The Transformation And Metabolic Analysis Of L-serine Uptake Pathway In Escherichia Coli

L-serine is a non-essential amino acid which has been widely used in pharmaceutimcals industry,food industry,cosmetics industry and so on.Microbial fermentation is a cheap and efficient way to produce L-serine which has a good prospect.At present,the way to constructe L-serine production strains is very clear and there are two methods to achieve this purpose: enhance L-serine synthesis pathway and impair L-serine degradation pathway.The researches about modifying L-serine synthesis and degradation pathway are very clear.Then the modification of transporting pathway has been take into account.And this research mainly focus on the L-serine uptake pathway,which is one part of L-serine transporting pathway.Four genes,sstT,cycA,sdaC and tdcC,have been reported to be involved in serine uptake in Escherichia coli.Gene sstT could encode a Na+/serine symporter which could also transport threonine.Gene cycA encode an alanine transporter but this transporter could also transport serine and glynine in E.coli.The production of gene sdaC and tdcC are both H+/serine symporters.And those two genes have a very high homology in the coding sequence,expect the sequence near the 3’end.Besides,sdaC encode a specific serine uptake protein,while tdcC encode a serine-serine transport system.The original strain SWCH-05(sdaA deleted,glyA mutated)have been used to constructed 4 single-gene deletion mutants and 6 double-gene deletion mutants in the early works.And the serine production of single-gene deletion mutants have been investgated.The result shows that gene sdaC deletion promoted the production mostly.In this study,4 triple-gene deletion mutants and 1 quadruple-gene deletion mutant were constructed by using Red recombination method.The L-serine uptake activities of those 5 mutants together with 10 ealry constructed mutants were tested to investigate the impact of uptake gene deletion.The L-serine uptake activity experiment of uptake gene overexpression strains were implement to verify the result of deletion mutants’ uptake activity;At last,shake-flask fermenation was used to test the L-serine production.The content of this study are focus on the following aspects:1)4 triple-gene deletion mutants SWCH-71 、 SWCH-72 、 SWCH-73 and SWCH-74 and a quadruple-gene deletion mutant SWCH-08 were constructed based on the double-gene deletion mutants SWCH-61、SWCH-62 and SWCH-64 by using Red recombination method.2)In order to investigate the impact of uptake genes deletion to the L-serine uptake ability,we tested the L-serine uptake activity of 15 L-serine uptake gene deletion mutants.The result shows that,among the single-gene deletion mutants,the L-serine uptake activity of sdaC mutant SWCH-51 was 0.798 nmol(mg dry weight)-1 after 30 min,which was decreased by 23.34 % compared with that of the parental strain SWCH-05.However,the L-serine uptake activities of the sstT,cycA and tdcC single-gene deletion mutants SWCH-53,SWCH-52 and SWCH-54 increased by 34.29 %,78.29 % and 48.03 % respectively.In double and triple-gene deletion strains,the L-serine uptake activities were decreased only in strains including sdaC deletion.When all four L-serine uptake genes were deleted,the L-serine uptake activity of sstT·cycA·sdaC·tdcC quadruple-gene deletion mutant SWCH-08 dropped to near zero.3)Taking the rising L-serine uptake activity of parital mutants into account,we constructed 4 L-serine uptake gene recombination plasmids pSC-sstT、pSC-cycA、pSC-sdaC and pSC-tdcC by using double digested test.And those plasmids were inducted into the original strain SWCH-05 respectively and obtained 4 L-serine uptake gene overexpressing strans SWCH-05/pSC-sstT、SWCH-05/pSC-cycA 、SWCH-05/pSC-sdaC and SWCH-05/pSC-tdcC.The serine uptake activity result of those 4 strains shows that,gene sstT、cycA、sdaC and tdcC were really taken part in L-serine uptake and were truly L-serine uptake genes.And the strain SWCH-05/pSC-cycA has the highest serine uptake activity which is 2.983 nmol(mg dry weight)-1,and the strain SWCH-05/pSC-sdaC followed.4)The shake-flask culture result revealed that,in all 11 multi-gene deletion mutants,sstT·sdaC double-gene deletion mutant SWCH-62/pSC-05,sstT·sdaC·tdcC and sstT·cycA·sdaC triple-gene deletion mutants SWCH-73/pSC-05 and SWCH-71/pSC-05 had higher L-serine production than the original strain and their L-serine production were 271.11 、 312.58 and 322.57 mg/L,respectively.Besides,the sstT·cycA·sdaC·tdcC quadruple-gene deletion mutant SWCH-08/pSC-05 achieved the highest L-serine production of 450.58 mg/L,which was 6-fold when compared to that of the original strain.In addition,the cell growth of double-and triple-gene deletion mutants were similar or above to that of the original strain.But the cell growth of the quadruple-gene deletion mutant decreased remarkably,whose OD600 was 28 % lower than that of the original strain.