Metabolic Engineering Of Escherichia Coli To Increase Ectoine Production

Ectoine has significant biological stability characteristics,and is widely used as a stabilizer and protector in fields such as skin care,food processing,and medical treatment,with high added value.In industrial production,halophilic bacteria are used to synthesize ectoine under the cultivation of high salt concentration.Hence,the fermentation process is relatively complex.In this study,a mutant strain Escherichia coli MWZ003/p FT28-ect ABC-Eclys C*-asp DH-ppc3 which has been constructed the heterologous synthesis pathway for ectoine was systematically metabolized.First,the competitive pathways of precursor substances were deleted and dynamically regulated to increase the intracellular accumulation of precursor substances and expand the metabolic flow of ectoine.Subsequently,the shaking flask fermentation conditions of Escherichia coli MWL009/p FT28-ect ABC-Eclys C*-asp DH-ppc3 were optimized to further improve the yield and production efficiency of ectoine.The main research conclusions are as follows:（1）The ectoine production was increased by deleting genes frd A,pfl B,pox B,adh E,and aro G.Four genes pfl B encoding the pyruvate formate-lyase,pox B encoding the pyruvate oxidase,adh E encoding the alcohol dehydrogenase,and aro G encoding the 3-deoxy-7-phosphoheptulonate synthase were deleted from MWZ003,resulting in the strain MWL007.After 36 h batch fermentation,comparing with the control MWZ003/p FT28-ect ABC-Eclys C*-asp DH-ppc3,ectoine production in MWL007/p FT28-ect ABC-Eclys C*-asp DH-ppc3 was 17.30 g·L^-1which increased 21.0%with a yield of 0.43 g·g^-1 glucose.Four genes pfl B,adh E,aro G and frd A encoding the fumarate reductase flavoprotein subunit,were deleted from MWZ003,resulting in the strain MWL008.After 36 h batch fermentation,comparing with the control MWZ003/p FT28-ect ABC-Eclys C*-asp DH-ppc3,ectoine production in MWL008/p FT28-ect ABC-Eclys C*-asp DH-ppc3 was 16.81 g·L^-1and increased 18.0%with a yield of 0.42 g·g^-1 glucose.（2）The ectoine production was further increased by deleting gene msc S.The gene msc S encoding the small conductance mechanosensitive channel Msc S was deleted from MWL007 and MWL008,resulting in the strain MWL009 and MWL010,respectively.After 36 h batch fermentation,comparing with the control MWZ003/p FT28-ect ABC-Eclys C*-asp DH-ppc3,ectoine production in MWL009/p FT28-ect ABC-Eclys C*-asp DH-ppc3 was 18.28 g·L^-1which increased 28.0%with a yield of 0.46 g·g^-1 glucose;ectoine production in MWL010/p FT28-ect ABC-Eclys C*-asp DH-ppc3 was17.70 g·L^-1which increased 23.7%with a yield of 0.44 g·g^-1 glucose.（3）The expression of glt A was inhibited to increase the production of ectoine.The glt A encodes the citrate synthase,which is an essential gene for acetyl-Co A to participate in the TCA cycle.CRISPRi was used to inhibit the expression of glt A to balance the intracellular accumulation of oxaloacetate and acetyl-Co A.RT-q PCR were conducted to validate the efficiency of gene glt A expression in the mutant strain MWZ003/p FT28-ect ABC-Eclys C*-asp DH-ppc3,p BAD33-d Cas9-g RNA-glt A2,which decreased by 45.0%compared to the control strain.After 36 h batch fermentation,comparing with the control strain,ectoine production in MWZ003/p FT28-ect ABC-Eclys C*-asp DH-ppc3,p BAD33-d Cas9-g RNA-glt A2 was increased 6.0%.（4）Conducted a series of single factor experiments to gradually optimize the shake flask fermentation conditions and further improved the production of ectoine and glucose conversion rate in the engineering strain MWL009/p FT28-ect ABC-Eclys C*-asp DH-ppc3.First,after 36 h batch fermentation,ectoine production was reached 18.45 g·L^-1after optimizing the induction of plasmid expression time for 4 h.Secondly,after 36 h batch fermentation,ectoine production was reached23.24 g·L^-1after optimizing the addition of sodium glutamate for 15 g·L^-1.Thirdly,after 42 h batch fermentation,ectoine production was reached 25.30 g·L^-1with a yield of 0.63 g·g^-1 glucose after optimizing the addition of ammonium sulfate for 15 g·L^-1.Finally,after 60 h fed-batch fermentation,the titer of ectoine in MWL009/p FT28-ect ABC-Eclys C*-asp DH-ppc3 reached 34.27 g·L^-1 with the conversion rate of 0.34 g·g^-1 glucose after optimizing the initial addition of glucose for 40 g·L^-1.