High Efficient Synthesis Of L-alanyl-L-glutamine By Whole-cell Catalysis Of Recombinant Microorganism

L-Alanyl-L-glutamine(Ala-Gln)is a novel nutritional supplement to replace L-glutamine(Gln)with advantages in solubility,thermal stability and high decomposition rate,and has been widely used in fields of food,health products,and medicine.Ala-Gln can be directly synthesized from L-alanine methyl ester hydrochloride(AlaOMe)and Gln by?-amino acid ester acyl transferase(Aet).Microbial synthesis based on the expression of Aet is suitable for industrial production of Ala-Gln because of its cheap raw materials,few reaction steps,mild reaction conditions,little environmental pollution,and few by-products.However,problems such as long reaction time and low molar conversion rate in the Aet catalysis process limit its application in the industrial production of Ala-Gln.In this study,the whole-cell catalysts heterologously expressing SsAet derived from Sphingobacterium siyangensis were constructed in Escherichia coli and Saccharomyces cerevisiae to synthesize Ala-Gln,respectively,which solved the above problems by cells reuse and flocculation self-immobilization technology.This study finally laid a good foundation for the safe,clean and efficient industrial production of Ala-Gln.The main results were listed as follows.Firstly,high-performance recombinant E.coli expressing SsAet was constructed to efficiently synthesize Ala-Gln.BL21(DE3)-pET29a-SsAET(BPA)was constructed with the most commonly used BL21(DE3).It was found that BPA expressed a large number of inclusion bodies,which affected its catalytic activity.The solubility of the protein was improved by replacing the host that facilitated the formation of disulfide bonds,with Origami 2(DE3)-pET29a-SsAET(OPA)having the highest soluble SsAet and 1.4 times the catalytic activity that of BPA.Furthermore,the optimal induction condition for OPA(induction temperature 16?;induction OD620=0.25;induction time 12 h;IPTG concentration 0.6 mmol/L)and the optimal condition for whole-cell catalytic reaction(reaction temperature 25?;reaction pH 8.5;reaction solution was borate buffer;substrate molar ratio of AlaOMe/Gln=1/2)were obtained.Based on the optimum condition,using a high-performance OPA as the whole-cell catalyst for the production of Ala-Gln,the reaction completed within 5 minutes,achieved a conversion rate of up to 94%and a specific production intensity of 1.78 g/L·min-1·OD620-1.This result is the highest level reported in current publications.Simultaneously,OPA maintained a catalytic efficiency of more than 80%in 5 cycle batches with satisfactory reusability,realizing high-efficiency production of Ala-Gln.However,at the same cell concentration and reaction time,the catalytic efficiency of whole-cell was reduced by 21%compared with the lysate,which proved the existence of diffusion resistance.Secondly,recombinant S.cerevisiae displaying SsAet on the surface was constructed.To overcome substrate diffusion resistance and potential biosafety threat of the E.coli expression system,biosafe S.cerevisiae was used as the host,and SsAet was displayed on the surface of yeast cells by N-terminal and C-terminal anchoring.SsAet had been successfully immobilized on the cell wall by immunofluorescence.Considering the growth and catalytic activity,N-terminal anchoring was selected as the method for SsAet to be displayed on the surface of cell.Using codon optimization,we found that the yield of Ala-Gln by codon-optimized EBY100-pYD1-SsAETS(EPagaAs)was 1.7 times that of non-codon optimized strain at 20 min;the reaction endpoint was advanced from 40 to 30 min which means that the production efficiency was improved by 33%.However,EPagaAs had a longer reaction time and the conversion molar rate was only 58%of OPA.It is possible that the post-translational modifications in yeast expression system affect SsAet catalytic activity.Furthermore,the post-translational modification effect in the yeast expression system was identified.By codon optimization,GS115-pPIC9-SsAETP(GPAp)with a secreting SsAet was constructed using Pichia pastoris with low glycosylation as the expression host.The induction conditions of recombinant P.pastoris and the reaction conditions for the synthesis of Ala-Gln were optimized.The optimal culture condition(induction temperature 26?;induction time 3 d;methanol supplementation 1.5%)and the optimal reaction condition(reaction temperature 28?;reaction pH 8.5;reaction substrate AlaOMe;substrate molar ratio of AlaOMe/Gln=2/1)were obtained as the standard for future experiments.The conversion molar rate of GPAP was only 71%that of OPA under the optimum conditions,which confirmed the influence of protein post-modification on the catalytic activity of SsAet.Then,the recombinant strains with the intracellular and secretory expression of SsAet were constructed.It was found that the catalytic activity of intracellularly expressed SsAet was significantly higher than that of secreted expression.The highest molar conversion rate of KM71-pPIC3.5K-SsAETP1800(KP3.5KAP1800)with intracellularly expressed SsAet and removal of endogenous signal peptides was 80%,which was more than 90%that of OPA and 41%higher than that of the control strain.Intracellular expression SsAet and removal of endogenous signal peptides showed the significance in improving the catalytic activity of SsAet in the yeast expression system.The molecular weight of secreted SsAet increased was confirmed by SDS-PAGE and Western blotting.The presence of glycosylation in the secreted SsAet was proved by the removal of the deglycosylation enzyme.It was found that the glycosylation occurred on the 442-bit Asn residue of the secreted SsAet by LC-MS,and the change of the secondary structure of glycosylated modified SsAet were identified by CD.These results demonstrated that glycosylation modification is an important factor affecting SsAet catalytic activity.Finally,based on the above results,the self-immobilized recombinant S.cerevisiae was constructed to efficiently synthesize Ala-Gln.EBY100-pRS424-SsAETs1800(EP424AS1800)with the intracellular expression of SsAet and removal of endogenous signal peptide was constructed using a high copy number plasmid.By the optimization of the culture and reaction conditions,EP424As1800 achieved a conversion rate of up to 74%within 5 min,which was 83%that of OPA and 1.44 times that of the surface display recombinant EPagaAs.Besides,the reaction time was shortened by more than 80%,the production efficiency was increased by 5 times,and a specific production intensity was 1.28 g/L·min-1·OD600-1.EP424AS1800-FLO1 with millimeter-scale flocculent particles and negative influence on catalytic activity was obtained by introducing the flocculation gene FLO1,which realized self-immobilization in the reactor.After 10 cycles of catalysis,EP424AS1800-FLO1 maintained stable catalytic performance and achieved continuous and efficient production of Ala-Gln.The research in this study lays the foundation for safe,clean and efficient industrial production of Ala-Gln.