Construction Of Escherichia Coli Producing L-serine From Glycerol

Glycerol is an alternative carbon source that is attracting a lot of attentation because it is a renewable resource generated as by-product from the biodiesel production process.How to use glycerol to produce high value-added products attracts growing attention.L-serine is widely used in pharmaceutical and cosmetic industries,the direct fermentative production of L-serine from renewable carbon source becomes more interesting.However,wild-type Escherichia coli are not able to produce L-serine by using glycerol as carbon source.In this study,the recombinant strain was constructed by using metabolic engineered methods,including L-serine synthesis pathway,degradation pathway,transportation pathway and glycerol utilization pathway,the recombinant strain can produce L-serine by using glycerol as the sole carbon source.Moreover,in order to enhance L-serine yield,medium optimization was taken in this study.The main results are as follows:(1)L-serine is the primary metabolite,it can convert to pyruvate by L-serine deaminase.Three L-serine deaminase genes(tdcG,sdaA and sdaB)were deleted in turn,with tdcG deleted alone or in combination with sdaA,resulting in minimal L-serine accumulation.However,when sdaB was also deleted,0.06 g/L L-serine accumulated,OD600 was 8.1 reduced by 12.9%.The glycerol was consumed all after 18 h.(2)PGDH,the first enzyme in the L-serine biosynthesis pathway in E.coli,is allosterically inhibited by L-serine.Removal of feedback inhibition by L-serine through mutation of 3-phosphoglycerate dehydrogenase,resulting in strain 4W with an L-serine titre of 1.1 g/L at 54 h.However,glycerol utilization was decreased significantly and consumed all after 42 h.OD600 was 4.8 reduced by 40.7%.In high concentration,L-serine may be toxic to the strains.(3)Adaptive evolution was therefore performed to improve glycerol utilization,resulting in strain 4WA.The glycerol was consumed all after 30 h.Tolerance to L-serine was enhanced by overexpressing cysteine/acetyl serine transporter encoded by eamA,resulting in strain 4WE,1.96 g/L L-serine was obtained.OD600 was 5.87,and the glycerol was consumed all after 24 h.(4)EamA was overexpressed in an evolution strain 4WA to improve L-serine production,further,resulting in the strain 4WAE.2.36 g/L L-serine was obtained,OD600 was 6.1,and the glycerol was consumed all after 24 h.With medium optimization,the strain 4WAE accumulated 3.0 g/L L-serine.In batch fermentation,4WAE consumed all glycerol at 18 h,OD600 was 9.4 and L-serine reached 3.23 g/L.In fed batch fermentation,OD600 was 15.48 and L-serine reached 7.53 g/L.