Escherichia coli for efficiently utilizing sucrose to produce L-lactic acid and its application

Abstract

The present invention discloses Escherichia coli and applications for efficiently utilizing sucrose to produce L-lactic acid, and belongs to the field of biotechnology. The Escherichia coli of the present invention is Escherichia coli HBUT-SL with a deposit number of CCTCC NO: M 20221463 or Escherichia coli HBUT-SLE with a deposit number of CCTCC NO: M 20221464. Compared with the starting strain HBUT-L, Escherichia coli HBUT-SL has an additional copy of the glpD gene. Escherichia coli HBUT-SLE is further transferred with the ptrc99a-glpD plasmid expressing glpD on the basis of Escherichia coli HBUT-SL. Compared with the starting strain HBUT-L, the Escherichia coli HBUT-SL and HBUT-SLE of the present invention have a significant improvement in lactic acid yield, average production intensity and maximum biomass. The present invention provides valuable strain resources for the utilization of sucrose molasses.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to Escherichia coli that efficiently utilizes sucrose to produce L-lactic acid and its application. Background Art

[0002] L-lactic acid is an important three-carbon organic acid, which is widely used in food, medicine, tobacco and chemical industries. L-lactic acid can be used as a monomer to synthesize the biodegradable material polylactic acid (PLA), which is one of the most valuable biodegradable materials. In the context of the "plastic ban", due to its excellent comprehensiveness, it can be widely used in medicine, construction, agriculture, forestry and clothing industries. The market potential of L-lactic acid will also increase in the future.

[0003] There are three methods for producing L-lactic acid: chemical method, enzymatic method and fermentation method. The product produced by chemical synthesis method is racemic lactic acid, namely DL-lactic acid. This product will cause the heat resistance of the material to deteriorate when used to synthesize polylactic acid. L-lactic acid can also be obtained by enzymatic method, but the cost of enzymatic synthesis of L-lactic acid using raw materials such as pyruvate is too high. The production of lactic acid by fermentation method has the characteristics of low production cost, high product optical purity and wide source of raw materials, and has become the main method for producing L-lactic acid in recent years. Among them, L-lactic acid produced by Escherichia coli fermentation has the characteristics of high optical purity and low nutritional requirements for microbial culture, and has attracted much attention in the industry. In addition to selecting suitable microorganisms to ferment L-lactic acid, low-cost and easily available raw materials are also important factors that can promote the reduction of L-lactic acid fermentation costs. For example, corn pulp (Chinese patent CN104450585A), cassava (Chinese patent CN101805759A; CN102304480), industrial / agricultural waste containing pentose or hexose (Chinese patent CN102690764A; CN102174602A; CN105506005A), xylose (Chinese patent CN102433293A), a mixture of organic waste and residual activated sludge (Chinese patent 103923952) and molasses (Chinese patent CN104178438A) have all been used in L-lactic acid fermentation. In recent years, the number of patent applications for producing L-lactic acid from lignocellulose has gradually increased (Chinese patents CN107988274A; CN109837316A; CN112662710A; CN 112501218A; CN 112941117 A).

[0004] Compared with lignocellulose, molasses is not expensive and is known as the cheapest carbon source among fermentation raw materials (CN104178438A). It also has the advantages of relatively simple raw material pretreatment and more convenient raw material collection. Its main components are sucrose, glucose and fructose. However, strains that can efficiently utilize sucrose and produce high optical purity L-lactic acid are still to be selected. Chinese patent CN104178438A "A strain of Lactobacillus delbrueckii suitable for producing high-purity L-lactic acid by molasses fermentation and fermentation method and application" uses Lactobacillus delbrueckii, which is difficult to obtain. In addition, the Lactobacillus delbrueckii isolated by other researchers has D-lactic acid as the main product (Zheng Zuoxing, Liu Zutong. Research on the production of lactic acid by fermenting corn flour with Lactobacillus delbrueckii ZL-513 [J]. Food and Fermentation Industry, 1992 (4): 5).

[0005] E. coli has a clear genetic background and mature genetic modification technology. Escherichia coli K1, B and C strains cannot utilize sucrose, but some E. coli with sucrose utilization ability can still be found, such as E. coli B62 (Tsunekawa H, Azuma S, Okabe M, et al. Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry [J]. Applied & Environmental Microbiology, 1992, 58 (6): 2081-2088.). It is also possible to introduce sucrose utilization-related genes of other Escherichia coli, such as Escherichia coli W strain, into the genome by transposition (Carruthers DN, TESaleski, Scholz SA, et al. Random chromosomal integration and screening yields E. coliK-12derivatives capable of efficient sucrose utilization[J]. ACS SyntheticBiology, 2020, 9(12).). Of course, exogenous sucrose phosphorylation or non-phosphorylation transport systems can also be introduced through plasmid expression (CN 112501105 A; He X, Li Y, TaoY, et al. Discovering and efficiently promoting the extracellular secretory expression of Thermobacillus sp. ZCTH02-B1 sucrose phosphorylase in Escherichia coli [J].International Journal of Biological Macromolecules, 2021, 173:532–540).

[0006] Escherichia coli W, as the starting strain (ATCC 9637), carries genes related to sucrose utilization and has a good potential for utilizing sucrose to produce lactic acid. How to combine genetic engineering methods with traditional strain breeding methods to further select Escherichia coli engineered strains that efficiently utilize sucrose to produce L-lactic acid will undoubtedly play a key role in promoting the utilization of molasses and the low-cost production of L-lactic acid. Summary of the invention

[0007] The purpose of the present invention is to overcome the current situation that the efficiency of existing strains in producing L-lactic acid using sucrose is low, and to provide an Escherichia coli engineered bacteria and its application that can efficiently produce L-lactic acid using sucrose obtained by adaptive domestication and reverse biology. The purpose of the present invention is also to provide a method for improving the ability of Escherichia coli to produce L-lactic acid using sucrose.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] An Escherichia coli that efficiently utilizes sucrose to produce L-lactic acid, which is one of the following Escherichia coli:

[0010] Escherichia coli HBUT-SL with a deposit number of CCTCC NO: M 20221463;

[0011] The deposit number of Escherichia coli HBUT-SLE is CCTCC NO: M 20221464.

[0012] The Escherichia coli HBUT-SL is obtained by adaptive domestication of Escherichia coli HBUT-L (recorded in the literature Engineering and adaptive evolution of Escherichia coli W for L-lactic acid fermentation from molasses and .corn steep liquor without additional nutrients, Bioresource Technology, 2013, 184:394-400) as the starting strain in a sucrose-rich environment. The gene encoding fumarate reductase has been knocked out in Escherichia coli HBUT-L. frdBC , gene encoding alcohol dehydrogenase adhE , genes encoding phosphate acetyltransferase pta , Gene encoding pyruvate formate lyase pflB , encoding sucrose promoter repressor gene cscR , Gene encoding acetaldehyde dehydrogenase aldA , gene encoding D-lactate dehydrogenase ldhA , while inserting Pediococcus acidilactici The gene encoding L-lactate dehydrogenase ldhL Compared with E. coli HBUT-SL and E. coli HBUT-L, the genome of E. coli HBUT-SL has one more copy of glycerol-3-phosphate dehydrogenase glpD Gene.

[0013] Adaptation of E. coli HBUT-L to sucrose raw materials: HBUT-L was adapted in a 5L fermenter. The strain was streaked on a plate containing an inorganic salt medium and cultured overnight at 35-37°C. Then 4-10 HBUT-L colonies grown on the plate were selected and inoculated into 50 mL of an inorganic salt medium containing 2% sucrose. After culturing at 35-37°C and 150-200 r / min for 12-16 h, the inoculation amount was transferred to 3L of an inorganic salt medium containing 10% sucrose at a rate of 5%-10%. The fermentation conditions were: 35-37°C and a stirring paddle speed of 150-200 r / min. The pH value of the fermentation liquid was controlled to 6.5 by adding 18-22% (w / w) Ca(OH)2. After fermentation for 18-24 h, the inoculation amount was transferred to another tank containing 3 L of fresh inorganic salt medium containing 10% sucrose at a rate of 10%. The fermentation was repeated for 6 generations. The transferred fermentation broth was diluted with sterile water and then spread on LB plates to isolate single colonies. Several single colonies were randomly selected and named. The strain with the best sucrose utilization effect was named HBUT-SL.

[0014] The E. coli HBUT-SLE was further transformed into the expression glpD Plasmid pTrc99a- glpD .

[0015] When E. coli HBUT-SL was cultured in an inorganic salt medium containing 10% sucrose and 0.5% corn steep liquor powder, the L-lactic acid production was 90.85 g / L, the fermentation time was 36 h, and the average production intensity was 2.52 g·L -1 h -1 , the maximum biomass was 19.55, and its lactic acid yield, average production intensity and maximum biomass were increased by 1.10, 1.83 and 2.02 times compared with the undomesticated E. coli HBUT-L, respectively, and the fermentation time was shortened by 24 h. When E. coli HBUT-SLE was cultured in the above medium, the L-lactic acid yield was 95.30 g / L, the fermentation time was 24 h, and the average production intensity was 3.97 g·L -1 h -1 , the maximum biomass was 16.28, and its lactic acid yield, average production intensity and maximum biomass were increased by 1.15, 2.88 and 1.68 times compared with the undomesticated Escherichia coli HBUT-L, respectively, and the fermentation time was shortened by 36 h.

[0016] The Escherichia coli that efficiently utilizes sucrose to produce L-lactic acid is used in producing L-lactic acid.

[0017] A method for producing L-lactic acid comprises the following steps: culturing the above-mentioned Escherichia coli in a culture medium containing sucrose and corn steep liquor dry powder.

[0018] A method for improving the ability of Escherichia coli to produce L-lactic acid using sucrose, for overexpressing the L-lactic acid-producing Escherichia coli glpD Gene.

[0019] Advantages and beneficial effects of the present invention:

[0020] (1) The present invention combines single gene modification and domestication breeding to achieve efficient use of sucrose to produce L-lactic acid by E. coli. In the past, the idea of ​​modifying E. coli to produce L-lactic acid was mainly to knock out the gene encoding fumarate reductase. frdBC , the gene encoding alcohol dehydrogenase adhE , a gene encoding phosphate acetyltransferase pta , the gene encoding pyruvate formate lyase pflB , encoding a sucrose promoter repressor gene cscR , the gene encoding acetaldehyde dehydrogenase aldA , the gene encoding D-lactate dehydrogenase ldhA, to avoid the production of other impurities such as acetic acid, D-lactic acid, formic acid, succinic acid, etc. To improve its utilization of sucrose, the main method is to knock out the gene encoding the sucrose promoter inhibitor cscR However, E. coli often has multiple transport pathways for the utilization of various carbon sources, and the utilization of carbon sources also involves multiple genes. Therefore, the traditional idea of ​​relying on single gene modification may not necessarily achieve the goal of efficiently utilizing sucrose to produce L-lactic acid. The present invention combines the method of targeted domestication to further explore the ability of E. coli to utilize sucrose, making up for the shortcomings of the original method.

[0021] (2) With the help of the idea of ​​reverse genomics, we have discovered glpD Gene, it is speculated that it may affect glycolysis and relieve fructokinase (encoding gene cscK) or sucrose hydrolase (encoding gene cscB ) to enhance sucrose utilization by overexpressing glpD The gene preliminarily confirmed the speculation, and a new engineered strain HBUT-SLE was constructed for efficient utilization of sucrose to produce L-lactic acid. The lactic acid yield, average production intensity and maximum biomass of the strain were increased by 1.15, 2.88 and 1.68 times compared with the undomesticated Escherichia coli HBUT-L, respectively, and the fermentation time was shortened by 36 h, providing a valuable strain resource for the utilization of sucrose molasses. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Comparison of the genomes of the strain HBUT-L before domestication and the strain HBUT-SL after domestication.

[0023] Figure 2 Comparison of the glpD gene in the genomes of the strain HBUT-L before domestication and the strain HBUT-SL after domestication.

[0024] Figure 3 This is the ptrc99a-glpD plasmid construction map.

[0025] Strain deposit information:

[0026] (1) Escherichia coli HBUT-SL, deposited in China Center for Type Culture Collection; deposited at Wuhan University, Wuhan, China; deposited on September 20, 2022; classification and nomenclature: Escherichia coli HBUT-SL; Deposit number: CCTCC NO: M 20221463.

[0027] (2) Escherichia coli HBUT-SLE, deposited in China Center for Type Culture Collection; deposited at Wuhan University, Wuhan, China; deposited on September 20, 2022; classification and nomenclature: Escherichia coliHBUT-SLE; Deposit number: CCTCC NO: M 20221464. DETAILED DESCRIPTION

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] The Escherichia coli HBUT-SL and Escherichia coli HBUT-SLE in the following examples were deposited by the applicant in the China Center for Type Culture Collection on September 20, 2022, with the deposit numbers CCTCC NO: M 20221463 and CCTCC NO: M20221464, and their classification names are Escherichia coli HBUT-SL and Escherichia coli HBUT-SLE.

[0031] The present invention is further described in detail below by way of examples.

[0032] Example 1 Construction of Engineering Bacteria

[0033] The starting strain was Escherichia coli HBUT-L, which was stored in our laboratory and has NCBI access number CP104442-CP104443 (described in the literature Engineering and adaptive evolution of Escherichia coli W for L-lactic acid fermentation from molasses and .corn steep liquor without additional nutrients, Bioresource Technology, 2013, 184:394-400). The gene encoding fumarate reductase has been knocked out in this bacterium. frdBC , the gene encoding alcohol dehydrogenase adhE , a gene encoding phosphate acetyltransferase pta , the gene encoding pyruvate formate lyase pflB , encoding a sucrose promoter repressor gene cscR , the gene encoding acetaldehyde dehydrogenase aldA , the gene encoding D-lactate dehydrogenase ldhA , while inserting Pediococcus acidilactici The gene encoding L-lactate dehydrogenase ldhL .

[0034] Example 2 Domestication of engineering bacteria

[0035] Overnight culture, then select 4 HBUT-L colonies grown on the plate and inoculate them into 50 mL of inorganic salt medium containing 2% sucrose. After culturing at 37℃ and 200 r / min for 12 h, transfer the inoculation volume to 3L inorganic salt medium containing 10% sucrose at 5% inoculation volume. The fermentation conditions are: 37℃ and 200 r / min stirring paddle speed. The pH value of the fermentation broth is controlled to 6.5 by adding 22% (w / w) Ca(OH)2. After 24 h of fermentation, transfer the inoculation volume to another 3L fresh inorganic salt medium containing 10% sucrose at 10% inoculation volume, and repeat the fermentation transfer for 6 generations. Samples of each batch of fermentation broth were taken at regular intervals, and the lactic acid content and biomass of the fermentation for 72 h were determined. The results are shown in Table 1. The fermentation broth of the last transfer was diluted with sterile water and spread on the LB plate to isolate single colonies. Single colonies were randomly selected and named. The strain with the best sucrose utilization effect was named HBUT-SL. The inorganic salt culture medium formula is: 3.5 g KH2PO4, 5.0 g K2HPO4, 3.5 g (NH4)2HPO4, 0.25 g MgSO4·7H2O, 15 mg CaCl2·2 H2O, 0.5 mg thiamine, 1 mL trace element mother solution per liter. The trace element mother solution composition is: add 1.6 g FeCl3, 0.2 g CoCl2·6H2O, 0.1 g CuCl2, 0.2 g ZnCl2·4H2O, 0.2 g NaMoO4, 0.05 g H3BO3 per liter in 0.1 mol / L HCl.

[0036] Table 1 Lactic acid production results of each batch of fermentation broth during the acclimation process (results of fermentation for 72 h)

[0037]

[0038] Example 3 Differences in the genome of Escherichia coli engineered bacteria before and after domestication

[0039] The determination of gene sequence and genetic characteristics of engineered bacteria after domestication is carried out in the following steps:

[0040] (1) The domesticated strain that can efficiently utilize sucrose to produce L-lactic acid (HBUT-SL in Example 2) and the strain before domestication (HBUT-L in Example 1) were sent to relevant companies for second-generation and third-generation genome sequencing. The genome resequencing was completed by Shenzhen BGI Genomics Co., Ltd. and Beijing Qingke Biotechnology Co., Ltd. Wuhan Branch. The reference sequence for sequence analysis was the genome sequence of ATCC 9637 ( https: / / ftp.ncbi.nlm.nih.gov / genomes / all / GCF / 008 / 868 / 305 / GCF_008868305.1_ASM886830v1 / ),sequencing depth is 100 times.

[0041] (2) The genome annotation work has been done by the sequencing company using prokka 1.14.6 ( https: / / github.com / tseemann / prokka ) is completed and the corresponding gbk file is generated.

[0042] (3) After annotation, use Mauve to perform genome sequence alignment to find differentially expressed genes. Preferably, Mauve (version 2.3.0, 32-bit java version 1.8.0_131 for Windows 8 system) is used. The results of genome alignment are shown in Table 2.

[0043] Table 2 Comparison of differentially expressed genes in the genome of strains before and after domestication

[0044]

[0045] Find genes related to sucrose utilization, transport or glycolysis, and find that HBUT-SL has one more copy of glycerol-3-phosphate dehydrogenase than HBUT-L glpD Gene. For comparison of related genomes, see Figure 2 .

[0046] The same method was used to perform SNP analysis on the two genomes, but no base mutations or deletions were found in genes related to sucrose utilization, transport or glycolysis.

[0047] Example 4 glp D gene overexpression

[0048] Speculation glpD Genes that promote glycolysis and thus reduce sucrose utilization csc K and csc A inhibits the product of the related enzyme encoded by A, thereby promoting the further utilization of sucrose. Based on this, glpD Genes improve the ability of E. coli to produce lactic acid using sucrose. The specific steps are as follows:

[0049] (1) First, amplify the glpD gene and cloned it into the pTrc99a plasmid to obtain the new plasmid pTrc99a- glpD .

[0050] Take 1 μL of plasmid ptrc99a As a template for PCR amplification, the ptrc99a-P1 (shown in SEQ ID NO.1) and ptrc99a-P1 (SEQ ID NO.2) was used as primer for PCR amplification. The PCR product was cut and recovered to obtain the linearized plasmid ptrc99a .

[0051] Using the colony PCR method, a small amount of HBUT-L colonies were used as templates for PCR amplification. glpD-P1 (shown in SEQ ID NO.3) and glpD- P2 (sequence shown in SEQ ID NO.4) was used as primer for PCR amplification, and the PCR product was recovered using a PCR product recovery kit.

[0052] The ptrc99a vector fragment and glpD The gene fragments were added at a molar ratio of 1:3, and seamless cloning was performed using the GeneArt™ Gibson Assembly EX cloning kit. The reaction products were transformed into DH5α competent cells by heat shock. After cell recovery, they were spread on LB solid plates containing 50 mg / L ampicillin resistance and cultured at 37°C for 16 h before picking single colonies to verify the clones. glpD- P3 (SEQ ID NO.5) and glpD -P4 (sequence as shown in SEQID NO.6) was used as the verification primer, and colony PCR verification was performed. The successfully verified colonies were further extracted with plasmids and sent to a sequencing company for sequencing verification. The verified plasmids were named ptrc99a- glpD , whose sequence is shown in SEQ ID NO.7.

[0053] (2) Plasmid pTrc99a- glpD Transformed into Escherichia coli HBUT-SL by chemical transformation 。 Escherichia coli HBUT-SL competent cells were prepared by the CaCl2 method, and ptrc99a- glpD The plasmid was transformed into E. coli HBUT-SL competent cells by heat shock method, and the revived bacterial solution was spread on LB solid medium containing 50 mg / L ampicillin resistance and cultured in a 37°C incubator for 16 h. A single colony was picked and streaked in LB solid medium containing 50 mg / L ampicillin resistance, and the strain was named HBUT-SLE.

[0054] Table 3 Primers used for cloning of glpD gene

[0055]

[0056] Example 5 Comparison of the results of L-lactic acid fermentation by engineered bacteria

[0057] When evaluating the ability of Escherichia coli to produce L-lactic acid using sucrose, the ability of HBUT-L, HBUT-SL and HBUT-SLE to produce L-lactic acid using sucrose was compared in a fermenter. The following fermentation method was used: 6 Escherichia coli colonies on the plate were inoculated into 50 mL of 2% sucrose inorganic salt medium, and cultured at 37℃ and 200 r / min for 12 h, and then transferred to 3 L inorganic salt medium containing 10% sucrose and 0.5% corn syrup dry powder at a 10% inoculum amount. The fermentation conditions were: 37℃ and the stirring paddle speed was 200 r / min. The pH value of the fermentation liquid was controlled to 6.5 by feeding 22% (w / w) Ca(OH)2, and the fermentation was continued for 72 h. Among them, for the strain HBUT-SLE with the plasmid of ptrc99a, 50 mg / L ampicillin was added to all culture media, and 0.1 M IPTG was added for induction after 6 h of fermentation.

[0058] The method of sampling fermentation broth and detecting L-lactic acid is as follows: the bacterial concentration is measured by UV spectrophotometer at OD value at wavelength 600 nm. Glucose or sucrose and organic acids are analyzed by high performance liquid chromatography, the chromatograph is waters e2695, the chromatographic column is Welch Xtimate Sugar-H (7.8×300mm, 8μm), the mobile phase is 5 mmol / L H2SO4, the flow rate is 1 mL / min, the column temperature is 45℃, the detector is PDA detector (waters 2998), and the detection wavelength is 210 nm. The optical purity of lactic acid is analyzed by high performance liquid chromatography, the chromatograph is waters e2695, the chromatographic column is chiral column SCAS Sumichiral OA-500 (4.6×150mm, 5 μm), the mobile phase is 2 mmol / L CuSO4 with 5% isopropanol, the flow rate is 1 mL / min, the column temperature is 40℃, the detector is PDA detector (waters 2998), and the detection wavelength is 254 nm.

[0059] As shown in Table 4, the domesticated E. coli strain HBUT-SL, when cultured in an inorganic salt medium containing 10% sucrose and 0.5% corn steep liquor powder, had an L-lactic acid yield of 90.85 g / L, a fermentation time of 36 h, and an average production intensity of 2.52 g·L -1 h -1 The maximum biomass was 19.55, and its lactic acid yield, average production intensity and maximum biomass were increased by 1.10, 1.83 and 2.02 times compared with the undomesticated E. coli HBUT-L, respectively, and the fermentation time was shortened by 24 h. glpThe E. coli HBUT-SLE obtained by using the D gene had an L-lactic acid yield of 95.30 g / L, a fermentation time of 24 h, and an average production intensity of 3.97 g·L -1 h -1 , the maximum biomass was 16.28, and its lactic acid yield, average production intensity and maximum biomass were increased by 1.15, 2.88 and 1.68 times compared with the undomesticated Escherichia coli HBUT-L, respectively, and the fermentation time was shortened by 36 h.

[0060] Table 4 Results of lactic acid production using 10% sucrose fermentation

[0061]

[0062] Note: The fermentation cycle is the fermentation time from the start of inoculation to the time when residual sugar in the fermentation liquid cannot be detected.

[0063] In summary, the present invention overcomes the current situation that the efficiency of existing strains in producing L-lactic acid using sucrose is low, and provides a method and corresponding strain for obtaining an engineered Escherichia coli that efficiently utilizes sucrose through adaptive domestication combined with reverse biological means, which provides a new idea for producing L-lactic acid using low-cost sucrose molasses resources.

[0064] It should be understood that the implementation methods of the present invention are not limited to the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An Escherichia coli for producing L-lactic acid using sucrose, characterized in that: The Escherichia coli is one of the following Escherichia coli: (1) Escherichia coli HBUT-SL with a deposit number of CCTCC NO: M 20221463; (2) Escherichia coli HBUT-SLE with a deposit number of CCTCC NO: M 20221464.

2. Use of the Escherichia coli according to claim 1 in producing L-lactic acid.

3. A method for producing L-lactic acid, characterized in that: The method comprises the following steps: culturing the Escherichia coli described in claim 1 in a culture medium containing sucrose and corn steep liquor dry powder.

4. A method for improving the ability of Escherichia coli to produce L-lactic acid using sucrose, characterized in that: The method is to overexpress the L-lactic acid-producing Escherichia coli gL Gene; the L-lactic acid producing Escherichia coli is the Escherichia coli HBUT-SL described in claim 1.

Images