Investigation Of L-serine Exporter In Corynebacterium Glutamicum

L-serine is an important kind of active pharmaceutical ingredient(API)of amino acid,and it is the top 30 most attractive building block biochemical.L-serine has a broad market and application prospects.Microbial production of L-serine from carbohydrate feedstocks has attracted attention,because of rich raw materials,sustainable production process,and environment-friendly.In the early stage,the L-serine producing strain Corynebacterium glutamicum was subjected to conventional mutagenesis and metabolic engineering to obtain the high-yield L-serine producing strain ?SSAAI.However,the production efficiency of C.glutamicum ?SSAAI is still not high,and improvement of its titer encountered a bottleneck,which may be caused by inefficient efflux rate of L-serine in the recombinant strain.Enhancement of L-serine secretion is beneficial to increase the L-serine titer.However,in the case of L-serine exporters in C.glutamicum,the only report is that the L-threonine exporter ThrE also functions as L-serine exporter.Therefore,the excavation and utilization of L-serine exporters is of great significance,which provides new ideas for L-serine producing strains improvement by metabolic engineering.In this study,the L-serine producing strain ?SSAAI was used as the research object.Firstly,the role of ThrE on L-serine efflux was investigated in C.glutamicum ?SSAAI.And then the previous genome sequencing information and comparison with the gene sequence were used to find new L-serine exporters.The main contents were as follows:(1)The role of exporter ThrE on L-serine efflux was investigated in C.glutamicum ?SSAAI.It was reported that the L-threonine exporter ThrE functions as L-serine exporter.The fact was found that the overexpression or knockout of ThrE had no significant effect on the accumulation of L-serine in C.glutamicum ?SSAAI,which indicated that ThrE was not the major L-serine exporter in C.glutamicum ?SSAAI,and there were unexplored L-serine exporters in C.glutamicum ?SSAAI.(2)Using comparative genomics information to explore the effect of differential genes on the L-serine efflux.The results of pre-genome sequencing and comparative genomics analysis showed that the proteins encoded by the four genes SD36_RS14680,SD36_RS02065,SD36_RS15070,and SD36_RS14970 may be related to L-serine efflux.It was found that the knockout of four variant genes had no significant effect on the accumulation of L-serine by ?SSAAI,indicating that the proteins had no releationship with L-serine export in C.glutamicum ?SSAAI.(3)Using gene sequence alignment to excavate new L-serine exporters.The L-cysteine exporter EamA could secrete L-serine in Escherichia coli.In order to detect new L-serine exporters,three eamA homologs were found in C.glutamicum by gene sequence alignment: NCgl2065,NCgl2050,NCgl0580.Knockout of NCgl2065,NCgl2050 did not affect the L-serine production of C.glutamicum ?SSAAI.However,C.glutamicum ?SSAAI with NCgl0580 deletion showed 60.0% decrease in L-serine titer.(4)The function and application of gene NCgl0580 was studied.NCgl0580 expression with plasmid in C.glutamicum ?SSAAI?N0580 restored the mutant's ability to produce L-serine.Using enhanced green fluorescent protein(EGFP)as a reporter protein,a membrane protein encoded by NCgl0580 was found.The function of protein encoded by NCgl0580 as a novel L-serine exporter was further confirmed by peptide feeding approaches,and overexpression of NCgl0580 increased L-serine production.C.glutamicum ?SSAAI-N0580 could accumulate 28.67 g/L L-serine,which was increased by 10.5% when compared with control C.glutamicum ?SSAAI.(5)The regulation of NCgl0580 was studied.The upstream gene NCgl0581 encodes a Lys R family transcriptional regulator.C.glutamicum ?SSAAI with NCgl0581 deletion showed 57.4% decrease in L-serine titer.The fluorescence expression of the probe plasmid showed that NCgl0581 was the positive regulator of NCgl0580,which laid the foundation for further exploration of the regulatory mechanism of L-serine expoter expression.