| The phosphoenolpyruvate:glucose phosphotransferase system on Escherichia coli is the most important way to transport glucose,which need consume phosphoenolpyruvate.Weakening of PTS system is conducive to the accumulation of PEP and increases L-threonine production.In this study,the CRISPR gene editing technology of Escherichia coli was used.Wild-type E.coli MG1655 was used as the starting bacterium.Four genes?ptsG,ptsH,crr and ptsI?related to the transport of glucose in the PTS system,rsd related to both the RNA polymerase and the PTS system and iclR related to glyoxalate were knocked out.Two genes?galP and glk?associated with glucose transporter and the gene gltA associated with the TCA cycle were overexpressed by strong promoter trc in genome.Then,an L-threonine producing strain was constructed by introducing plasmid pFW01-thrA*BC-rhtC harboring the key genes for L-threonine biosynthesis and secretion.The main research results are as follows:?1?Key genes affecting L-threonine production in the PTS system of E.coli were identified by gene knockout.The key genes ptsG,ptsH,crr,and ptsI related to glucose transporter in PTS system of wild-type E.coli MG1655 were deleted,resulting in mutant strains WMZ002,WMZ003,WMZ004 and WMZ005.Then,plasmid pFW01-thrA*BC-rhtC was transferred into these strains separately.Compared with MG1655/pFW01-thrA*BC-rhtC,the production of threonine by that four recombinant strains were improved,and WMZ004/pFW01-thrA*BC-rhtC produced the highest yield,the yield was 5.67 g·L-11 after fermentation in shake flask for 24 h.The knockout of ptsG and crr can increase the rate of glucose uptake.The knockout of ptsH can inhibit the uptake of glucose.The knockout of ptsI severely inhibits the growth of bacteria,the production of threonine and the absorption of glucose.?2?The deletion of two or more genes in ptsG,ptsH,crr and ptsI all reduced the threonine yield of the original single mutant.The mutant WMZ012,WMZ013 and WMZ014 were obtained by knockout of ptsGH,ptsHcrr and ptsHcrr in wild-type MG1655.Then,plasmid pFW01-thrA*BC-rhtC was transferred into these strains.Compared with the control bacterium WMZ004/pFW01-thrA*BC-rhtC,the threonine production of the above three recombinant strains decreased,among which WMZ013/pFW01-thrA*BC-rhtC produced the highest yield,and the yield of flask fermentation at 36 h was only 4.41 g·L-1.?3?The deletion of rsd gene can improve the L-threonine production of wild E.coli and ptsH mutant.rsd gene was both deleted in MG1655 and WMZ003,resulting in mutant strains WMZ001 and WMZ007.Then,plasmid pFW01-thrA*BC-rhtC was transferred into these strains.Compared with the control strain MG1655/pFW01-thrA*BC-rhtC,the threonine yield of WMZ001/pFW01-thrA*BC-rhtC in flask fermentation for 36 h was 1.85 g·L-1,which was1.76 times higher.Compared with WMZ003/pFW01-thrA*BC-rhtC,the yield of WMZ007/pFW01-thrA*BC-rhtC reached 4.33 g·L-1 at 36 h,which was 33.23%higher.?4?Overexpression of galP gene can increase the production of L-threonine and sugar consumption of ptsH-knockout mutant.galP gene was overexpressed in WMZ007,obtaining the mutant WMZ010.Then,plasmid pFW01-thrA*BC-rhtC was transferred into it.Compared with WMZ007/pFW01-thrA*BC-rhtC,the fermentation yield of WMZ007/pFW01-thrA*BC-rhtC reached 5.35 g·L-1 at 24 h,which was 23.56%higher.?5?The deletion of iclR gene can improve the L-threonine yield of crr mutant strain,and on this basis,the over-expression of gltA gene can further improve the L-threonine yield.iclR gene was deleted in WMZ004,resulting in mutant strains WMZ015.In WMZ015,the gltA gene was overexpressed by the strong promoter trc,and the mutant WMZ016 was obtained.Then,plasmid pFW01-thrA*BC-rhtC was transferred into these strains.Compared with WMZ007/pFW01-thrA*BC-rhtC,the fermentation yield of WMZ015/pFW01-thrA*BC-rhtC reached 6.63 g·L-1 at 36 h,which was 16.93%higher.Compared with WMZ015/pFW01-thrA*BC-rhtC,the fermentation yield of WMZ016/pFW01-thrA*BC-rhtC reached 9.21 g·L-1at 24 h,which was 38.91%higher.?6?The optimal glucose concentration for WMZ016/pFW01-thrA*BC-rhtC was 50 g·L-1,reaching a maximum conversion rate of 0.35 g·g-1 glucose.The mutant was fermented for 36 h in medium with 30 g·L-1,40 g·L-1,50 g·L-1 and 60 g·L-1 glucose,and the threonine production was 9.33 g·L-1,13.29 g·L-1,17.23 g·L-1 and 17.98 g·L-1,respectively. |
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