Construction And Efficient Synthesis Of L-Cysteine-Producing-Strains

L-cysteine is a sulfhydryl amino acid that is widely used in medicine,chemical industries and food processing.At present,L-cysteine is mainly produced by chemical synthesis and extraction.However,these methods have problems such as complicated process,low yield and environmental pollution.As an efficient and eco-friendly process,the bio-fermentation method has become the preferred process for the production of various amino acids in recent years.Hwoever,there are also problems such as low titer of L-cysteine due to insufficient precursor of L-serine.In this study,the L-serine-producing strain E.coli 4WG and C.glutamicum A36 were selected as the starting strain,and key enzymes in the L-cysteine metabolic pathway,transporter and degradation pathway were modified to construct a series of recombinant strains.Afterwards,the sulfur source was optimized to improve L-cysteine titer of the recombinant C.glutamicum.The main results were as follows:（1）The L-serine-producing strain E.coli 4WG(2.01 g·L^-1)was selected as the starting strain,which had been constructed in our laboratory.Four recombinant strains 4WG-Eccys E^Fr,4WG-Eccys E^Fr-yde D,4WG-Δtna A-Eccys E^Frand 4WG-Δtna A-Eccys E^Fr-yde D were constructed with glycerol by overexpressing of the key enzyme of the L-cysteine synthesis pathway（encoding gene cys E）,and the key enzyme of transport pathway（encoding gene yde D）.Moreover,the key enzyme of the degradation pathway（encoding gene tna A）were deleted.The L-cysteine titer of the strain 4WG-Δtna A-Eccys E^Fr-yde D was highest(313.4mg·L^-1)among the four recombinant strains,which was 1.3 times higher than 4WG-Eccys E^Fr.In addition,the original strain E.coli 4WG did not accumulate L-cysteine.（2）Then,C.glutamicum A36 in our laboratory was seleced as the starting strain,which could produce L-serine with 30.57 g·L^-1.The recombinant strain S-C-1 was constructed by overexpressing of L-serine-O-acetyltransferase（encoded by cys E）derived from E.coli.The recombinant strain S-C-2 was constructed by overexpressing of cys E^Frderived from Arabidopsis thaliana.The recombinant strains S-C-3 and S-C-4 were constructed by overexpressing of OAS thiolase-A（encoded by cys K）in strains S-C-1 and S-C-2 respectively.The fermentation results of the four recombinant strains showed that the L-cysteine titer of strains S-C-3 and S-C-4 decreased by 17.8%and 65.5%compared with S-C-1 and S-C-2.In order to further increase the L-cysteine production,the recombinant strains S-C-5 and S-C-6were constructed by overexpressing the key enzymes（encoded by bcr）of the transport pathway in strains S-C-1 and S-C-2.The recombinant strains S-C-7,S-C-8,S-C-9 and S-C-10were constructed by knocking out the key enzyme of L-cysteine degradation pathway（encoded by aec D）in strains S-C-1,S-C-2,S-C-5 and S-C-6.Then the fermentation of the recombinant strains were performed,and the results showed that the L-cysteine titer of S-C-7was the highest,reached 286.7 mg·L^-1.（3）The sulfur source was optimized to increase L-cysteine titer of the recombinant strain S-C-7 further,the results showed that when the sulfur source was sodium thiosulfate with the concentration of 12 g·L^-1.And the addition time was 24 h,the L-cysteine titer could reach581.6 mg·L^-1which was 1.0 times higher than that before optimization.Finally,in 5-L bioreactor,the L-cysteine titer of strain S-C-7 could reach 1.2 g·L^-1which was the highest titer of L-cysteine produced by C.glutamicum up to now.