A method for synthesizing lactic acid monomer using sucrose as a raw material

Abstract

The present invention relates to the technical fields of fermentation engineering and genetic engineering, and particularly relates to a method for synthesizing lactic acid monomer using sucrose as a raw material. Through the genetic recombination technology of site-directed gene replacement, the promoter sequence of the sucrose metabolic operon in the production strain is corrected, enabling it to maintain a high level of expression throughout the production process of lactic acid monomer, demonstrating an obvious ability to highly catabolize sucrose to synthesize lactic acid monomers with high optical purity and high chemical purity (D-lactic acid or L-lactic acid). For example, strains DSAPW5 and LSAPW5 are fermented for 28-30 h under the condition of 25-50 °C, and 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%. The present invention can be applied to the industrial production of lactic acid monomer using sucrose or sucrose-containing raw materials.

Description

Technical Field

[0001] The present invention relates to the technical fields of fermentation engineering and genetic engineering, and particularly relates 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 the molecular formula C2H5OCOOH. In addition to its applications in food, feed, and pharmaceutical additives, highly optically and chemically pure lactic acid monomers can also be used as precursors for biodegradable polymers such as polylactic acid (PLA). Using glucose (starch) as a 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 metabolic engineering bacteria (mainly Escherichia coli modified by metabolic engineering), which is the main production method of lactic acid monomers today.

[0003] Due to the expected increase in the demand for lactic acid monomers in PLA manufacturing, finding raw materials for large-scale and stable production is crucial for its sustainable development (Wang Zhengxiang. Research on the Development Status and Countermeasures of China's Polylactic Acid Industry. Engineering Sciences in China, 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 biological manufacturing process. However, most of the commonly used starting strains - Escherichia coli in the biological manufacturing of lactic acid monomers cannot directly utilize sucrose (Jahreis, K., et al. Adaptation of sucrose metabolism in the Escherichia coli wild-type strain EC3132. Journal of Bacteriology, 2002, 184(19): 5307-5316), or although they can utilize it, the utilization efficiency is low and it does not have industrial application value.

[0004] Multiple studies have been conducted on the problem of low sucrose utilization efficiency in Escherichia coli. By knocking out the sucrose metabolism negative regulatory coding genes in strains with the ability to utilize sucrose cscR the growth of the strain can be improved (Arifin, Y., et al. Deletion of 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, through genetic engineering techniques, the genes related to sucrose transport and hydrolysis in Escherichia coli strain WcscA (encoding invertase), cscK (encoding fructokinase), and cscB (encoding sucrose transporter) expressed in an Escherichia coli strain unable to metabolize sucrose can confer the ability of sucrose metabolism on the recombinant strain (Bruschi, M., et al. A transferable sucrose utilization approach for non-sucrose-utilizing Escherichiacoli strains. Biotechnology Advances, 2012, 30(5):1001-10; Carruthers, D. N., 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. Research reports show that the recombinant Escherichia coli HBUT-L capable of metabolizing sucrose can produce L-lactic acid after 96 h of fermentation of metabolized sucrose, indicating that it is feasible to synthesize lactic acid monomers using sucrose as a 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. Study on the production of L-lactic acid by an engineered Escherichia coli strain using sugarcane molasses. Hubei Agricultural Sciences, 2016).

[0005] To solve the problem that Escherichia coli cannot effectively metabolize sucrose into lactic acid monomers, by adding invertase that hydrolyzes sucrose to the culture system, after sucrose is hydrolyzed into glucose and fructose, lactic acid can be produced from sucrose by the method of simultaneous saccharification and fermentation (Zhang Huayu et al. Simultaneous saccharification and fermentation of lactic acid monomers using sucrose as a raw material. Sugarcane and Canesugar 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). Heterologous expression of invertase in lactic acid monomer-producing strains to endow them with the ability to hydrolyze sucrose, hydrolyzing sucrose into glucose and fructose first, and then metabolizing and synthesizing them into lactic acid monomers by the strains 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 more efficiently and simply carry out the biological fermentation of lactic acid monomers under anaerobic conditions, based on the clarification that the activation of the initial process of sucrose metabolism in Escherichia coli is an aerobic behavior and cannot meet the anaerobic requirements necessary for lactic acid monomer biosynthesis, the present invention creatively selects, based on genomic transcriptional feature analysis, promoters that can initiate gene transcription under anaerobic conditions, namely so-called anaerobic promoters, and then uses gene replacement technology to replace the promoter of the original sucrose-initiated metabolic gene in the strain with an anaerobic promoter. The newly obtained strain can efficiently complete the aerobic metabolic growth of sucrose and, under the established new fermentation process, efficiently initiate anaerobic sucrose metabolism, thereby realizing the efficient biological synthesis of sucrose into lactic acid monomers, which can be applied to the industrial production of efficiently preparing lactic acid monomers from sucrose or sucrose-containing raw materials. Summary of the Invention

[0007] The object of the present invention is to genetically modify the promoter mediating sucrose metabolism in the genome of lactic acid monomer-producing strains through site-directed gene replacement genetic recombination technology, and use a promoter that can still initiate high-level transcription under anaerobic acid-producing conditions to replace the original promoter at a fixed point, so as to obtain a new strain with a high-intensity ability to metabolize sucrose and efficiently synthesize lactic acid monomers, thereby improving the efficiency of producing lactic acid monomers from sucrose raw materials, simultaneously significantly simplifying the production process and promoting the reduction of production costs.

[0008] In order to achieve the above object, the present invention adopts the following technical route:

[0009] 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. The method is to replace the promoter of the sucrose metabolism operon in the genome of lactic acid monomer-producing strains cscAKB with an anaerobic promoter;

[0010] Further, the anaerobic promoter includes but is not limited to: 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 etc.;

[0011] Preferably, the anaerobic promoter is: P gapA , P gadA , P pflB , P eno , P cysK ;

[0012] Furthermore, the promoter P gapA , the nucleotide sequence is as shown in SEQ ID NO.1;

[0013] Furthermore, the promoter P gadA , the nucleotide sequence is as shown in SEQ ID NO.2;

[0014] Furthermore, the promoter P pflB , the nucleotide sequence is as shown in SEQ ID NO.3;

[0015] Furthermore, the promoter P eno , the nucleotide sequence is as shown in SEQ ID NO.4;

[0016] Furthermore, the promoter P cysK , the nucleotide sequence is as shown in SEQ ID NO.5.

[0017] The second technical solution provided by the present invention is an Escherichia coli recombinant bacterium capable of efficiently synthesizing lactic acid monomer using sucrose. The recombinant bacterium is obtained by gene recombination and gene modification techniques. It uses an Escherichia coli strain that can efficiently synthesize lactic acid monomer from the glycolytic intermediate pyruvate as the starting strain, and replaces the promoter of the sucrose metabolism operon cscAKB in the genome with an anaerobic promoter;

[0018] Furthermore, the anaerobic promoter includes but is not limited to: 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 etc.;

[0019] Preferably, the anaerobic promoter is: P gapA , P gadA , P pflB , P eno , P cysK ;

[0020] Furthermore, the starting strain used for the recombinant bacterium is Escherichia coli ( Escherichia coli ), CGMCC No.11059, or Escherichia coli ( Escherichia coli ), CGMCC No.11060.

[0021] The third technical solution provided by the present invention is the application of the recombinant strain described in the second technical solution, particularly the application in the production of lactic acid monomer;

[0022] Furthermore, it is the application in the synthesis of lactic acid monomer using sucrose as a raw material;

[0023] Furthermore, the method for fermenting and producing lactic acid monomer using the above recombinant bacterium is as follows:

[0024] Shake flask fermentation test: Inoculate into the fermentation medium at an inoculum size with an initial OD 600 value of 0.015 - 0.075, an initial sucrose concentration of 0.5% - 1% (w / v), a fermentation temperature of 25°C - 37°C, a pH of 6.5 - 7.5, a shaker speed of 100 - 250 r / min, culture until the OD 600 is 1.8 - 3.0, add sucrose with a final concentration of 1% - 7% (w / v) and calcium carbonate with a concentration of 1% - 5% (w / v), and then statically culture at 37°C - 50°C for 12 - 24 h;

[0025] Fermenter fermentation test: Inoculate the seed liquid into the fermenter containing the fermentation medium at an inoculum size with an initial OD 600 value of 0.1 - 1.0, an initial sucrose concentration of 1% - 7% (w / v), and the fermentation is carried out according to a two - stage fermentation method. In the cell growth stage, the temperature is controlled at 25°C - 37°C, the pH is maintained at 6.5 - 7.5, the rotation speed is 1 - 1000 r / min, and the dissolved oxygen is controlled at 20% - 80%; when the cell concentration reaches an OD 600 of 10 - 50, enter the anaerobic lactic acid fermentation stage, the temperature is controlled at 37°C - 50°C, the stirring speed is adjusted to 1 - 200 r / min, and sucrose solution with a concentration of 30% - 70% (w / v) and calcium hydroxide suspension with a concentration of 5% - 35% (w / v) are respectively fed, maintaining the sugar concentration in the fermentation broth at 0.5% - 4% and the pH at 6.5 - 7.5; the total fermentation time is 24 - 36 h.

[0026] The composition of the fermentation medium 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 liquor 0.1 - 1.5 mL / L;

[0027] The composition of the trace element mother liquor is (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.

[0028] Beneficial effects:

[0029] The present invention creatively determines an anaerobic promoter that can efficiently initiate and guide the sucrose metabolic pathway. Applying this discovery to strain improvement, the new strain obtained can metabolize sucrose as efficiently as glucose and synthesize and accumulate lactic acid monomers.

[0030] Using the recombinant bacteria of the present invention, in the production process of lactic acid, other raw materials (such as invertase, etc.) do not need to be added, and lactic acid monomers can be efficiently synthesized using sucrose as the 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 using sucrose as the raw material, and can be applied to the large-scale production of lactic acid monomers using sucrose as the raw material, which can further improve the economic benefits and industrial structure of sugarcane planting and beet planting. The present invention can also be used for the development of efficiently producing other biological products using sucrose as the raw material by Escherichia coli.

[0031] The recombinant bacteria of the present invention grow rapidly using sucrose at 25 - 37 °C to form bacterial cells; at 37 - 50 °C, lactic acid monomers with high optical purity and high chemical purity (D-lactic acid or L-lactic acid) are rapidly synthesized using sucrose. It can directly and efficiently metabolize sucrose to synthesize lactic acid monomers, and has an obvious ability to strongly catabolize sucrose to synthesize lactic acid monomers with high optical purity and high chemical purity (D-lactic acid or L-lactic acid). For example, strains DSAPW5 and LSAPW6 ferment for 28 - 30 h under the condition of 25 - 50 °C, and 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%. Description of the drawings

[0032] Figure 1 For the selection of anaerobic promoters for sucrose metabolism, the transcriptional intensities of the genes initiated by each promoter are arranged from high to low.

[0033] Figure 2 For sucrose metabolism genescscAKB Flow chart of replacement of the promoter region sequence

[0034] Figure 3 PCR identification electrophoresis patterns of the new sucrose-metabolizing strains DSAPW5 and LSAPW6

[0035] Among them, lane M is: marker; lane 1 is: PCR product of the wild-type strain; lanes 2 and 3 are respectively: PCR products of the mutant strains DSAPW5 and LSAPW6 Specific implementation manners

[0036] In order to make the objectives, technical solutions and advantages of this patent clearer, the following further elaborates on this patent in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this patent and are not used to limit the present invention

[0037] The starting strains adopted in the present invention are metabolic engineering Escherichia coli ( Escherichia coli ), CGMCC No.11059 and Escherichia coli ( Escherichia coli ), CGMCC No.11060, which are D-lactic acid monomer-producing bacteria and L-lactic acid monomer-producing bacteria respectively, and have been deposited in the China General Microbiological Culture Collection Center (abbreviation: CGMCC), and the deposit dates are both July 7, 2015

[0038] The method for dynamic analysis of the whole genome transcriptional level adopted in the present invention: Using the conventional lactic acid fermentation method in the laboratory, sequencing samples are prepared in a 5 L fermenter with an initial fermentation medium volume of 2.5 L and a sucrose concentration of 30 g / L. Samples are taken at three fermentation stages: culturing to the mid-logarithmic phase (cell density (OD 600 )) reaching about 10) under aerobic conditions, culturing for 3 h under anaerobic conditions, and restoring aerobic growth (about 3 h). The cell samples are collected by centrifugation at 5000 ×g for 5 min, quickly frozen with liquid nitrogen, and stored at -70°C. The mRNA of samples at different fermentation stages is prepared by Guangzhou Gene Denovo Biotechnology Co., Ltd., and transcriptome analysis sequencing is completed

[0039] The main experimental methods adopted in the present invention are as follows

[0040] 1. Gene cloning, gene recombination and construction of recombinant plasmids

[0041] Conventional molecular cloning operations are carried out with reference to the method in the literature (Sambrook. et al. Molecular Cloning: A Laboratory Manual, 1989)

[0042] 2. Extraction of chromosomal DNA

[0043] The method for extracting Escherichia coli chromosomal DNA was carried out according to the literature (Zhuge Jian, Wang Zhengxiang. Handbook of Industrial Microbiology Experiment Techniques, China Light Industry Press, 1994).

[0044] 3. Extraction of Plasmid DNA

[0045] After lysing the cell wall with a certain concentration of lysozyme, the extraction of plasmid DNA was carried out using the plasmid mini-prep kit from Sigma.

[0046] 4. Gene Amplification

[0047] DNA amplification was carried out in a 0.2 mL PCR thin-walled tube. The 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). Depending on different amplification lengths, the extension temperature and time of the PCR reaction were different. Unless otherwise specified, all PCR reactions were carried out using Pfu DNA polymerase.

[0048] 5. Gene Deletion and Site-Specific Integration in Escherichia coli

[0049] The method described in the literature (Zhou Li et al. Method for Deleting Multiple Genes in Escherichia coli Based on the Red Recombination System and the Xer Recombination System. Microbiology Bulletin, 2010, 37: 923−928) was referred to. The main steps were as follows: (1) Using Escherichia coli genomic DNA as a template, the target gene sequence was amplified by PCR and cloned into a suitable vector. The foreign gene expression cassette to be expressed was cloned into it by selecting appropriate restriction enzyme sites for digestion; further, through the restriction enzyme sites existing outside the expression cassette, the difEry fragment (coding sequence as listed) was cloned into it, thus obtaining a site-specific integration sequence with a foreign gene expression cassette (i.e., homologous arm 1 - expression cassette - dif - Ery - dif - homologous arm 2). (2) The above site-specific integration expression cassette sequence was prepared by PCR amplification technology. After purifying this fragment, it was electrotransformed into the recipient strain containing the helper plasmid pKD46. (3) Under the action of the Red recombinase produced by the helper plasmid pKD46, the above DNA fragment underwent double exchange with the target gene on the chromosome, replacing the target gene while introducing the target gene expression cassette sequence. The recombinant transformants could be screened out using the erythromycin resistance marker carried on the mutation cassette. This mutant strain then underwent recombination at two dif sites under the action of the Xer recombinase produced by itself, circularizing and removing the antibiotic resistance gene. The correct transformants were verified by methods such as colony PCR verification, plasmid extraction and restriction digestion, and fermentation verification of function.

[0050] 6. Evaluation of the growth of recombinant bacteria

[0051] It is carried out in a 250 mL Erlenmeyer flask containing 50 - 100 mL of fermentation medium. According to the inoculum size with an initial OD 600 value of 0.015 - 0.075, the sugar concentration is 0.1% - 1.0%, the fermentation temperature is 30°C - 37°C, the pH is 6.5 - 7.5, the shaker speed is 100 - 250 r / min, culture for 12 - 24 h, and samples are taken regularly to measure OD 600 , and observe its growth situation.

[0052] 7. Lactic acid fermentation test

[0053] The composition of the fermentation medium 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 liquor 0.1 - 1.5 mL / L;

[0054] The composition of the trace element mother liquor is (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.

[0055] ① Shake - flask fermentation test: The fermentation process is carried out in a 250 mL Erlenmeyer flask containing 50 - 100 mL of fermentation medium. According to the inoculum size with an initial OD 600 value of 0.015 - 0.075, the initial sucrose concentration is 0.5% - 1% (w / v), the fermentation temperature is 25°C - 37°C, the pH is 6.5 - 7.5, the shaker speed is 100 - 250 r / min, culture until OD 600 reaches 1.8 - 3.0, add sucrose with a final concentration of 1% - 7% (w / v) and calcium carbonate with a concentration of 1% - 5% (w / v), and then statically culture at 37°C - 50°C for 12 - 24 h. Samples are taken regularly to measure the cell mass, residual sugar, and the content of L - lactic acid or D - lactic acid in the fermentation broth.

[0056] 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 600 The 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.

[0057] 8. Analysis of fermentation broth components:

[0058] 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.

[0059] 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.

[0060] ② Determination of sucrose concentration: It was determined by HPLC method. Chromatographic analysis conditions: The chromatographic column was graceprevail carbohydrate ES 5u liquid chromatographic 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.

[0061] ③ Determination of the contents of organic acids such as D-lactic acid and L-lactic acid: It was carried out by HPLC. The chromatographic detection conditions were as follows: 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 were the average values of the results of 3 parallel experiments.

[0062] ④ Determination of the optical purity of lactic acid monomer: It was carried out by HPLC. 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.

[0063] The present invention will be further explained and illustrated below through specific examples.

[0064] Example 1 Preparation of samples based on sucrose metabolism transcriptome analysis

[0065] Cell samples at different stages were prepared by sucrose metabolism fermentation in a 5 L fermenter. Take a single colony of the test strain CGMCC No. 11059 or CGMCC No. 11060, inoculate it into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium, and culture it at 37 °C and a stirring speed of 200 r / min until the OD 600 reached 3.0 to prepare the primary seed liquid; inoculate the primary seed liquid into a 500 mL Erlenmeyer flask containing 100 mL of fermentation medium with a sucrose concentration of 5 g / L, and culture it at 37 °C and a stirring speed of 200 r / min until the OD 600 reached 3.0 to prepare the secondary seed liquid; inoculate the secondary seed liquid into a 5 L fermenter containing fermentation medium according to the inoculation amount with an initial OD 600 value of 0.3, the liquid loading volume was 2.5 L, and the initial sucrose concentration was 30 g / L, and the fermentation temperature was 37 °C.

[0066] The fermentation medium composition (g / L) is as follows: Na2HPO4·12H2O 15.11, KH2PO4 3, NH4Cl 1, NaCl 0.5, MgSO4 0.12, trace element mother liquor 1 mL / L;

[0067] The composition of the trace element mother liquor (g / L) is: FeCl3·6H2O 2.4, CoCl2·6H2O 0.3, CuCl2·2H2O 0.15, ZnCl2 0.3, Na2MO4·2H2O 0.3, H3BO3 0.075, MnCl2·4H2O 0.495.

[0068] The preparation of samples for transcriptome analysis is carried out by sampling in three stages. The first stage (S1) is the cell growth stage: using ammonia water to maintain pH 7.0, adjusting the rotation speed and air flux, maintaining the dissolved oxygen above 60%, aerobically culturing the cells until the mid-logarithmic phase (OD 600 value is about 10), taking 100 mL of the fermentation broth, centrifuging at 4°C, 5000 × g for 5 min, discarding the supernatant to collect the cells, and immediately freezing the collected cells in liquid nitrogen and storing them at -70°C; The second stage (S2) is the anaerobic fermentation stage: closing the ventilation, controlling the rotation speed at 200 r / min, using ammonia water to maintain pH 7.0, enabling the cells to enter the anaerobic fermentation state (about 3 h), taking 100 mL of the fermentation broth, centrifuging at 4°C, 5000 × g for 5 min, discarding the supernatant to collect the cells, and immediately freezing the collected cells in liquid nitrogen and storing them at -70°C; The third stage (S3) is the recovery growth stage: restoring ventilation, adjusting the rotation speed, maintaining the dissolved oxygen above 60%, using 1 M hydrochloric acid to maintain pH 7.0, enabling the cells to re-enter the aerobic growth state (about 3 h), taking 100 mL of the fermentation broth, centrifuging at 4°C, 5000 × g for 5 min, discarding the supernatant to collect the cells, and immediately freezing the collected cells in liquid nitrogen and storing them at -70°C.

[0069] Selection of promoters that can mediate sucrose metabolism under anaerobic conditions in Example 2

[0070] The transcriptome samples were prepared by the above fermentation method. The mRNA of the strains in the three fermentation stages (S1 - S3) was prepared by Guangzhou Gene Denovo Biotechnology Co., Ltd., and then sequenced on the Illumina sequencing platform. In this invention, the complete genome sequence of Escherichia coli W (NCBI database; CP002185.1) was used as the reference genome. TopHat2 was used to align the filtered Clean reads with the reference genome to obtain the position information on the reference genome, and the number of mapped reads and the number of unmapped reads were obtained. Subsequent bioinformatics analysis was carried out based on the number of mapped reads. According to the position information of the number of mapped reads on the reference genome, the FPKM method was used to quantify the expression levels of the sample transcripts and genes. The screening criteria for differentially expressed genes were set as Fold Change (FC) ≥ 2 and False Discovery Rate (FDR) < 0.01, and they were divided into up-regulated genes and down-regulated genes according to the relative expression levels. At the same time, functional annotation and enrichment analysis of multiple databases such as GO and KEGG were carried out on the differential genes.

[0071] The promoter selection in this invention was carried out according to the following criteria: 1) the promoters of genes involved in "carbohydrate metabolism" in the genome; 2) the promoters of genes with increased gene expression levels in the anaerobic fermentation stage (S2) compared with the aerobic growth stage (S1); 3) the top 20 genes with relatively high expression levels. Taking the simultaneous compliance with the above selection criteria as the selection standard, 20 gene corresponding promoters were screened and obtained, which were 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 , and the expression intensity comparison is as Figure 1 .

[0072] Example 3 Construction of a new strain for sucrose metabolism

[0073] Select the 20 promoters with the highest transcriptional intensity as candidate promoters, which are P gapA , PgadA , 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 original promoter sequence of the sucrose metabolism operon in the genome of Escherichia coli CGMCC No. 11059 or CGMCC No. 11060 cscAKB was deleted and replaced with a candidate promoter, and the experimental procedure was as Figure 2 shown. Since there is a bidirectional promoter between cscAKB in the natural sucrose metabolism operon cscA and cscK genes, which can simultaneously participate in the co-transcription of cscKB gene and the reverse transcription of cscA gene, in this embodiment, the candidate promoter was reversely spliced by gene splicing to construct a promoter that can bidirectionally regulate gene transcription. Taking the replacement of P gapA as an example, the experimental steps are as follows.

[0074] Using the genome of Escherichia coli CGMCC No. 11059 or CGMCC No. 11060 as a template, the upstream homologous sequence of the original promoter in the sucrose metabolism operon cscAKB (i.e., cscA gene partial fragment) was amplified by PCR with primers P1 and P2. At the same time, the sequence P gapA -1 and P gapA -2 were used to amplify the sequence P cscA for initiating the transcription of gapA gene by PCR. Then, primers P1 and P gapA -2 were used to perform overlap PCR on the two fragments to obtain a gene sequence cscA '-P gapA with the upstream homologous sequence and the corresponding promoter, and this fragment was cloned into the plasmid pSKsym (published in Overhage J. et al. Biotransformation of eugenol tovanillin by a mutant of Pseudomonassp. 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 .

[0075] 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.

[0076] 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 ' ::difEryTransform this fragment into Escherichia coli CGMCC No.11059 and CGMCC No.11060, screen the transformants on erythromycin-resistant (160 μg / mL) LB medium, and then subculture on non-selective LB medium to screen out the transformants with lost erythromycin resistance. Extract the chromosomal DNA of the transformants and verify it by PCR (using the verification primers P1 and P5 in Table 1. The band size of the wild-type strain is about 800 bp, and the band size of the mutant strain is about 1500 bp. The verification results are as Figure 3 ). Obtain the mutant strains Escherichia coli DSAPW5 and LSAPW6.

[0077] Use the above similar method to replace the other 19 promoters, and prepare the expression cassettes P 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 ,P pfkA - 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 respectively transformed into Escherichia coli CGMCC No.11059 and CGMCC No.11060 to obtain 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.

[0078] Table 1 List of primers used in this case

[0079]

[0080] Table 2 Genetic characteristics of new sucrose-metabolizing strains

[0081]

[0082] Example 4 Fermentation of recombinant bacteria to produce lactic acid monomer using sucrose as raw material

[0083] Using the recombinant bacteria in Table 2 as fermentation strains, the ability to ferment sucrose raw material to produce lactic acid monomer was evaluated in a 5 L fermenter.

[0084] Pick a single colony of the fermentation strain and inoculate it into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium. Culture it at a culture temperature of 37°C and a rotation speed of 200 r / min until OD 600 reaches 3.0 to prepare the primary seed liquid; inoculate the primary seed liquid into a 500 mL Erlenmeyer flask containing 100 mL of fermentation medium with a sucrose concentration of 5 g / L. Culture it at a culture temperature of 37°C and a rotation speed of 200 r / min until OD 600 reaches 3.0 to prepare the secondary seed liquid. The secondary seed liquid was inoculated according to the initial OD 600An inoculum size of approximately 0.3 was used to inoculate the seed solution into a 5-L fermenter containing 2.5 L of fermentation medium. The initial sucrose concentration was 3%, the fermentation temperature was 37 °C, the air flow rate and rotation speed were adjusted to control the dissolved oxygen not lower than 60%, and ammonia water was used to maintain the pH at 7.0 for aerobic cultivation until the OD 600 value was approximately 30. Then, the temperature of the fermenter was set to 40 °C, the aeration was turned off, and the stirring speed was 200 r / min to enter the anaerobic fermentation stage. A 60% sucrose solution was fed continuously to maintain the sugar concentration in the fermentation broth at 1%, and a 25% calcium hydroxide suspension was fed continuously to maintain the pH at 7.0. 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.

[0085] The composition of the fermentation medium was (g / L): Na2HPO4·12H2O 15.11, KH2PO4 3, NH4Cl 1, NaCl 0.5, MgSO4 0.12, and trace element mother liquor 1 mL / L;

[0086] The composition of the trace element mother liquor was (g / L): FeCl3·6H2O 2.4, CoCl2·6H2O 0.3, CuCl2·2H2O 0.15, ZnCl2 0.3, Na2MO4·2H2O 0.3, H3BO3 0.075, and MnCl2·4H2O 0.495.

[0087] 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 with the anaerobic promoter replaced were shown in Table 3.

[0088] Table 3 Production of lactic acid monomer using sucrose as raw material

[0089]

[0090] As can be seen from the results in Table 3, the effects of replacing 20 promoters were very different, and the yields were in the range of 100 - 380% of the original strain. It can be seen that not all replacements of anaerobic promoters can lead to an increase in lactic acid production. Among them, replacing the original promoter with P cscAKB P gapA P gadA P pflB P eno P cysK significantly improved the lactic acid production and achieved unexpected technical effects.

[0091] Example 5 High-efficient production of lactic acid monomer by recombinant bacteria fermenting sucrose under different fermentation processes

[0092] The seed solutions of DSAPW5, DSYSW5, LSAPW6, and LSYSW6 were respectively inoculated according to the initial OD600 Inoculum with a value of 0.1 was inoculated into a 5 L fermenter containing 2.5 L of fermentation medium. The initial sucrose concentration was 2.5% (w / v). The fermentation was carried out according to a two-stage fermentation method. During the cell 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 cell concentration reached OD 600 of 25, it entered the anaerobic lactic acid fermentation stage. The temperature was controlled at 40 °C, the stirring speed was adjusted to 100 r / min, and a 40% (w / v) sucrose solution and a 20% (w / v) calcium hydroxide suspension were added dropwise respectively to maintain the sugar concentration in the fermentation broth at 2% and the pH at 7.0; the total fermentation time was 32 h.

[0093] The fermentation medium composition was (g / L): Na2HPO4·12H2O 15.11, KH2PO4 3, NH4Cl 1, NaCl 0.5, MgSO4 0.12, trace element mother liquor 1 mL / L;

[0094] The composition of the trace element mother liquor was (g / L): FeCl3·6H2O 2.4, CoCl2·6H2O 0.3, CuCl2·2H2O 0.15, ZnCl2 0.3, Na2MO4·2H2O 0.3, H3BO3 0.075, MnCl2·4H2O 0.495.

[0095] The fermentation results are shown in Table 4 below.

[0096] Table 4

[0097]

[0098] Example 6 High-efficient production of lactic acid monomer by recombinant bacteria fermentation of sucrose under different fermentation processes

[0099] The seed solutions of DSAPW5, DSYSW5, LSAPW6, and LSYSW6 were respectively inoculated into a 5 L fermenter containing 2.5 L of fermentation medium according to an inoculum with an initial OD 600 value of 0.5. The initial sucrose concentration was 4% (w / v). The fermentation was carried out according to a two-stage fermentation method. During the cell 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 cell concentration reached OD 600 of 30, it entered the anaerobic lactic acid fermentation stage. The temperature was controlled at 42 °C, the stirring speed was adjusted to 150 r / min, and a 60% (w / v) sucrose solution and a 25% (w / v) calcium hydroxide suspension were added dropwise respectively to maintain the sugar concentration in the fermentation broth at 0.5% and the pH at 6.8; the total fermentation time was 28 h.

[0100] The fermentation medium composition (g / L) is as follows: 15.11 of Na2HPO4·12H2O, 3 of KH2PO4, 1 of NH4Cl, 0.5 of NaCl, 0.12 of MgSO4, and 1 mL / L of trace element mother liquor;

[0101] The composition of the trace element mother liquor (g / L) is as follows: 2.4 of FeCl3·6H2O, 0.3 of CoCl2·6H2O, 0.15 of CuCl2·2H2O, 0.3 of ZnCl2, 0.3 of Na2MO4·2H2O, 0.075 of H3BO3, and 0.495 of MnCl2·4H2O.

[0102] The fermentation results are shown in Table 5 below.

[0103] Table 5

[0104]

[0105] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions, and variations in the forms and details of 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 replace the promoter of the sucrose metabolism operon in the genome of the lactic acid monomer-producing strain cscAKB with an anaerobic promoter; The anaerobic promoter is selected from: P gapA , P gadA , P pflB , P eno , or P cysK ; The lactic acid monomer-producing strain is Escherichia coli ( Escherichia coli ), CGMCC No. 11059, or Escherichia coli ( Escherichia coli ), CGMCC No. 11060; The promoter P gapA , the nucleotide sequence is as shown in SEQ ID NO.1; the promoter P gadA , the nucleotide sequence is as shown in SEQ ID NO.2; the promoter P pflB , the nucleotide sequence is as shown in SEQ ID NO.3; the promoter P eno , the nucleotide sequence is as shown in SEQ ID NO.4; the promoter P cysK , the nucleotide sequence is as shown in SEQ ID NO.

5.

2. A recombinant Escherichia coli strain capable of synthesizing lactic acid monomers using sucrose, characterized in that, The recombinant bacterium uses an Escherichia coli strain capable of efficiently synthesizing lactic acid monomers from the sugar metabolism intermediate pyruvate as the starting strain, and replaces the promoter of the sucrose metabolism operon in the genome cscAKB with an anaerobic promoter; The anaerobic promoter is selected from: P gapA , P gadA , P pflB , P eno , or P cysK ; The starting strain used for the recombinant bacterium is Escherichia coli ( Escherichia coli ), CGMCC No. 11059, or Escherichia coli ( Escherichia coli ), CGMCC No. 11060; The promoter P gapA , the nucleotide sequence is as shown in SEQ ID NO.1; the promoter P gadA , the nucleotide sequence is as shown in SEQ ID NO.2; the promoter P pflB , the nucleotide sequence is as shown in SEQ ID NO.3; the promoter P eno , the nucleotide sequence is as shown in SEQ ID NO.4; the promoter P cysK , the nucleotide sequence is as shown in SEQ ID NO.

5.

6. Use of the recombinant bacterium according to claim 2 in the production of lactic acid monomer.

4. The application according to claim 3, wherein It is the use in the production of lactic acid monomer using sucrose as a raw material.

5. The application according to claim 3, wherein The shake flask fermentation method is as follows: Inoculate the production strain into the fermentation medium at an inoculum size with an initial OD 600 value of 0.015 - 0.075, with an initial sucrose concentration of 0.5% - 1%, and culture at a fermentation temperature of 25°C - 37°C until the OD 600 reaches 1.8 - 3.

0. Then add sucrose with a final concentration of 1% - 7% and calcium carbonate with a final concentration of 1% - 5%, and then statically culture at 37°C - 50°C for 12 - 24 h.

6. The application according to claim 3, characterized in that The fermentation method in the fermenter is as follows: inoculate the seed liquid into the fermentation medium at an inoculum size with an initial OD 600 value of 0.1 - 1.0, with an initial sucrose concentration of 1% - 7%. The fermentation is carried out according to a two-stage fermentation method. During the cell growth stage, the temperature is controlled at 25°C - 37°C and the dissolved oxygen is 20% - 80%. When the cell concentration reaches OD 600 of 10 - 50, enter the anaerobic lactic acid fermentation stage. The temperature is controlled at 37°C - 50°C, and sucrose solution and calcium hydroxide suspension are added dropwise respectively to maintain the sugar concentration in the fermentation broth at 0.5% - 4% and the pH at 6.5 - 7.5; The total fermentation time is 24 - 36 h.

7. The application according to claim 5 or 6, characterized in that, The composition of the fermentation medium is as follows (in 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 liquor 0.1 - 1.5 mL / L.

8. The application according to claim 7, characterized in that, The composition of the trace element mother liquor is as follows (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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