| Ala-Gln(AQ)is a functional dipeptide with high water solubility,good thermal stability and high bioavailability.It is widely used in clinical medicine,post-operative rehabilitation,sports health care and other fields.At present,chemical synthesis is mainly adapt in the production of AQ.Because of the complicated steps,time-consuming,laborconsuming,low yield and by-products,it is urgent to establish a green and efficient method for synthesizing AQ.In this study,glutamine synthesis module and AQ synthesis module were constructed by means of metabolic engineering.Glutamine synthase(GlnA)and Lamino acid-ligase(BacD)with excellent catalytic properties were obtained through screening.Through RBS optimization and copy number optimization,recombinant Escherichia coli capable of efficiently catalyzing glutamic acid(L-Glu)and alanine(L-Ala)to synthesize AQ was constructed.The results are as follows:1?The recombinant strain pYB1a-BsbacD/BW was obtained,and the AQ synthesis module was constructed by expression of L-amino acid-ligase(BacD)in Escherichia coli BW25113.The recombinant bacteria were used for whole cell catalysis with alanine and glutamine as substrates.The synthesis of AQ was initially realized.2?Escherichia coli AQ09 which effectively alleviated the degradation of AQ was obtained by knocking out the gene pepABDN encoding dipeptide degrading enzyme and the gene cluster dppA-F encoding dipeptide transporter in Escherichia coli BW by CRISPR technology.After the recombinant strain pYB1a-BsbacD/AQ09 reacted in whole-cell catalytic system with L-Gln and L-Ala as substrates for 18 hours.The AQ yield reached 3.3 mM,which was 65.0% higher than that of the starting strain pYB1a-BsbacD/BW(yield 2.0 mM).3?In order to reduce the cost of raw materials,the Gln synthesis module was constructed: A glutamine synthase CgGlnA capable of efficiently transforming L-Glu into L-Gln was screened out.The recombinant strain expressing CgGlnA was catalyzed by LGlu as substrate.The yield of L-Gln reached 22.4 mM and the molar yield was 44.8%.The recombinant strain AQ06 was obtained by knocking out the glutaminase-encoding genes glsA,glsB and the genes encoding glnA subunit-modifying enzymes glnE and glnB in BW,and pYB1a-CgglnA was introduced into AQ06 to obtain recombinant strain pYB1aCgglnA/AQ06.The yield of L-Gln reached 46.5 mM,which was 107.6% higher than that of the original strain pYB1a-CgglnA/BW.4?Escherichia coli AQ10 with chassis AQ06 and AQ09 knockout traits was obtained by superimposing the AQ synthesis module and the Gln synthesis module,and the plasmid pYB1s-CgglnA-BsbacD co-expressing CgglnA and BsbacD was introduced into AQ10 to obtain the strain pYB1s-CgglnA-BsbacD/AQ10.The strain catalyzed for 6 hours in a whole-cell catalytic system by L-Glu and L-Ala as substrates,and the AQ yield was 23.6 Mm,which was 1080% higher than the original strain pYB1a-BsbacD/BW.5?Further optimization of synthetic pathway: The recombinant strain YGS176300 was obtained by optimizing the number of RBS and plasmid copies to regulate the expression of BacD protein.The recombinant strain reacted in whole-cell catalytic system with L-Glu and L-Ala as substrates for 6 hours.The AQ yield reached 32.4 mM,which was 79.1% higher than that of pYB1s-CgglnA-BsbacD/AQ10.Furthermore,the recombinant strain LGS176300 was obtained by replacing plasmid vector with medium copy plasmid pLB1 s.The AQ yield of the strain reached 22.8mM,which was only 9.0% lower than that of YGS176300,but the concentration of the bacterial solution increased by twice after culture.6?Optimizing the Conditions of Whole Cell Catalytic Synthesis of AQ: Optimizing the catalytic reaction system by exploring the whole cell catalytic conditions,such as temperature,pH,sugar supplementation,etc.the optimum catalytic conditions were obtained as follows: 100 mL-Glu and 125 mL-Ala,50 mM of glucose(added every three hours,five times),pH 9.0,30 C,AQ yield reached 65.6 mM and molar yield of L-Glu was 65.6%. |
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