WO2022151700A1 - 一种能提高生产效率的无菌毛大肠杆菌的构建及应用 - Google Patents

一种能提高生产效率的无菌毛大肠杆菌的构建及应用 Download PDF

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WO2022151700A1
WO2022151700A1 PCT/CN2021/108590 CN2021108590W WO2022151700A1 WO 2022151700 A1 WO2022151700 A1 WO 2022151700A1 CN 2021108590 W CN2021108590 W CN 2021108590W WO 2022151700 A1 WO2022151700 A1 WO 2022151700A1
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escherichia coli
coli
wqm026
phb
fermentation
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王小元
乔君
檀昕
任鸿宇
吴铮
胡晓清
李烨
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

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  • the invention relates to the construction and application of a sterile hairy Escherichia coli capable of improving production efficiency, and belongs to the fields of genetic engineering and fermentation engineering.
  • Bacterial fimbriae are large, long, and thin supramolecular protein appendages that appear on cells and are responsible for biofilm formation, chemotaxis, adhesion, and DNA transmembrane transfer. Pili are also an important part of bacterial biofilms and have multiple adverse effects on industrial activities, such as increasing the risk of product spoilage or contamination, causing serious infections, forming dormant cells to increase antibiotic resistance, depleting bacteria of energy and substrates , block the diffusion of nutrients, form biological fouling, reduce heat transfer, increase corrosion, and shorten the service life of fermentation equipment.
  • PHAs Polyhydroxyalkanoates
  • PHAs Polyhydroxyalkanoates
  • PHAs Polyhydroxyalkanoates
  • PHAs have special properties caused by functional groups, such as biodegradability, biocompatibility, gas barrier properties, piezoelectricity, and nonlinear optical activity.
  • the properties of PHAs determine their use as biodegradable plastics, tissue engineering scaffolds, and many other potential applications.
  • Poly-3-hydroxybutyrate (PHB) is a kind of PHA, and Escherichia coli is often used in the industrial production of PHB.
  • L-Threonine is an essential nutritional amino acid for the human body and an important part of protein synthesis. Widely used in human food, cosmetics, medicine, animal feed and health care products.
  • the key to the synthesis of PHB and L-threonine in Escherichia coli is to balance the product and cell growth, not only to make the cell grow well, but also to enhance the metabolic flux of its synthetic pathway, and to increase the yield by controlling the expression level of the product formation pathway.
  • the synthesis of PHB is mainly improved by optimizing fermentation conditions, optimizing metabolic pathways, increasing intracellular coenzyme concentration, and optimizing expression plasmids.
  • the efficient synthesis of L-threonine mainly focuses on the modification of metabolic pathways, such as fatty acids. Blocking, phosphotransferase system, substrate redistribution, etc.
  • the substrates acetyl-CoA and oxaloacetate for the synthesis of PHB and L-threonine come from the glycolysis pathway and the citric acid cycle pathway, respectively. If PHB and L-threonine are to be highly produced, a large amount of carbon sources will be consumed, and serious problems will occur. affect bacterial growth. This makes the increase of PHB production unable to meet the needs of industrial production, and the growth of L-threonine fermentation is slow. Therefore, to provide a new method for promoting the growth of strains and improving the synthesis of PHB and L-threonine is of great significance for further improving the synthesis of PHB and L-threonine.
  • the present invention knocks out 64 genes of the fimbriae gene cluster on the Escherichia coli genome in Escherichia coli to obtain mutant strain WQM026.
  • the WQM026 strain grows faster in a nutrient deficient (M9) medium, and the thallus total increase.
  • M9 nutrient deficient
  • the genes related to the synthesis of PHB and L-threonine were respectively transformed into the reduced strain WQM026, and the obtained recombinant strains WQM026/pBHR68 and WQM026/pFW01-thrA*BC-rhtC could synthesize PHB and threonine efficiently.
  • the first object of the present invention is to provide a method for promoting the growth of Escherichia coli and improving the yield of fermented products, the method is to knock out the fimbriae gene cluster on the Escherichia coli genome; the pili gene cluster is: yagV-Z, gltF-yhcF, fimA-H, sfmA-F, ycbQ-F, ydeQ-T, yraH-K, yadC-N, yehA-D, ybgO-D, yfcO-V and/or ygiL-I.
  • the pilus gene cluster contains 64 genes, in the order of yagV, yagW, yagX, yagY, yagZ, gltF, yhcA, yhcD, yhcE, yhcF, fimA, fimI, fimC, fimD, fimF, fimG, fimH, sfmA, sfmC, sfmD, sfmH, sfmF, ycbQ, ycbR, ycbS, ycbT, ycbU, ycbV, ycbF, ydeQ, ydeR, ydeS, ydeT, yraH, yraI, yraJ, yraK, yadC, yadK, yadL, yadM, htrE, yad
  • the E. coli is E. coli MG1655.
  • the second object of the present invention is to provide recombinant Escherichia coli constructed and obtained by applying any of the above methods.
  • the recombinant E. coli further contains plasmid pBHR68 or plasmid pFW01-thrA*BC-rhtC.
  • the third object of the present invention is the use of the recombinant E. coli in adverse environments.
  • the unfavorable environment is M9 medium lacking amino acids
  • the medium composition comprises: glucose 4g/L, Na 2 HPO 4 12H 2 O 17.1g/L, KH 2 PO 4 4g/L, NH 4 Cl 3g/L, NaCl 0.5g/L, MgSO 4 0.24g/L and CaCl 2 0.011g/L.
  • the fourth object of the present invention is to provide the use of the recombinant Escherichia coli for synthesizing metabolites in the absence of amino acids.
  • the recombinant E. coli containing plasmid pBHR68 produces PHB in the absence of amino acids.
  • the method for producing PHB by the recombinant Escherichia coli is as follows: the strain is activated and cultured on an LB plate for 10-15 hours, inoculated into the medium, placed at 35-38° C., and cultured at 150-250rpm for 5- Seed liquid was obtained in 10 h, and 5% (v/v) inoculum was inoculated into the fermentation medium, and cultured at 35-38° C. and 150-250 rpm for 40-50 h.
  • the fermentation medium for producing PHB comprises: glucose 15-20 g/L, Na 2 HPO 4 ⁇ 12H 2 O 15-20 g/L, KH 2 PO 4 1-8 g/L , NH 4 Cl 1-8g/L, NaCl 0.2-0.8g/L, MgSO 4 0.1-0.5g/L and CaCl 2 0.01-0.05g/L.
  • the recombinant E. coli containing plasmid pFW01-thrA*BC-rhtC produces L-threonine in the absence of amino acids.
  • the method for producing L-threonine by the recombinant Escherichia coli is as follows: the strain is activated and cultured on an LB plate for 10-15 hours, inoculated into the medium, placed at 35-38° C., 150-250 rpm Cultivated for 5-10 hours to obtain seed liquid, inoculated with 10% (v/v) inoculum in the fermentation medium, placed at 35-38° C. and cultured at 150-250 rpm for 30-40 hours.
  • the fermentation medium for producing L-threonine comprises: yeast powder 1-5g/L, citric acid 1-5g/L, (NH 4 ) 2 SO 4 20-30g /L, KH 2 PO 4 5-10g/L, Glucose 25-35g/L, MgSO 4 ⁇ 7H 2 O 1-5g/L, FeSO4 ⁇ 7H 2 O 2-8mg/L, MnSO4 ⁇ 4H 2 O 2- 8mg/L and CaCO3 15-25g /L.
  • the present invention also provides the method for promoting the growth of Escherichia coli and effectively increasing the yield of fermented products or the application of the recombinant Escherichia coli in the fields of pharmaceutical preparation, materials or environmental protection.
  • the invention knocks out 64 genes of the fimbriae gene cluster on the E. coli genome in E. coli to obtain mutant strain WQM026, the WQM026 strain grows faster in a nutrient-deficient (M9) medium, and the total amount of bacteria increases. Subsequently, the genes related to the synthesis of PHB and L-threonine were transformed into the reduced strain WQM026, respectively.
  • the obtained recombinant strains WQM026/pBHR68 and WQM026/pFW01-thrA*BC-rhtC could efficiently synthesize PHB and L-threonine, respectively.
  • the synthesized PHB accounted for 87.87% of the dry cell weight, which was 3.44 times that of the wild-type control strain MG1655/pBHR68.
  • the yield of L-threonine was 2.49 g/L, which was 3.66 times that of MG1655/pFW01-thrA*BC-rhtC.
  • Figure 1 Pili synthesis and assembly gene clusters.
  • Figure 2 Knockout of each fimbriae gene cluster promotes growth curves in LB and M9 medium.
  • Figure 3 Microscopic examination of the synthesis of PHB in M9G medium by PHB-synthesizing strains knocking out the pilus gene cluster alone.
  • Figure 4 Microscopic examination of the synthesis of PHB in LBG medium by PHB-synthesizing strains knocking out the pilus gene cluster alone.
  • Figure 5 Characterization of fimbriae deletion E. coli mutant WQM026.
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • the fermentation broth was centrifuged at 4°C and 4000 rpm/min for 20 min, the supernatant was discarded, and the cells were frozen and freeze-dried. A certain amount of freeze-dried cells was weighed for methyl esterification, and methyl esterification was carried out by the reported conventional methyl esterification method, and finally conventional gas chromatography was performed for quantification.
  • E. coli cells were grown on solid LB plates for 12 h and imaged with transmission electron microscopy (TEM).
  • TEM transmission electron microscopy
  • E. coli cells were centrifuged at 4000 rpm/min for 5 min, washed twice with phosphate buffered saline (PBS buffer), and fixed with 2.5% glutaraldehyde solution for at least 72 h. Images were acquired with a G2spirit transmission electron microscope at 100 kV using a Gatan US4000 4kx4k CCD.
  • E. coli cells carrying pBHR68 were collected by centrifugation, washed twice with pH 7.4 PBS buffer, and after centrifugation, the cells were transferred to a pre-weighted centrifuge tube, wrapped with plastic wrap, and pricked with multiple small holes , so that the water can be evaporated to dryness, and the bacteria liquid can be prevented from spraying out, freeze-dried in a vacuum freeze dryer for 48 hours to completely dry, generally touch the bottom of the tube to be at room temperature, indicating that it is completely dry, or flick the bottom of the tube with your finger, the bacteria The cells are considered to be completely dry if the cells can be dispersed and easily detached from the tube wall. Weigh and calculate dry weight.
  • the extraction and quantification of intracellular ATP were carried out according to the instructions of the ATP detection kit, and the ATP analysis was carried out with an agilent1260 series HPLC system.
  • the analytical column was a 250mm ⁇ 4.0mm ODS-2HYPERSIL C18 chromatographic column.
  • E. coli MG1655 and WQM026 were grown to mid-exponential in M9 medium, harvested at 12000 rpm for 3 min, washed twice with PBS buffer, and snap frozen in liquid nitrogen. Extraction, construction and sequencing of RNA libraries were performed by GENEWIZ Biotechnology. The MG1655 genome was used as the reference sequence for sequence reading, alignment and analysis. Differential gene expression was calculated according to their expression levels and P-value ⁇ 0.05 by FIESTAViewer v.1.0 software.
  • Agilent 1200 or 1260 series high performance liquid chromatography system equipped with Thermo 250mm ⁇ 4.0mm ODS-2HYPERSIL C18 chromatographic column, is used to detect the concentration of L-threonine.
  • concentration of L-threonine was obtained by pre-column derivatization of o-phthalaldehyde (Koros, A., Varga, Z., Molnar-Perl, I. 2008. Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9 -fluorenylmethyl chloroformate derivatives in cheese by high-performance liquid chromatography. J Chromatogr A, 1203(2), 146-52.) for quantitative detection.
  • LB medium yeast powder 5, peptone 10 and NaCl 10.
  • M9 medium (g/L): Glucose 4, Na 2 HPO 4 ⁇ 12H 2 O 17.1, KH 2 PO 4 4, NH 4 Cl 3, NaCl 0.5, MgSO 4 0.24 and CaCl 2 0.011.
  • LBG medium (g/L): glucose 20, yeast powder 5, peptone 10 and NaCl 10.
  • M9G medium (g/L): glucose 20, Na 2 HPO 4 ⁇ 12H 2 O 17.1, KH 2 PO 4 4, NH 4 Cl 3, NaCl 0.5, MgSO 4 0.24 and CaCl 2 0.011.
  • PHB fermentation medium (g/L): glucose 20, Na 2 HPO 4 ⁇ 12H 2 O 17.1, KH 2 PO 4 4, NH 4 Cl 3, NaCl 0.5, MgSO 4 0.24 and CaCl 2 0.011.
  • L-threonine fermentation medium g/L: yeast powder 2, citric acid 2, (NH 4 ) 2 SO 4 25, KH 2 PO 4 7.46, glucose 30, MgSO 4 7H 2 O 2 , FeSO 4 . 7H2O 0.005, MnSO4 4H2O 0.005 and CaCO3 20 .
  • fimbriae deletion engineered bacteria The specific construction process of fimbriae deletion engineered bacteria is as follows:
  • Plasmid pTargetF01 uses pTargetF as a template, uses primers F-sgRNA-yagV-Z and R-sgRNA, and uses sequencing primers T-yagV-Z-F and T-sgRNA-R to confirm the correctness of the plasmid.
  • plasmids pTargetF02, pTargetF03, pTargetF04, pTargetF05, pTargetF06, pTargetF07, pTargetF08, pTargetF09, pTargetF10, pTargetF11, pTargetF12, pTargetF27, pTarget,F28, pTargetF29 and pTargetF08 were constructed in the same way as ., Sun, B., Yang, J., Yang, S. 2015. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol, 81(7), 2506-14.), and using the corresponding the primers.
  • CRISPR-Cas9 mutant strains There are 12 fimbriae synthesis and assembly gene clusters yagVWXYZ, gltFyhcADEF, fimAICDFGH, sfmACDHF, ycbQRSTUVF, ydeQRST, yraHIJK, yadCKLMhtrEecpDyadN, yehABCD, ybgOPQD and yfcOPQRSTUV in the E. coli MG1655 genome.
  • the pili synthesis and assembly gene cluster is further divided into 64 genes, namely yagV, yagW, yagX, yagY, yagZ, gltF, yhcA, yhcD, yhcE, yhcF, fimA, fimI, fimC, fimD, fimF, fimG, fimH, sfmA, sfmC, sfmD, sfmH, sfmF, ycbQ, ycbR, ycbS, ycbT, ycbU, ycbV, ycbF, ydeQ, ydeR, ydeS, ydeT, yraH, yraI, yraJ, yraK, yadC, yadK, yadL, yadM, htrE,
  • WQM001 is a knockout of the yagVWXYZ gene cluster from the genome.
  • the upstream and downstream homology arms were amplified with primer pairs F1-yagV-Z/R1-yagV-Z and F2-yagV-Z/R2-yagV-Z, these two PCR products were recovered with a gel recovery kit, and the primer pairs F1-yagV-Z/R2-yagV-Z was subjected to overlapping PCR to obtain overlapping homology arms.
  • the purified overlapping homology arm (400 ng) and pTargetF01 (100 ng) were mixed and electroporated into 80 ⁇ L of MG1655/pCas competent cells.
  • strains without pTargetF01 and pCas plasmids were used for subsequent studies.
  • the other 11 mutant strains WQM002, WQM003, WQM004, WQM005, WQM006, WQM007, WQM008, WQM009, WQM010, WQM011, and WQM012 were constructed by the same method.
  • WQM026 was constructed by knocking out all 12 pilus operons one by one using this method.
  • the strains, plasmids and primers involved in this section are shown in Tables 1 and 2.
  • E. coli contains 12 gene clusters that synthesize and assemble fimbriae (Figure 1). Pili not only increase the pathogenicity of bacteria, but also consume a large amount of energy and carbon sources during their synthesis and assembly. Therefore, in theory, removing these fimbriae could improve the biosafety and production efficiency of E. coli.
  • the empty vector pBSK and pBHR68 containing PHA synthetic gene were electroporated into MG1655 and 12 mutant strains to obtain MG1655/pBSK, WQM001/pBSK, WQM002/pBSK, WQM003/pBSK, WQM004/ pBSK, WQM005/pBSK, WQM006/pBSK, WQM007/pBSK, WQM008/pBSK, WQM009/pBSK, WQM010/pBSK, WQM011/pBSK, WQM010/pBSK, WQM011/pBSK and WQM012/pBSK; MG1655/pBHR688, WQM002/pBHR68 ⁇ WQM003/pBHR68 ⁇ WQM004/pBHR68 ⁇ WQM005/pBHR68 ⁇ WQM006/p
  • the OD value of WQM026 at 18h was about 3 times that of MG1655. This suggests that removing all 12 fimbriae gene clusters in E. coli favors cell growth, especially in nutrient-poor environments. Electron microscope observation of MG1655 and WQM026 cells showed that the surface of MG1655 cells was covered with a large number of fimbriae (Fig. 5B), but the surface of WQM026 was smooth with no signs of hairs (Fig. 5C).
  • the citrate concentration in WQM026 was 28.78% lower than that in MG1655 (Fig. 5D).
  • Citrate is a key metabolite in the TCA cycle, and its lower concentration in WQM026 suggests that the saved carbon does not flow into the TCA cycle, further suggesting that the absence of pili reduces energy expenditure.
  • intracellular ATP concentrations in WQM026 containing MG1655 were also determined (Fig. 5D).
  • the ATP concentrations in WQM026 and MG1655 were 0.741 and 0.402 ⁇ mol/g, respectively, and the ATP concentration in WQM026 was 1.8 times higher than that in MG1655. This suggests that removing the fimbriae can save energy.
  • WQM026 Compared with the control MG1655, WQM026 had the advantages of good growth and high accumulation of ATP and acetyl-CoA. Therefore, the application in fermentation industry is worth exploring.
  • the plasmid pBHR68 containing the PHA synthesis pathway was electrotransformed into WQM026 to obtain WQM026/pBHR68.
  • Ultrathin section electron microscopy analysis revealed that giant PHA particles filled WQM026/pBHR68 cells (Fig. 6B), but only a very small amount of PHA was observed in MG1655/pBHR68 cells (Fig. 6A). This indicates that WQM026 can synthesize PHA efficiently.
  • PHA can be synthesized from a variety of substrates and form intracellular insoluble spherical inclusions or PHA particles. There are over 90 different PHA monomers.
  • Introduction of pBHR68 in E. coli usually produces poly-3-hydroxybutyrate (PHB), but other types of PHA are also produced when pBHR68 is introduced. Therefore, the type and yield of PHA were determined by GC/MS method.
  • the strain was activated and cultured on LB plate for 12h, picked 1 macrocycle moss and inoculated into a 250mL conical flask containing 50mL LB, placed at 37°C, and cultivated at 200rpm for 6h to obtain seed liquid, 5% (v/v) inoculation The amount was inoculated into 50 mL of fermentation medium, and cultured at 37 °C and 200 rpm for 48 h.
  • the fermentation broth of the above-mentioned strain WQM026/pBHR68 was subjected to PHA extraction, methyl esterification and GC/MS analysis. Only one peak was observed in the GC spectrum, and its retention time was 5.508 min, which was exactly the same as that of standard PHB (Fig. 6C). Mass spectrometry analysis of PHA produced by WQM026/pBHR68 also showed the same pattern as standard PHB (Fig. 6D). This confirms that the PHA produced by WQM026/pBHR68 is PHB.
  • the strain was activated and cultured on LB plate for 12h, picked 1 macrocycle moss and inoculated into a 200mL conical flask containing 50mL LB, placed at 37°C, and cultivated for 4h under the condition of 200rpm to obtain seed liquid, 10% (v/v) inoculation
  • the amount was inoculated into 80 mL of fermentation medium, placed at 37 °C, and cultivated at 200 rpm for 36 h.
  • the content of L-threonine in the fermentation broth was determined.

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Abstract

提供了一种促进大肠杆菌生长并提高发酵产品产量的方法，其特征在于，敲除大肠杆菌基因组上菌毛基因簇；所述菌毛基因簇为：yagV-Z，gltF-yhcF，fimA-H，sfmA-F，ycbQ-F，ydeQ-T，yraH-K，yadC-N，yehA-D，ybgO-D，yfcO-V和/或ygiL-I。还提供了由上述方法获得的重组大肠杆菌以及利用该大肠杆菌发酵生产PHB和L-苏氨酸的方法。

Description

一种能提高生产效率的无菌毛大肠杆菌的构建及应用 技术领域
本发明涉及一种能提高生产效率的无菌毛大肠杆菌的构建及应用，属于基因工程和发酵工程领域。
背景技术
细菌菌毛是出现在细胞上的大而长、薄的超分子蛋白质附属物，负责生物膜的形成、趋化、粘附和DNA跨膜转移。菌毛也是细菌生物膜的重要组成部分，对工业活动有多种不利影响，如增加产品变质或污染的风险、引起严重感染、形成休眠细胞以增加抗生素耐药性、消耗细菌的能量和底物，阻断营养物质扩散，形成生物污垢，减少传热，增加腐蚀，缩短发酵设备的使用寿命。
聚羟基烷酸盐(PHAs)是由多种羟基酰基辅酶a作为底物合成的，并作为不溶性球形包裹体或PHA颗粒保留在细胞内。一般认为，PHAs在降低碳当量和储存多余碳方面起着重要作用，这可以提高细胞在饥饿时的抗应力能力。PHAs具有生物降解性、生物相容性、气体阻隔性、压电性、非线性光学活性等由官能团引起的特殊性质。PHAs的性质决定了它可以用作生物降解塑料、组织工程支架和许多其他潜在的应用。聚3-羟基丁酸酯(PHB)是PHA中的一种，大肠杆菌常用于PHB的工业生产。L-苏氨酸是人体必需的营养氨基酸，是蛋白质合成的重要组成部分。广泛应用于人类食品、化妆品、医药、动物饲料和保健品。
大肠杆菌合成PHB和L-苏氨酸的关键在于平衡产物和细胞生长，既要使得细胞生长好，又要增强其合成途径的代谢流，并通过控制产物形成途径的表达水平来提高产量。现有报道中主要通过优化发酵条件、优化代谢途径、提高胞内辅酶浓度和优化表达质粒等手段来改善PHB的合成；L-苏氨酸的高效合成主要集中在代谢途径的修饰上，如脂肪酸阻断、磷酸转移酶系统、底物再分配等方面。而合成PHB和L-苏氨酸的底物乙酰辅酶A和草酰乙酸分别来自糖酵解途径和柠檬酸循环途径，若要高产PHB和L-苏氨酸会消耗大量的碳源，并严重影响菌体生长。这使得PHB产量的提高无法满足工业上生产的需求，L-苏氨酸发酵生长缓慢。因此，提供一种新的促进菌株生长并提高PHB和L-苏氨酸合成的方法，对于进一步提高PHB和L-苏氨酸的合成具有十分重大的意义。
发明内容
为了解决上述存在的技术问题，本发明在大肠杆菌中敲除大肠杆菌基因组上菌毛基因簇的64个基因得到突变菌WQM026，WQM026菌株在营养缺乏(M9)培养基中生长变快，菌体总量增多。随后将PHB和L-苏氨酸合成的相关基因分别转化到精简菌株WQM026中，得 到的重组菌WQM026/pBHR68和WQM026/pFW01-thrA*BC-rhtC，可以高效合成PHB和苏氨酸。
本发明的第一个目的是提供一种促进大肠杆菌生长并提高发酵产品产量的方法，所述方法是敲除大肠杆菌基因组上菌毛基因簇；所述菌毛基因簇为：yagV-Z，gltF-yhcF，fimA-H，sfmA-F，ycbQ-F，ydeQ-T，yraH-K，yadC-N，yehA-D，ybgO-D，yfcO-V和/或ygiL-I。
在一种实施方式中，所述菌毛基因簇含有64个基因，依次为yagV，yagW，yagX，yagY，yagZ，gltF，yhcA，yhcD，yhcE，yhcF，fimA，fimI，fimC，fimD，fimF，fimG，fimH，sfmA，sfmC，sfmD，sfmH，sfmF，ycbQ，ycbR，ycbS，ycbT，ycbU，ycbV，ycbF，ydeQ，ydeR，ydeS，ydeT，yraH，yraI，yraJ，yraK，yadC，yadK，yadL，yadM，htrE，yadV，yadN，yehA，yehB，yehC，yehD，ybgO，ybgP，ybgQ，ybgD，yfcO，yfcP，yfcQ，yfcR，yfcS，yfcT，yfcU，yfcV，ygiL，yqiG，yqiH，yqiI；其序列的NCBI登录号依次为946631，947349，947606，948806，948759，947746，947741，947738，4056032，947735，948838，948841，948843，948844，948845，948846，948847，945522，945367，945160，945407，944977，948306，946773，946934，947185，945561，945562，945559，946050，946049，946047，946042，947658，947657，947656，947654，944837，944835，944829，944828，944819，944859，944841，946642，946617，946621，946619，947550，945110，946537，945325，946620，946788，946779，946818，946418，1450268，1450268，949109，947522，947529，947531，947535。
在一种实施方式中，所述大肠杆菌为大肠杆菌MG1655。
本发明的第二个目的是提供应用上述任一方法构建获得的重组大肠杆菌。
在一种实施方式中，所述重组大肠杆菌还含有质粒pBHR68或质粒pFW01-thrA*BC-rhtC。
本发明的第三个目的是所述重组大肠杆菌在不利环境中的应用。
在一种实施方式中，所述不利环境是缺乏氨基酸的M9培养基，培养基组成包括：葡萄糖4g/L，Na 2HPO 4·12H 2O 17.1g/L，KH 2PO 44g/L，NH 4Cl 3g/L，NaCl 0.5g/L，MgSO 40.24g/L和CaCl 20.011g/L。
本发明的第四个目的是提供所述重组大肠杆菌在缺乏氨基酸的条件下合成代谢产物的应用。
在一种实施方式中，所述含有质粒pBHR68的重组大肠杆菌在缺乏氨基酸的条件下生产PHB。
在一种实施方式中，所述重组大肠杆菌生产PHB的方法是：菌株在LB平板上活化培养10-15h，接种到培养基中，置于35-38℃，150-250rpm条件下培养5-10h得到种子液，5％(v/v)接种量接种于发酵培养基，置于35-38℃，150-250rpm条件下培养40-50h。
在一种实施方式中，所述生产PHB的发酵培养基，其组成包括：葡萄糖15-20g/L，Na 2HPO 4·12H 2O 15-20g/L，KH 2PO 41-8g/L，NH 4Cl 1-8g/L，NaCl 0.2-0.8g/L，MgSO 40.1-0.5g/L和CaCl 20.01-0.05g/L。
在一种实施方式中，所述含有质粒pFW01-thrA*BC-rhtC的重组大肠杆菌在缺乏氨基酸的条件下生产L-苏氨酸。
在一种实施方式中，所述重组大肠杆菌生产L-苏氨酸的方法是：菌株在LB平板上活化培养10-15h，接种到培养基中，置于35-38℃，150-250rpm条件下培养5-10h得到种子液，10％(v/v)接种量接种于发酵培养基，置于35-38℃，150-250rpm条件下培养30-40h。
在一种实施方式中，所述生产L-苏氨酸的发酵培养基，其组成包括：酵母粉1-5g/L，柠檬酸1-5g/L，(NH 4) 2SO 420-30g/L，KH 2PO 45-10g/L，葡萄糖25-35g/L，MgSO 4·7H 2O 1-5g/L，FeSO4·7H 2O 2-8mg/L，MnSO4·4H 2O 2-8mg/L和CaCO 315-25g/L。
本发明还提供所述促进大肠杆菌生长及有效提高发酵产品产量的方法或所述重组大肠杆菌在药物制备、材料或环保领域的应用。
有益效果
本发明在大肠杆菌中敲除大肠杆菌基因组上菌毛基因簇的64个基因得到突变菌WQM026，WQM026菌株在营养缺乏(M9)培养基中生长变快，菌体总量增多。随后将PHB和L-苏氨酸合成的相关基因分别转化到精简菌株WQM026中，得到的重组菌WQM026/pBHR68和WQM026/pFW01-thrA*BC-rhtC可以分别高效合成PHB和L-苏氨酸。合成的PHB占细胞干重的87.87％，是野生型对照菌MG1655/pBHR68的3.44倍。L-苏氨酸产量为2.49g/L，是MG1655/pFW01-thrA*BC-rhtC的3.66倍。
附图说明
图1：菌毛合成和组装基因簇。
图2：敲除每个菌毛基因簇促进在LB和M9培养基中的生长曲线。
图3：单独敲除菌毛基因簇的PHB合成菌株在M9G培养基中合成PHB镜检。
图4：单独敲除菌毛基因簇的PHB合成菌株在LBG培养基中合成PHB镜检。
图5：菌毛缺失大肠杆菌突变株WQM026的特性分析。
图6：WQM026生产PHB和L-苏氨酸的应用。
具体实施方式
(1)基因的敲除方法
采用CRISPR/Cas9敲除系统对大肠杆菌进行“无痕”基因敲除。首先向大肠杆菌(Escherichia coli str.K-12 substr.MG1655)中电转入pCas，通过L-阿拉伯糖诱导，使重组酶 Gam、Bet和Exo表达。然后将同源臂片段和含有特定N20序列的pTargetF质粒同时电转入MG1655/Cas9感受态细胞。涂布于卡那霉素和壮观霉素双抗性平板，30℃培养18h后，菌落PCR筛选突变菌株。
获得突变菌株后，向培养基中添加异丙基-β-D-硫代半乳糖苷(IPTG)，诱导pCas转录形成sgRNA-pMB1，结合Cas9对pTargetF的pMB1复制子进行切割破坏，去除pTargetF。而含有pCas的菌株可直接进行下一轮的敲除，将含有pCas的菌株于42℃下过夜培养，去除温敏质粒pCas。
(2)PHB产量测定方法
将发酵液在4℃，4000rpm/min下离心20min，弃去上清后，冷冻菌体，并冷冻干燥。称取一定量冷冻干燥菌体进行甲酯化，采用已报道的常规甲酯化方法进行甲酯化，最后进行常规气相色谱定量。
(3)细胞显微观察
为了观察细胞形态，大肠杆菌细胞在固体LB板上生长12h，并用透射电子显微镜(TEM)成像。为了观察细胞内PHA颗粒，在PHA发酵生产24h后，用1％结晶紫制备细菌悬浮液，并用油浸透镜(放大倍数＝100×)在明亮视野显微镜下观察。同时，将大肠杆菌细胞以4000rpm/min的速度离心5min，用磷酸盐缓冲液(PBS缓冲液)洗涤两次，用2.5％戊二醛溶液固定至少72h。用G2spirit透射电子显微镜在100千伏电压下使用Gatan US4000 4kx4k CCD获得图像。
(4)PHA的提取、定性和定量分析。
离心收集携带pBHR68的大肠杆菌细胞5mL，采用pH 7.4的PBS缓冲液洗涤两次，离心收集后，将细胞转移到预先准确称重的离心管中，包上保鲜膜，并扎出多个小洞，使得水分可以蒸干，又避免菌液喷出，在真空冷冻干燥机中冻干48h至完全干燥，一般触摸管底为常温状态，表明已经完全干透，或者用手指轻弹管底，菌体可以散开并轻易脱离管壁，则视为菌体完全干燥。称重量，计算出干重。
称取1-10mg完全干燥的干菌体，同时称取PHB标准样品约10mg转移至预先准确称重的酯化管中，以便准确计算称得的样品重量。然后进行酯化操作，加入2mL甲醇(含有3％硫酸)和2mL氯仿，加上酯化管盖子并盖紧，期间通过超声将样品散开使得酯化更彻底，沸水浴6h以上。大约0.5h后，即可观察到PHB标样一般以粉末形式迅速溶解。若未观察到0.5h后标准品的形态变化与溶解，则应更换整套试剂，甲醇和氯仿的变质都会导致酯化无法正常进行，而更换新开封的试剂往往可以解决上述问题，经过6h沸水浴后，在通风橱中冷却充分后，小心打开酯化管并加入1mL去离子水，这时需要分别将盖子旋紧并激烈震荡至体系完全充分混 匀，此时将酯化管置于通风处中静置3h以上以分相，分相后，上层为水相，下层为有机相，取适量有机相加入到气相样品瓶中，盖紧盖子保持密封，保存在-80℃中。
气相色谱是定量PHB含量的准确灵敏的手段。采用Scion-SQ-456-GC模块，配备DB-5MS熔融石英毛细管柱(30m×0.25mm×0.25μm)，对提取物进行分析，以确定其PHA组成和准确含量。气相色谱用70ev的电离能获得了正电子电离(EI)，并用扫描间隔为0.5s的m/z50到m/z650的离子对质谱进行了编程。PHA产生量(wt％)以干细胞重量百分比(DCW)表示。气相定量采用岛津GC 2010气相色谱，使用安捷伦DB WAX 30m-0.32mm气相色谱柱和火焰离子化检测器，进样温度为250℃。以不同量的商业化PHB作为标样绘制各标准样品的标准曲线。
(5)ATP和乙酰辅酶A的提取与测定
大肠杆菌细胞在M9培养基中培养至对数生长早期或中期(OD 600＝0.8-1.0)，用ATP和乙酰辅酶A测定试剂盒收集细胞内ATP和乙酰辅酶A水平。根据ATP检测试剂盒说明书进行细胞内ATP的提取和定量，用agilent1260系列HPLC系统进行ATP分析，分析柱为250mm×4.0mm ODS-2HYPERSIL C18色谱柱。
(6)MG1655和WQM026的转录组分析
将大肠杆菌MG1655和WQM026在M9培养基中培养至指数中期，在12000rpm下收获3min，用PBS缓冲液洗涤两次，在液氮中快速冷冻。RNA文库的提取、构建和测序由GENEWIZ生物技术公司执行。以MG1655基因组为参考序列，进行序列读取、比对和分析。通过FIESTAViewer v.1.0软件，根据它们的表达水平和P值≤0.05计算差异基因表达。
(7)L-苏氨酸浓度分析：
Agilent 1200或1260系列高效液相色谱系统，配备Thermo 250mm×4.0mm ODS-2HYPERSIL C18色谱柱，用于检测L-苏氨酸浓度。L-苏氨酸浓度采用邻苯二甲醛柱前衍生法(Koros,A.,Varga,Z.,Molnar-Perl,I.2008.Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by high-performance liquid chromatography.J Chromatogr A,1203(2),146-52.)进行定量检测。
(8)培养基：
LB培养基(g/L)：酵母粉5，蛋白胨10和NaCl 10。
M9培养基(g/L)：葡萄糖4，Na 2HPO 4·12H 2O 17.1，KH 2PO 44，NH 4Cl 3，NaCl 0.5，MgSO 40.24和CaCl 20.011。
LBG培养基(g/L)：葡萄糖20，酵母粉5，蛋白胨10和NaCl 10。
M9G培养基(g/L)：葡萄糖20，Na 2HPO 4·12H 2O 17.1，KH 2PO 44，NH 4Cl 3，NaCl 0.5，MgSO 40.24和CaCl 20.011。
PHB发酵培养基(g/L)：葡萄糖20，Na 2HPO 4·12H 2O 17.1，KH 2PO 44，NH 4Cl 3，NaCl 0.5，MgSO 40.24和CaCl 20.011。
L-苏氨酸发酵培养基(g/L)：酵母粉2，柠檬酸2，(NH 4) 2SO 425，KH 2PO 47.46，葡萄糖30，MgSO 4·7H 2O 2，FeSO4·7H 2O 0.005，MnSO4·4H2O 0.005和CaCO 320。
实施例1菌毛缺失工程菌构建
菌毛缺失工程菌具体构建过程如下：
(1)MG1655/pCas感受态细胞细胞制备：
接种带有Red重组辅助质粒pCas9(Jiang,Y.,Chen,B.,Duan,C.,Sun,B.,Yang,J.,Yang,S.2015.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.Appl Environ Microbiol,81(7),2506-14.)的大肠杆菌MG1655，于含100μg/mL卡那霉素的LB液体培养基中，30℃，200rpm过夜培养。按2％接种量转接100mL LB液体培养基，30℃，200rpm培养至OD 600＝0.2时加入L-阿拉伯糖(终浓度为30mmol/L)诱导重组酶的表达，继续培养至OD 600＝0.5，将培养液冰浴半小时后转入预冷的50mL离心管中，4℃，8000rpm离心10min收集菌体，沉淀用预冷的10％甘油洗涤3次，最后用1mL 10％甘油悬浮，每管80μL分装至预冷的无菌EP管中备用。
(2)pTargetF质粒构建：
pTargetF系列质粒的构建引物列于表2。质粒pTargetF01由pTargetF作为模板，利用引物F-sgRNA-yagV-Z和R-sgRNA，使用测序引物T-yagV-Z-F和T-sgRNA-R证实质粒的正确性。其他质粒pTargetF02、pTargetF03、pTargetF04、pTargetF05、pTargetF06、pTargetF07、pTargetF08、pTargetF09、pTargetF10、pTargetF11、pTargetF12、pTargetF27、pTargetF28、pTargetF29和pTargetF30采用相同的方法构建(Jiang,Y.,Chen,B.,Duan,C.,Sun,B.,Yang,J.,Yang,S.2015.Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.Appl Environ Microbiol,81(7),2506-14.)，并使用相应的引物。
(3)CRISPR-Cas9构建突变株：大肠杆菌MG1655基因组中共有12个菌毛合成和组装基因簇yagVWXYZ，gltFyhcADEF，fimAICDFGH，sfmACDHF，ycbQRSTUVF，ydeQRST，yraHIJK，yadCKLMhtrEecpDyadN，yehABCD，ybgOPQD和yfcOPQRSTUV，这12个菌毛合成和组装基因簇又分为64个基因，分别为yagV，yagW，yagX，yagY，yagZ，gltF，yhcA，yhcD，yhcE，yhcF，fimA，fimI，fimC，fimD，fimF，fimG，fimH，sfmA，sfmC，sfmD， sfmH，sfmF，ycbQ，ycbR，ycbS，ycbT，ycbU，ycbV，ycbF，ydeQ，ydeR，ydeS，ydeT，yraH，yraI，yraJ，yraK，yadC，yadK，yadL，yadM，htrE，yadV，yadN，yehA，yehB，yehC，yehD，ybgO，ybgP，ybgQ，ybgD，yfcO，yfcP，yfcQ，yfcR，yfcS，yfcT，yfcU，yfcV，ygiL，yqiG，yqiH，yqiI，其序列的NCBI登录号依次为946631，947349，947606，948806，948759，947746，947741，947738，4056032，947735，948838，948841，948843，948844，948845，948846，948847，945522，945367，945160，945407，944977，948306，946773，946934，947185，945561，945562，945559，946050，946049，946047，946042，947658，947657，947656，947654，944837，944835，944829，944828，944819，944859，944841，946642，946617，946621，946619，947550，945110，946537，945325，946620，946788，946779，946818，946418，1450268，1450268，949109，947522，947529，947531，947535。
用CRISPR-Cas9方法，分别从大肠杆菌MG1655基因组中敲除12个菌毛合成和组装基因簇yagVWXYZ，gltFyhcADEF，fimAICDFGH，sfmACDHF，ycbQRSTUVF，ydeQRST，yraHIJK，yadCKLMhtrEecpDyadN，yehABCD，ybgOPQD和yfcOPQRSTUV，构建了相应突变株WQM001，WQM002，WQM003，WQM004，WQM005，WQM006，WQM007，WQM008，WQM009，WQM010，WQM011和WQM012。例如，WQM001是从基因组中敲除yagVWXYZ基因簇。上游和下游同源臂用引物对F1-yagV-Z/R1-yagV-Z和F2-yagV-Z/R2-yagV-Z扩增，这两个PCR产物用胶回收试剂盒回收，并用引物对F1-yagV-Z/R2-yagV-Z进行重叠PCR，得到重叠同源臂。纯化后的重叠同源臂(400ng)和pTargetF01(100ng)混合后，电转入80μL MG1655/pCas感受态细胞中。电转后细胞在30℃下复苏45min，接着涂布于含有50μg/mL壮观霉素和卡那霉素的双抗性平板。在30℃下培养36h后，用引物F1-yagV-Z/R2-yagV-Z进行菌落PCR并挑选出目的菌株。接着将突变株接入含有IPTG(终浓度为0.5mmol/L)的LB培养基中过夜培养，去除pTargetF01质粒。pCas质粒含有温敏复制子，在42℃下培养24h去除pCas质粒。不含pTargetF01和pCas质粒的菌株用于后续研究。其他11个突变株WQM002，WQM003，WQM004，WQM005，WQM006，WQM007，WQM008，WQM009，WQM010，WQM011，WQM012采用相同方法构建。WQM026是采用此方法逐个敲除所有12个菌毛操纵子构建的。本部分涉及的菌株、质粒和引物如表1和2所示。
表1菌株和质粒表
Figure PCTCN2021108590-appb-000001
Figure PCTCN2021108590-appb-000002
Figure PCTCN2021108590-appb-000003
Figure PCTCN2021108590-appb-000004
表2引物序列表
Figure PCTCN2021108590-appb-000005
Figure PCTCN2021108590-appb-000006
Figure PCTCN2021108590-appb-000007
实施例2敲除单个菌毛基因簇促进生长和PHB合成
大肠杆菌含有12个合成和组装菌毛的基因簇(图1)。菌毛不仅能增加细菌的致病性，而且在其合成和组装过程中消耗了大量的能量和碳源。因此，从理论上讲，去除这些菌毛可以提高大肠杆菌的生物安全性和生产效率。
从大肠杆菌MG1655的染色体上分别去除了12个菌毛合成和组装基因簇，得到的菌株WQM001、WQM002、WQM003、WQM004、WQM005、WQM006、WQM007、WQM008、WQM009、WQM010、WQM011和WQM012。这些菌株分别在LB和M9培养基中培养。每隔2h检测菌液OD值并绘制得到菌株的生长曲线，如图2所示：在LB培养基中，所有12株突变株的生长曲线与对照株MG1655相似，表明去除大肠杆菌中12个菌毛合成和组装基因簇中的任何一个都不会影响或会略微改善细胞生长；在M9培养基中，12株突变株的生长状况均好于对照株MG1655。菌毛的生物合成和组装需要消耗大量的氨基酸，在M9培养基中 不含有任何氨基酸，在M9培养基中生长的大肠杆菌细胞必须自身合成所有20种氨基酸才能合成蛋白质。因此，减少菌毛合成和组装使突变体中保存的氨基酸可用于促进细胞生长。这表明，去除大肠杆菌中的菌毛有利于细胞生长，特别是在营养缺乏的条件下。
为了检测菌毛突变体的生产效率，将空载体pBSK和含有PHA合成基因的pBHR68电转导入MG1655和12个突变菌株中，得到MG1655/pBSK、WQM001/pBSK、WQM002/pBSK、WQM003/pBSK、WQM004/pBSK、WQM005/pBSK、WQM006/pBSK、WQM007/pBSK、WQM008/pBSK、WQM009/pBSK、WQM010/pBSK、WQM011/pBSK、WQM010/pBSK、WQM011/pBSK和WQM012/pBSK；MG1655/pBHR68、WQM001/pBHR68、WQM002/pBHR68、WQM003/pBHR68、WQM004/pBHR68、WQM005/pBHR68、WQM006/pBHR68、WQM007/pBHR68、WQM008/pBHR68、WQM009/pBHR68、WQM010/pBHR68、WQM011/pBHR68、WQM010/pBHR68、WQM011/pBHR68和WQM012/pBHR68。这些重组菌株在M9G和LBG培养基中生长，在显微镜下观察细胞及其载体对照(图3和4)。
当在M9G培养基中生长时(图3)，所有13个空载菌株都显示出相似的大小，并且没有产生任何PHA。其中MG1655/pBHR68细胞体积略有增大，仅有少数细胞产生PHA。然而，含有PHA合成基因的pBHR68的12个突变菌株的细胞大小明显增大，几乎所有细胞都产生PHA(细胞内的白色颗粒)。
在LBG培养基中生长时(图4)，所有13个空载菌株大小相似，均未产生PHA。MG1655/pBHR68的大小与其对照相似，且不产生PHA。12个含有pBHR68的突变体细胞大小增大，产生PHA的程度小于M9G培养基中的相应菌株。这表明去除菌毛有利于大肠杆菌产生PHA。
实施例3 WQM026的形态与代谢物分析
实施例2结果可以看出，每一个菌毛合成和组装基因簇的去除都能促进大肠杆菌细胞的生长和PHA的生物合成，因此大肠杆菌MG1655染色体上的12个菌毛合成和组装基因簇全部被删除，从而构建菌毛缺陷菌株WQM026(构建方式见实施例1)。每隔2h检测菌液OD值并绘制得到菌株的生长曲线，如图5A所示：WQM026在LB培养基中的生长略好于MG1655，在M9培养基中的生长远好于MG1655，在培养至18h时WQM026的OD值约是MG1655的3倍。这表明去除大肠杆菌中的所有12个菌毛基因簇有利于细胞生长，特别是在缺乏营养的环境中。电镜观察MG1655和WQM026细胞，MG1655细胞表面布满大量菌毛(图5B)，但WQM026表面光滑，无菌毛迹象(图5C)。
为了探讨菌毛缺失使大肠杆菌产生更多PHA的原因，测定了WQM026中乙酸和乙酰辅 酶A的浓度。乙酰辅酶A是PHA合成的直接前体，而乙酸是由乙酰辅酶A产生的。WQM026的乙酸和乙酰辅酶A水平显著高于MG1655，分别提高了23.57％和79.18％(图5D)。菌毛的缺失提高了大肠杆菌WQM026体内乙酸和乙酰辅酶A水平，有利于大肠杆菌产生PHA。此外，WQM026中的柠檬酸盐浓度比MG1655低28.78％(图5D)。柠檬酸盐是TCA循环中的关键代谢物，其在WQM026中的较低浓度表明节省的碳并未流入TCA循环，进一步说明菌毛的缺失减少了能量消耗。由于各种菌毛的生物合成和组装需要能量，因此还测定了含MG1655的WQM026中的细胞内ATP浓度(图5D)。WQM026和MG1655中的ATP浓度分别为0.741和0.402μmol/g，WQM026较MG1655中的ATP浓度提高了1.8倍。这表明去除菌毛可以节省能源。
实施例4 WQM026在合成PHB的应用
与对照MG1655相比，WQM026具有生长良好、ATP和乙酰辅酶A积累量高的优点。因此，在发酵工业中的应用值得探索。
将含有PHA合成途径的质粒pBHR68电转导入WQM026，得到WQM026/pBHR68。超薄切片电镜分析显示，巨大的PHA颗粒填充WQM026/pBHR68细胞(图6B)，但在MG1655/pBHR68细胞中只观察到极少量的PHA(图6A)。这表明WQM026可以有效地合成PHA。
PHA可由多种底物合成并形成细胞内不溶性球形包涵体或PHA颗粒。有超过90种不同的PHA单体。在大肠杆菌中引入pBHR68通常会产生聚3-羟基丁酸酯(PHB)，但当pBHR68被引入时，也会产生其他类型的PHA。因此，采用GC/MS法测定PHA的种类和产量。
菌株在LB平板上活化培养12h，挑取1大环菌苔接种到含有50mL LB的250mL锥形瓶中，置于37℃，200rpm条件下培养6h得到种子液，5％(v/v)接种量接种于50mL发酵培养基，置于37℃，200rpm条件下培养48h。
对上述菌株WQM026/pBHR68的发酵液进行PHA提取、甲基酯化和GC/MS分析，在GC光谱中只观察到一个峰，其保留时间为5.508min，与标准PHB的保留时间完全相同(图6C)。WQM026/pBHR68产生的PHA的质谱分析也显示了与标准PHB相同的模式(图6D)。这证实了WQM026/pBHR68产生的PHA是PHB。如图6E所示，与对照MG1655/pBHR026相比，WQM026/pBHR68的PHB和干细胞重量百分比(DCW)产量均显著增加，分别达到3.31g/L和87.87％，较对照MG1655/pBHR026分别提高了倍2和3.44倍。
实施例5 WQM026在合成L-苏氨酸的应用
由于大肠杆菌已被开发用于生产L-苏氨酸，WQM026中高效生产L-苏氨酸的潜力也被进一步研究。将含有L-苏氨酸生物合成和转运关键基因的质粒pFW01-thrA*BC-rhtC分别转入MG1655和WQM026，得到MG1655/pFW01 thrA*BC-rhtC和WQM026/pFW01 thrA*BC-rhtC。
菌株在LB平板上活化培养12h，挑取1大环菌苔接种到含有50mL LB的200mL锥形瓶中，置于37℃，200rpm条件下培养4h得到种子液，10％(v/v)接种量接种于80mL发酵培养基，置于37℃，200rpm条件下培养36h。测定发酵液中L-苏氨酸的含量，与对照菌株MG1655/pFW01 thrA*BC rhtC相比，WQM026/pFW01 thrA*BC rhtC合成L-苏氨酸的产量达到2.49g/L，相比对照菌株MG1655/pFW01 thrA*BC rhtC提高了266.18％(3.66倍)(图6F)。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

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  1. 一种促进大肠杆菌生长并提高发酵产品产量的方法，其特征在于，敲除大肠杆菌基因组上菌毛基因簇；所述菌毛基因簇为：yagV-Z，gltF-yhcF，fimA-H，sfmA-F，ycbQ-F，ydeQ-T，yraH-K，yadC-N，yehA-D，ybgO-D，yfcO-V和/或ygiL-I。
  2. 根据权利要求1所述的方法，其特征在于，所述菌毛基因簇含有64个基因，依次为yagV，yagW，yagX，yagY，yagZ，gltF，yhcA，yhcD，yhcE，yhcF，fimA，fimI，fimC，fimD，fimF，fimG，fimH，sfmA，sfmC，sfmD，sfmH，sfmF，ycbQ，ycbR，ycbS，ycbT，ycbU，ycbV，ycbF，ydeQ，ydeR，ydeS，ydeT，yraH，yraI，yraJ，yraK，yadC，yadK，yadL，yadM，htrE，yadV，yadN，yehA，yehB，yehC，yehD，ybgO，ybgP，ybgQ，ybgD，yfcO，yfcP，yfcQ，yfcR，yfcS，yfcT，yfcU，yfcV，ygiL，yqiG，yqiH，yqiI；其序列的NCBI登录号依次为946631，947349，947606，948806，948759，947746，947741，947738，4056032，947735，948838，948841，948843，948844，948845，948846，948847，945522，945367，945160，945407，944977，948306，946773，946934，947185，945561，945562，945559，946050，946049，946047，946042，947658，947657，947656，947654，944837，944835，944829，944828，944819，944859，944841，946642，946617，946621，946619，947550，945110，946537，945325，946620，946788，946779，946818，946418，1450268，1450268，949109，947522，947529，947531，947535。
  3. 根据权利要求1～2任一所述的方法，其特征在于，所述大肠杆菌为大肠杆菌MG1655。
  4. 应用权利要求1～3任一所述方法构建获得的重组大肠杆菌。
  5. 根据权利要求4所述的重组大肠杆菌，其特征在于，还含有质粒pBHR68或质粒pFW01-thrA*BC-rhtC。
  6. 权利要求4或5所述重组大肠杆菌在缺乏氨基酸的环境下合成代谢产物的应用。
  7. 一种发酵生产PHB的方法，其特征在于，所述方法是使用权利要求5所述的重组大肠杆菌在缺乏氨基酸的环境下进行发酵。
  8. 根据权利要求7所述的方法，其特征在于，所述缺乏氨基酸的环境是菌株生产PHB的发酵培养基，其组成包括：葡萄糖15-20g/L，Na 2HPO 4·12H 2O 15-20g/L，KH 2PO 41-8g/L，NH 4Cl 1-8g/L，NaCl 0.2-0.8g/L，MgSO 40.1-0.5g/L和CaCl 20.01-0.05g/L。
  9. 一种发酵生产L-苏氨酸的方法，其特征在于，所述方法是使用权利要求5所述的重组大肠杆菌在缺乏氨基酸的环境下进行发酵。
  10. 权利要求1～3任一所述方法，或权利要求4或5所述重组大肠杆菌在发酵、药物制备、材料或环保领域的应用。
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