JP2023535865A - Construction and use of fimbriated E. coli to improve production efficiency - Google Patents

Abstract

The present invention provides a method for promoting the growth of E. coli and increasing the yield of its fermentation products, comprising knocking out a pilus gene cluster in the E. coli genome, said pilus gene cluster being 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 shall be. The present invention also provides a recombinant E. coli obtained by the above method, and a method for fermentatively producing PHB and L-threonine using the E. coli. [Selection diagram] Figure 1

Description

The present invention relates to the construction and use of pililess E. coli to improve production efficiency, and belongs to the fields of genetic engineering and fermentation engineering.

Bacterial pili are large, long, thin supramolecular protein appendages that appear on cells and are responsible for biofilm formation, chemotaxis, adhesion, and transmembrane DNA movement. Pili are also important components of bacterial biofilms and have various adverse effects on industrial operations. For example, increasing the risk of product spoilage and contamination, causing severe infections, forming dormant cells and increasing resistance to antibiotics, consuming bacterial energy and substrates, and spreading nutrients. formation of biofouling, reduced heat transfer, increased corrosion, shortened useful life of fermentation equipment, and the like.

Polyhydroxyalkanoates (PHAs) are synthesized using various hydroxyacyl coenzymes a as substrates and retained in cells as insoluble spherical inclusion bodies or PHA particles. In general, PHAs play an important role in reducing carbon equivalents and storing excess carbon, which could improve the resistance of cells to starvation stress. PHAs have special properties due to their functional groups, such as biodegradability, biocompatibility, gas barrier properties, piezoelectricity, and nonlinear optical activity. Used for scaffolding, and many other potential uses. Poly-3-hydroxybutyrate (PHB) is a kind of PHA, and E. coli is often used for industrial production of PHB. L-threonine is an essential nutritional amino acid for the human body and an important component of protein synthesis. Widely used in human food, cosmetics, medicine, animal feed and health food.

In the synthesis of PHB and L-threonine by Escherichia coli, it is important to balance the product and cell growth. Specifically, the cells are grown well, and at the same time, the metabolic flux of the synthetic pathway is enhanced, and the expression level of the synthetic pathway of the product is controlled to increase the yield. Existing reports mainly improve PHB synthesis by optimizing fermentation conditions, optimizing metabolic pathways, increasing intracellular coenzyme concentrations, and optimizing expression plasmids, and the efficiency of L-threonine is improved. Biological synthesis focuses on modifications of metabolic pathways such as fatty acid blockade, the phosphotransferase system, and substrate redistribution. Acetyl coenzyme A and oxaloacetate, which are synthetic substrates for PHB and L-threonine, are obtained from the glycolytic system and the citric acid cycle, respectively. It has a significant effect on the growth of fungi. Therefore, the increase in raw yield of PHB cannot satisfy the industrial production demand, and the fermentative growth of L-threonine slows down. Therefore, it is very important to enhance the growth of strains and provide new methods to improve PHB and L-threonine synthesis to further improve PHB and L-threonine synthesis.

In order to solve the above technical problems, the present invention provides mutant strain WQM026 by knocking out 64 genes of the pilus gene cluster in the E. coli genome from E. coli. The WQM026 strain grew faster on nutrient-deficient (M9) medium and increased the total amount of cells. Subsequently, the recombinant bacteria WQM026/pBHR68 and WQM026/pFW01-thrA*BC-rhtC obtained by transforming the strain WQM026 after compacting the genes related to the synthesis of PHB and L-threonine, respectively, were transformed into PHB and L-threonine. It can efficiently synthesize threonine.

A first object of the present invention is to provide a method for promoting the growth of E. coli and increasing the yield of its fermentation product, the method comprising knocking out the pilus gene cluster in the E. coli genome. wherein the pilus gene cluster comprises 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.

In one embodiment of the invention, said pilus gene cluster comprises 64 genes, said 64 genes being, in order, 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, y RaI , yraJ, yraK, yadC, yadK, yadL, yadM, htrE, yadV, yadN, yehA, yehB, yehC, yehD, ybgO, ybgO, ybgP, ybgQ, ybgD, yfcO, yfcP, yfcQ, yfcR, yfcS, yfcT, yfcU, yfc V. , ygiL, yqiG, yqiH, yqiI, and the NCBI accession numbers for their sequences are 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, 94 6047, 946042, 947658, 947657, 947656, 947654, 944837, 944835, 944829, 944828, 944819, 944859, 944841, 946642, 946617, 946621, 946619, 947550, 945110, 946537, 945325, 946620, 946788, 946779, 946818, 94 6418, 1450268, 1450268, 949109, 947522, 947529, 947531, 947535 be.

In one embodiment of the invention, the E. coli is E. coli MG1655.

A second object of the present invention is to provide a recombinant E. coli obtained by constructing by any one of the above methods.

In one embodiment of the invention, said recombinant E. coli further comprises plasmid pBHR68 or plasmid pFW01-thrA*BC-rhtC.

A third object of the present invention is to provide use of said recombinant E. coli in hostile environments.

In one embodiment of the present invention, said unfavorable environment is M9 medium deficient in amino acids, and the composition of said medium is glucose 4 g/L, Na ₂ HPO _{4.12H 2} O 17.1 g/L, KH ₂ PO. ₄₄ g/L, _NH4Cl 3 g/L, NaCl 0.5 g/L, _MgSO4 0.24 g/L and CaCl2 _0.011 g/L.

A fourth object of the present invention is to provide the use of said recombinant E. coli in the synthesis of metabolites under conditions of amino acid deficiency.

In one embodiment of the present invention, recombinant E. coli containing the plasmid pBHR68 described above produce PHB under conditions of amino acid depletion.

In one embodiment of the present invention, the method for producing PHB by the recombinant E. coli is as follows. Specifically, the strain is activated and cultured on an LB plate for 10-15 hours, inoculated into the medium, placed under the conditions of 35-38° C. and 150-250 rpm, and cultured for 5-10 hours to obtain an inoculum, followed by fermentation. The medium was inoculated with an inoculum of 5% (v/v) and cultured at 35-38°C, 150-250 rpm for 40-50 hours.

In one embodiment of the present invention, the composition of said PHB-producing fermentation medium is: 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 1-8 g/L, NaCl 0.2-0.8 g/L, MgSO ₄ 0.1-0.5 g/L and CaCl ₂ 0.01-0.05 g/L.

In one embodiment of the present invention, recombinant E. coli containing the plasmid pFW01-thrA*BC-rhtC described above produces L-threonine under conditions of amino acid deficiency.

In one embodiment of the present invention, the method for producing L-threonine by the recombinant E. coli is as follows. Specifically, the strain is activated and cultured on an LB plate for 10-15 hours, inoculated into the medium, and cultured at 35-38 ° C. and 150-250 rpm for 5-10 hours to obtain an inoculum solution and a fermentation medium. was inoculated at an inoculum of 10% (v/v), placed at 35-38°C, 150-250 rpm, and cultured for 30-40 hours.

In one embodiment of the present invention, the composition of the fermentation medium for producing L-threonine is: yeast powder powder 1-5 g/L, citric acid 1-5 g/L, (NH ₄ ) ₂ SO ₄ 20-30 g/L L, KH _{2 PO 5-10} g/L, glucose 25-35 g/L, MgSO 4.7H ₂ O 1-5 g/L, FeSO _{4.7H 2} O 2-8 mg/L, MnSO _{4.4H 2} O 2 -8 mg/L and CaCO ₃ 15-25 g/L.

The present invention further provides a method for promoting the growth of said E. coli and effectively increasing the yield of its fermentation product, or the use of said recombinant E. coli in the fields of fermentation, pharmaceutical preparation, materials or environmental protection.

[advantageous effect]
The present invention knocks out 64 genes of the pilus gene cluster in the E. coli genome from E. coli to obtain mutant strain WQM026. The WQM026 strain grew faster on nutrient-deficient (M9) medium and increased the total amount of cells. Subsequently, the genes associated with the synthesis of PHB and L-threonine were transformed into compacted strain WQM026, respectively, and the resulting recombinant strains WQM026/pBHR68 and WQM026/pFW01-thrA*BC-rhtC were transformed into PHB and threonine, respectively. can be efficiently synthesized. The synthesized PHB accounted for 87.87% of the dry weight of the cells, 3.44 times that of the wild-type control MG1655/pBHR68. The yield of L-threonine is 2.49 g/L, which is 3.66 times that of MG1655/pFW01-thrA*BC-rhtC.

A group of genes involved in the synthesis and assembly of bacterial pili. Growth curves for knocking out each bacterial pilus gene cluster to promote growth in LB and M9 media. Microscopic examination of PHB synthesis in M9G medium by a PHB-synthesizing strain with a single knockout of the pilus gene cluster. Microscopic examination of PHB synthesizing in LBG medium by a PHB-synthesizing strain with a single knockout of the pilus gene cluster. Characterization of the pilus-deficient E. coli mutant strain WQM026. Use of WQM026 in the production of PHB and L-threonine.

(1) Gene Knockout Method Using the CRISPR/Cas9 knockout system, E. coli genes are knocked out without a trace. First, pCas was electrotransformed in Escherichia coli str. K-12 substr. MG1655 and induced with L-arabinose to express the recombinant enzymes Gam, Bet and Exo. The homology arm fragments and the specific N20 sequence-containing pTargetF plasmid were then co-electrotransformed in MG1655/Cas9 competent cells. After plating on kanamycin and spectinomycin double resistant plates and culturing at 30° C. for 18 hours, mutated strains were screened by colony PCR.

After the mutated strain is obtained, isopropyl-β-D-thiogalactoside (IPTG) is added in the medium to induce transcription of pCas to form sgRNA-pMB1, which is combined with Cas9 to form the pMB1 replicon of pTargetF. was destroyed to remove pTargetF. The pCas containing strain was used as such for subsequent knockdowns and the pCas containing strain was incubated overnight at 42°C to remove the temperature sensitive plasmid pCas.

(2) Method for measuring PHB yield The fermentation broth was centrifuged at 4°C and 4000 rpm/min for 20 minutes, the supernatant was discarded, and the cells were frozen and lyophilized. A fixed amount of freeze-dried cells was weighed, methyl-esterified by a conventionally reported conventional methyl-esterification method, and finally quantified by a conventional gas chromatography.

(3) Microscopic Observation of Cells To observe cell morphology, E. coli cells were grown on solid LB plates for 12 hours and imaged with a transmission electron microscope (TEM). In order to observe intracellular PHA particles, after fermenting and producing PHA for 24 hours, a bacterial cell suspension was prepared with 1% crystal violet and examined with a bright field microscope using an oil immersion lens (magnification = 100x). Observed. At the same time, E. coli cells were centrifuged at 4000 rpm/min for 5 minutes, washed twice with phosphate buffer (PBS buffer), and then fixed with 2.5% glutaraldehyde solution for at least 72 hours. Images were acquired with a G2 spirit transmission electron microscope using a Gatan US4000 4kx4k CCD at a voltage of 100 kV.

(4) Extraction, Qualitative and Quantitative Analysis of PHA E. coli cells harboring pBHR68 were collected by 5 mL centrifugation and washed twice with PBS buffer (pH 7.4). After centrifugation and collection, the cells were transferred to a precisely pre-weighed centrifugal tube, the centrifugal tube was wrapped with a cling film, and multiple small holes were made in the cling film so as not to evaporate the water and blow out the cells. opened. Lyophilized for 48 hours in a vacuum freeze dryer until completely dry. Complete dryness is indicated by touching the bottom of the tube at room temperature, or by gently flicking the bottom of the tube with a finger and the cells separate and easily detach from the wall of the tube. I decided. Weigh and calculate dry weight.

In order to accurately calculate the weight of the weighed sample, weigh 1 to 10 mg of completely dried dry cells and about 10 mg of the PHB standard sample, and transfer them to an esterification tube that has been accurately weighed in advance. . Then, add 2 mL of methanol (containing 3% sulfuric acid) and 2 mL of chloroform, seal the esterification tube with a cap, disperse the sample with ultrasonic waves to make the esterification more complete, and place in a boiling water bath for 6 hours or more. Carry out esterification. Fast dissolution of the PHB sample, which is generally powdery, is observed after about 0.5 hours. If the standard does not change form or dissolve after 0.5 hours, all reagents should be replaced. Degradation of methanol and chloroform can lead to poor esterification, while replacement with freshly opened reagents generally solves the above problem. After 6 hours of boiling water bath and allowing to cool sufficiently in a fume hood, carefully open the esterification tube and add 1 mL of deionized water, this time sealing the cap separately until the system is thoroughly mixed. Vigorously shaken, at which time the esterification tube was left in the fume hood for 3 hours or more to allow the phases to separate. Then, the upper layer was made into the water phase and the lower layer was made into the organic phase. An appropriate amount of the organic phase was added to the gas phase sample bottle, and the bottle was sealed and stored at -80°C.

Gas chromatography is an accurate and sensitive means of quantifying PHB content. The extract was analyzed using a Scion-SQ-456-GC module equipped with a DB-5MS fused silica capillary column (30 m x 0.25 mm x 0.25 μm) to determine its PHA composition and precise content. For gas chromatography, positron ionization (EI) was obtained with an ionization energy of 70 ev and mass spectra were programmed with ions from m/z 50 to m/z 650 with a scan interval of 0.5 sec. PHA production (wt%) was expressed as percent by weight of stem cells (DCW). For gas phase determination, Shimadzu GC2010 gas chromatography was used. An Agilent DB WAX 30m-0.32mm GC column and flame ionization detector are used and the sample injection temperature is 250°C. For each standard sample, a standard curve was plotted using different amounts of commercially available PHB as a standard sample.

(5) Extraction and measurement of ATP and acetyl-coenzyme A E. coli cells were cultured in M9 medium until early logarithmic growth or mid-logarithmic growth (OD600 = 0.8-1.0), The amounts of intracellular ATP and acetyl-coenzyme A were collected using ATP and acetyl-coenzyme A assay kits. Intracellular ATP was extracted and quantified according to the instructions of the ATP assay kit, and ATP analysis was performed using an Agilent 1260 series HPLC system, and the analytical column was a 250 mm×4.0 mm ODS-2HYPERSIL C18 column.

(6) Transcriptome analysis of MG1655 and WQM026 E. coli MG1655 and WQM026 were cultured in M9 medium to mid-exponential growth, harvested at 12000 rpm for 3 min, washed twice with PBS buffer, and then washed with liquid nitrogen. Quick frozen. RNA library extraction, construction and sequencing were performed by GENEWIZ Biotechnology. Sequence reading, comparison and analysis were performed using the MG1655 genome as a reference sequence. Differences in gene expression were analyzed using FIESTA Viewer v.1. 1.0 software, based on their expression levels and P-value ≤ 0.05.

(7) Concentration Analysis of L-Threonine The concentration of L-threonine was measured using an Agilent 1200 series or 1260 series HPLC system equipped with a Thermo 250 mm×4.0 mm ODS-2HYPERSIL C18 column. The concentration of L-threonine was determined by the pre-column derivatization method with o-phthalaldehyde (Koros, A., Varga, Z., Molnar-Perl, I. 2008. Simultaneous analysis of amino acids and amino acids as their o-phthalaldehyde). anethiol- 9-fluorenylmethyl chloroformate derivatives in cheese by high-performance liquid chromatography. J Chromatogr A, 1203(2), 146-52.).

(8) Medium LB medium (g/L): yeast powder 5, peptone 10 and NaCl 10.
M9 medium (g/L): Glucose 4, _{Na2HPO4.12H2O 17.1} , _{KH2PO4 4} , _NH4Cl3 , NaCl 0.5 _{, MgSO4} 0.24 and _CaCl2 0.011 .
LBG medium (g/L): glucose 20, yeast powder 5, peptone 10 and NaCl 10.
M9G medium (g/L): glucose 20, _{Na2HPO4.12H2O 17.1} , _{KH2PO4 4} , _NH4Cl3 , NaCl 0.5 _{, MgSO4} 0.24 and _CaCl2 0.011 .
PHB fermentation medium (g/L) _: Glucose 20, _{Na2HPO4.12H2O 17.1} , _{KH2PO4 4} , _NH4Cl3 , NaCl 0.5, _MgSO4 0.24 and CaCl2 _0.1 . 011.
L-threonine fermentation medium ₍ g/L): yeast powder 2, citric acid 2, ( _NH4 ) _2SO4 25, _{KH2PO4 7.46} , glucose ₃₀ , _MgSO4.7H2O2 , _FeSO4 . _7H2O 0.005, _{MnSO4.4H2O 0.005} and _CaCO3 20.

Example 1 Construction of pilus-deficient engineered bacterium A specific construction process of pilus-deficient engineered bacterium is as follows.
(1) Cell preparation of MG1655/pCas competent cells Red recombinant helper plasmid 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. in liquid medium , 30° C., 200 rpm overnight. This culture solution was inoculated into 100 mL of LB liquid medium at 2% inoculum, and when cultured at 30 ° C. and 200 rpm until OD600 = 0.2, L-arabinose (final concentration 30 mmol / L) was added to induce recombinase expression. did. Subsequently, culture is performed until D600 = 0.5, and the culture solution is left in an ice bath for 30 minutes, placed in a pre-chilled 50 mL centrifuge tube, and centrifuged at 8000 rpm at 4°C for 10 minutes to collect the cells. did. The precipitate was washed three times with prechilled 10% glycerol and finally suspended in 1 mL of 10% glycerol. Aliquot 80 μL of each tube into pre-chilled sterile EP tubes and save.

(2) Construction of pTargetF plasmid Table 2 shows primers for construction of pTargetF series plasmids. Plasmid pTargetF01 uses pTargetF as a template, uses primers F-sgRNA-yagVZ and R-sgRNA, and uses sequencing primers T-yagVZF and T-sgRNA-R to confirm the correctness of the plasmid. . Other plasmids pTargetF02, pTargetF03, pTargetF04, pTargetF05, pTargetF06, pTargetF07, pTargetF08, pTargetF09, pTargetF10, pTargetF11, pTargetF12, pTargetF27, pTargetF28, pT targetF29 and pTargetF30 were prepared by the same method (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), 2 506-14.), Corresponding primers were used.

(3) CRISPR-Cas9 construction mutant E. coli MG1655 genome contains 12 fimbriae synthesis and assembly gene groups yagVWXYZ, gltFyhcADEF, fimAICDFGH, sfmACDHF, ycbQRSTUVF, ydeQRST, yraHIJK, yadCKLMhtrEecpDyadN, yehABCD , ybgOPQD and yfcOPQRSTUV are present are doing. These 12 pili synthesis/assembly gene clusters are subdivided into 64 genes, yagV, yagW, yagX, yagY, yagZ, gltF, yhcA, yhcD, yhcE, yhcF, fimA, fimI, fimC, Fimd, Fimf, FIMG, FIMG, SFMA, SFMC, SFMD, SFMD, SFMF, SFMF, YCBQ, YCBR, YCBT, YCBT, YCBV, YCBF, YDEQ, YDET, YDET, YR AH, 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, yq iH, yqiI and the NCBI accession numbers for their sequences are 946631, 947349, 947606, 948806, 948759, 947746, 947741, 947738, 4056032, 947735, 948838, 948841, 948843, 948844, 9 48845, 948846, 948847, 945522 ,945367,945160,945407,944977,948306,946773,946934,947185,945561,945562,945559,946050,946049,946047,946042,947658,947657,947657,9 47656, 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.

Using the CRISPR-Cas9 method, from the E. coli MG1655 genome, 12 fimbriae synthesis/assembly gene clusters yagVWXYZ, gltFyhcADEF, fimAICDFGH, sfmACDHF, ycbQRSTUVF, ydeQRST, yraHIJK, yadCKLMhtrEecpDyadN, yehABCD, ybg OPQD and yfcOPQRSTUV were knocked out, respectively, and the corresponding Mutants WQM001, WQM002, WQM003, WQM004, WQM005, WQM006, WQM007, WQM008, WQM009, WQM010, WQM011 and WQM012 were constructed. For example, WQM001 knocked out the yagVWXYZ gene cluster from the genome. The two PCR products obtained by amplifying F1-yagVZ/R1-yagVZ and F2-yagVZ/R2-yagVZ with the upstream homology arm primer and the downstream homology arm primer are collected by gel. Collected with a kit, overlapping PCR was performed on F1-yagVZ/R2-yagVZ using primers to obtain overlapping homology arms. The purified overlapping homology arms (400 ng) and pTargetF01 (100 ng) were mixed and then electrotransformed in 80 μL of MG1655/pCas competent cells. After electrotransformation, cells were resuscitated at 30° C. for 45 minutes and then plated on double resistant plates containing spectinomycin and kanamycin at 50 μg/mL. After culturing at 30° C. for 36 hours, colony PCR was performed using primers F1-yagVZ/R2-yagVZ to select the target strain. Mutants were then inoculated into LB medium containing IPTG (0.5 mmol/L final concentration) and incubated overnight to remove the pTargetF01 plasmid. The pCas plasmid contains a temperature sensitive replicon and was incubated at 42°C for 24 hours to remove the pCas plasmid. Strains that do not contain the pTargetF01 and pCas plasmids are used for subsequent studies. Eleven other mutant strains WQM002, WQM003, WQM004, WQM005, WQM006, WQM007, WQM008, WQM009, WQM010, WQM011, WQM012 were constructed by the same method. WQM026 was constructed by knocking out all 12 pilus operons one by one using this method. Tables 1 and 2 show strains, plasmids and primers related to this part.

Example 2 Enhancing Proliferation and PHB Synthesis by Knocking Out a Set of Pili Genes E. coli contains 12 gene clusters for synthesizing and assembling pili (Fig. 1). Pili not only enhance bacterial virulence, but also consume large amounts of energy and carbon sources during their synthesis and assembly. Theoretically, therefore, removal of these pili could improve the biosafety and productivity of E. coli.

Strains WQM001, WQM002, WQM003, WQM004, WQM005, WQM006, WQM007, WQM008, WQM009, WQM010, WQM011 and WQM012 were obtained by removing 12 pili synthesis/assembly gene groups from the chromosome of E. coli MG1655. Ta. These strains were cultured in LB medium and M9 medium, respectively. As shown in FIG. 2, the OD value of the bacterial solution was measured every 2 hours and the growth curve of the strain was plotted. In LB medium, the growth curves of all 12 mutant strains are almost the same as the control strain MG1655, so any one of the 12 fimbriae synthesis/assembly gene clusters in E. coli Removal was shown to have no effect on or slightly improve cell growth. In M9 medium, all 12 mutant strains grew better than the control strain MG1655. Pili biosynthesis and assembly requires the consumption of large amounts of amino acids, but since M9 medium does not contain any amino acids, E. coli cultured and grown in M9 medium synthesizes proteins. Therefore, it was necessary to synthesize all 20 kinds of amino acids by oneself. Thus, by reducing pilus synthesis and assembly, amino acids conserved in the mutants became available to promote cell proliferation. From this, it was found that removal of E. coli fimbriae, especially in the case of nutritional deficiencies, is advantageous for cell growth.

To test the productivity of the pilus mutants, the empty vector pBSK and the vector pBHR68 containing the synthetic gene for PHA were electrotransformed in MG1655 and the 12 mutant strains, 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/pBHR68, WQM001/pBHR68, WQM002/pBHR68, WQM003/pBHR68, WQM004/pBHR68, WQM005/pBHR68, WQM006/pBHR68, WQM007/pBHR6 8, WQM008/pBHR68, WQM009/pBHR68, WQM010/pBHR68, WQM011/ pBHR68, WQM010/pBHR68, WQM011/pBHR68 and WQM012/pBHR68 were obtained. These recombinant strains were grown in M9G and LBG media and cells and their vector controls were observed microscopically (Figs. 3, 4).

When grown in M9G medium (Fig. 3), all 13 empty vector strains showed similar size and did not produce PHA. Of these, MG1655/pBHR68 had slightly larger cell volumes and only a few cells produced PHA. However, the 12 mutant strains of pBHR68 containing the PHA synthesis gene were significantly increased in their cell size and almost all cells produced PHA (intracellular white granules).

When grown in LBG medium (Fig. 4), all 13 empty vector strains were of similar size and none produced PHA. MG1655/pBHR68 was similar in size to its control and did not produce PHA. The 12 mutant cells containing pBHR68 increased in size and produced less PHA than the corresponding strains in M9G medium. This suggested that removal of fimbriae was advantageous for E. coli to produce PHA.

Example 3 Analysis of Morphology and Metabolites of WQM026 From the results of Example 2, removal of a set of pilus synthesis/assembly gene clusters both promoted E. coli cell growth and PHA biosynthesis. A pilus-deficient strain WQM026 was constructed by deleting all 12 pilus synthesis/assembly gene clusters in the MG1655 chromosome of . The bacterial fluid OD values were measured every 2 hours and the growth curve of the strain was plotted as shown in Figure 5A. WQM026 grew slightly better than MG1655 in LB medium, much better than MG1655 in M9 medium, and after culturing up to 18 hours, the OD value of WQM026 was about three times that of MG1655. Met. This indicates that removal of all 12 pilus gene clusters in E. coli is advantageous for cell growth, especially under nutrient-poor environments. When MG1655 and WQM026 cells were observed under an electron microscope, the cell surface of MG1655 was heavily covered with pili (Fig. 5B), whereas the surface of WQM026 was smooth and no trace of pili was observed. (Fig. 5C).

To investigate why E. coli produces more PHA due to pili deletion, the concentrations of acetate and acetyl-coenzyme A in WQM026 were measured. Acetyl-coenzyme A is the immediate precursor for PHA synthesis and acetic acid is produced from acetyl-coenzyme A. The amounts of acetic acid and acetyl-coenzyme A in WQM026 were significantly higher than in MG1655, increasing by 23.57% and 79.18%, respectively (Fig. 5D). Pili deletion increases the levels of acetate and acetyl-coenzyme A in E. coli WQM026, favoring E. coli for PHA production. Also, the concentration of citrate in WQM026 was 28.78% lower than that in MG1655 (Fig. 5D). Citrate is a key metabolite of the TCA cycle and its low concentration in WQM026 indicates that the conserved carbon is not flowing into the TCA cycle, furthermore, loss of pilus. indicated a reduction in energy consumption. Since energy is required for the biogenesis and assembly of the various pili, ATP concentrations in cells of WQM026 containing MG1655 were also measured (Fig. 5D). The ATP concentrations in WQM026 and MG1655 were 0.741 and 0.402 μmol/g, respectively, and the ATP concentration in WQM026 increased 1.8-fold over that in MG1655. This indicated that pili removal could save energy.

Example 4 Use of WQM026 in PHB Synthesis WQM026 has the advantage of better growth and higher accumulation of ATP and acetyl coenzyme A compared to the control MG1655. Its use in the fermentation industry is therefore worth considering.

Plasmid pBHR68 containing the PHA synthesis pathway was electrotransformed in WQM026 to give WQM026/pBHR68. Electron microscopy analysis of ultrathin sections showed that WQM026/pBHR68 cells were loaded with large PHA particles (Fig. 6B), whereas only trace amounts of PHA were observed in MG1655/pBHR68 cells. (Fig. 6A). This suggested that WQM026 could efficiently synthesize PHA.

PHAs can be synthesized from a variety of substrates and form insoluble spherical inclusion bodies or PHA particles inside cells. There are more than 90 types of PHA monomers. Introduction of pBHR68 into E. coli normally produces poly-3-hydroxybutyrate (PHB), but introduction of pBHR68 also produces other types of PHA. Therefore, the type and yield of PHA were measured by the GC/MS method.

After incubating the strain on an LB plate for 12 hours, one large mossy ring of bacteria was picked and inoculated into a 250 mL conical flask containing 50 mL of LB. After culturing for 6 hours under these conditions, an inoculum solution was obtained. 5% (v/v) inoculum was inoculated into 50 mL of fermentation medium and cultured at 37° C. and 200 rpm for 48 hours.

PHA extraction, methyl esterification, and GC/MS analysis were performed on the fermentation broth of the above strain WQM026/pBHR68. Only one peak that was exactly the same was observed (Fig. 6C). The mass spectrum of PHA produced by WQM026/pBHR68 showed the same pattern as standard PHB (Fig. 6D). This proved that the PHA produced by WQM026/pBHR68 was PHB. As shown in FIG. 6E, the PHB and stem cell weight percent (DCW) yields of WQM026/pBHR68 were both significantly increased compared to control MG1655/pBHR026 to 3.31 g/L and 87.87%, respectively. 2-fold and 3.44-fold improvement over the control MG1655/pBHR026, respectively.

Example 5 Use of WQM026 in L-Threonine Synthesis Since Escherichia coli was developed for L-threonine production, the possibility of efficiently producing L-threonine with WQM026 was further investigated. Plasmid pFW01-thrA*BC-rhtC containing key genes for L-threonine biosynthesis and transport was transformed in MG1655 and WQM026 respectively to obtain MG1655/pFW01 thrA*BC-rhtC and WQM026/pFW01 thrA*BC-rhtC. .

After incubating the strain on an LB plate for 12 hours, one large mossy ring of bacteria was picked and inoculated into a 250 mL conical flask containing 50 mL of LB. After culturing under these conditions for 4 hours, an inoculum solution was obtained. 80 mL of fermentation medium was inoculated with an inoculum amount of 10% (v/v) and cultured for 36 hours at 37° C. and 200 rpm. When the L-threonine content in the fermentation broth was measured, compared with the control strain MG1655/pFW01 thrA*BC rhtC, the synthetic yield of L-threonine by WQM026/pFW01 thrA*BC rhtC was 2.49 g/L. reached 266.18% (3.66-fold) compared to the control strain MG1655/pFW01 thrA*BC rhtC (Fig. 6F).

Although the present invention is disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Any changes and modifications can be made by those skilled in the art without departing from the spirit and scope of the invention. The scope of rights of the present invention shall be based on the scope defined by the claims.

Claims (10)

A method of promoting growth and increasing the yield of fermentation products of E. coli comprising knocking out pilus genes in the E. coli genome, wherein the pilus genes are 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 comprises 64 genes, which are in order yagV, yagW, yagX, yagY, yagZ, gltF, yhcA, yhcD, yhcE, yhcF, fimA, fimI, fimC, fimD , Fimf, FIMG, FIMH, SFMA, SFMD, SFMD, SFMD, SFMF, SFMF, SFMF, YCBR, YCBT, YCBS, YCBV, YCBV, YDEQ, YDER, YDET, YRAH, YRAH, YRAH, YRAH, YRAH, YRAH, YRAH RAI, YRAJ, YRAK, YADC, YADK . qiI and the NCBI accession numbers for those sequences are, in order: 5, 948846, 948847, 945522, 945367, 945160, 945407, 944977, 948306, 946773, 946934, 947185, 945561, 945562, 945559, 946050, 946049, 946047, 946042, 947658, 947657, 94 7656, 947654, 944837, 944835, 944829, 944828, 944819, 944859, 944841, 946642, 946617, 946621, 946619, 947550, 945110, 946537, 945325, 946620, 946788, 946779, 946818, 946418, 1450268, 1450268, 949109, Claims characterized by 947522, 947529, 947531, 947535 Item 1. The method according to item 1. 3. The method according to claim 1 or 2, wherein the E. coli is E. coli MG1655. A recombinant E. coli constructed by the method according to any one of claims 1-3. 5. The recombinant E. coli according to claim 4, further comprising plasmid pBHR68 or plasmid pFW01-thrA*BC-rhtC. Use of the recombinant E. coli according to claim 4 or 5 in metabolite synthesis under an amino acid-deficient environment. A method for fermentative production of PHB, characterized in that the recombinant Escherichia coli according to claim 5 is fermented under an amino acid-deficient environment. The amino acid-deficient environment is a fermentation medium for producing PHB by the strain, and the composition of the fermentation medium is 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 1-8 g/L, NaCl 0.2-0.8 g/L, MgSO ₄ 0.1-0.5 g/L and CaCl ₂ 0.01-0.05 g/L 8. The method of claim 7, comprising L. A method for fermentative production of L-threonine, wherein the recombinant E. coli according to claim 5 is fermented under an amino acid-deficient environment. Use of the method according to any one of claims 1 to 3 or the recombinant E. coli according to claims 4 or 5 in fermentation, drug preparation, materials or environmental protection.

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