biotechnol Bioeng 111: 2580-2586 (2014)], incorporated herein by reference, with the following modifications. Each cell extract (equivalent to 0.05 mg of total protein) for PcaI, PcaJ, PaaH1, Ech, Ter (either egTer or tdTer), Ptb, and Buk1 was added into a reaction mixture (50 mM potassium phosphate buffer, 0.4 mM succinyl-CoA, 4 mM NADH, 2 mM ADP, and 0.5 g/L β-ketoadipic acid, pH 7.0, final concentrations) to a final volume of 0.2 mL and incubated for 24 h at room temperature. Subsequently, each sample was mixed with 0.2 mL of 1 M HCl and internal standard (1,14-tetradecanedioic acid) to a volume of 0.5 mL and vortexed thoroughly for 30 s. After the addition of 0.5 mL of ethyl acetate, the sample was vortexed thoroughly for 1 min, followed by centrifugation at 13,000 rpm for 1 min. Then, 0.35 mL of the ethyl acetate fraction was aliquoted and evaporated by using a rotary evaporator, followed by resuspension in 0.04 mL of ethyl acetate. The resuspended sample was mixed with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) at a 1:1 (v/v) ratio and derivatized at room temperature for 24 h. The formation of adipic acid was analyzed using GC-MS.
Overnight seed cultures were diluted 1:100 (v/v) into 50 mL of M9 medium supplemented with 0.2% (w/v) glucose, 0.2% (w/v) casamino acids and the appropriate antibiotics (100 μg/L carbenicillin, 50 μg/L kanamycin, and 25 μg/L chloramphenicol) in 250 mL baffled flasks and cultured at 30° C. and 225 rpm. The engineered E. coli cultures were induced with 0.2% (w/v) L-arabinose at OD600 1.2-1.5, which was followed by the addition of the p-coumaric acid substrate to a final concentration of 0.1% (w/v). Sampling was performed at 18 h and 36 h. The formation of adipic acid was analyzed by using GC-MS.
Engineered E. coli MG1655 cells were first grown in M9 medium (supplemented with 0.2% (w/v) glucose and 0.2% (w/v) casamino acids) to the exponential phase at OD600 1.0. The inoculums were added to shaken flasks (37° C., 225 rpm) to a final concentration of OD600 0.01, with each flask containing 50 mL of M9 medium supplemented with 0.2% (w/v) glucose as the carbon source, 0.2% (w/v) casamino acids and the relevant lignin derivatives as substrates. p-Coumaric acid (Sigma-Aldrich, U.S.A.) was first dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 10% (w/v) before being added to the M9 medium to a final concentration of 0.1% (w/v). The L-arabinose system and HA-controlled system were induced with 0.2% (w/v) L-arabinose or 0.1% (w/v) p-coumarate, respectively, after reaching OD600 1.0. One milliliter of the biotransformation culture was extracted at each time point (18 h and 36 h) for GC-MS measurements.
OPEFB depolymerized lignin cocktail was reconstituted based on the identified concentrations of aromatic compounds reported in Mohamad Ibrahim et al., [Mohamad Ibrahim et al., CLEAN—Soil, Air, Water 2=36: 287-291 (2008)]. In brief, individual compounds of OPEFB were prepared separately and subsequently mixed together to give the final concentrations stated in Table 2.
All the individual compounds were purchased from Sigma-Aldrich with a purity of >97% and prepared in DMSO at a concentration that limits DMSO to 1% (v/v) in the final lignin cocktail solution. All the stock solutions of the compounds were kept at 4° C. in aliquots prior to use. Ten milliliter of lignin cocktail was prepared to comprise; 1.8 g of vanillin (Cat. No. 94752), 1 g of p-coumaric acid (≥98% (HPLC), Cat. No. C9008), 320 mg of p-hydroxybenzaldehyde (4-hydroxybenzoic acid; ≥99%, Cat. No. 240141), 110 mg of vanillic acid (4-hydroxy-3-methoxybenzoic acid; ≥97% (HPLC), Cat. No. 94770), 18 mg of p-hydroxybenzoic acid (3,4-dihydroxybenzoic acid; ≥98%, Cat. No. 37580) and 13 mg of ferulic acid (trans-ferulic acid; 99%, Cat. No. 128708) in DMSO. The solution was vortexed to ensure homogenized mixture. The cocktail was diluted 100-fold in the reaction volume to result in the final substrate concentration and referred as ‘1×OPEFB’.
Batch fermentation was carried out in a 5 L working volume BIOSTAT® B-DCU II benchtop bioreactor (Sartorius Stedim) for bioconversion to generate adipic acid or levulinic acid. The temperature was maintained at 30° C., and the pH was controlled at 7.0 by automated addition of acid (1 M H2SO4) and base solutions (1 M NaOH). Oxygen was constantly supplied at 10 L/min, and the impeller speed was set to 400 rpm to ensure homogenous aeration. Antifoam agent (200 μL) was added to the culture to prevent excessive foaming. A seed culture of relevant engineered E. coli MG1655 cells was cultured at 30° C. overnight and subsequently transferred into 1 L of fresh culture media containing 3 antibiotics (100 mg/L carbenicillin, 50 mg/L kanamycin, and 25 mg/L chloramphenicol). For fermentation of engineered E. coli with an L-arabinose-controlled circuit, substrate (OPEFB lignin cocktail) and inducer (0.2% (w/v) L-arabinose) were added 4 h post-inoculation as the culture reached late log phase. For fermentation of engineered E. coli with an HA-controlled circuit, the substrate was fed into the vessel immediately following inoculation. Aliquots of samples were taken at 18 h, 36 h and 42 h for further analysis using GC-MS and absorbance measurements at 600 nm. Batch fermentations were run in duplicate, and the results are reported as the average with standard deviation.
Ferulic acid, p-coumaric acid, vanillin, vanillic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and protocatechuic acid quantification were carried out using the protocol adopted by Barghini et al. [Barghini et al., Microbial Cell Factories 6: 13 (2007)], incorporated herein by reference, with modifications. First, 0.4 mL of the extracted batch culture was filter-sterilized with a 0.22 μm filter (Sartorius Stedim, Germany) before analysis by an Agilent 1260 HPLC apparatus equipped with an Inertsil ODS3 C18 reverse-phase column (length 250 mm, diameter 4.6 mm and particle size 5 μm) and a diode array detector (DAD). Compounds in the filtered culture were eluted with an isocratic pressure of 150 bars, a mobile phase comprising an aqueous solution of 35% methanol and 1% acetic acid, and a flow rate of 1 mL/min. Detection was performed at UV wavelengths of 300 nm (ferulic acid, p-coumaric acid, vanillin, vanillic acid, p-hydroxybenzaldehyde) and 254 nm (p-hydroxybenzoic acid, protocatechuic acid) with a sample injection volume of 10 μl. The retention times of the samples were compared with those of purified standards (Sigma-Aldrich, U.S.A.) for identification and quantification.
To extract organic acids (β-ketoadipic acid, adipic acid and levulinic acid) for detection, 500 μL of 1 M HCl, 300 μL of ethyl acetate and 100 μL of an internal standard (1,14-tetradecanedioic acid) were added to 1 mL of a cell culture sample. Subsequently, cells were disrupted by bead beating (FastPrep-24™ 5G and acid-washed beads (≤106 μm) running at 6.5 m/s and 1 min interval 4 times) and centrifuged for 10 min at 20,000×g at 4° C. to separate the organic phase. The ethyl acetate extracts were incubated with a derivatization agent (BSTFA with 1% trimethylchlorosilane (TCMS)) overnight prior to analysis by gas-liquid chromatography (GC) using an Agilent 7890B GC system equipped with an HP-5MS column (Agilent) coupled to a mass spectrometer (Agilent 5977).
As a first step to convert the depolymerized OPEFB lignin to value-added chemicals, a 9-enzyme pathway (
First, a converging pathway was constructed that comprises feruloyl-CoA synthetase (Fcs), enoyl-CoA hydratase (Ech), vanillin dehydrogenase (Vdh), vanillate O-demethylase (VanAB), and p-hydroxybenzoate hydroxylase (PobA) (
When the OPEFB lignin derivatives (formulated in the ratio that is naturally found after the pre-treatment) were tested, up to 400 mg/L protocatechuic acid was detected, reaching 11.5% of theoretical yield. The less-than-expected yield is mostly due to the inefficient utilization of vanillin, where only 2.7% molar conversion to protocatechuic acid was observed despite its high starting concentration (1.8 g/L). As a high concentration of vanillin has been reported to inhibit bacterial growth [Zaldivar et al., Biotechnology and Bioengineering 65: 24-33 (1999)], one possible approach to improve vanillin utilization would be to oxidize the depolymerized OPEFB lignin mixture, especially vanillin [Fargues et al., Chemical Engineering & Technology 19: 127-136 (1996)], to the less toxic compound vanillic acid prior to its feeding to the engineered cells. As vanillic acid has shown complete conversion, this approach may improve the yield of bioproduction and reduce toxicity to the host cells. However, these approaches have not been fully explored in this study, as the aim of this work is to first demonstrate the feasibility of direct conversion of the OPEFB lignin cocktail.
After the successful production of protocatechuic acid from the OPEFB lignin derivatives, a de-aromatization pathway involving protocatechuate 3,4-dioxygenase (PcaGH), 3-carboxy-cis,cis-muconate cycloisomerase (PcaB), 4-carboxymuconolactone decarboxylase (PcaC) and β-ketoadipate enol-lactone hydrolase (PcaD) (
A direct biosynthesis of adipic acid from carbon sources in E. coli has been reported [Yu et al., Biotechnol Bioeng 111: 2580-2586 (2014); Cheong et al., Nat Biotechnol 34: 556-561 (2016); Zhao et al., Metabolic Engineering 47: 254-262 (2018)] where artificial adipic acid synthesis pathways were constructed to convert glucose or glycerol to adipic acid. In a recent study, Niu et al. [Niu et al., Metabolic Engineering 59: 151-161(2020)] successfully demonstrated adipic acid production from β-ketoadipic acid in Pseudomonas putida KT2440. Adapted from these findings, an adipic production pathway was constructed and validated (
Unlike the adipic acid pathway, levulinic acid production involves a single decarboxylation step from β-ketoadipic acid. This reaction was catalyzed by acetoacetate decarboxylase (Adc) from Clostridium acetobutylicum [Cheong et al., Nat Biotechnol 34: 556-561 (2016)]. The level of levulinic acid exceeded 60 mg/L within 36 h of bioconversion under shake-flask conditions (
To facilitate the conversion of β-ketoadipic acid, repurposing of the existing native metabolic pathway in E. coli was required to channel reduction and decarboxylation pathways (
For adipic acid production, acyl-CoA dehydrogenase (fadE) and long-chain fatty acid CoA ligase (fadD) or both (fadED) genes were targeted, as these are involved in fatty acid metabolism, which can potential utilize six-carbon dicarboxylic acid (adipic acid) for β-oxidation [Lennen et al., Biotechnol Bioeng 106: 193-202 (2010); Sathesh-Prabu and Lee, J Agric Food Chem 63: 8199-8208 (2015)]. This metabolism can potentially utilize six-carbon dicarboxylic acid (adipic acid) for β-oxidation [Smit et al., Biotechnol Lett 27: 859-864 (2005)]. However, the deletion of these genes did not significantly improve adipic acid production. With the focus of channeling the flux towards β-ketoadipyl-CoA, AtoDA (AtoD; SEQ ID NO: 48 and AtoA; SEQ ID NO: 50) which shares ˜50% amino acid sequence similarity to the engineered PcaIJ (PcaI; SEQ ID NO: 26 and PcaJ; SEQ ID NO: 28) was inactivated. However, presence of the atoDA gene was found to play a pivotal role in initiating this new pathway, as deletion resulted in complete ablation of adipic acid production. As succinyl-CoA is an important cofactor in the formation of β-ketoadipyl-CoA, sucCD (sucC; SEQ ID NO: 53 and sucD; SEQ ID NO: 55, which encode subunits of succinyl-CoA synthetase) were deleted to minimize the competing conversion of succinyl-CoA towards succinate [Birney et al., J Bacteriol 178: 2883-2889 (1996); Zhao et al., Metabolic Engineering 47: 254-262 (2018)]. β-Ketoadipyl-CoA thiolase (paaJ; SEQ ID NO: 51) was also targeted for deletion due to its role in the reversible catalysis of β-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA [Yu et al., Biotechnol Bioeng 111: 2580-2586 (2014); Babu et al., Process Biochemistry 50: 2066-2071 (2015)]. Among the list of genes that can potentially shunt intermediates from the introduced reduction reactions, sucCD deletion resulted in the greatest improvement in adipic acid production. The sucCD mutant was able to convert the substrates at an approximately 3-fold higher efficiency than other mutants, as shown by the higher production observed at an early time point (18 h) (
For levulinic acid production, as acetoacetate decarboxylase (Adc)[Cheong et al., Nat Biotechnol 34: 556-561 (2016)] was used to convert β-ketoadipic acid, the atoDA genes from the acetoacetic acid degradation pathway in E. coli were targeted for deletion. The atoDA genes were targeted because they share >50% amino acid sequence similarity with the pca/J-encoded enzyme based on sequence alignment (
Overall, the outcome of the host engineering approaches indicates that the sucCD-deleted host is suitable for adipic acid production and that the atoDA-deleted host is suitable for levulinic acid production. These two strains were used in subsequent experiments for downstream optimization.
During pathway validation and host engineering experiments, an induction system based on the L-arabinose [Guzman et al., J Bacteriol 177: 4121-4130 (1995)] inducer was used to regulate the expression of pathway enzymes in a dose-dependent manner. The role of the genetic controller is to modulate downstream gene transcription through the expression of the bacteriophage-based T7 polymerase (
Although the L-arabinose controller (pBAD) is an effective genetic device, it requires additional external resources, i.e., L-arabinose acts as an inducer, thus increasing the deployment cost of the biocatalytic cells. To improve the economics of OPEFB lignin utilization, we considered employing the hydroxycinnamic acid (HA) controller system reported by Lo et al., in 2016 [Lo et al., Cell Syst 3: 133-143 (2016)]. The HA controller system is inducible by HAs such as ferulic acid and p-coumaric acid, which are present in the depolymerized OPEFB lignin.
For comparison, we tested both L-arabinose (pBAD; SEQ ID NO: 41) and HA (SEQ ID NO: 42) controllers for adipic and levulinic acid production in optimized host strains (ΔsucCD and ΔatoDA, respectively) under shake-flask conditions at 30° C. and using p-coumaric acid (final concentration, 1 g/L) as the substrate (Table 1). The fermentation temperature was set at 30° C. instead of the commonly used 37° C. based on two reasons: 1) less energy is required to maintain a lower temperature while not impacting the growth of the engineered E. coli, and 2) the unstable compound β-ketoadipic acid can have a longer half-life for enzymatic conversion at the lower temperature. The L-arabinose-induced controller performed better than the HA controller in terms of the product yield: a 2-fold higher titer was observed for both adipic and levulinic acid production (
In an attempt to further boost the yield, the inherent problems faced by shake-flask experiments, such as limited oxygen levels and uncontrolled pH, which can affect the productivity of microbial hosts was overcome by using a controlled bioreactor which can regulate these parameters during the fermentation process. Bioreactors with oxygen (pO2) and pH sensors and their relevant pumps to maintain these parameters at target values were used for OPEFB lignin conversion (
Under controlled conditions, the respective optimized host strains (ΔsucCD and ΔatoDA) bearing the HA controller performed similar to, if not slightly better, than the strains bearing the L-arabinose controller: ˜1.8-fold higher titer was observed for levulinic acid production (455.7 mg/L vs. 253.5 mg/L per 1×OPEFB lignin at 36 h) and ˜23% higher for adipic acid production (9.5 mg/L vs 7.8 mg/L per 1×OPEFB lignin at 18 h) in the HA controller strains (
Overall, this is the first report of autonomous cell-based production of adipic and levulinic acid from an OPEFB lignin cocktail without the need for upstream separation into individual derivatives before conversion and costly chemical inducers. Herein, we have demonstrated methods to produce adipic acid and levulinic acid from OPEFB lignin, primarily because both are industrially relevant chemicals that can be derived from the versatile dearomatized precursor β-ketoadipic acid.
In this study, we demonstrate direct utilization of unfractionated depolymerized OPEFB lignin to produce commodity chemicals using an engineered E. coli strain. E. coli was engineered to have 3 genetic modules for the following functions: 1. genetic control for autonomous activation, 2. conversion of depolymerized lignin derivatives into β-ketoadipic acid by pathway enzymes, and 3. conversion of β-ketoadipic acid to commodity chemicals by pathway enzymes (
| Number | Date | Country | Kind |
|---|---|---|---|
| 10202002037R | Mar 2020 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2021/050114 | 3/5/2021 | WO |
