One-step Efficient Synthesis Of Agmatine And 1,4-butanediamine By Metabolically Engineering Corynebacterium Crenatum

Agmatine and 1,4-butanediamine are amino acid derivatives and biological polyamines,which have important applications in many fields.With L-arginine as a precursor,agmatine was synthesized by decarboxylation in the presence of arginine decarboxylase,and agmatine is catalyzed to 1,4-butanediamine through agmatinase.In this study,the synthesis pathway of agmatine and 1,4-butanediamine was constructed in Corynebacterium crenatum SYPA5-5 by the methods of synthetic biology and metabolic engineering,and their yield was increased.The main contents are as follows:（1）Two arginine decarboxylases derived from E.coli K-12 were overexpressed in C.crenatum SYPA5-5,and the recombinant strain PC2（C.crenatum/pXMJ19-speA）with the expression of biosynthetic arginine decarboxylation was selected for agmatine production by comparison of the enzyme activity and the yield of shake flask fermentation.The 39.4 g·L^-1 of agmatine was produced by the fed-batch fermentation with PC2,and the conversion rate was0.26 g·g^-1 glucose.（2）Two ornithine decarboxylase,biosynthetic arginine decarboxylation and agmatinase derived from E.coli K-12 were heterologously expressed in C.crenatum SYPA5-5 to construct three strains producing 1,4-butanediamine.The results of shake flask fermentation showed that PC5（C.crenatum/pXMJ19-speA-speB）had a higher production potential of 1,4-butanediamine,but the intermediate product agmatine was accumulated.The expression level of agmatinase was regulated by RBS sequences with different intensities.It was found that the agmatinase activity of recombinant strain PC5-6（C.crenatum/pXMJ19-speA-R6speB）was 1.8 times higher than that of control strain PC5.The whole-cell transformation yield of 1,4-butanediamine by recombinant strain PC5-6 was 30.3%higher than that of the control strain,reaching 67.9 g·L^-1.The shake flask fermentation yield of 1,4-butanediamine with PC5-6reached 14.9 g·L^-1,which was 44.8%higher than that of the control strain.（3）Metabolic engineering of the chassis strain reduced the degradation of 1,4-butanediamine and increased the extracellular transport capacity of 1,4-butanediamine to further increase the production of 1,4-butanediamine.By knocking out the N-acetyltransferase gene sna A reduced the acetylation of 1,4-butanediamine,and the 1,4-butanediamine yield of 18.2 g·L^-1 was produced by single knock strain PC7（C.crenatumΔsna A/pXMJ19-speA-R6speB）in shake flask fermentation,which is 25.3%higher than that of the control strain PC5-6.By knockout of the spermidine synthase gene speE to reduce the downstream metabolism of 1,4-butanediamine,and the production of 1,4-butanediamine in double-knockout strain PC9（C.crenatumΔsna AΔspeE/pXMJ19-speA-R6speB）was 7.8%higher than that in PC7,reaching 19.6 g·L^-1.The repressor gene cgm R of the transporter Cgm A was knocked out to enhance the extracellular transport capacity of 1,4-butanediamine,and the yield of 1,4-butanediamine was further increased by 10.3%to 21.7 g·L^-1 by strain PC11（C.crenatumΔsna AΔspeEΔcgm R/pXMJ19-speA-R6speB）.The recombinant strain PC11 was obtained by metabolic engineering,the 1,4-butanediamine yield of 21.7 g·L^-1 was produced with glucose as substrate by PC11,which was 49.1%higher than that of the control strain PC5-6.（4）The xylose metabolic pathway derived from E.coli K-12 was constructed in strain PC11 by a double plasmid system to expand the range of substrate.PC14（PC11/p EC-XK99E-xyl AB）,which co-overexpressed xylose isomerase and xylulokinase,was screened by a growth performance assay and shake flask fermentation with xylose as carbon source.The results showed that the PC14 can produce 18.5 g·L^-1 and 17.0 g·L^-1 1,4-butanediamine in shake flask fermentation with mixed sugar and simulated wheat straw hydrolysate as carbon source,respectively.（5）Finally,the fed-batch culture of PC11 and PC14 were performed in 5 L bioreactor,and the strain PC11 was fermented with glucose as substrate for 72 h to produce 41.5 g·L^-1 of1,4-butanediamine,with a conversion rate of 0.18 g·g^-1 glucose.The strain PC14 was fermented for 72 h with mixed sugar as substrate,and produced 36.8 g·L^-1 of 1,4-butanediamine,and the conversion rate was 0.15 g·g^-1 total sugar.The strain PC14 was fermented for 72 h with simulated wheat straw hydrolysate as substrate,and produced 33.4g·L^-1 of 1,4-butanediamine,and the conversion rate was 0.14 g·g^-1 total sugar.