Method for synthesizing lactic acid monomer by taking cane sugar as raw material

Abstract

The invention relates to the technical field of fermentation engineering and genetic engineering, in particular to a method for synthesizing a lactic acid monomer by taking cane sugar as a raw material. According to the method, a promoter sequence of a sucrose metabolism operon in a production strain is corrected through a genetic recombination technology of site-directed gene replacement, so that high-level expression is maintained in the whole lactic acid monomer production process; the strain DSAPW5 and the strain LSAPW5 have obvious capability of high-intensity catabolism of sucrose to synthesize lactic acid monomers (D-lactic acid or L-lactic acid) with high optical purity and high chemical purity, for example, when the strains DSAPW5 and LSAPW5 are fermented for 28-30 hours at 25-50 DEG C, the fermentation levels of the D-lactic acid and the L-lactic acid can respectively reach 158.58 g / L and 159.65 g / L, and the optical purity can reach 99.94% and 99.95%. The method can be applied to industrial production of lactic acid monomers using cane sugar or cane sugar-containing raw materials.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering and genetic engineering, and in particular to a method for synthesizing lactic acid monomers using sucrose as a raw material. Background Art

[0002] Lactic acid, also known as α-hydroxypropionic acid, has a molecular formula of C2H5OCOOH. In addition to its application in food, feed and pharmaceutical additives, high optical and chemical purity lactic acid monomers can also be used as precursors for biodegradable polymers such as polylactic acid (PLA). Using glucose (starch) as raw material, the large-scale production of lactic acid monomers (D-lactic acid or L-lactic acid) has been achieved through the biological metabolism of metabolically engineered bacteria (mainly metabolically engineered Escherichia coli), which is the main production method of lactic acid monomers today.

[0003] Due to the expected increase in demand for lactic acid monomers in PLA manufacturing, finding raw materials for large-scale stable production is crucial to its sustainable development (Wang Zhengxiang. Research on the current status and countermeasures of the development of my country's polylactic acid industry. China Engineering Science, 2021, 23(6): 155-166). In this context, sucrose has emerged as a viable and sustainable chemical production raw material, highlighting its importance in the biomanufacturing process. However, most of the starting strains used in the biomanufacturing of lactic acid monomers, Escherichia coli, cannot directly utilize sucrose (Jahreis, K., et al. Adaptation of sucrosemetabolism in the Escherichia coli wild-type strain EC3132. Journal of Bacteriology, 2002, 184(19):5307-5316), or although it can be used, the utilization efficiency is low and it has no industrial application value.

[0004] Many studies have been conducted to address the problem of low sucrose utilization efficiency in E. coli. cscR Deletion of can improve the growth of strains (Arifin, Y., et al. cscR in Escherichia coli W improves growth and poly-3-hydroxybutyrate (PHB) production from sucrose in fed batch culture. Journal of Biotechnology, 2010, 156(4): 27-278). Further, the genes related to sucrose transport and hydrolysis in Escherichia coli W strain were modified by genetic engineering technology. cscA (encoding sucrose invertase), cscK (encoding fructokinase) and cscB (encoding sucrose transporter) was expressed in an Escherichia coli strain that cannot metabolize sucrose, which can endow the recombinant bacteria with sucrose metabolism ability (Bruschi, M., et al. A transferable sucrose utilization approach for non-sucrose-utilizing Escherichiacoli strains. Biotechnology Advances, 2012, 30(5):1001-10; Carruthers, DN, et al. Random chromosomal integration and screening yields E. coli K-12 derivatives capable of efficient sucrose utilization. ACS Synth Biol, 2020, 9(12):3311-3321), but its sucrose metabolism efficiency is low and it also has no industrial application value. A study reported that the recombinant Escherichia coli HBUT-L that can metabolize sucrose was obtained by recombinant technology, and it can metabolize sucrose to produce L-lactic acid after 96 h of fermentation, indicating that it is feasible to synthesize lactic acid monomers using sucrose as raw material, but the conversion rate is only 74.0%, the yield is only 60 g / L, and the production intensity is only 0.389 g / (L·h) (Zhao Jinfang et al. Research on the production of L-lactic acid by fermentation of sugarcane molasses by Escherichia coli engineering bacteria. Hubei Agricultural Sciences, 2016).

[0005] In order to solve the problem that Escherichia coli cannot effectively metabolize sucrose into lactic acid monomers, by adding sucrase to the culture system, sucrose is hydrolyzed into glucose and fructose, and then lactic acid can be produced from sucrose by simultaneous saccharification and fermentation (Zhang Huayu et al. Synchronous saccharification and fermentation of lactic acid monomers using sucrose as raw material. Sugarcane Sugar Industry, 2021, 50(4):77-84; Wang Zhengxiang, Tian Kangming, Niu Dandan, Lu Fuping, Chinese invention patent, application number 202011610612.4, application date December 30, 2020). By heterologously expressing sucrase in the lactic acid monomer production strain, it is given the ability to hydrolyze sucrose, and the sucrose is first enzymatically hydrolyzed into glucose and fructose, and then metabolized and synthesized into lactic acid monomers by the bacteria. This is also an effective method for bioconverting sucrose into lactic acid monomers (Wang Zhengxiang, Tian Kangming, Niu Dandan, Lu Fuping, Chinese invention patent, application number 202011610612.4, application date December 30, 2020).

[0006] In order to enable Escherichia coli to directly utilize sucrose and to more efficiently and simply perform lactic acid monomer biofermentation under anaerobic conditions, the present invention, based on the understanding that the activation of the initial process of sucrose metabolism in Escherichia coli is an aerobic behavior and cannot be applied to the anaerobic requirements required for the biosynthesis of lactic acid monomers, creatively selects a promoter that can play a gene transcription initiation function under anaerobic conditions, i.e., a so-called anaerobic promoter, based on genome transcription feature analysis, and then uses gene replacement technology to replace the original promoter of the sucrose initial metabolism gene in the strain with an anaerobic promoter. The obtained new strain can efficiently complete the aerobic metabolic growth of sucrose and efficiently start anaerobic sucrose metabolism under the established new fermentation process, thereby realizing the efficient biosynthesis of sucrose to lactic acid monomers, and can be applied to the industrial production of efficiently preparing lactic acid monomers with sucrose or sucrose-containing raw materials. Summary of the invention

[0007] The purpose of the present invention is to genetically correct the promoter that mediates sucrose metabolism in the genome of the lactic acid monomer production strain through the genetic recombination technology of site-specific gene replacement, and to replace the original promoter with a promoter that can still initiate high-level transcription under anaerobic acid production conditions, so as to obtain a new strain with high-intensity sucrose metabolism ability and efficient synthesis of lactic acid monomers, thereby improving the efficiency of producing lactic acid monomers from sucrose raw materials, and simultaneously significantly simplifying the production process and promoting the reduction of production costs.

[0008] In order to achieve the above purpose, the present invention adopts the following technical route: One of the technical solutions provided by the present invention is a method for improving the ability of Escherichia coli to synthesize lactic acid monomers using sucrose, wherein the sucrose metabolism operon in the genome of the lactic acid monomer production strain is modified. cscAKB The promoter of was replaced with an anaerobic promoter; Furthermore, the anaerobic promoter includes but is not limited to: gapA , P gadA , P pflB , P eno , P cysK , P glnA , P pgk , P manX , P adhE , P pfkA , P acnA , P hchA , P agp , P serA , P melA , P tpiA , P pykA , P fruK , P ackA , P yqhD wait; Preferably, the anaerobic promoter is: gapA, P gadA , P pflB , P eno , P cysK ; Furthermore, the promoter P gapA , the nucleotide sequence is shown in SEQ ID NO.1; Furthermore, the promoter P gadA , the nucleotide sequence is shown in SEQ ID NO.2; Furthermore, the promoter P pflB , the nucleotide sequence is shown in SEQ ID NO.3; Furthermore, the promoter P eno , the nucleotide sequence is shown in SEQ ID NO.4; Furthermore, the promoter P cysK , the nucleotide sequence is shown in SEQ ID NO.5.

[0009] The second technical solution provided by the present invention is a recombinant Escherichia coli that can efficiently synthesize lactic acid monomers from sucrose. The recombinant bacterium is obtained by gene recombination and gene modification technology. It uses an Escherichia coli strain that can efficiently synthesize lactic acid monomers from pyruvic acid, an intermediate of sugar metabolism, as a starting strain, and the sucrose metabolism operon in the genome is modified. cscAKB The promoter was replaced with an anaerobic promoter; Furthermore, the anaerobic promoter includes but is not limited to: gapA , P gadA , P pflB , P eno , P cysK , P glnA , P pgk , P manX , P adhE , P pfkA , P acnA , P hchA , P agp , P serA , P melA , P tpiA , P pykA , P fruK , P ackA , P yqhD wait; Preferably, the anaerobic promoter is: gapA , P gadA , P pflB , P eno , P cysK ; Furthermore, the starting strain of the recombinant bacteria is Escherichia coli ( Escherichia coli ) CGMCC No.11059, or Escherichia coli ( Escherichia coli )CGMCC No.11060.

[0010] The third technical solution provided by the present invention is the application of the recombinant strain described in the second technical solution, especially in the production of lactic acid monomers; Further, it is used in the synthesis of lactic acid monomer using sucrose as raw material; Furthermore, the method for producing lactic acid monomer by fermentation using the above recombinant bacteria is as follows: Shake flask fermentation test: according to the starting OD 600 The inoculum size of 0.015-0.075 was added to the fermentation medium, the initial sucrose concentration was 0.5%-1% (w / v), the fermentation temperature was 25°C-37°C, the pH was 6.5-7.5, the shaking speed was 100-250 r / min, and the culture was continued until OD 600 The pH value was 1.8-3.0, sucrose and calcium carbonate were added at a final concentration of 1%-7% (w / v), and then cultured at 37°C-50°C for 12-24 h; Fermentation test in fermenter: the seed solution was added according to the initial OD 600 The inoculum with an inoculum size of 0.1-1.0 was inoculated into a fermentation tank filled with fermentation medium. The initial sucrose concentration was 1%-7% (w / v). The fermentation was carried out according to a two-stage fermentation method. During the bacterial growth stage, the temperature was controlled at 25°C-37°C, the pH was maintained at 6.5-7.5, the rotation speed was 1-1000 r / min, and the dissolved oxygen was controlled at 20%-80%. When the bacterial concentration reached OD 600 The temperature was controlled at 37°C~50°C, the stirring speed was adjusted to 1~200r / min, and 30%~70% (w / v) sucrose solution and 5%~35% (w / v) calcium hydroxide suspension were added respectively to maintain the sugar concentration in the fermentation liquid at 0.5%~4% and the pH at 6.5~7.5; the total fermentation time was 24~36 h.

[0011] The fermentation medium composition is (g / L): Na2HPO4·12H2O 5-25, KH2PO4 1-10, NH4Cl 0.1-5, NaCl 0.2-1, MgSO4 0.01-0.5, trace element mother solution 0.1-1.5mL / L; The composition of trace element mother solution is (g / L): FeCl3·6H2O 0.1~5, CoCl2·6H2O 0.1~0.5, CuCl2·2H2O 0.01~0.2, ZnCl20.1~0.5, Na2MO4·2H2O 0.1~0.5, H3BO30.01~0.1, MnCl2·4H2O 0.1~1.

[0012] Beneficial effects: The present invention creatively identifies an anaerobic promoter that can efficiently start and guide the sucrose metabolic pathway. This discovery is applied to bacterial strain improvement, and the obtained new bacterial strain can metabolize sucrose as efficiently as glucose and synthesize and accumulate lactic acid monomers.

[0013] By using the recombinant bacteria of the present invention, the production process of lactic acid does not need to add other raw materials (such as sucrase, etc.), and can achieve efficient synthesis of lactic acid monomers using sucrose as raw material. The present invention helps to expand the raw material supply for large-scale production of lactic acid monomers, optimize the production technology of lactic acid monomers produced from sucrose raw materials, and is applied to the large-scale production of lactic acid monomers using sucrose as raw material, which can further improve the economic benefits and industrial structure of sugarcane and beet planting industries. The present invention can also be used for the development of other biological products efficiently produced by Escherichia coli using sucrose as raw material.

[0014] The recombinant bacteria of the present invention rapidly grow using sucrose at 25-37°C to form bacteria, and rapidly synthesize lactic acid monomers (D-lactic acid or L-lactic acid) with high optical purity and high chemical purity using sucrose at 37-50°C. The recombinant bacteria directly and efficiently metabolize sucrose to synthesize lactic acid monomers, and have the ability to significantly decompose sucrose to synthesize lactic acid monomers (D-lactic acid or L-lactic acid) with high optical purity and high chemical purity. For example, when strains DSAPW5 and LSAPW6 are fermented at 25-50°C for 28-30 h, the fermentation levels of D-lactic acid and L-lactic acid can reach 158.58 g / L and 159.65 g / L, respectively, and the optical purity can reach 99.94% and 99.95%. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 In order to select anaerobic promoters for sucrose metabolism, the transcription intensity of genes initiated by each promoter was arranged from high to low.

[0016] Figure 2 Sucrose metabolism gene cscAKB Flowchart of the boot region sequence replacement.

[0017] Figure 3 Electrophoresis patterns of sucrose metabolizing new strains DSAPW5 and LSAPW6 identified by PCR Among them, lane M is: marker; lane 1 is: wild-type strain PCR product; lanes 2 and 3 are: mutant strains DSAPW5, LSAPW6 PCR products. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0019] The starting strain used in the present invention is metabolically engineered Escherichia coli ( Escherichia coli ) CGMCC No.11059 and Escherichia coli ( Escherichia coli ) CGMCC No.11060, respectively, are D-lactic acid monomer producing bacteria and L-lactic acid monomer producing bacteria, which have been deposited in China General Microbiological Culture Collection Center (referred to as CGMCC), and the preservation date is July 7, 2015.

[0020] The present invention is used for the dynamic analysis method of the whole genome transcription level: using the conventional laboratory lactic acid fermentation method, the sequencing sample is prepared in a 5 L fermentation tank with an initial fermentation medium volume of 2.5 L and a sucrose concentration of 30 g / L. The cells are cultured under aerobic conditions to the mid-logarithmic phase (cell density (OD 600 Sampling was performed at three fermentation stages: culture under anaerobic conditions for 3 h and recovery of aerobic growth (about 3 h). Cell samples were collected by centrifugation at 5000 × g for 5 min, quick-frozen in liquid nitrogen, and stored at -70°C. The mRNA of samples at different fermentation stages was prepared by Guangzhou Kidio Biotechnology Co., Ltd., and transcriptome analysis and sequencing were completed.

[0021] The main experimental methods used in the present invention are as follows: 1. Gene cloning, gene recombination and construction of recombinant plasmids Conventional molecular cloning procedures were performed according to the literature method (Sambrook et al. Molecular Cloning: A Laboratory Manual, 1989).

[0022] 2. Extraction of Chromosome DNA The method for extracting Escherichia coli chromosomal DNA was carried out according to the literature (Zhuge Jian, Wang Zhengxiang. Industrial Microbiology Experimental Technology Manual, China Light Industry Press, 1994).

[0023] 3. Extraction of plasmid DNA Plasmid DNA was extracted using a plasmid extraction kit from Sigma after cell walls were lysed with a certain concentration of lysozyme.

[0024] 4. Gene Amplification DNA amplification was performed in 0.2 mL PCR thin-wall tubes. PCR amplification conditions were: 1× (95°C 5 min); 30× (94°C 10 s, 58°C 30 s, 72°C 30-300 s); 1× (72°C 10 min). The extension temperature and time of the PCR reaction varied depending on the amplification length. Unless otherwise specified, all PCR reactions were performed using Phew DNA polymerase.

[0025] 5. E. coli gene deletion and site-directed integration The method was carried out according to the literature (Zhou Li et al. Multi-gene deletion method of Escherichia coli based on Red recombination system and Xer recombination system. Microbiology Bulletin, 2010, 37:923−928). The main steps are as follows: (1) Using E. coli genomic DNA as a template, PCR amplify the target gene sequence and clone it into a suitable vector. Select appropriate restriction sites to cut the exogenous gene expression cassette to be expressed; further, use the restriction sites on the outside of the expression cassette to clone the target gene sequence into the vector. difEry The fragment (coding sequence as shown in the table) is cloned into it, thereby obtaining a site-specific integration sequence with an exogenous gene expression cassette (i.e., homology arm 1-expression cassette- dif - Ery - dif - homology arm 2). (2) The above-mentioned site-specific integration expression cassette sequence is prepared by PCR amplification technology, and the fragment is purified and then electroporated into the recipient strain containing the auxiliary plasmid pKD46. (3) Under the action of the Red recombinase produced by the auxiliary plasmid pKD46, the above-mentioned DNA fragment is double-exchanged with the target gene on the chromosome, replacing the target gene while bringing in the target gene expression cassette sequence. The recombinant transformant can be screened out using the erythromycin resistance marker on the mutation cassette. The mutant strain is then subjected to two dif The recombination of the site and the removal of antibiotic resistance genes by cyclization. The correct transformants were verified by colony PCR, plasmid extraction and enzyme digestion, and fermentation verification function.

[0026] 6. Evaluation of the growth of recombinant bacteria The fermentation was carried out in a 250 mL Erlenmeyer flask containing 50-100 mL of fermentation medium. 600 The inoculum size was 0.015-0.075, the sugar concentration was 0.1%-1.0%, the fermentation temperature was 30°C-37°C, the pH was 6.5-7.5, the shaking speed was 100-250r / min, the culture was 12-24 h, and the OD was measured at regular intervals. 600 , and observe its growth.

[0027] 7. Lactic acid fermentation test The composition of the fermentation medium is (g / L): Na2HPO4·12H2O 5~25, KH2PO41~10, NH4Cl 0.1~5, NaCl0.2~1, MgSO40.01~0.5, trace element mother solution 0.1-1.5 mL / L; Composition of trace element mother solution (g / L): FeCl3·6H2O 0.1~5, CoCl2·6H2O 0.1~0.5, CuCl2·2H2O0.01~0.2, ZnCl20.1~0.5, Na2MO4·2H2O 0.1~0.5, H3BO30.01~0.1, MnCl2·4H2O 0.1~1.

[0028] ① Shake flask fermentation test: The fermentation process was carried out in a 250 mL Erlenmeyer flask containing 50-100 mL of fermentation medium. 600 The inoculum size was 0.015-0.075, the initial sucrose concentration was 0.5%-1% (w / v), the fermentation temperature was 25°C-37°C, the pH was 6.5-7.5, the shaking speed was 100-250 r / min, and the culture was continued until OD 600 The pH value was 1.8-3.0, sucrose with a final concentration of 1%-7% (w / v) and calcium carbonate with a final concentration of 1%-5% (w / v), and then cultured at 37°C-50°C for 12-24 h. Samples were taken regularly to determine the amount of bacteria, residual sugar, and the content of L-lactic acid or D-lactic acid in the fermentation broth.

[0029] ② Fermentation test in a fermenter: Take a single colony of the fermentation strain and inoculate it into 50 mL of LB liquid medium. Incubate it at 25°C~37°C and 100~250 r / min shaking for 5~15 h as the first-level seed liquid. Inoculate the first-level seed liquid into 100 mL of fermentation medium with sucrose added as the carbon source. The initial sugar concentration is 0.1%~1.0%. Incubate it at 25°C~37°C and 100~250 r / min shaking for 5~15 h as the second-level seed liquid. 600 The inoculum with a value of 0.1~1.0 was inoculated into a fermentation tank containing a fermentation medium, with an initial sucrose concentration of 1~7% (w / v). The initial volume of the fermentation tank after inoculation was 25%~60% of the working volume, and the fermentation was carried out according to the two-stage fermentation method. During the bacterial growth stage of the fermentation process, the temperature was controlled at 25°C~37°C, the pH was maintained at 6.5~7.5, the rotation speed was 1~1000 r / min, and the dissolved oxygen was controlled at 20%~80%; when the bacterial concentration reached OD 600The temperature was 10-50, and the anaerobic lactic acid fermentation stage was entered. The temperature was controlled at 37°C-50°C, the stirring speed was adjusted to 1-200 r / min, and 30%-70% (w / v) sucrose solution and 5%-35% (w / v) calcium hydroxide suspension were added respectively to maintain the sugar concentration in the fermentation broth at 0.5%-4%, the pH at 6.5-7.5, and the total fermentation time was 24-36 h. Samples were taken regularly during the fermentation process to analyze cell density, sugar consumption, lactic acid yield, main metabolic intermediates and other organic acid products.

[0030] 8. Analysis of fermentation broth components: Sample pretreatment: 1 mL of fermentation broth was mixed with 50 μL of 10 mol / L sulfuric acid and centrifuged at 12000 r / min for 5 min. An appropriate amount of supernatant was taken and an equal volume of anhydrous ethanol was added. After mixing, the mixture was allowed to stand at 4°C for 4 h and centrifuged at 12000 r / min for 5 min. The supernatant was appropriately diluted with ddH2O and filtered through a 0.22 μm organic microporous filter membrane for analysis and determination of relevant components.

[0031] ① Glucose concentration determination: After the sample is appropriately diluted, the glucose concentration is determined using an SBA-40C biosensor, and the average of three parallel data is taken.

[0032] ② Determination of sucrose concentration: HPLC method was used for determination, and the chromatographic analysis conditions were as follows: the chromatographic column was graceprevail carbohydrate ES 5u liquid chromatography column, the column temperature was 30°C, the column pressure was 90 bar, the mobile phase was 65% acetonitrile (v / v), the flow rate was 1.0 mL / min, the detector drift tube temperature was 90°C, the air carrier gas flow rate was 2.2 mL / min, and the injection volume was 15 μL.

[0033] ③ Determination of organic acid content such as D-lactic acid and L-lactic acid: HPLC was used, and the chromatographic detection conditions were: the chromatographic column was HPX-87H organic acid analysis column, the column temperature was 65°C, the detection wavelength was 210 nm, the mobile phase was 5 mmol / L sulfuric acid solution, the flow rate was 0.6 mL / min, and the injection volume was 20 μL. All data are the average of 3 parallel test results.

[0034] ④ Determination of optical purity of lactic acid monomer: HPLC was used, and the chromatographic detection conditions were as follows: the chromatographic column was Astec CLC-L optical purity analysis column, the column temperature was 25°C, the detection wavelength was 254 nm, the mobile phase was 5 mmol / L copper sulfate solution, the flow rate was 1 mL / min, and the injection volume was 10 μL.

[0035] The present invention is further explained below by means of specific examples.

[0036] Example 1 Sample preparation based on sucrose metabolism transcriptome analysis Sucrose metabolism and fermentation were performed in a 5 L fermenter to prepare cell samples at different stages. A single colony of the strain CGMCC No.11059 or CGMCC No.11060 was inoculated into a 250 mL Erlenmeyer flask containing 50 mL LB liquid medium and cultured at 37°C and 200 r / min until OD 600 The first-level seed solution was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of fermentation medium, the sucrose concentration was 5 g / L, and the culture was carried out at 37°C and a stirring speed of 200 r / min until the OD 600 is 3.0, prepare the secondary seed solution; according to the initial OD 600 The value is 0.3. The inoculation amount is to inoculate the secondary seed liquid into a 5 L fermentation tank filled with fermentation medium, the liquid volume is 2.5 L, the initial sucrose concentration is 30 g / L, and the fermentation temperature is 37°C.

[0037] The composition of the fermentation medium was (g / L): Na2HPO4·12H2O 15.11, KH2PO43, NH4Cl 1, NaCl 0.5, MgSO40.12, trace element mother solution 1 mL / L; Composition of trace element mother solution (g / L): FeCl3·6H2O 2.4, CoCl2·6H2O 0.3, CuCl2·2H2O 0.15, ZnCl20.3, Na2MO4·2H2O 0.3, H3BO30.075, MnCl2·4H2O 0.495.

[0038] The preparation of samples for transcriptome analysis was divided into three stages. The first stage (S1) was the bacterial growth stage: pH 7.0 was maintained with ammonia water, the rotation speed and air flux were adjusted, the dissolved oxygen was maintained above 60%, and the bacteria were aerobically cultured to the mid-logarithmic phase (OD 600The value is about 10), take 100 mL of fermentation liquid, centrifuge at 4°C, 5000 × g for 5 min, discard the supernatant to collect the bacteria, and immediately freeze the collected bacteria in liquid nitrogen and store at -70°C; the second stage (S2) is the anaerobic fermentation stage: turn off the ventilation, control the speed at 200 r / min, use ammonia water to maintain pH7.0, so that the bacteria enter the anaerobic fermentation state (about 3 h), take 100 mL of fermentation liquid, centrifuge at 4°C, 5000 × g for 5 min, discard the supernatant to collect the bacteria, and immediately freeze the collected bacteria in liquid nitrogen and store at -70°C; the third stage (S3) is the recovery growth stage: restore ventilation, adjust the speed, maintain the dissolved oxygen above 60%, use 1 M hydrochloric acid to maintain pH7.0, so that the bacteria re-enter the aerobic growth state (about 3 h), take 100 mL of fermentation liquid, centrifuge at 4°C, 5000 × g for 5 min, discard the supernatant, collect the cells, and immediately freeze the collected cells in liquid nitrogen and store at -70°C.

[0039] Example 2 Selection of promoters that can mediate sucrose metabolism under anaerobic conditions Transcriptome samples were prepared according to the above fermentation method, and the mRNA of the strains in the three fermentation stages (S1-S3) was prepared by Guangzhou Kidio Biotechnology Co., Ltd. and then sequenced on the Illumina sequencing platform. The present invention uses the whole genome sequence of Escherichia coli W (NCBIdatabase; CP002185.1) as the reference genome, and uses TopHat2 to align the Clean reads obtained after filtration with the reference genome, obtain the position information on the reference genome, obtain the number of reads on the alignment and the number of reads on the non-alignment, and perform subsequent bioinformatics analysis based on the number of reads on the alignment. Through the position information of the number of reads on the reference genome, the FPKM method is used to quantify the expression levels of sample transcripts and genes, and the difference multiple (Fold Change, FC) ≥ 2 and the false discovery rate (False Discovery Rate, FDR) < 0.01 are used as the screening criteria for differentially expressed genes, and are divided into up-regulated genes and down-regulated genes according to the relative expression, and the differential genes are simultaneously annotated and enriched by various databases such as GO and KEGG.

[0040] The present invention selects promoters according to the following criteria: 1) promoters of genes involved in "carbohydrate metabolism" in the genome; 2) promoters of genes whose gene expression levels in the anaerobic fermentation stage (S2) are higher than those in the aerobic growth stage (S1); 3) the top 20 genes with relatively high expression levels. Based on the selection criteria that meet the above selection criteria at the same time, 20 corresponding gene promoters were screened, namely P gapA , P gadA , P pflB, P eno , P cysK , P glnA , P pgk , P manX , P adhE , P pfkA , P acnA , P hchA , P agp , P serA , P melA , P tpiA , P pykA , P fruK , P ackA , P yqhD , the expression intensity comparison is Figure 1 .

[0041] Example 3 Construction of a new strain of sucrose metabolizer The 20 promoters with the highest transcription initiation strength were selected as candidate promoters, namely P gapA , P gadA , P pflB , P eno , P cysK , P glnA , P pgk , P manX , P adhE , P pfkA , P acnA , P hchA , P agp , P serA , P melA , P tpiA , P pykA , P fruK , P ackA , P yqhD Based on the above gene deletion and integration method, the sucrose metabolism operon in the genome of Escherichia coli CGMCC No.11059 or CGMCC No.11060 was cscAKB The original promoter sequence was deleted and replaced with the candidate promoter. The experimental process is as follows Figure 2 As shown. Due to the natural sucrose metabolism operon cscAKB exist cscA and cscK There is a bidirectional promoter between genes that can participate in cscKB Co-transcription of genes and cscA In this embodiment, the candidate promoter is reversely spliced ​​by gene splicing to construct a promoter that can bidirectionally regulate gene transcription. gapA Taking the replacement as an example, the experimental steps are as follows.

[0042] Using the genome of E. coli CGMCC No.11059 or CGMCC No.11060 as template, PCR amplified the sucrose metabolism operon with primers P1 and P2. cscAKB The original promoter upstream homologous sequence (ie: cscA gene fragment), and primer P gapA -1 and P gapA -2 PCR amplification for priming cscA Gene transcription sequence P gapA . Then use primers P1 and P gapA -2 Overlap PCR was performed on the two fragments to obtain the gene sequence with upstream homologous sequence and corresponding promoter cscA '-P gapA The fragment was cloned into the plasmid pSKsym (published in Overhage J. et al. Biotransformation of eugenol tovanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene. Appl Microbiol Biotechnol, 1999,52, 820-828) Sma Ⅰ restriction site, and obtain the recombinant plasmid pSK- cscA '-P gapA .

[0043] Using the genome of E. coli CGMCC No.11059 or CGMCC No.11060 as template, primers P gapA -2 and P gapA -3 PCR amplification for priming cscK and cscB Gene transcription sequence P gapA , primers P3 and P4 were used to amplify the sucrose metabolism operon by PCR cscAKB The downstream homologous sequence of the original promoter (i.e.: cscK Then use primer P gapA -3 and P4 were overlapped by PCR to obtain the gene sequence P with downstream homologous sequence and corresponding promoter. gapA - cscK ', the fragment and plasmid pSK- cscA '-P gapA Use restriction enzyme Sma Ⅰ and Bam The recombinant plasmid pSK-P was obtained by double restriction digestion with HⅠ and ligation.gapA -cscAK ', the plasmid contains two promoters P gapA , can be started simultaneously cscA and cscKB Transcription of genes.

[0044] Will have dif Erythromycin resistance gene fragment difEry (shown in SEQ ID NO.6) was cloned into the above recombinant plasmid pSK-P gapA -cscAK 'of Sma Ⅰ restriction site, and obtain the recombinant plasmid pSK - P gapA - cscAK ' ::difEry Using the recombinant plasmid as a template, PCR amplification was performed with primers P1 and P5 to obtain the mutation cassette P gapA - cscAK ' ::difEry The fragment was transformed into E. coli CGMCC No.11059 and CGMCC No.11060, and the transformants were selected on erythromycin resistance (160 μg / mL) LB medium, and then subcultured on non-selective LB medium to select transformants that lost erythromycin resistance. The chromosomal DNA of the transformants was extracted and verified by PCR (using the verification primers P1 and P5 in Table 1, the wild-type strain band size is about 800 bp, and the mutant strain band size is about 1500 bp. The verification results are shown in Figure 3 ). The mutant strains of Escherichia coli DSAPW5 and LSAPW6 were obtained.

[0045] The other 19 promoters were replaced using a similar method as described above, and the expression cassette P was prepared by replacing the corresponding primers (as shown in Table 1). gadA - cscAK ' ::difEry , P pflB - cscAK ' ::difEry , P eno - cscAK ' ::difEry , P cysK - cscAK ' ::difEry , P glnA - cscAK ' ::difEry , P pgk - cscAK ' ::difEry , P manX - cscAK ' ::difEry , P adhE - cscAK ' ::difEry , PpfkA - cscAK ' ::difEry , P acnA - cscAK ' ::difEry , P hchA - cscAK ' :: difEry , P agp - cscAK ' ::difEry , P serA - cscAK ' ::difEry , P melA - cscAK ' ::difEry , P tpiA - cscAK ' ::difEry , P pykA - cscAK ' ::difEry , P fruK - cscAK ' ::difEry , P ackA - cscAK ' ::difEry and P yqhD - cscAK ' ::difEry The expression cassettes were transformed into Escherichia coli CGMCC No.11059 and CGMCC No.11060, respectively, to obtain the mutant strains DSADW5 / LSADW6, DSPLW5 / LSPLW6, DSENW5 / LSENW6, DSYSW5 / LSYSW6, DSLNW5 / LSLNW6, DSPGW5 / LSPGW6, DSANW5 / LSANW6, DSDHW5 / LSDHW6, DSFKW5 / LSFKW6, DSCNW5 / LSCNW6, DSCHW5 / LSCHW6, DSAGW5 / LSAGW6, DSERW5 / LSERW6, DSELW5 / LSELW6, DSPIW5 / LSPIW6, DSYKW5 / LSYKW6, DSRUW5 / LSRUW6, DSCKW5 / LSCKW6 and DSQHW5 / LSQHW6, as shown in Table 2.

[0046] Table 1 List of primers used in this case

[0047] Table 2 Genetic characteristics of new strains capable of sucrose metabolism

[0048] Example 4 Recombinant bacteria fermented sucrose to produce lactic acid monomer The recombinant bacteria listed in Table 2 were used as fermentation strains to evaluate the ability of sucrose raw material to produce lactic acid monomer in a 5 L fermenter.

[0049] Take a single colony of the fermentation strain and inoculate it into a 250 mL Erlenmeyer flask containing 50 mL LB liquid medium. Cultivate at 37°C and 200 r / min until OD reaches 0. 600 The first-level seed solution was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of fermentation medium, and the sucrose concentration was 5 g / L. The culture temperature was 37°C and the rotation speed was 200 r / min until the OD 600 The secondary seed solution was prepared at an initial OD of 3.0. 600 The seed solution was inoculated into a 5 L fermentation tank containing 2.5 L fermentation medium with an inoculum size of about 0.3. The initial sucrose concentration was 3%, the fermentation temperature was 37°C, the air flux and the rotation speed were adjusted to control the dissolved oxygen to not less than 60%, and the pH was maintained at 7.0 with ammonia water. Aerobic culture was performed until OD 600 The value was about 30, the fermenter temperature was set to 40°C, the ventilation was turned off, the stirring speed was 200 r / min, and the anaerobic fermentation stage was entered. 60% sucrose solution was added to maintain the sugar concentration in the fermentation liquid at 1%, and 25% calcium hydroxide suspension was added to maintain the pH at 7.0. The fermentation was terminated after the lactic acid content no longer increased and the sucrose concentration was lower than 0.1%. The total fermentation time was 30 h.

[0050] The fermentation medium composition is (g / L): Na2HPO4·12H2O 15.11, KH2PO43, NH4Cl 1, NaCl0.5, MgSO40.12, trace element mother solution 1 mL / L; Composition of trace element mother solution (g / L): FeCl3·6H2O 2.4, CoCl2·6H2O 0.3, CuCl2·2H2O 0.15, ZnCl20.3, Na2MO4·2H2O 0.3, H3BO30.075, MnCl2·4H2O 0.495.

[0051] Using the above fermentation technology, at the end of fermentation, the accumulation concentrations of D-lactic acid and L-lactic acid, sugar-acid conversion rate, optical purity and chemical purity of the strain after anaerobic promoter replacement are shown in Table 3.

[0052] Table 3 Production of lactic acid monomers using sucrose as raw material

[0053] From the results in Table 3, we can see that the effects of the 20 promoter replacements are very different, and the yields are distributed in the range of 100-380% of the original strains. It can be seen that not all anaerobic promoter replacements can lead to an increase in lactic acid production. cscAKB The original promoter was replaced by P gapA , P gadA , P pflB , P eno , P cysK Afterwards, the lactic acid production was significantly increased, achieving unexpected technical results. Example 5: Efficient production of lactic acid monomers by fermentation of sucrose by recombinant bacteria under different fermentation processes The seed solutions of DSAPW5, DSYSW5, LSAPW6 and LSYSW6 were respectively 600 The inoculum size of 0.1 was inoculated into a 5 L fermenter containing 2.5 L fermentation medium. The initial sucrose concentration was 2.5% (w / v). The fermentation was carried out according to the two-stage fermentation method. During the bacterial growth stage, the temperature was controlled at 35°C, the pH was maintained at 7.0, the rotation speed was 200-800 r / min, and the dissolved oxygen was controlled at 40%. When the bacterial concentration reached OD 600 The temperature was controlled at 40 °C, the stirring speed was adjusted to 100 r / min, and 40% (w / v) sucrose solution and 20% (w / v) calcium hydroxide suspension were added respectively to maintain the sugar concentration in the fermentation liquid at 2% and pH 7.0; the total fermentation time was 32 h.

[0054] The composition of the fermentation medium was (g / L): Na2HPO4·12H2O 15.11, KH2PO43, NH4Cl 1, NaCl 0.5, MgSO40.12, trace element mother solution 1 mL / L; Composition of trace element mother solution (g / L): FeCl3·6H2O 2.4, CoCl2·6H2O 0.3, CuCl2·2H2O 0.15, ZnCl20.3, Na2MO4·2H2O 0.3, H3BO30.075, MnCl2·4H2O 0.495.

[0055] The fermentation results are shown in Table 4 below.

[0056] Table 4

[0057] Example 6 Efficient production of lactic acid monomer by fermentation of sucrose by recombinant bacteria under different fermentation processes The seed solutions of DSAPW5, DSYSW5, LSAPW6 and LSYSW6 were respectively 600The inoculum size of 0.5 was inoculated into a 5 L fermenter containing 2.5 L fermentation medium. The initial sucrose concentration was 4% (w / v). The fermentation was carried out according to the two-stage fermentation method. During the bacterial growth stage, the temperature was controlled at 37°C, the pH was maintained at 6.8, the rotation speed was 200-800 r / min, and the dissolved oxygen was controlled at 50%. When the bacterial concentration reached OD 600 The temperature was controlled at 42 °C, the stirring speed was adjusted to 150 r / min, and 60% (w / v) sucrose solution and 25% (w / v) calcium hydroxide suspension were added respectively to maintain the sugar concentration in the fermentation liquid at 0.5% and pH 6.8; the total fermentation time was 28 h.

[0058] The composition of the fermentation medium was (g / L): Na2HPO4·12H2O 15.11, KH2PO43, NH4Cl 1, NaCl 0.5, MgSO40.12, trace element mother solution 1 mL / L; Composition of trace element mother solution (g / L): FeCl3·6H2O 2.4, CoCl2·6H2O 0.3, CuCl2·2H2O 0.15, ZnCl20.3, Na2MO4·2H2O 0.3, H3BO30.075, MnCl2·4H2O 0.495.

[0059] The fermentation results are shown in Table 5. Table 5

[0060] Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for improving the ability of Escherichia coli to synthesize lactic acid monomers using sucrose, characterized in that: The method is to insert the sucrose metabolism operon in the genome of the lactic acid monomer production strain cscAKB The promoter of was replaced with an anaerobic promoter; The anaerobic promoter includes but is not limited to: gapA , P gadA , P pflB , P eno , P cysK .

2. A recombinant Escherichia coli capable of synthesizing lactic acid monomers using sucrose, characterized in that: The recombinant bacteria uses an Escherichia coli strain that can efficiently synthesize lactic acid monomers from pyruvic acid, an intermediate of sugar metabolism, as a starting strain, and the sucrose metabolism operon in the genome is cscAKB The promoter was replaced with an anaerobic promoter; The anaerobic promoter includes but is not limited to: gapA , P gadA , P pflB , P eno , P cysK .

3. The recombinant Escherichia coli capable of synthesizing lactic acid monomers using sucrose as claimed in claim 2, characterized in that: The promoter P gapA , the nucleotide sequence is shown in SEQ ID NO.1; the promoter P gadA , the nucleotide sequence is shown in SEQ ID NO.2; the promoter P pflB , the nucleotide sequence is shown in SEQ ID NO.3; the promoter P eno , the nucleotide sequence is shown in SEQ ID NO.4; the promoter P cysK , the nucleotide sequence is shown in SEQ ID NO.

5.

4. The recombinant Escherichia coli capable of synthesizing lactic acid monomers using sucrose as claimed in claim 2, characterized in that: The starting strain used in the recombinant bacteria is Escherichia coli ( Escherichia coli ) CGMCC No.11059, or Escherichia coli ( Escherichia coli )CGMCC No.11060.

5. Use of the recombinant bacteria according to any one of claims 2 to 4 in the production of lactic acid monomers.

6. The use according to claim 5, characterized in that It is used in the production of lactic acid monomer using sucrose as raw material.

7. The use according to claim 5, characterized in that The shake flask fermentation method is as follows: the production strain is divided into 600 The inoculum size of 0.015-0.075 was added to the fermentation medium, the initial sucrose concentration was 0.5%-1%, and the fermentation temperature was 25°C-37°C and cultured until the OD 600 The pH value was 1.8-3.0, sucrose with a final concentration of 1%-7% and calcium carbonate with a final concentration of 1%-5%, and then cultured at 37°C-50°C for 12-24 h.

8. The use according to claim 5, characterized in that The fermentation method of the fermentation tank is as follows: the seed liquid is heated to 400 °C according to the initial OD 600 The inoculum with an inoculum size of 0.1-1.0 was inoculated into the fermentation medium, and the initial sucrose concentration was 1%-7%. The fermentation was carried out according to the two-stage fermentation method. During the bacterial growth stage, the temperature was controlled at 25°C-37°C and the dissolved oxygen was 20%-80%. When the bacterial concentration reached OD 600 The temperature is 10-50, and then the anaerobic lactic acid fermentation stage is entered. The temperature is controlled at 37°C-50°C, and sucrose solution and calcium hydroxide suspension are added separately to maintain the sugar concentration in the fermentation liquid at 0.5%-4% and the pH at 6.5-7.5; The total fermentation time is 24~36 h.

9. The use according to claim 7 or 8, characterized in that The fermentation medium composition is calculated by g / L as follows: Na2HPO4·12H2O 5-25, KH2PO4 1-10, NH4Cl 0.1-5, NaCl 0.2-1, MgSO4 0.01-0.5, and trace element mother solution 0.1-1.5 mL / L.

10. The use according to claim 9, characterized in that The composition of the trace element mother solution is calculated in g / L: FeCl3·6H2O 0.1~5, CoCl2·6H2O 0.1~0.5, CuCl2·2H2O 0.01~0.2, ZnCl2 0.1~0.5, Na2MO4·2H2O 0.1~0.5, H3BO3 0.01~0.1, MnCl2·4H2O 0.1~1.

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