Chiral l-lactic acid production engineering bacteria capable of selectively degrading racemic d-lactic acid and application thereof

Abstract

The application belongs to the technical field of microbial application, and relates to a chiral L-lactic acid production engineering bacterium capable of selectively degrading racemic D-lactic acid and application of the chiral L-lactic acid production engineering bacterium. Pediococcus acidilactici The strain is classified and named as Pediococcus acidilactici LJ2071 and is preserved in the China Center for Type Culture Collection, has a preservation number of CCTCC M 20251230 and a preservation date of May 30, 2025. The strain has the ability to efficiently metabolize various types of biomass-derived monosaccharides and synthesize high-concentration and high-chirality L-lactic acid, has a certain degradation capacity for D-lactic acid contained in the fermentation raw material itself, and can use raw material containing a certain amount of racemic lactic acid to produce high-chirality L-lactic acid. When the concentration of racemic D-lactic acid in the fermentation liquor is not more than 1.5 g / L, the chirality of the L-lactic acid product is greater than 99.5%, and the L-lactic acid product can be used as a poly-lactic acid material polymerization monomer.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology and relates to a chiral L-lactic acid producing engineered bacterium capable of selectively degrading racemic D-lactic acid and its application. Background Technology

[0002] Lactic acid (LA), also known as 2-hydroxypropionic acid, with the molecular formula C3H6O3, is an organic acid widely used in food, medicine, and cosmetics. Lactic acid has two optical isomers, L(+)-lactic acid and D(-)-lactic acid. In recent years, with the increasing scarcity of fossil resources and growing environmental awareness, biodegradable materials, represented by polylactic acid (PLA), have become a research hotspot. Therefore, the demand for lactic acid monomers used in the synthesis of PLA has also increased significantly. Generally, the chirality of lactic acid used in the synthesis of PLA needs to be above 99.5%.

[0003] In previous work in the laboratory, a strain of lactic acid bacteria was isolated from straw biomass. Pediococcus acidilactici ZB1 (deposited at the China Center for Type Culture Collection, accession number CCTCC M 2023150, deposit date February 20, 2023). Subsequently, the D-lactate dehydrogenase gene of this bacterium was knocked out. ldhD Knockout of phosphotransketase gene pkt Knockout of the acetate kinase gene ackA2 Integration of heterologous transketolase gene tkt Integration of heterologous aldolase genes tal Integral xylose isomerase xylA Integrating heterologous xylanokinase xylB A highly chiral L-lactic acid-producing strain was constructed by site-directed mutagenesis of the global transcriptional regulator CcpA protein (T65I) and the Sigma54-dependent transcriptional regulator LevR protein (E159G). Pediococcus acidilactici Z1 (deposited at the China Center for Type Culture Collection, accession number CCTCC M 20252274, deposit date October 21, 2025). This bacterium produced 130.8 ± 1.6 g / L of L-lactic acid during dry biorefining using 30% wheat straw as raw material, with a chiral purity of 99.6% for the product L-lactic acid.

[0004] Recent laboratory research indicates that various lactic acid bacteria are commonly found on the surface of lignocellulose raw materials. These bacteria may utilize the small amount of fermentable monosaccharides present in the raw materials to simultaneously produce L-lactic acid and D-lactic acid. Therefore, when lignocellulose raw materials are used as a carbon source for chiral L-lactic acid fermentation, the small amount of racemic D-lactic acid they contain will result in a lower optical purity of the L-lactic acid product (Guo et al., 2024, Biotechnol Bioeng, 121: 670–682). It has been reported that inexpensive nitrogen source corn steep liquor contains approximately 14%-20% L-lactic acid and D-lactic acid (Zhang et al., 2022, Fer, 8(10): 546-558); Okano et al. determined that the contents of L-lactic acid and D-lactic acid in corn steep liquor dry powder were 0.05 g / g and 0.09 g / g, respectively (Okano et al., 2022, Biotechnol J, 17: 2100331-2100339). Therefore, by reducing or even completely removing the small amount of racemic D-lactic acid present in the fermentation raw materials themselves, the production cost of chiral L-lactic acid will be greatly reduced and its optical purity will be further improved.

[0005] Currently, there are few reports on the application of lactate-degrading enzymes (including lactate oxidase and lactate dehydrogenase) in the production of chiral lactate. Okano et al. used genetic engineering methods to... L. plantarum KOLP7 secretion expression originates from Enterococcus L-lactic acid oxidase of sp. NBRC 3427 achieved L. plantarum KOLP achieved highly chiral D-lactic acid production in corn steep liquor containing L-lactic acid (optical purity exceeding 99.99%) (Okano et al., 2022, Biotechnol J, 17: 2100331-2100339). Wang Zhou et al. from Hubei University of Technology overexpressed an endogenous membrane-bound D-lactic acid dehydrogenase in the engineered Escherichia coli D-lactic acid strain HBUT-D, enabling the strain to produce L-lactic acid (optical purity exceeding 99.9%) using corn steep liquor or molasses containing a small amount of L-lactic acid as fermentation feedstock (Wang Zhou et al., 2024, Biotechnology Bulletin, 40(05):290-299). Both of these reports involve removing the L-lactic acid racemate for highly chiral D-lactic acid production. Chauliac et al., in order to remove 1-10% D-lactic acid from L-lactic acid slurry, knocked out the L-lactic acid dehydrogenase encoding gene... E. coliAdaptive evolution was performed. The adaptively evolved strains exhibited strong D-lactic acid removal capabilities, completely degrading 8.7 g / L of D-lactic acid from 135 g / L total lactic acid, with an optical purity of L-lactic acid exceeding 99.99% (Chauliac et al., 2015, Biotechnol Lett, 37: 2411–2418). However, *E. coli* showed low tolerance to lignocellulose hydrolysate, making it unsuitable for industrial production. Shapira et al. utilized organic waste for… Bacillus coagulan During L-lactic acid fermentation, purified D-lactic acid oxidase GOX2071 or crude enzyme solution was added to the fermentation system at the beginning, middle and late stages of fermentation, respectively, to achieve the production of L-lactic acid with high chiral purity (optical purity exceeding 99.5%) (US Patent Application No. 17 / 602,642.2022). However, this method requires the use of protease to treat the fermentation broth, which is costly.

[0006] The above four studies are the only reports to date of utilizing lactate oxidase or lactate dehydrogenase to degrade L-lactic acid / D-lactic acid to achieve chiral separation of D-lactic acid / L-lactic acid. In the future, research on genetically engineering L-lactic acid / D-lactic acid producing strains to possess D-lactic acid / L-lactic acid degradation capabilities will become an important direction in lactic acid biorefining research. However, currently, integrated expression of D-lactic acid dehydrogenase in L-lactic acid industrial production strains has not been achieved, and there is a lack of L-lactic acid industrial production strains capable of degrading racemic D-lactic acid.

[0007] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] In view of the above-mentioned deficiencies in strains, production technologies, and microbial resources, this invention aims to provide a chiral L-lactic acid production engineered strain capable of selectively degrading racemic D-lactic acid. This strain possesses the ability to efficiently metabolize various biomass-derived monosaccharides, especially lignocellulose-derived sugars, while simultaneously degrading some D-lactic acid in the fermentation feedstock, ultimately synthesizing high-concentration and highly chiral L-lactic acid. The fermentation product can be used for subsequent purification and recovery of chiral L-lactic acid and serves as a precursor for polylactic acid polymerization, demonstrating significant industrial application potential.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution: A chiral L-lactic acid-producing engineered bacterium that selectively degrades D-lactic acid has been classified and named *Pediococcus lactis*. Pediococcus acidilacticiLJ2071 is deposited at the China Center for Type Culture Collection, accession number CCTCCM 20251230, on May 30, 2025.

[0010] Furthermore, Pediococcus lactis Pediococcus acidilactici The LJ2071 strain was constructed by using *Glucobacterium oxyphylla*. Gluconobacter oxydans The D-lactate oxidase GOX2071 encoding gene from 621H (purchased from the American Center for Type Culture Collection) was ligated into the temperature-sensitive shuttle plasmid pSET4E, and then transformed into... Pediococcus acidilactici In Z1, integration was achieved through homologous recombination. ack This was achieved at site A.

[0011] That is, this strain was developed through the use of engineered Pediococcus lactis. Pediococcus acidilactici The genome of Z1 (deposited at the China Center for Type Culture Collection, accession number CCTCC M 20252274, deposit date October 21, 2025) ack Site A integration originates from *Glucosamine oxidans*. Gluconobacter oxydans 621H (purchased from the American Center for Type Culture Collection) encodes the gene for D-lactate oxidase. gox2071 Obtained.

[0012] The engineered strain of *Pediococcus lactis* Pediococcus acidilactici Z1 is derived from wild-type Pediococcus lactis. Pediococcus acidilactici ZB1 was obtained through metabolic engineering. Wild-type *Pediococcus lactis*. Pediococcus acidilactici ZB1, screened from straw biomass raw materials, exhibits excellent lactic acid fermentation performance. Its accession number is CCTCC M 2023150, the accession date is February 20, 2023, and the accession address is the China Center for Type Culture Collection. Pediococcus acidilactici The metabolic engineering performed on ZB1 included knocking out the D-lactate dehydrogenase gene. ldhD Knockout of phosphotransketase gene pkt Knockout of the acetate kinase gene ackA2 Integration of heterologous transketolase gene tkt Integration of heterologous aldolase genes tal Integral xylose isomerase xylA Integrating heterologous xylanokinase xylB The global transcriptional regulator CcpA protein underwent site-directed mutagenesis at T65I, and the Sigma54-dependent transcriptional regulator LevR protein underwent site-directed mutagenesis at E159G.

[0013] A chiral L-lactic acid production engineered strain that selectively degrades racemic D-lactic acid exhibits a certain ability to degrade the D-lactic acid contained in the fermentation feedstock during the production of high-concentration L-lactic acid.

[0014] Furthermore, Pediococcus lactis Pediococcus acidilactici LJ2071 can utilize a wide range of carbon sources, including but not limited to refined sugars such as glucose, xylose, mannose, galactose, and arabinose, as well as monosaccharides from sources such as grain starch, agricultural waste, energy crops, forestry waste, and industrial biomass.

[0015] Furthermore, the various nitrogen sources used in the fermentation production of this strain include, but are not limited to, yeast extract, hydrolyzed vegetable protein, and corn steep liquor.

[0016] Furthermore, this strain requires the addition of certain nutrients during the production of high-concentration and high-chirality L-lactic acid, including but not limited to yeast extract and corn steep liquor at 5-25 g / L, ammonium sulfate at 2-10 g / L, and manganese sulfate monohydrate at 0.1-0.5 g / L.

[0017] This invention provides the use of Pediococcus lactis Pediococcus acidilactici Specific steps for producing high-concentration and high-chirality L-lactic acid using LJ2071: (1) Activation of bacterial strains: Pediococcus lactis [[ID=3D]]Pediococcus acidilactici LJ2071 was inoculated into MRS liquid medium and cultured at 37-45℃ with shaking until OD reached. 600 When the pH reaches 3.5 or higher, an activated culture medium is obtained.

[0018] (2) Seed culture: The activated culture solution was transferred to MRS liquid medium at an inoculation rate of 5-20% (v / v) and cultured at 37-45 ℃ with shaking until the OD600 reached 3.5 or higher to obtain the seed culture solution.

[0019] (3) Fermentation culture: The seed culture solution is inoculated into a culture medium containing various biomass sugars at an inoculation rate of 5-20% (v / v) to produce highly chiral L-lactic acid. During the fermentation process, the pH is controlled at 4-6 using neutralizing agents such as sodium hydroxide or calcium hydroxide solution, the fermentation temperature is 37-45 ℃, and the fermentation lasts for 2-3 days.

[0020] Preferably, the composition and concentration of the MRS liquid culture medium used in steps (1) and (2) are as follows: glucose 5-30 g / L, peptone 5-15 g / L, yeast extract 5-15 g / L, sodium acetate 2-10 g / L, ammonium citrate 1-4 g / L, dipotassium hydrogen phosphate 0.1-2 g / L, magnesium sulfate heptahydrate 0.1-1 g / L and manganese sulfate monohydrate 0.1-0.5 g / L.

[0021] Preferably, the fermentation medium used in step (3) consists of two parts: biomass raw materials and nutrients. The carbon source used includes, but is not limited to, monosaccharides from various grain starches, agricultural waste, energy crops, forestry waste, and industrial biomass. More specifically, it is wheat straw or corn straw. The nitrogen source used includes, but is not limited to, yeast extract, hydrolyzed plant protein, and corn steep liquor.

[0022] Preferably, the various biomass used in step (3) needs to undergo appropriate pretreatment and hydrolysis to release free fermentable monosaccharides. Furthermore, wheat straw or corn straw is subjected to dilute acid pretreatment, biological detoxification, and saccharification.

[0023] As a preferred option, the nutrient composition and concentration of cellulose L-lactic acid fermentation in step (3) are as follows: ammonium sulfate about 10 g / L, manganese sulfate monohydrate about 0.25 g / L, industrial-grade yeast extract and / or corn steep liquor, and the total amount added is about 25 g / L based on the mass of dry matter.

[0024] Preferably, the fermentation method in step (3) is simultaneous saccharification and co-fermentation: the biomass raw material has a solid content of 20-35% (w / w), the amount of cellulase is 4-5 mg protein / g dry straw, and pre-saccharification is carried out at 45-50 ℃ for 6-12 h; after the pre-saccharification is completed, liquid biological detoxification is carried out, and after the detoxification is completed, *Pediococcus lactis* is inoculated. Pediococcus acidilactici LJ2071 seed culture medium was used for lactic acid fermentation at 42 ℃, 150-200 rpm and pH 5.5.

[0025] The present invention describes a chiral L-lactic acid production engineered strain capable of selectively degrading racemic D-lactic acid. This strain can produce L-lactic acid with a chirality of 99.48% by replacing 40% of the organic nitrogen source yeast extract with corn steep liquor (containing racemic D-lactic acid) in shake flask fermentation, and L-lactic acid with a chirality of 99.63% by producing it in a 3L bioreactor.

[0026] This invention provides an application of a chiral L-lactic acid-producing engineered bacterium capable of selectively degrading racemic D-lactic acid. This strain possesses the ability to selectively degrade racemic D-lactic acid from fermentation carbon sources, nitrogen sources, and nutrients. The *Pediococcus lactis* strain described in this invention... Pediococcus acidilactici LJ2071 is used to produce high-concentration and high-chirality L-lactic acid. The concentration of L-lactic acid produced by this strain is generally not less than 80 g / L. When the concentration of D-lactic acid introduced into the fermentation system does not exceed 1.5 g / L, the chirality of the L-lactic acid product is not less than 99.5%.

[0027] Compared with the prior art, the present invention has the following positive effects: The present invention provides Pediococcus lactis Pediococcus acidilactici LJ2071 can efficiently convert monosaccharides from various biomass sources to produce high-concentration and high-chirality L-lactic acid. Even when corn steep liquor containing a high concentration of D-lactic acid replaces 40% of the organic nitrogen source, yeast extract, it can still produce cellulose L-lactic acid with a chirality of 99.63%. This strain overcomes the limitation that fermentation feedstocks cannot contain D-lactic acid and has great potential in reducing the cost of fermentation feedstocks.

[0028] Preservation instructions: Classified as Pediococcus lactis Pediococcus acidilactici LJ2071 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20251230 and deposit date of May 30, 2025.

[0029] Pediococcus acidilactici Pediococcus acidilactici Z1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20252274 and deposit date of October 21, 2025.

[0030] Wild-type Pyrococcus lactis Pediococcus acidilactici ZB1, The accession number is CCTCC M 2023150, the accession date is February 20, 2023, and the accession address is China Center for Type Culture Collection. Attached Figure Description

[0031] Figure 1 : Pediococcus acidilactici Pediococcus acidilactici The content of D-lactic acid and the chirality of L-lactic acid in the fermentation broth during shake-flask fermentation of LJ2071 using corn steep liquor as a nitrogen alternative.

[0032] Figure 2 : Pediococcus acidilactici Pediococcus acidilactici Z1 shows the D-lactic acid content and L-lactic acid chirality in the fermentation broth during shake-flask fermentation to produce L-lactic acid using corn steep liquor as a nitrogen alternative.

[0033] Figure 3 : Pediococcus acidilactici Pediococcus acidilactici LJ2071 uses corn steep liquor as an alternative nitrogen source to produce L-lactic acid through fermentation in a 3L bioreactor.

[0034] Figure 4 : Pediococcus acidilactici Pediococcus acidilactici Z1 uses corn steep liquor as an alternative nitrogen source to produce L-lactic acid through fermentation in a 3L bioreactor.

[0035] Figure 5 : Pediococcus acidilactici Pediococcus acidilacticiThe content of D-lactic acid and the chirality of L-lactic acid in the fermentation broth during the production of L-lactic acid in a 3L bioreactor using corn steep liquor as a nitrogen alternative.

[0036] Figure 6 : Pediococcus acidilactici Pediococcus acidilactici Z1 shows the D-lactic acid content and L-lactic acid chirality in the fermentation broth during L-lactic acid production in a 3L bioreactor using corn steep liquor as a nitrogen alternative. Detailed Implementation Plan

[0037] The following embodiments provide a further detailed description of the present invention, but the implementation of the present invention is not limited to the scope defined by these embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0038] Pediococcus acidilactici Pediococcus acidilactici The construction of strain LJ2071 specifically involved using strains derived from *Gluconobacterium oxysporum*. Gluconobacter oxydans The D-lactate oxidase encoding gene of 621H (purchased from the American Center for Type Culture Collection) gox2071 Connecting both sides ackA Homologous arms of approximately 750 bp upstream and downstream of the gene were ligated to the temperature-sensitive shuttle plasmid pSET4E using the seamless cloning enzyme HB-infusion Master mix (Hanheng Biotechnology Shanghai Co., Ltd.). The constructed knockout plasmid was then transformed into a Gene Pulser Xcell electroporator (Bio-Rad, USA) at 2300V, 200Ω, and 25μF. Pediococcus acidilactici In Z1, integration into [the target cell] is achieved through a two-step homologous recombination method. ack Obtained from site A. Pediococcus acidilactici LJ2071 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20251230 and deposit date of May 30, 2025. Pediococcus acidilactici Z1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20252274 on October 21, 2025.

[0039] The aforementioned two-step homologous recombination method specifically refers to recombinating plasmids containing the aforementioned knockout plasmids. Pediococcus acidilacticiStrain Z1 was inoculated into liquid MRS medium containing 5 μg / mL erythromycin (20 g / L glucose, 10 g / L peptone, 10 g / L yeast extract, 5 g / L sodium acetate, 2 g / L ammonium hydrogen citrate, 22 g / L dipotassium hydrogen phosphate, 0.58 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate monohydrate) and cultured at 28 °C for 24 h. The resulting bacterial culture was then spread onto solid MRS medium (with 20% agar powder added) containing 5 μg / mL erythromycin and cultured at 42 °C until single colonies appeared, completing the first recombination. Single colonies were picked and cultured in liquid MRS medium at 28 °C, and then transferred to fresh liquid MRS medium every 24 h, repeating this process 10 times. Each time, the bacterial culture was spread onto solid MRS medium and cultured at 42 °C until single colonies appeared. Colony PCR was then performed on the single colonies to determine the amplified colonies. ackA The size of the gene fragment is used to determine whether gene integration is complete. Successfully integrated strains are named... Pediococcus acidilactici LJ2071.

[0040] The engineered strain of *Pediococcus lactis* Pediococcus acidilactici Z1, specifically, is derived from wild-type Pediococcus lactis. Pediococcus acidilactici ZB1 was obtained through metabolic engineering. Wild-type *Pediococcus lactis*. Pediococcus acidilactici ZB1, screened from straw biomass raw materials, exhibits excellent lactic acid fermentation performance. Its accession number is CCTCC M 2023150, the accession date is February 20, 2023, and the accession address is the China Center for Type Culture Collection. Pediococcus acidilactici The metabolic engineering performed on ZB1 included knocking out the D-lactate dehydrogenase gene. ldhD Knockout of phosphotransketase gene pkt Knockout of the acetate kinase gene ackA2 Integration of heterologous transketolase gene tkt Integration of heterologous aldolase genes tal Integral xylose isomerase xylA Integrating heterologous xylanokinase xylB The global transcriptional regulator CcpA protein underwent site-directed mutagenesis at T65I, and the Sigma54-dependent transcriptional regulator LevR protein underwent site-directed mutagenesis at E159G.

[0041] The aforementioned Pediococcus acidilactici The gene knockout, integration, and site-directed protein mutation performed on ZB1 are all achieved through steps similar to the two-step homologous recombination method described above.

[0042] Example 1: Pediococcus lactis Pediococcus acidilacticiLJ2071 uses liquid corn steep liquor containing racemic D-lactic acid to partially replace shake-flask fermentation of yeast extract.

[0043] Frozen Pediococcus lactis in a -80°C freezer Pediococcus acidilactici LJ2071 bacterial culture was inoculated at a rate of 10% (v / v) into shake flasks containing MRS liquid medium and activated in a shaker at 42 °C and 150 rpm until OD was reached. 600 The concentration was increased to 3.5 or higher to obtain the activated strain solution. The activated strain solution was transferred at an inoculum rate of 10% (v / v) to a shake flask containing fresh MRS liquid medium and incubated at 42 °C with constant temperature shaking at 150 rpm until the OD value was reached. 600 The seed culture was obtained when the pH was above 3.5. The seed culture was inoculated at a rate of 10% (v / v) into 250 mL shake flasks containing 50 mL of culture medium. The fermentation medium consisted of 80 g / L glucose, 40 g / L xylose, 5 g / L sodium acetate, 2 g / L ammonium citrate, 2 g / L potassium dihydrogen phosphate, 0.58 g / L magnesium sulfate heptahydrate, and 0.25 g / L manganese sulfate monohydrate. The organic nitrogen source was industrial-grade yeast extract and / or corn steep liquor, with a total addition of 25 g / L based on dry matter mass. The liquid corn steep liquor had the following specifications: total nitrogen content 2.59%, ammonia nitrogen content 0.37%, dry matter content 37.53%, and pH 4.08. Fermentation conditions were 42 ℃ and 150 rpm, with the pH adjusted to 5.5 using 0.6 g CaCO3 / g sugar.

[0044] As a preferred method, high performance liquid chromatography (HPLC) was used to detect the contents of glucose, xylose, total lactic acid and D-lactic acid in the fermentation broth.

[0045] Fermentation results as follows Figure 1 and 2 As shown, when 0%, 40%, and 80% corn steep liquor were used as the organic nitrogen source to replace yeast extract, *Pediococcus lactis*... Pediococcus acidilactici The D-lactic acid content in the fermentation broth of LJ2071 was consistently lower than that of the parent strain. Pediococcus acidilactici Z1, and its chirality far exceeds that of the parent strain. When the corn steep liquor substitution rate is 40%, the residual D-lactic acid concentration is only 0.49 g / L, with a chirality as high as 99.48%, basically meeting the requirements for polymerization-grade L-lactic acid, with an L-lactic acid concentration of 81.94 g / L. Furthermore, the *Pediococcus lactis* strain in this invention... Pediococcus acidilactici LJ2071 can replace up to 40% of the yeast extract with corn steep liquor.

[0046] Example 2: Pediococcus lactis Pediococcus acidilacticiLJ2071 uses a 3L bioreactor to produce cellulose L-lactic acid by partially replacing yeast extract with liquid corn steep liquor containing racemic D-lactic acid.

[0047] After being washed and dust-removed, wheat straw was dried and then ground in a pulverizer. The pulverized straw was then sieved (sieve diameter 10 mm). According to the NREL two-step acid hydrolysis method, the cellulose content in the wheat straw was 31.2%, the xylan content was 24.3%, and the lignin and ash contents were 19.4% and 9.6%, respectively. The specific method for dry acid pretreatment was as follows: sulfuric acid was added at 2.5% of the dry straw mass, and the mixture was kept at 175 °C for 3 minutes.

[0048] The simultaneous saccharification and co-fermentation of wheat straw to produce cellulose L-lactic acid involves several steps. First, the wheat straw is saccharified to release fermentable sugars. The specific steps are as follows: pretreated wheat straw with a solid content of 30% (w / w) is added in multiple batches to a 5L bioreactor. Cellulase is added at a dosage of 4 mg protein / g dry matter, and pre-saccharification is carried out at 50 °C and 200 rpm for 12 h. The resulting slurry is immediately transferred to a 3L bioreactor, where the temperature is set at 37 °C, and detoxified bacterial strains are introduced. Paecilomyces variotii After inoculating FN89 spores at a 10% (v / v) inoculum, liquid detoxification was initiated with a stirring speed of 750 rpm and an aeration rate of 1 vvm until inhibitors such as acetic acid, 5-hydroxymethylfurfural, and furfural were completely removed. After liquid detoxification, aeration was turned off, the stirring speed was reduced to 300 rpm, and the temperature was raised to 42 °C. L-lactic acid producing strains were then inoculated at a 10% (v / v) inoculum. P. acidilactici LJ2071 seed culture and nutrients. Nutrients refer to: corn steep liquor and / or yeast extract at a final concentration of 25 g dry matter / L, 10 g / L ammonium sulfate, and 0.25 g manganese sulfate monohydrate. During fermentation, a 25% (w / w) Ca(OH)₂ solution was used as a neutralizing agent, the fermentation pH was controlled at 5.5, and fermentation lasted 72 h. The determination of glucose, xylose, total lactic acid, and D-lactic acid was the same as in Example 1.

[0049] Fermentation results as follows Figure 3 and 4 As shown, under conditions where corn steep liquor replacement rates were 100%, 60%, 40%, and 0%, *Pediococcus lactis*... Pediococcus acidilactici LJ2071 and its parent strain Pediococcus acidilactici Both Z1 can produce cellulose L-lactic acid using wheat straw as a carbon source and corn steep liquor and yeast extract as organic nitrogen sources. The lactic acid production capacity is not significantly different, both exceeding 100 g / L.

[0050] The degradation results of D-lactic acid during fermentation are as follows: Figure 5 and6 As shown, Pediococcus lactis Pediococcus acidilactici The D-lactic acid content in the cellulose lactic acid produced by LJ2071 fermentation is always lower than that of the parent strain. Pediococcus acidilactici Z1 strain consistently exhibits higher chirality than its parental strain. When using *Pediococcus lactis* at a corn steep liquor substitution rate of 40%,... Pediococcus acidilactici LJ2071 can produce cellulose L-lactic acid with a chirality of 99.63%, which meets the requirements for polymer-grade L-lactic acid. The residual D-lactic acid concentration is only 0.42 g / L, and the L-lactic acid concentration is as high as 112.3 g / L.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the concept of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A chiral L-lactic acid-producing engineered bacterium that selectively degrades racemic D-lactic acid, characterized in that, This strain was classified and named *Pediococcus lactis*. Pediococcus acidilactici LJ2071 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20251230 and deposit date of May 30, 2025.

2. The chiral L-lactic acid-producing engineered bacterium that selectively degrades racemic D-lactic acid according to claim 1, characterized in that, This strain was developed from engineered Pediococcus lactis. Pediococcus acidilactici Z1 genome ack Site A integration originates from *Glucosamine oxidans*. Gluconobacter oxydans The gene 621H encodes D-lactate oxidase. gox2071 The engineered Pediococcus lactis obtained; Pediococcus acidilactici Z1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20252274 and deposit date of October 21, 2025.

3. The chiral L-lactic acid-producing engineered bacterium that selectively degrades racemic D-lactic acid according to claim 2, characterized in that, The engineered strain of *Pediococcus lactis* Pediococcus acidilactici Z1 is derived from wild-type Pediococcus lactis. Pediococcus acidilactici ZB1 was obtained through metabolic engineering. Wild-type Pyrococcus lactis Pediococcus acidilactici ZB1, screened from straw biomass raw materials, exhibits excellent lactic acid fermentation performance. Its accession number is CCTCC M 2023150, the accession date is February 20, 2023, and the accession address is the China Center for Type Culture Collection. Pediococcus acidilactici The metabolic engineering performed on ZB1 included knocking out the D-lactate dehydrogenase gene. ldhD Knockout of phosphotransketase gene pkt Knockout of the acetate kinase gene ackA2 Integration of heterologous transketolase gene tkt Integration of heterologous aldolase genes tal Integral xylose isomerase xylA Integrating heterologous xylanokinase xylB The global transcriptional regulator CcpA protein underwent site-directed mutagenesis at T65I, and the Sigma54-dependent transcriptional regulator LevR protein underwent site-directed mutagenesis at E159G.

4. The chiral L-lactic acid-producing engineered bacterium that selectively degrades racemic D-lactic acid according to claim 1, characterized in that, This strain has a certain ability to degrade D-lactic acid contained in the fermentation raw materials when producing high concentrations of L-lactic acid.

5. The application of a chiral L-lactic acid-producing engineered bacterium according to any one of claims 1-4 in the production of high-concentration and high-chirality L-lactic acid.

6. The application of the chiral L-lactic acid-producing engineered bacterium that selectively degrades racemic D-lactic acid according to claim 5, characterized in that, Pediococcus acidilactici Pediococcus acidilactici LJ2071 can utilize a wide range of carbon sources, including but not limited to refined sugars such as glucose, xylose, mannose, galactose, and arabinose, as well as monosaccharides from sources such as grain starch, agricultural waste, energy crops, forestry waste, and industrial biomass.

7. The application of the chiral L-lactic acid-producing engineered bacterium that selectively degrades racemic D-lactic acid according to claim 5, characterized in that, The various nitrogen sources used in the fermentation production of this strain include, but are not limited to, yeast extract, hydrolyzed vegetable protein, and corn steep liquor.

8. The application of the chiral L-lactic acid-producing engineered bacterium that selectively degrades racemic D-lactic acid according to claim 5, characterized in that, This strain requires the addition of certain nutrients during the production of high-concentration and high-chirality L-lactic acid, including but not limited to yeast extract and corn steep liquor at 5-25 g / L, ammonium sulfate at 2-10 g / L, and manganese sulfate monohydrate at 0.1-0.5 g / L.

9. The application of the chiral L-lactic acid-producing engineered bacterium that selectively degrades racemic D-lactic acid according to claim 5, *Pediococcus lactis*. Pediococcus acidilactici LJ2071 is a method for producing high-concentration and high-chirality L-lactic acid, characterized in that... The specific steps include: (1) Activation of strain: Pediococcus lactis Pediococcus acidilactici LJ2071 was inoculated into MRS liquid medium and cultured at 37-45 °C with shaking until OD200. 600 When the concentration reaches 3.5 or higher, the strain activation solution is obtained; (2) Seed culture: The activated culture solution was transferred to MRS liquid medium at an inoculation rate of 5-20% (v / v), and the strain was cultured at 37-45℃ with shaking until OD. 600 When the pH reaches 3.5 or higher, a seed culture solution is obtained; (3) Fermentation culture: The seed culture solution is inoculated into a culture medium containing monosaccharides from various biomass sources at an inoculation rate of 5-20% (v / v) to produce highly chiral L-lactic acid. During the fermentation process, the pH value is controlled at 4-6 using sodium hydroxide or calcium hydroxide solution as a neutralizing agent, and fermentation is carried out at 37-45 ℃ for 2-3 days.

10. The application of the chiral L-lactic acid-producing engineered bacterium that selectively degrades racemic D-lactic acid according to claim 9, characterized in that, The lactic acid cocci Pediococcus acidilactici The concentration of L-lactic acid produced by LJ2071 is generally not less than 80 g / L; when the concentration of D-lactic acid introduced into the fermentation system does not exceed 1.5 g / L, the chirality of the L-lactic acid product is not less than 99.5%.

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