CN115011536B

CN115011536B - Engineering bacterium for producing high optical purity D-lactic acid by double anaerobic promoters and preparation method and application thereof

Info

Publication number: CN115011536B
Application number: CN202210669063.0A
Authority: CN
Inventors: 王金华; 赵筱; 王永泽; 王周; 刘宗求
Original assignee: Hubei University of Technology
Current assignee: Jiangsu Cambrian Biological Cell Science Co ltd
Priority date: 2022-06-14
Filing date: 2022-06-14
Publication date: 2023-06-23
Anticipated expiration: 2042-06-14
Also published as: CN115011536A

Abstract

The invention provides an engineering bacterium for producing high optical purity D-lactic acid by double anaerobic promoters, a preparation method and application thereof, and the preservation number is CCTCC NO: m2022448. The invention also provides application of the engineering bacteria for producing high-optical-purity D-lactic acid in fermentation production of high-optical-purity D-lactic acid; the engineering bacteria can complete fermentation of 12% glucose within 23h of anaerobic batch fermentation, the D-lactic acid content in fermentation liquor is 104-105g/L, and the sugar acid conversion rate is more than 95%. After 1 g/L-lactic acid is added into the culture solution for fermentation, the optical purity of the D-lactic acid can reach 99.99 percent, which is obviously higher than 99.25 percent of the original strain. The invention can effectively eliminate the L-lactic acid in the fermentation tank while fermenting by enhancing the capability of the D-lactic acid engineering bacteria for converting the L-lactic acid into the pyruvic acid under the anaerobic condition, so that the optical purity of the target product D-lactic acid is improved.

Description

Engineering bacterium for producing high optical purity D-lactic acid by double anaerobic promoters and preparation method and application thereof

Technical Field

The invention relates to the technical fields of microbiology, biochemistry and fermentation engineering, in particular to engineering bacteria for producing high optical purity D-lactic acid, a preparation method and application thereof.

Background

Lactic acid is used as an important raw material in the industries of food, medicine, chemical industry, cosmetics and the like, and has huge production economic value. Global production of lactic acid was reported to reach 122 ten thousand tons in 2016 and increased at a rate of 16.2% per year (Xuejiao Tian, hao Chen, hao Liu, jihong Chen, recent advances in lactic acid production by lactic acid bacteria, appl Biochem Biotechnol,2021,193 (12): 4151-4171). D-lactic acid is a chiral intermediate for synthesizing pesticides and is also a precursor for synthesizing polylactic acid materials with high heat resistance, so that the production and the application of the D-lactic acid are widely focused. It has been proved that the optical purity of D-lactic acid is a key factor in determining the synthesis of highly heat-resistant polylactic acid (PLA) and chiral pesticides.

The microbial fermentation method has become the main method for producing D-lactic acid at home and abroad due to the advantages of wide sources of raw materials, low production cost, high optical purity, high safety and the like. D-lactic acid engineering bacteria and production bacteria are not few, and mainly concentrate on lactic acid bacteria, saccharomycetes, escherichia coli and the like. The optical purity of the optically pure D-lactic acid prepared by the method in the prior art needs to be improved. The production of D-lactic acid requires not only its yield but also optical purity of not less than 99.5% in general, and the higher the better.

Therefore, in order to improve the optical purity of D-lactic acid, it is necessary to develop an engineering bacterium which is induced by a double anaerobic promoter to produce high optical purity D-lactic acid and a method for producing high optical purity D-lactic acid by fermentation.

Disclosure of Invention

The invention aims to provide an engineering bacterium for producing high optical purity D-lactic acid by inducing a double anaerobic promoter, a preparation method and application thereof, and the engineering bacterium has high capability of converting L-lactic acid into pyruvic acid under the anaerobic condition, so that the L-lactic acid in raw materials can be eliminated in the fermentation process, and each batch of fermentation production of the high optical purity D-lactic acid is ensured.

In a first aspect of the invention, an engineering bacterium for producing high optical purity D-lactic acid by a double anaerobic promoter is provided, wherein the engineering bacterium for producing high optical purity D-lactic acid by the double anaerobic promoter is Escherichia coli HBUT-D-DNL, and the preservation number is: cctccc NO: m2022448.

In a second aspect of the invention, the use of the engineering bacteria in the fermentative production of high optical purity D-lactic acid is provided.

In a third aspect of the present invention, there is provided a fermentation broth comprising:

fermenting the engineering bacteria which produce the high optical purity D-lactic acid by inducing the double anaerobic promoters to obtain fermentation liquor;

or spray drying the fermentation liquor to obtain the dry powder microbial inoculum.

In a fourth aspect of the present invention, there is provided a method for producing the engineering bacterium, the method comprising:

amplifying the genome of the wild E.coli W serving as a template by using a primer pair shown in SEQ ID NO.3-SEQ ID NO.4 to obtain a fragment 1;

amplifying the genome of the wild E.coli W serving as a template by using a primer pair shown in SEQ ID NO.5-SEQ ID NO.6 to obtain a fragment 2;

amplifying the fragment 1 and the fragment 2 serving as templates by using primer pairs shown in SEQ ID NO.7-SEQ ID NO.8 to obtain a fragment 3; performing TA cloning on the fragment 3 and screening positive clones to obtain a plasmid pJH-nlldD;

amplifying the plasmid pJH-nlldD serving as a template by using a primer pair shown in SEQ ID NO.9-SEQ ID NO.10, and recovering and purifying by cutting gel to obtain a fragment 4;

plasmid pAGI02 was digested with restriction enzyme HindIII, and purified by gel cutting to obtain linearized plasmid fragment with pflBp 6;

performing seamless cloning on the fragment 4 and the linearized plasmid fragment with pflBp6 to obtain plasmids pJH-pnlldD of double anaerobic promoters pflBp6 and nirBp regulatory lldD genes;

and transferring the plasmid pJH-pnlldD into a strain E.coli HBUT-D15 to obtain the engineering bacterium.

In a fifth aspect of the present invention, there is provided a method for fermenting high-yield optically pure D-lactic acid using the engineering bacterium, the method comprising:

inoculating the engineering bacteria which are induced by the double anaerobic promoters to produce high optical purity D-lactic acid into a seed culture medium for seed culture to obtain activated bacteria liquid;

inoculating the activated bacterial liquid into a fermentation culture medium for fermentation culture to obtain the optically pure D-lactic acid.

Further, the formula of the seed culture medium is as follows: LB medium with 4wt% glucose added; the seed culture conditions are that the temperature is 37+/-0.5 ℃ and the rotating speed is 200+/-20 r/min.

Further, the formula of the fermentation medium is as follows: 1/5LB medium, 12wt% glucose; the conditions of the fermentation culture are that the temperature is 37+/-0.5 ℃ and 200+/-20 r/min, and the anaerobic fermentation is adopted in the fermentation culture: i.e. 23wt% calcium hydroxide was added.

Further, the OD of the activated bacterial liquid ₆₀₀ Inoculating to fermentation medium for fermentation culture at 1-1.5 times.

Further, an inoculum size of 10% was used in the fermentation culture.

One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:

1. the invention stably enhances the expression of the L-lactate dehydrogenase under anaerobic conditions through double promoters, and the effect of the double promoters is obviously better than that of a single anaerobic promoter. The single anaerobic promoter increases the unit enzyme activity of L-lactate dehydrogenase to 4.2 times and the double anaerobic promoter increases the unit enzyme activity to 6.29 times through enzyme activity detection.

2. The double-promoter expression system adopted by the invention is a self-induction expression system, does not need to add any inducer and does not need to increase extra cost; the induction condition is only anaerobic, and is suitable for the anaerobic fermentation process.

3. When the invention is applied to the fermentation production of D-lactic acid, the L-lactic acid in the fermentation tank can be effectively eliminated under the whole-course anaerobic fermentation condition, so that the optical purity of the target product D-lactic acid is obviously improved. The engineering bacteria E.coli HBUT-D-DNL can be fermented in 23h, and the fermentation liquor contains 104-105g/L of D-lactic acid, and the sugar acid conversion rate is more than 95%. When L-lactic acid of 1g/L is added, the optical purity of D-lactic acid after fermentation of the initial strain HBUT-D15 is 99.25%, and the E.coli HBUT-D-DNL can reach 99.99%.

The preservation date of the engineering bacteria for inducing the production of high optical purity D-lactic acid by the double anaerobic promoters is 2022, 4 and 21, and the preservation number is CCTCC NO: m2022448. The classification is Escherichia coli HBUT-D-DNL, the preservation unit is China center for type culture Collection, and the address is the university of Wuhan in Wuhan, hubei province, china, post code: 430072.

drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a physical diagram of plasmid pJH-pnlldD;

FIG. 2 is a PCR-validated electrophoresis of plasmid pJH-pnlldD constructs.

Detailed Description

The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.

Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control. Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present invention are commercially available or may be obtained by existing methods.

The discovery process of the technical problem of the invention is as follows:

in the early stage of the laboratory, D-lactic acid engineering bacteria HBUT-D are constructed by carrying out gene modification on E.coli W, knocking out adhE, frdABCD, pta, pflB, aldA and cscR genes, and the method comprises the following steps ofThe strain can be used for producing 85g/L D-lactic acid by fermenting 100g/L sucrose in a 7L fermenter, and the optical purity reaches 98.3 percent (Yongze Wang, tian Tian, jinfang, jinhua Wang, tao Yan, liyuan Xu, liu Zao, erin Garza, andrew Iverson, ryan Manow, chris Finan, shaping Zhou, homofermentative production of D-lactic acid from sucrose by a metabolically engineered Escherichia coli, biotechnol Lett,2012,34 (11): 2069-2075); meanwhile, after optimization by a neutralizer, the strain can also be used for producing 127g/L D-lactic acid by using 130g/L glucose for fermentation, the conversion rate is 93%, and the average production strength is 6.35g/L.h (Ye Liu, wa Gao, xiao Zhao, jinhua Wang, erin Garza, ryan Manow, shengde Zhou, pilot scale demonstration of D-lactic acid fermentation facilitated by Ca (OH) ₂ using a metabolically engineered Escherichia coli,Bioresour Tecchnol,2014,169:559-565)。

HBUT-D15 obtained after multi-generation domestication can be fermented in a 7L fermentation tank by using 200g/L glucose to produce 184-191g/L D-lactic acid, and the optical purity reaches 99.9%; in a 3 ton fermenter, 146-150g/L D-lactic acid can be produced by 160g/L glucose fermentation with an optical purity of 99.8% (Xiangmin Fu, yongze Wang, jinhua Wang, erin Garza, ryan Manow, shangde Zhou, semi-industrial scale (30 m) ² )fed-batch fermentation for the production of D-lactate by Escherichia coli strain HUBT-D15,J Ind Microbiol Biotechnol,2017,44(2):221-228)。

However, in practical industrial-scale fermentative production of HBUT-D15 strain, we found that different batches of fermentation, due to the use of fermentation raw materials (such as corn steep liquor and industrial-scale glucose water containing more L-lactic acid) and the variation of environmental conditions outside the fermentation, tend to result in a decrease in optical purity of the product of 0.1-0.2%.

Therefore, the general idea of the invention for solving the technical problems is as follows:

(1) Constructing a plasmid pJH-pnlldD of an L-lactate dehydrogenase gene (lldD gene) with a double anaerobic promoter;

(1) PCR (polymerase chain reaction) amplification of a fragment nirBp of 136bp (including a promoter region thereof) upstream of the full length of the escherichia coli lldD gene and the (nirB gene) coding region of the nitrate reductase subunit B;

(2) ligating the nirBp and lldD genes by ligation PCR;

(3) TA cloning and sequencing the connecting fragment nirBp+lldD, and naming the plasmid as pJH-nlldD;

(4) the fragment nirBp+lldD was ligated downstream of the anaerobic promoter pflBp6 of plasmid pAGI02 by a method of cloning based on T5exonuclease, and a double anaerobic promoter regulatory plasmid pJH-pnlldD was constructed.

(2) The plasmid pJH-pnlldD is transferred into a strain E.coli HBUT-D15 by a transformation method to obtain the strain E.coli HBUT-D-DNL. The strain is used for producing the high-gloss pure D-lactic acid by fermentation.

The invention constructs a plasmid of a double anaerobic promoter regulation and control lldD gene (the L-lactic dehydrogenase coded by the gene can convert L-lactic acid into pyruvic acid, but the conversion process can only occur under aerobic condition and is influenced by carbon metabolism inhibition), and converts the plasmid into engineering bacteria HBUT-D15, aiming at increasing the capability of converting L-lactic acid into pyruvic acid under anaerobic condition, thereby eliminating L-lactic acid in raw materials in the fermentation process, ensuring that each batch of fermentation produces high-optical-purity D-lactic acid and improving the use value of the D-lactic acid.

The following will describe in detail the engineering bacteria induced by the double anaerobic promoters to produce high optical purity D-lactic acid, and the preparation method and application thereof by combining the examples and experimental data.

Example 1 construction of plasmid pJH-pnlldD for controlling the lldD Gene by double anaerobic promoters

The specific operation steps for constructing the plasmid pJH-pnlldD are as follows:

(1) The genome of wild E.coli W is used as a template, and the lldD-P1 and the lldD-P2 are used as primers to amplify the lldD gene sequence. The wild type e.coli W is e.coli W3110 wild type, commercially available from the biont of the hundred-ohm bordetella, platform No.: bio-82057.

TABLE 1 PCR primer sequences in the present invention

(2) The genome of the wild E.coli W is used as a template, and the nirBp-P1 and the nirBp-P2 are used as primers to amplify the nirBp of 136pb sequence (including promoter sequence) on the upstream of the nirB gene.

(3) The nirBp and the lldD genes are connected through connecting PCR, and the specific operation steps are as follows:

the PCR products of (1) and (2) were each used as templates, and the nirBp and the lldD fragment were ligated by PCR reaction using the nlldD-P1 and nlldD-P2 as primers.

(4) TA cloning was performed on the linker fragment nirBp+lldD of (3). And sequencing and verifying positive clones obtained by screening the resistance plate, and naming the plasmid with correct verification as pJH-nlldD. Wherein the nucleotide sequence of the connecting fragment nirBp+lldD is shown in SEQ ID NO. 1.

(5) Plasmid pAGI02 was digested with restriction enzyme HindIII, and purified by gel cutting to obtain linearized plasmid fragment with pflBp 6; the plasmid pAGI02 in the examples was constructed with the PUC19 plasmid as the basic skeleton, and has the optimized promoter region of the pyruvate formate lyase gene (pflBp 6). Plasmid pAGI02 is described in: iverson A, garza E, zhao J, wang Y, zhao X, wang J, manow R, zhou S.Increate reducing power output (NADH) of glucose catabolism for reduction of xylose to xylitol by genetically engineered Escherichia coli AI05.world J Microbiol Biotechnol.2013,29 (7): 1225-1232.

(6) And (3) carrying out PCR (polymerase chain reaction) amplification by taking the obtained plasmid pJH-nlldD as a template and taking nlldD-P3 and nlldD-P4 as primers, and recovering and purifying amplified fragments by cutting gel. Plasmids and strains used in examples 1 and 2 of the present invention are shown in Table 2.

TABLE 2 strains and plasmids used in the present invention

(7) The fragment purified in step (6) and the linearized plasmid purified in step (5) were added in a molar ratio of 3:1 in appropriate volumes, T5 Exoneclease 1. Mu.L, 10X Reaction Buffer 1. Mu.L, and water was added to a total volume of 10. Mu.L, and after 5min of reaction at 4℃they were immediately transferred into DH 5. Alpha. Competent cells by heat shock and positive clones were screened by resistance plates.

(8) And (3) taking the pnlldD-P1 and the pnlldD-P2 as primers, and verifying positive clones by PCR (shown in figure 2) to obtain a plasmid pJH-pnlldD (shown in figure 1) with double anaerobic promoters pflBp6 and nirBp for regulating the lldD genes. The nucleotide sequences of the two promoter +lldD genes (namely pflBp6+nirBp+lldD) are shown in SEQ ID NO. 2.

Example 2 obtaining Strain E.coli HBUT-D-DNL

1. Initial strain HBUT-D15

(1) Knocking out genes of wild E.coli W strains adhE, frdABCD, pta, pflB, aldA and cscR, and constructing to obtain the D-lactic acid engineering bacteria HBUT-D, wherein the details are shown in the literature: yongze Wang, tian Tian, jinfang, jinhua Wang, tao Yan, liyuan Xu, liu Zao, erin Garza, andrew Iverson, ryan Manow, chris Finan, shaping Zhou, homofermentative production of D-lactic acid from sucrose by a metabolically engineered Escherichia coli, biotechnol Lett,2012,34 (11): 2069-2075.

(2) Carrying out multi-generation culture, fermentation and domestication on the D-lactic acid engineering bacteria HBUT-D to obtain an initial strain HBUT-D15, wherein the details are shown in the literature: xiangmin Fu, yongze Wang, jinhua Wang, erin Garza, ryan Manow, shengde Zhou, semi-inductive scale (30 m) ² )fed-batch fermentation for the production of D-lactate by Escherichia coli strain HUBT-D15,J Ind Microbiol Biotechnol,2017,44(2):221-228.

2. The plasmid pJH-pnlldD is transferred into a strain E.coli HBUT-D15 by a transformation method to obtain the strain E.coli HBUT-D-DNL. The preservation date is 2022, 4 and 21, and the preservation number is CCTCC NO: m2022448. The classification is Escherichia coli HBUT-D-DNL, the preservation unit is China center for type culture Collection, and the address is the university of Wuhan in Wuhan, hubei province, china, post code: 430072.

comparative example 1

The plasmid pJH-nlldD in example 1 was transformed into the strain HBUT-D15 by the transformation method to obtain the strain HBUT-D-DL.

Experimental example 1 enzyme Activity detection of L-lactate dehydrogenase (lldD)

1. Extracting crude enzyme solution: 2-3 single colonies were picked from the plate and inoculated into 50mL of liquid LB medium, cultured overnight at 37℃at 200r/min, transferred to a 250mL shake flask containing 150mL of liquid LB medium at 1% of the inoculum size, cultured for 12 hours at 37℃at 200r/min, and collected by centrifugation. Adding 15mL buffer solution (containing 0.5% Triton X-100), resuspending, ultrasonic crushing (ultrasonic power is 300W, working for 15s, interval is 15s, time is 10 min) to obtain cell lysate, 12000r/min, centrifuging for 5min, and collecting supernatant to obtain crude enzyme solution. The total protein content of the crude enzyme solution was measured by Lowry method using BSA as a protein standard.

2. Enzyme activity detection: the reaction system was 1mL: add 10. Mu.L of 10mg/L PMS, 10. Mu.L of 10umol/mL FMN-Na ₂ 50. Mu.L of MTT 5mg/mL, 500. Mu.L of L-lactic acid 1mg/mL, 50. Mu.L of a properly diluted crude enzyme solution, and the total volume was made up to 1mL with 0.067mol/L, pH 7.0 potassium phosphate buffer. The reaction was terminated at 37℃and pH 7.0 for 20min, followed by ice bath for 5min, and the absorbance at 570nm was measured. The inactivated enzyme solution was used as a control. The enzyme activity unit (u) is defined as: the amount of enzyme required to reduce 1 mol MTT per minute at 37℃and pH 7.0 was defined as 1 unit.

The results of the L-lactate dehydrogenase (LldD) enzyme activities are shown in Table 3.

TABLE 3 enzymatic Activity of L lactate dehydrogenase (LldD)

The enzyme activity results of Table 3 above show that the unit enzyme activity of HBUT-D-DL was increased to 4.2 times and the unit enzyme activity of HBUT-D-DNL was increased to 6.29 times relative to the starting strain HBUT-D15. The anaerobic promoter has obvious up-regulation effect on the expression of the lldD gene, and the regulation effect of double-promoter tandem connection is better than that of single-promoter.

Experimental example 2 production of D-lactic acid by fermentation

The capacity of three strains (original strain HBUT-D15, strain E.coli HBUT-D-DNL of example 2, strain E.coli HBUT-D-DL of comparative example 1) to produce D-lactic acid by fermentation was compared by fermentation experiments; the specific operation steps are as follows:

(1) A single colony was picked from the plate and inoculated into an anaerobic tube containing 10mL of seed culture solution and cultured overnight at 37 ℃. Inoculating 2mL of bacterial liquid into 300mL of seed liquid, and culturing OD at 37 ℃ at 200r/min ₆₀₀ To 1-1.5. Wherein, seed culture medium: LB medium, 4% glucose.

(2) The bacterial liquid is inoculated into 5L fermentation medium with 10% (v/v) inoculation amount, placed in a 7L fermentation tank Sartorius BB-8846880 (Germany Sartorius Stedim Biotech company) with an automatic regulating system, cultured and fermented at 37 ℃ for 200r/min, and the pH is controlled to 7.0 by taking 23% calcium hydroxide as a neutralizer. Culturing with 12% glucose as substrate until fermentation is completed. Wherein, fermentation medium: 1/5LB medium (i.e., 1/5 of the standard LB medium composition) and 12% glucose (1 g/L L-lactic acid added).

(3) The samples were taken at regular intervals, and the cell concentration, glucose, lactic acid, the concentration of its metabolite and the optical purity of lactic acid were measured.

The concentration of the bacterial cells is measured by acidolysis with 3mol/L HCl solution, and then OD value is measured at 600nm wavelength by a visible light spectrophotometer. Glucose, xylose, arabinose and organic acid were analyzed by high performance liquid chromatography Waters e2695 (Waters Co., USA), chromatographic column Bio-Rad HPX 87H, mobile phase 4mmol/L H ₂ SO ₄ The flow rate is 0.5mL/min, the column temperature is 40 ℃, and the detector is a PDA or ELS detector. The optical purity of lactic acid is analyzed by high performance liquid chromatograph Waters e2695 (Waters Co., USA), the chromatographic column is CHIRAL column EC 250/4NUCLEOSIL CHIRAL-1, and the mobile phase is 2mmol/L CuSO ₄ The flow rate is 0.5mL/min, the column temperature is 40 ℃, and the detector is a PDA detector. The fermentation results are shown in Table 4.

TABLE 4 fermentation results

As shown in Table 4, the engineering bacteria HBUT-D-DNL completed fermentation within 23h, and the fermentation broth contained 104-105g/L lactic acid, and the sugar acid conversion rate reached more than 95%. When 1g/L L-lactic acid is added into the culture medium, the final optical purity of D-lactic acid produced by fermentation of the original strain HBUT-D15, the strain HBUT-D-DL with the single anaerobic promoter regulation plasmid and the strain HBUT-D-DNL with the double anaerobic promoter regulation plasmid is 99.25%,99.88% and 99.99%, respectively. The strain HBUT-D-DNL can effectively eliminate L-lactic acid in the fermentation process, so that the optical purity of a target product D-lactic acid is stabilized and improved, and the use value of the product is improved.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

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gatgcgcatt caggtatgag cggcccgaac gcggcaatgc gccgctactt gcaagcggtg 960

acacatccgc aatgggcgtg ggatgtgggc ctgaacggtc gtccacatga tttaggtaat 1020

atctcagctt atctcggcaa accgaccgga ctggaagatt acatcggctg gctggggaat 1080

aacttcgatc cgtccatctc atggaaagac cttgaatgga tccgcgattt ctgggatggc 1140

ccgatggtga tcaaagggat cctcgatccg gaagatgcgc gcgatgcagt acgttttggt 1200

gctgatggaa ttgtggtttc taaccacggt ggccgccagc tggacggtgt actctcttcc 1260

gcccgtgcac tgcctgctat tgcagatgcg gtgaaaggtg atatagccat tctggcggat 1320

agcggaattc gtaacgggct tgatgtcgtg cgtatgattg cgctcggtgc cgacaccgta 1380

ctgctgggtc gtgctttctt gtatgcgctg gcaacagcgg gccaggcggg tgtagctaac 1440

ctgctaaatc tgatcgaaaa agagatgaaa gtggcgatga cgctgactgg cgcgaaatcg 1500

atcagcgaaa ttacgcaaga ttcgctggtg caggggctgg gtaaagagtt gcctgcggca 1560

ctggctccca tggcgaaagg gaatgcggca tag 1593

<210> 3

<211> 42

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gaaaagaaat cgaggcaaaa atgattattt ccgcagccag cg 42

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

acatacagcg ccgaacggtc 20

<210> 5

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

ccgtgactta agaaaattta tac 23

<210> 6

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

ctggctgcgg aaataatcat ttttgcctcg atttcttttc 40

<210> 7

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

cagcaatata cccattaagg agtat 25

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

aacgactatg ccgcattc 18

<210> 9

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gcaaccactg gcacaggcac cagcaatata cccattaagg agtat 45

<210> 10

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

gaccatgatt acgccaagct ctatgccgca ttccctttcg 40

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gaactgcagg acgaggcagc 20

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gcaaccccgc ttattgatgt aaatc 25

Claims

1. The engineering bacterium for producing high optical purity D-lactic acid by the double anaerobic promoters is characterized in that the engineering bacterium for producing high optical purity D-lactic acid by the double anaerobic promoters is Escherichia coli HBUT-D-DNL, and the preservation number is: cctccc NO: m2022448.

2. An application of the engineering bacteria in fermenting high-yield optical pure D-lactic acid.

3. A fermentation broth, the fermentation broth comprising:

fermenting the engineering bacteria which are induced to produce the high optical purity D-lactic acid by the double anaerobic promoters according to claim 1 to obtain fermentation liquor;

4. A method for fermenting high-yield optically pure D-lactic acid by using the engineering bacteria of claim 1, comprising:

inoculating the engineering bacteria induced by the double anaerobic promoters to produce high optical purity D-lactic acid in a seed culture medium for seed culture to obtain an activated bacterial liquid;

inoculating the activated bacterial liquid into a fermentation culture medium for fermentation culture to obtain the D-lactic acid with high optical purity.

5. The method of claim 4, wherein the seed medium is formulated as follows: LB medium supplemented with 4wt% glucose; the seed culture conditions are that the temperature is 37+/-0.5 ℃ and the rotating speed is 200+/-20 r/min.

6. The method of claim 4, wherein the fermentation medium is formulated as follows: 1/5LB medium, 12wt% glucose; the conditions of the fermentation culture are that the temperature is 37+/-0.5 ℃ and 200+/-20 r/min, the whole-course anaerobic fermentation is adopted in the fermentation culture, and the neutralizer is 23wt% of calcium hydroxide.

7. The method according to claim 4, wherein the OD600 of the activated bacterial liquid is 1-1.5, and the activated bacterial liquid is inoculated into a fermentation medium for fermentation culture.

8. The method of claim 4, wherein 10% inoculum size is used in the fermentation culture.