Improving The Efficiency Of L-Threonine Synthesis Based On Modification Of Betaine Synthesis And Transport In Escherichia Coli

With the betaine add to the medium,it upregulates the expression of glucose-6-phosphate dehydrogenase(zwf),could increase the NADPH,which is beneficial for L-threonine production.In Escherichia coli,betaine can be taken through ProP encoded by proP or ProVWX encoded by proVWX.ProP is a H+-osmolyte symporter,whereas ProVWX is an ABC transporter.Betaine can also be synthesized in E.coli by enzymes encoded by betABIT.However,the influence of ProP,ProVWX and betABIT on L-threonine production in E.coli has not been investigated.This study improved the L-threonine synthesis efficiency through metabolic engineering modifications based on the study of the effect of betaine synthesis and transport on L-threonine production in Escherichia coli.The main conclusions are as follows:(1)The addition of betaine improved the growth and L-threonine synthesis of E.coli strain TWF001.The addition of 1-1.5 g/L betaine resulted in an increase in L-threonine production of approximately 1 g/L.Deletion of betABIT retarded the growth of TWF001 and slightly decreased the L-threonine production.Due to the lack of betaine synthesis,E.coli is unable to use betaine as an osmoprotective agent,resulting in a decrease in biomass and L-threonine production.Deletion of proP or/and proVWX significantly increased the L-threonine production.When proP was deleted,the L-threonine production increased 33.3%;when proVWX was deleted,the L-threonine production increased 40.0%.When both proP and proVWX were deleted(TSW003),the resulting strain TSW003 produced 23.5 g/L L-threonine after 36 h flask cultivation.(2)The genes betABIT,proC,fad R,crr and pts G were individually deleted from TSW003 to determine their growth and threonine production.and it was found that further absence of either crr(TWS008)or pts G(TWS009)improved L-threonine production.TSW008 produced24.9 g/L L-threonine after 36 h flask cultivation with a yield of 0.62 g/g glucose and a productivity of 0.69 g/L/h.TSW009 produced 26 g/L L-threonine after 48 h flask cultivation with a yield of 0.65 g/g glucose and a productivity of 0.54 g/L/h,which is 116% increase compared to the control TWF001.(3)The modified strain TSW008 with the highest productivity undergoes fed-batch fermentation in a fermentor.After 48 h fed-batch fermentation,65.3 g/L L-threonine was produced,with a productivity of 1.36 g/L/h,sugar and acid The conversion rate was 0.343 g/g,which was 21.4% higher than the original wild strain producing L-threonine.