Recombinant bacterium for producing L-lactic acid through co-utilization of glucose and xylose as well as construction method and application of recombinant bacterium

Abstract

The invention discloses a recombinant bacterium for producing L-lactic acid through co-utilization of glucose and xylose as well as a construction method and application of the recombinant bacterium. The invention relates to the technical field of biology, in particular to recombinant bacteria for producing L-lactic acid through co-utilization of glucose and xylose as well as a construction method and application. Methylglyoxal synthase, and / or pyruvate formate lyase, and / or fumarate reductase and / or L-lactic dehydrogenase in receptor Escherichia coli Rec1.0 are modified to obtain recombinant Escherichia coli, the recombinant Escherichia coli is fermented for 24 h, L-lactic acid is the only metabolite, and the yield reaches 2458 g / L; the consumption rate of xylose is 0.76 g / g DCW / h, the consumption rate of glucose is 0.69 g / g DCW / h, and the consumption rate of total sugar reaches 1.45 g / g DCW / h. The recombinant Escherichia coli constructed by the invention can realize efficient utilization of lignocellulose hydrolysate (containing glucose and xylose), and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to recombinant bacteria for producing L-lactic acid by co-utilizing glucose and xylose, their construction methods, and applications. Background Technology

[0002] Lignocellulose is the world's most widely available renewable biomass resource. It is the richest biomass source for the renewable production of fuels and chemicals. Statistics show that lignocellulose accounts for approximately 50% of all biomass production annually, equivalent to about 10-50 billion tons. Utilizing lignocellulose hydrolysate for fermentation to produce bioenergy and bulk chemicals can partially reduce dependence on petroleum resources. Glucose and xylose are the main hydrolyzed monosaccharides in lignocellulose, with glucose accounting for 30%-50% and xylose for 5%-20%.

[0003] L-lactic acid is a chiral small molecule and a precursor for the synthesis of many chiral substances, with wide applications in medicine, pesticides, and chemicals. L-polylactic acid (PLA), synthesized from L-lactic acid, is biodegradable and an ideal green polymer material: it can replace many traditional plastic products, such as polyethylene (PE), polypropylene (PP), and polystyrene (PS), in the production of packaging materials, disposable tableware, plastic bags, plastic bottles, and plastic films; it can be used to manufacture fiber products, such as clothing, textiles, and carpets; it can be blended with other fiber materials to create biodegradable fibers, which are environmentally friendly; it can be used to manufacture biodegradable foam packaging materials, protective pads, and packaging films; and it can be used to manufacture medical devices and supplies, such as sutures, bone repair materials, and implants. Due to its biodegradability and biocompatibility, it is widely used in the medical field.

[0004] The efficient co-utilization of glucose and xylose to achieve the synthesis of L-lactic acid will promote the reuse of lignocellulose resources. Summary of the Invention

[0005] The problem to be solved by this invention is how to improve the co-utilization capacity of microorganisms of glucose and xylose to produce high levels of L-lactic acid.

[0006] To address the aforementioned problems, this invention first provides a method for constructing recombinant Escherichia coli.

[0007] The method for constructing recombinant Escherichia coli provided by the present invention includes modifying the methylglyoxal synthase (MgsA protein), and / or pyruvate formate lyase (PflB protein), and / or fumarate reductase (Frd protein), and L-lactate dehydrogenase (ldhL* protein) in the recipient Escherichia coli to obtain recombinant Escherichia coli.

[0008] In this invention, the L-lactate dehydrogenase is an L-lactate dehydrogenase mutant, abbreviated as ldhL* protein. In one specific embodiment, the amino acid sequence of the L-lactate dehydrogenase mutant is sequence 1 in the sequence listing.

[0009] In the above method, the MgsA protein in the modified recipient E. coli is used to inhibit the expression and / or activity of the MgsA protein; the PflB protein in the modified recipient E. coli is used to inhibit the expression and / or activity of the PflB protein; the Frd protein in the modified recipient E. coli is used to inhibit the expression and / or activity of the Frd protein; and the ldhL protein in the modified recipient E. coli is used to activate the expression and / or activity of the ldhL protein.

[0010] The activation of the expression and / or activity of the ldhL protein may be achieved by introducing the encoding gene of the L-lactate dehydrogenase mutant into a recipient microorganism, thereby activating the expression of the L-lactate dehydrogenase mutant.

[0011] In the above method, the methylglyoxal synthase (MgsA) can be any of the following proteins:

[0012] A1) The amino acid sequence is the protein with GenBank accession number WP_000424181.1 (updated January 5, 2022), positions 1-152; encoded by the nucleotide sequence 2883345-2883800 with GenBank accession number NC_010468.1 (updated May 16, 2023);

[0013] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1), which has more than 80% identity with the protein shown in A1) and has methylglyoxal synthase activity.

[0014] A3) is a fusion protein with methylglyoxal synthase activity obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0015] In the above method, the pyruvate formate lyase (PflB) can be any of the following proteins:

[0016] B1) The protein with amino acid sequence encoded by GenBank accession number WP_001292822.1 (updated February 28, 2022), numbers 1-760; and encoded by nucleotide sequences encoded by GenBank accession number NC_010468.1 (updated May 16, 2023), numbers 2956804-2959083.

[0017] B2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in B1), which has more than 80% identity with the protein shown in B1) and has pyruvate formate lyase activity.

[0018] B3) A fusion protein with pyruvate formate lyase activity obtained by attaching a tag to the N-terminus and / or C-terminus of B1) or B2).

[0019] In the above method, the fumarate reductase (FrdABCD) can be any of the following proteins:

[0020] C1) The protein containing the FrdA amino acid sequence in GenBank WP_001192973.1 (updated January 19, 2023), positions 1-602, is encoded by nucleotides 4214758-4216563 in GenBank NC_010468.1 (updated May 16, 2023); the protein containing the FrdB amino acid sequence in GenBank WP_000829498.1 (updated October 24, 2023), positions 1-244, is encoded by nucleotides 4216559-4217290 in GenBank NC_010468.1 (updated May 16, 2023). The sequences are encoded as follows: The protein with amino acid sequence FrdC, located at GenBank number WP_000208757.1 (updated January 19, 2023), is encoded by nucleotide sequences 4217304-4217696 in GenBank number NC_010468.1 (updated May 16, 2023); the protein with amino acid sequence FrdD, located at GenBank number WP_000609663.1 (updated July 4, 2023), is encoded by nucleotide sequences 4217710-4218066 in GenBank number NC_010468.1 (updated May 16, 2023).

[0021] C2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in C1) that has more than 80% identity with the protein shown in C1) and has fumarate reductase activity.

[0022] A fusion protein with fumarate reductase activity is obtained by attaching a tag to the N-terminus and / or C-terminus of C1) or C2).

[0023] In the above method, the L-lactate dehydrogenase mutant LdhL* can have an amino acid sequence as sequence 1 in the sequence listing.

[0024] Compared to L-lactate dehydrogenase from Lactobacillus plantarum ATCC 14917 (Genbank ID KRL33571.1, updated November 6, 2015, protein at positions 1-317), the L-lactate dehydrogenase mutant has two point mutations: threonine (T) at position 42 of the amino acid sequence of L-lactate dehydrogenase is replaced with alanine (A), and amino acid at position 255 is replaced with serine (S) instead of glycine (G). The mutated protein is named L-lactate dehydrogenase mutant (abbreviated as LdhL*).

[0025] The amino acid sequence of LdhL* is:

[0026] MPNHQKVVLVGDGAVGSSYAFAMAQQGIAEEFVIVDVVKDRAKGDALDLEDAQAFTAPKKIYSGEYSDCKDADL

[0027] VVITAGAPQKPGESRLDLVNKNLNILSSIVKPVVDSGFDGIFLVAANPVDILTYATWKFSGFPKDRVIGSGTSL

[0028] DSSRLRVALGKQFNVDPRSVDAYIMGEHGDSEFAAYSTATIGTRPVRDVAKEQGVSDEDLAKLEDGVRNKAYDI

[0029] INLKGATFYGIGTALMRISKAILRDENAVLPVSAYMDGQYGLNDIYIGTPAVIGGTGLKQIIESPLSADELKMQDSAATLKKVLNDGLAELENK*(sequence 1).

[0030] In this article, the encoding gene mgsA for methylglyoxal synthase can be any of the following:

[0031] E1) The nucleotide sequence is the DNA molecule with GenBank accession number NC_010468.1 (updated May 16, 2023) numbers 2883345-2883803;

[0032] The nucleotide sequence defined in E2) has more than 80% identity with that defined in E1) and encodes the DNA molecule of the methylglyoxal synthase;

[0033] E3) hybridizes under stringent conditions with any of the defined nucleotide sequences in E1)-E2) and encodes the DNA molecule of the methylglyoxal synthase.

[0034] In this article, the gene pflB encoding pyruvate-formate lyase may be any of the following:

[0035] F1) The nucleotide sequence is the DNA molecule with GenBank number NC_010468.1 (updated May 16, 2023) numbers 2956804-2959086;

[0036] The nucleotide sequence defined in F2) has more than 80% identity with that defined in F1) and encodes the DNA molecule of the pyruvate formate lyase.

[0037] F3) hybridizes under stringent conditions with any of the defined nucleotide sequences from F1) to F2) and encodes the DNA molecule of the pyruvate-formate lyase.

[0038] In this article, the gene encoding frdABCD for fumarate reductase can be any of the following:

[0039] G1) The frdA nucleotide sequence is from DNA molecule with GenBank number NC_010468.1 (updated May 16, 2023) 4214758-4216566; the frdB nucleotide sequence is from DNA molecule with GenBank number NC_010468.1 (updated May 16, 2023) 4216559-4217293; the frdC nucleotide sequence is from DNA molecule with GenBank number NC_010468.1 (updated May 16, 2023) 4217304-4217699; and the frdD nucleotide sequence is from DNA molecule with GenBank number NC_010468.1 (updated May 16, 2023) 4217710-4218069.

[0040] The nucleotide sequence defined in G2) has more than 80% identity with that defined in G1) and encodes the DNA molecule of the fumarate reductase;

[0041] G3) hybridizes under stringent conditions with any of the defined nucleotide sequences in G1)-G2) and encodes the DNA molecule of the fumarate reductase.

[0042] In this article, the gene encoding lactate dehydrogenase, ldhL*, can be any of the following:

[0043] D1) The gene sequence of the L-lactate dehydrogenase mutant is sequence 2 in the sequence listing. Compared with the L-lactate dehydrogenase gene of Lactobacillus plantarum ATCC 14917 (molecules 16296 to 17249 of GenBank with GenBank number AZEJ01000016.1, updated on November 6, 2015), it has two point mutations. Specifically, nucleotide 124 is changed from A to G and nucleotide 763 is changed from G to A. The mutated gene is named the L-lactate dehydrogenase mutant gene (abbreviated as gene ldhL*).

[0044] In this article, the promoter for the gene ldhL* that activates lactate dehydrogenase is M1-93, and the nucleotide sequence of promoter M1-93 is as follows:

[0045] 5'-TTATCTCTGGCGGTGTTGACAAGAGATAACAACGTTGATATAATTGAGCCCGTATTGTTAGCATGTACGTTTAAACCAGGAAACAGCT-3'.

[0046] Those skilled in the art can readily mutate the nucleotide sequences encoding methylglyoxal synthase, pyruvate-formate lyase, fumarate reductase, and L-lactate dehydrogenase of the present invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides having 75% or higher identity with the nucleotide sequences of methylglyoxal synthase, pyruvate-formate lyase, fumarate reductase, and L-lactate dehydrogenase of the present invention, provided they encode and function as methylglyoxal synthase, pyruvate-formate lyase, fumarate reductase, and L-lactate dehydrogenase, are all derived from and equivalent to the sequences of the present invention.

[0047] As used herein, the term "identity" refers to sequence similarity to a natural amino acid or nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequences encoding methylglyoxal synthase, pyruvate formate lyase, fumarate reductase, and L-lactate dehydrogenase of the present invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0048] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0049] In the above method, the recipient Escherichia coli can be Escherichia coli Rec1.0.

[0050] The *Escherichia coli* Rec1.0 strain is a strain obtained by restoring the knocked-out pgi and zwf genes in *Escherichia coli* AE1.0; the specific construction method of *Escherichia coli* AE1.0 has been described in: Zhu, XN, Fan, FY, Qiu, HN, Shao, MY, Li, D, Yu, Y, Bi, CH, Zhang, XL (2022). New xylose transporters support the simultaneous consumption of glucose and xylose in *Eescherichiacoli*. *Life*, 1(2), 156-170. https: / / doi.org / 10.1002 / mlf2.12021.

[0051] In the above method, the methylglyoxal synthase, and / or pyruvate formate lyase, and / or fumarate reductase in the modified recipient Escherichia coli are achieved by knocking out the coding genes of the methylglyoxal synthase, and / or pyruvate formate lyase, and / or fumarate reductase.

[0052] In one specific embodiment, the knockout of the coding gene for methylglyoxal synthase, and / or the coding gene for pyruvate formate lyase, and / or the coding gene for fumarate reductase can be achieved through homologous recombination.

[0053] In one specific embodiment, the gene encoding the L-lactate dehydrogenase mutant can be activated by homologous recombination.

[0054] The present invention also provides recombinant Escherichia coli constructed using the above method.

[0055] In one specific embodiment, the recombinant Escherichia coli can simultaneously enhance cell growth and lactic acid production capacity through evolutionary metabolism.

[0056] The present invention also provides a method for producing L-lactic acid, comprising culturing the aforementioned recombinant Escherichia coli to obtain a fermentation product, and obtaining L-lactic acid from the fermentation product.

[0057] The present invention also provides the above-described construction method or the recombinant Escherichia coli for any of the following applications:

[0058] P1. Applications in the production of L-lactic acid;

[0059] P2. Application in increasing L-lactic acid production;

[0060] P3, Application in hydrolyzed glucose and / or xylose;

[0061] P4. Applications in hydrolyzed lignocellulose.

[0062] This invention obtained strain GX003 by sequentially knocking out the coding genes for methylglyoxal synthase, pyruvate-formate lyase, and fumarate reductase in recombinant *E. coli* Rec1.0, and integrating the coding gene for L-lactate dehydrogenase. Using glucose and xylose as substrates, GX003 fermented for 24 hours, achieving a lactic acid yield of 24.58 g / L, a xylose consumption rate of 0.76 g / g DCW / h, a glucose consumption rate of 0.69 g / g DCW / h, and a total sugar consumption rate of 1.45 g / g DCW / h. The *E. coli* engineered strain constructed in this invention achieves the ability of microorganisms to share glucose and xylose to produce L-lactate, meeting the need for simultaneous and efficient utilization of glucose and xylose from biomass resources, and realizing the efficient utilization of lignocellulose hydrolysate. This provides an important platform for the efficient fermentation production of lignocellulose hydrolysate feedstock for other biofuels and biochemicals, and can effectively reduce production costs and environmental pollution problems caused by the extraction of chemicals from petroleum. It has broad application prospects. Attached Figure Description

[0063] Figure 1 The mgsA knockout validation fragment is shown in lane 1, which contains recombinant E. coli Rec1.0ΔmgsA, and lane 2 contains strain ATCC 8739.

[0064] Figure 2 For pflB knockout validation, lane 1 contains recombinant E. coli GX001ΔpflB, and lane 2 contains strain ATCC 8739.

[0065] Figure 3 For LdhL*, the frd validation fragment was integrated. Lane 1 contained recombinant E. coli GX002Δfrd-ldhL*, and lane 2 contained strain ATCC 8739.

[0066] Figure 4 Analysis of fermentation products of GX003 showed that glucose was the peak at 11.133 min, xylose at 11.965 min, and L-lactic acid at 16.363 min. Detailed Implementation

[0067] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0068] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0069] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0070] The pRedCas9 plasmid used in the following examples is described in: Zhu et al. 2017, The CRISPR / Cas9-facilitated multiplex pathway optimization (CFPO) technique and its application to improve the Escherichia coli xylose utilization pathway Metab. Eng. 43, 37-45. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0071] The placZ plasmid used in the following examples has been described in: Qiu Huanna et al., Construction of a strict promoter on the chromosome of Escherichia coli. 2018, Microbiology Bulletin, 45(8): 1693-1704. The public may obtain this biological material from the applicant. This biological material is only for repeating the experiments of this invention and may not be used for other purposes.

[0072] The pV4 plasmid used in the following examples is described in: Patent CN114058560A, Method for the Production of Glycine. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0073] The Escherichia coli ATCC 8739 in the following examples can be obtained from the American Type Culture Collection Center (ATCC) (https: / / www.atcc.org). This biological material is only for repeating the experiments of the present invention and should not be used for other purposes.

[0074] The Rec1.0 strain in the following examples is a strain obtained after restoring the knocked-out pgi and zwf genes of Escherichia coli AE1.0. Escherichia coli Rce1.0 and AE1.0 have been described at: Zhu, XN; Fan, FY; Qiu, HN; Shao, MY; Li, D.; Yu, Y.; Bi, CH; Zhang, XL, New xylose transporters support the simultaneous consumption of glucose and xylose in Eescherichia coli.mLife2022,1(2),156-170. The biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of this invention and may not be used for any other purpose.

[0075] The Slac007 strain in the following examples is an L-lactic acid-producing strain that integrates the ldhL* gene. *Escherichia coli* Slac007 is described in: Recombinant *Escherichia coli* for L-lactic acid production and its applications, authorization number CN112852693B, authorization date: November 16, 2021. Its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 19459. This biological material is available to the public from the applicant and is intended solely for repeating experiments of this invention and may not be used for any other purpose.

[0076] The pORF1 plasmid in the following examples is a bleomycin-containing anti-plasmid. pORF1 is described in: Zheng F, Kawabe Y, Murakami M, Takahashi M, Nishihata K, Yoshida S, Ito A, Kamihira M. LINE-1 vectors mediate recombinant antibody gene transfer by retrotransposition in Chinese hamster ovary cells. Biotechnol J. 2021 Jul;16(7):e2000620. doi:10.1002 / biot.202000620.Epub 2021May 20.PMID:33938150. This biological material is available to the public from Addgene#206025. This biological material is only for repeating the experiments of this invention and should not be used for other purposes.

[0077] The pKD46 plasmid used in the following examples is a recombinase-containing plasmid. pKD46 is described in: Datsenko, KA, Wanner, BL, 2000. One-step inactivation of chromosomal genes in Escherichiacoli K-12 using PCR products. Proc. Nat. Acad. Sci. USA 97, 6640–6645. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and shall not be used for any other purpose.

[0078] The promoters M1-93 used in the following examples are described in: Lu, J., Tang, JL, Liu, Y., Zhu, XN, Zhang, TC, Zhang, XL (2012). Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization. Appl Microbiol Biotechnol, 93(6), 2455-2462.

[0079] The strains used in this invention are shown in Table 1, the primers used are shown in Table 2, and the plasmids involved are shown in Table 3.

[0080] Table 1. Strains used in this invention

[0081]

[0082]

[0083] Table 2. Primers used in this invention

[0084]

[0085]

[0086] Table 3. Plasmids used and constructed in this invention

[0087]

[0088] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0089] Example 1: Construction of recombinant Escherichia coli GX001

[0090] Starting with recombinant E. coli Rec1.0, mgsA (nucleotide sequence is GenBank ID NC_010468.1, updated May 16, 2023, positions 2883345-2883803) was knocked out using the CRISPR / Cas9 dual plasmid gene editing system to obtain recombinant E. coli GX001. The specific steps are as follows:

[0091] (1) Construction of pV4-del-mgsA plasmid

[0092] The specific steps for constructing the pV4-del-mgsA plasmid are as follows:

[0093] The first step was to obtain pV4 plasmid backbone fragment A. Using pV4 plasmid as a template, reverse PCR was performed with primers N20-B-F1 and N20-B-R1 (specific sequences are shown in Table 2) to obtain a PCR product of approximately 4.1 kb, which is pV4 plasmid backbone fragment A. This fragment contains cat, P15A, and the self-cleaving element lacI-Ptrc-cat-N20-gRNA.

[0094] The amplification system consisted of: 10 μL New England Biolabs Phusion 5X buffer, 1 μL dNTPs (2.5 mM each), 20 ng DNA template, 2 μL primers (10 μM each), 0.5 μL Phusion High-Fidelity DNA polymerase (2.5 U / μL), and 33.5 μL distilled water, for a total volume of 50 μL.

[0095] The amplification conditions were as follows: 98℃ pre-denaturation for 2 minutes (1 cycle); 98℃ denaturation for 10 seconds, 56℃ annealing for 10 seconds, 72℃ extension for 2.5 minutes (30 cycles); 72℃ extension for 5 minutes (1 cycle).

[0096] The second step is to obtain the mgsA-N20-gRNA sequence fragment B. Using pV4 plasmid as a template, PCR amplification was performed using primers mgsA-N20-B-F2 and N20-B-R2 (specific sequences are shown in Table 2) to obtain a PCR product of about 400 bp, which is the DNA fragment E. The amplification system and amplification conditions are the same as the first step in Example 1(1).

[0097] The third step is to obtain the upstream and downstream homologous arm fragments C and D of the mgsA gene. Using the genomic DNA of *E. coli* ATCC 8739 as a template, PCR amplification was performed using primers mgsA-F1 and mgsA-R1 (specific sequences are shown in Table 2) to obtain the upstream homologous arm fragment C, approximately 480 bp. Similarly, PCR amplification was performed using primers mgsA-F2 and mgsA-R2 (specific sequences are shown in Table 2) to obtain the downstream homologous arm fragment D, approximately 480 bp. The amplification system and conditions are the same as in the first step of step (1).

[0098] In the fourth step, the pV4 plasmid backbone fragment A, the mgsA-N20-gRNA sequence fragment B, and the upstream and downstream homologous arms C and D of the mgsA gene were assembled using the Golden Gate technique (Engler, C., Kandzia, R., Marillonnet, S., 2008. A one-pot, one-step, precision cloning method with high throughput capability. PLoS One 3, e3647) (sequentially and seamlessly linked in the order of A, B, C, and D), and chemically transformed into competent TransT1 cells (Beijing TransGen Biotech Co., Ltd.). The obtained clones were verified by PCR using primers P15A-UP and mgsA-R2 (specific sequences are shown in Table 2). The band size was approximately 1.2 kb. Plasmid DNA was extracted from positive clones and sent for sequencing analysis to obtain the correct pV4-del-mgsA plasmid.

[0099] (2) mgsA gene knockout

[0100] Step 1, preparation of electrotransformation competent cells: (1) Pick single colonies from Luria-Bertani plates (LB medium: 1L containing 10g tryptone, 10g yeast extract and 5g sodium chloride; 1.5% agar), inoculate into 30mL LB liquid medium, and culture at 37℃ and 250r / min until OD. 550(1) Take about 0.5 and place in an ice bath for 10 minutes. (2) Transfer to a 50 mL sterile centrifuge tube and centrifuge at 4°C and 5000 r / min for 5 minutes. (3) Discard the supernatant, resuspend the precipitate in 20 mL sterile water, and centrifuge at 4°C and 5000 r / min for 5 minutes. (4) Discard the supernatant, resuspend the precipitate in 15 mL sterile water, and centrifuge at 4°C and 5000 r / min for 5 minutes. (5) Discard the supernatant, resuspend the precipitate in 10 mL 10% glycerol, and centrifuge at 4°C and 5000 r / min for 5 minutes. (6) After discarding the supernatant, resuspend the bacterial cells in the residual liquid and take 50 μL to a new 1.5 mL centrifuge tube. It can be used directly for electroporation or stored in a -80°C refrigerator.

[0101] The second step involved preparing Rec1.0 electrotransformable competent cells of *E. coli*. Plasmids pRedCas9 and pV4-del-mgsA were simultaneously transformed into the Rec1.0 electrotransformable competent cells. The cells were plated with kanamycin and chloramphenicol antibiotics and incubated overnight at 30°C. Single colonies were picked and incubated overnight at 30°C using 2 mL LB agar (containing kanamycin and chloramphenicol; 2.5% L(+)-arabinose) at 250 rpm to induce homologous recombination and cleave of non-recombinant DNA. The incubated DNA was diluted and plated on LB agar (containing kanamycin and chloramphenicol; 2.5% L(+)-arabinose) and incubated overnight at 30°C. Single colonies were then selected for colony PCR verification using primers mgsA-YZ-up and mgsA-YZ-down (specific sequences are shown in Table 2). The results are as follows: Figure 1 As shown, the positive clone was amplified to obtain PCR products of approximately 960 bp in size, and the positive clone was named recombinant Escherichia coli GX001.

[0102] Example 2: Construction of recombinant Escherichia coli GX002

[0103] Starting from recombinant Escherichia coli GX001, pflB (nucleotide sequence is GenBank number NC_010468.1, updated on May 16, 2023, positions 2956804-2959086) was knocked out using the CRISPR / Cas9 dual plasmid gene editing system to obtain recombinant Escherichia coli GX002.

[0104] (1) Construction of pV4-del-pflB plasmid

[0105] The specific steps for constructing the pV4-del-pflB plasmid are as follows:

[0106] The first step is to obtain pV4 plasmid backbone fragment A. Using pV4 plasmid as a template, reverse PCR amplification was performed using primers N20-B-F1 and N20-B-R1 (specific sequences are shown in Table 2) to obtain a PCR product of approximately 4.1 kb, which is pV4 plasmid backbone fragment A. This fragment contains cat, P15A, and the self-cleaving element lacI-Ptrc-cat-N20-gRNA. The amplification system and conditions are the same as in the first step of step (1).

[0107] The second step is to obtain the pflB-N20-gRNA sequence fragment E. Using pV4 plasmid as a template, PCR amplification is performed using primers pflB-N20-B-F2 and N20-B-R2 (specific sequences are shown in Table 2) to obtain a PCR product of approximately 400 bp, which is the DNA fragment H. The amplification system and amplification conditions are the same as the first step in step (1).

[0108] The third step is to obtain the upstream and downstream homologous arm fragments F and G of the pflB gene. Using genomic DNA from *E. coli* ATCC 8739 as a template, PCR amplification was performed using primers pflB-F1 and pflB-R1 (specific sequences are shown in Table 2) to obtain the upstream homologous arm fragment F, approximately 480 bp. Similarly, PCR amplification was performed using primers pflB-F2 and pflB-R2 (specific sequences are shown in Table 2) to obtain the downstream homologous arm fragment G, approximately 430 bp. The amplification system and conditions are the same as in the first step of step (1).

[0109] In the fourth step, the pV4 plasmid backbone fragment A, the pflB-N20-gRNA sequence fragment E, and the upstream and downstream homologous arms G and F of the pflB gene were assembled using the Golden Gate technique (sequentially and seamlessly linked in the order of A, E, G, and F), and then chemically transformed into competent TransT1 cells (Beijing TransGen Biotech Co., Ltd.). The obtained clones were verified by PCR using primers P15A-UP and pflB-R2, with band sizes of approximately 1.3 kb. Plasmid DNA was extracted from positive clones and sequenced for analysis, yielding the correct pV4-del-pflB plasmid.

[0110] (2) The specific steps for pflB gene knockout are as follows:

[0111] Starting with the engineered Escherichia coli strain GX001, plasmids pRedCas9 and pV4-del-pflB were simultaneously transformed into GX001 electrocompetent cells (the preparation technique for electrocompetent cells is described in step (2) of Example 1). The cells were plated with kanamycin and chloramphenicol antibiotics and incubated overnight at 30°C. Single colonies were picked and placed in 2 mL LB agar (containing kanamycin and chloramphenicol; 2.5% L(+)-arabinose), rotated at 250 r / min, and incubated overnight at 30°C to induce homologous recombination and cleave non-recombinant DNA. The DNA was diluted and plated on LB agar (containing kanamycin and chloramphenicol; 2.5% L(+)-arabinose) and incubated overnight at 30°C. Single colonies were picked for colony PCR verification using primers pflB-YZ-up and pflB-YZ-down. The results are as follows: Figure 2 As shown, the positive clone was amplified to obtain a PCR product of 880 bp in size, and the positive clone was named recombinant Escherichia coli GX002.

[0112] Example 3: Construction of recombinant Escherichia coli GX003

[0113] Starting from recombinant E. coli GX002, the ldhL* gene (nucleotide sequence of sequence 2 in the sequence listing) was integrated into the knockout frdABCD gene site using a one-step method. (The GenBank accession number for the frdA nucleotide sequence is NC_010468.1, updated on May 16, 2023, positions 4214758-4216566; the accession number for the frdB nucleotide sequence is NC_010468.1, updated on May 16, 2023.) In 2023, positions 4216559-4217293 (containing the frdC nucleotide sequence, GenBank ID NC_010468.1, updated May 16, 2023), and positions 4217304-4217699 (containing the frdD nucleotide sequence, GenBank ID NC_010468.1, updated May 16, 2023), recombinant *E. coli* GX003 was obtained. The specific experimental steps are as follows:

[0114] The first step involved using the genome of strain Slac007 (patent granted, patent number CN112852693B, grant date: 2021.11.16) as a template, and performing PCR amplification with primers frd-up / ldhL-R-CPEC. The resulting product was 1497 bp and named fragment K. This fragment contained the upstream homologous arm of the frd knockout gene, the ldhL* coding gene, and the M1-93 promoter. Using pORF1 plasmid as a template, PCR amplification was performed with primers BleR-up-CPEC / BleR-down-CPEC, resulting in a product of 511 bp and named fragment L. This fragment contained the bleomycin resistance gene BleR. Using the genome of strain Slac007 as a template, PCR amplification was performed with primers frd-F1-CPEC / frd-down, resulting in a product of 417 bp and named fragment M. This fragment contained the downstream homologous arm of the frd knockout gene. The PCR amplification system and conditions were as described in the first step of step (1).

[0115] The second step involves assembling fragments K, L, and M using the CPEC technique (Quan, J. and Tian, ​​J. (2009) Circular polymerase extension cloning of complex gene libraries and pathways PLoS One4, e6441) (sequentially and seamlessly linked in the order of K, L, and M) to obtain a fragment of 2374 bp (sequence 3 in the sequence listing).

[0116] The third step involves preparing GX002 electrocompetent cells according to the preparation method of step (2) in Example 1, transforming and integrating the pKD46 plasmid into the GX002 electrocompetent cells, growing them on ampicillin plates to obtain the intermediate strain GX002pKD46.

[0117] Step 4, preparation of electrocompetent cells of GX002pDK46: (1) Pick single clones from LB plates containing 50ug / ml ampicillin antibiotic, inoculate into 30mL LB liquid medium, and culture at 37℃ and 250r / min, containing 2.5% ampicillin. L+ arabinose, cultured to OD 550(1) Take about 0.5 and place in an ice bath for 10 minutes. (2) Transfer to a 50 mL sterile centrifuge tube and centrifuge at 4°C and 5000 r / min for 5 minutes. (3) Discard the supernatant, resuspend the precipitate in 20 mL sterile water, and centrifuge at 4°C and 5000 r / min for 5 minutes. (4) Discard the supernatant, resuspend the precipitate in 15 mL sterile water, and centrifuge at 4°C and 5000 r / min for 5 minutes. (5) Discard the supernatant, resuspend the precipitate in 10 mL 10% glycerol, and centrifuge at 4°C and 5000 r / min for 5 minutes. (6) After discarding the supernatant, resuspend the bacterial cells in the residual liquid and take 50 μL to a new 1.5 mL centrifuge tube. It can be used directly for electroporation or stored in a -80°C refrigerator.

[0118] Step 5: The 2374bp fragment assembled in step 2 was electroporated into GX002pDK46 competent cells, plated on LB agar plates containing bleomycin (50ug / ml), and the resulting clones were verified and sequenced using frdB-up / frdD-down. The clones were 2374bp in size. Figure 3 The clone that has been verified to be correct will be named GX003.

[0119] Example 4: Fermentation of recombinant Escherichia coli

[0120] Seed culture medium: NaCl: 10 g / L, Tryptone: 10 g / L, yeast extract: 5 g / L, solvent: water.

[0121] The fermentation medium consists of the following components:

[0122] Macroelements: Glucose 50g / L, Xylose 50g / L, NH4H2PO4 17.56g / L, (NH4)2HPO4 52.6g / L, MgSO4·7H2O 37g / L, Betaine-HCl 15.361g / L, KCl 18.75g / L.

[0123] Trace elements: FeCl3·6H2O 2.4g / L, CoCl2·6H2O 0.3g / L, CuCl2·2H2O 0.15g / L, ZnCl2 0.3g / L, Na2MoO4·2H2O 0.3g / L, MnCl2·4H2O 0.5g / L, H3BO3 0.072g / L.

[0124] Anaerobic fermentation of recombinant Escherichia coli includes the following steps:

[0125] (1) Seed culture: 30 mL of seed culture medium was prepared in a 250 mL Erlenmeyer flask and sterilized at 115 °C for 15 min. After cooling, single clones of recombinant Escherichia coli Rec1.0 and GX003 were picked from the plate and inoculated into the seed culture medium. The culture was carried out at 37 °C and 250 rpm for 12 hours to obtain the seed liquid, which was used for inoculation of the fermentation medium.

[0126] (2) Fermentation culture: The fermentation medium volume in the 500mL anaerobic tank is 250mL. The seed culture from step (1) is added according to the final concentration OD. 550nm An inoculum of 0.1 g was added to the fermentation medium, and fermentation was carried out at 37°C and 150 rpm for 1 day to obtain the fermentation broth. 6M KOH was used as the neutralizing agent to maintain the pH of the fermenter at 7.0. The fermentation broth contains all substances within the fermenter. No gas was introduced during the cultivation process.

[0127] Analytical methods: Components in the fermentation broth after 96 hours were determined using an Agilent-1200 high-performance liquid chromatograph. The concentrations of glucose, xylose, and organic acids in the fermentation broth were determined using a Biorad Aminex HPX–87H organic acid analytical column. The optical purity of lactic acid was analyzed using a Sumika Chemical Analysis Service (Japan) SUMICHIRALOA-6000 chiral column.

[0128] Glucose, xylose, and lactic acid standards were purchased from Merck Life Sciences, catalog numbers 50-99-7, 58-86-6, and 50-21-5, respectively. Specific detection procedures were performed in accordance with the following literature: Zhu, XN; Tan, ZG; Xu, HT; Chen, J.; Tang, JL; Zhang, XL, Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia. coli.Metab Eng 2014, 24 The material methods are described in sections 87-96. The results are shown in Table 4 and... Figure 4 .

[0129] Table 4. Evaluation of Anaerobic Fermentation of Recombinant Escherichia coli

[0130]

[0131] From Table 4 and Figure 4It can be seen that the recombinant Escherichia coli strain GX003, obtained by multi-step knockout of the metabolic pathway from the starting strain Rec1.0, produced L-lactic acid as the only metabolite after 24 hours of fermentation. Figure 4 The yield of the original strain Rec1.0 reached 24.58 g / L, with a xylose consumption rate of 0.76 g / g DCW / h, a glucose consumption rate of 0.69 g / g DCW / h, and a total sugar consumption rate of 1.45 g / g DCW / h. In contrast, the original strain Rec1.0 had an L-lactic acid yield of 0 g / L and a total sugar consumption rate of 1.34 g / g DCW / h.

[0132] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for constructing recombinant Escherichia coli, characterized in that, The method includes modifying the methylglyoxal synthase, and / or pyruvate lyase, and / or fumarate reductase, and / or L-lactate dehydrogenase in the recipient Escherichia coli to obtain recombinant Escherichia coli.

2. The method according to claim 1, characterized in that, The methylglyoxal synthase in the modified recipient *E. coli* is used to inhibit the expression and / or activity of the methylglyoxal synthase; the pyruvate lyase in the modified recipient *E. coli* is used to inhibit the expression and / or activity of the pyruvate lyase; the fumarate reductase in the modified recipient *E. coli* is used to inhibit the expression and / or activity of the fumarate reductase; and the L-lactate dehydrogenase in the modified recipient *E. coli* is used to activate the expression and / or activity of the L-lactate dehydrogenase.

3. The method according to claim 1 or 2, characterized in that, The methylglyoxal synthase is any of the following proteins: A1) The Genbank sequence number for the amino acid sequence is WP_000424181.1, updated on January 5, 2022, for protein positions 1-152; A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1), which has more than 80% identity with the protein shown in A1) and has methylglyoxal synthase activity. A3) A fusion protein with methylglyoxal synthase activity obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2). The pyruvate formate lyase is any one of the following proteins: B1) The GenBank number for the amino acid sequence is WP_001292822.1, updated on February 28, 2022, for proteins with amino acid sequences from position 1 to 760. B2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in B1), which has more than 80% identity with the protein shown in B1) and has pyruvate formate lyase activity. B3) A fusion protein with pyruvate formate lyase activity obtained by attaching a tag to the N-terminus and / or C-terminus of B1) or B2). The fumarate reductase FrdABCD is any one of the following proteins: C1) The Genbank number for the FrdA amino acid sequence is WP_001192973.1, updated on January 19, 2023, for the protein with positions 1-602. The GenBank number for the FrdB amino acid sequence is WP_000829498.1, updated on October 24, 2023, representing the protein from position 1 to 244. The FrdC amino acid sequence is represented by the Genbank number WP_000208757.1, updated on January 19, 2023, for the protein containing positions 1-131. The FrdD amino acid sequence is WP_000609663.1, updated on July 4, 2023, representing the protein from position 1 to 119. C2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in C1) that has more than 80% identity with the protein shown in C1) and has fumarate reductase activity. C3) A fusion protein with fumarate reductase activity is obtained by attaching a tag to the N-terminus and / or C-terminus of C1) or C2). The L-lactate dehydrogenase is a protein whose amino acid sequence is sequence 1 in the sequence listing.

4. The method according to any one of claims 1-3, characterized in that, The recipient Escherichia coli is Escherichia coli Rec1.

0.

5. The method according to any one of claims 1-4, characterized in that, The methylglyoxal synthase, and / or pyruvate-formate lyase, and / or fumarate reductase and / or L-lactate dehydrogenase in the modified recipient *E. coli* are achieved by knocking out the coding gene for methylglyoxal synthase, and / or the coding gene for pyruvate-formate lyase, and / or the coding gene for fumarate reductase, and / or activating the coding gene for L-lactate dehydrogenase.

6. Recombinant Escherichia coli constructed by the method of any one of claims 1-5.

7. A method for producing L-lactic acid, comprising culturing the recombinant Escherichia coli of claim 6 to obtain a fermentation product, and obtaining L-lactic acid from the fermentation product.

8. The method according to any one of claims 1-5 or the recombinant Escherichia coli according to claim 6, in any of the following applications: P1, Production of L-lactic acid; P2, Increase L-lactic acid production; P3, hydrolyzed glucose and / or xylose; P4, hydrolyzed lignocellulose.

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