Study On ?-Alanine Production From Fumaric Acid Using A Dual-enzyme Cascade

?-Alanine,which is known as the only?-amino acid occurring in nature,is the precursor for the synthesis of several vital functional compounds,such as inositol and pantothenic acid.As a result,it is widely used in medicine,food and many other areas.Compared with conventional chemical methods,the enzymatic conversion using L-aspartic acid as the substrate is in favor due to its mild reaction and high conversions.Unfortunately,serious mechanism-based inactivation and high cost of L-aspartic acid have limited its large-scale preparation.In the present study,we designed and constructed a dual-enzyme cascade consisting of AspA and PanD for one-pot transformation of cheap fumaric acid to?-alanine.The catalytic stability of PanD was improved by protein engineering.Furthermore,the expression level of AspA and PanD were fine-tuned.The major results are listed as follows:?1?Cascade reaction design and in vitro reconstruction of?-alanine biosynthesis.First,Laspartate-?-decarboxylase from Bacillus subtilis?BsPanD?and L-aspartase from Escherichia coli?EcAspA?were chosen based on the measurements of specific activity.Then,the synthesis of the target product?-alanine through in vitro cascade reaction was confirmed by mass spectrometry.Furthermore,serious mechanism-based inactivation of PanD,which resulted in poor catalytic stability,was identified by in vivo conversion experiments.Thus,it was identified as the rate-limiting enzyme in this cascade pathway.?2?Engineering of BsPanD to relieve mechanism-based inactivation.First,based on the inactivation pathway,it was speculated that the catalytic stability could be improved by increasing the incorrect protonation distance.Thus,a combinatorial strategy of conformation dynamics and structural alignment was applied,resulting in two positive mutation sites I46V and K104S.Additionally,C-terminal deletion method as well as the literature mutant site I88M were introduced,eventually generating an optimal variant Q5(BsPanD^{I46V/I88M/K104S/I126*}).The conversion experiments for Q5 in shake flask showed a?-alanine titer of 98.0 g·L^-11 in conversions with L-aspartic acid as substrate,which was 158%higher than that of Q0.Kinetic parameters of the BsPanD variants were determined,which were consistent with conversion experiments.Compared to Q0,the catalytic half-life in vivo conversions with whole-cells and in vitro reactions with purified enzymes of variant Q5 were increased by 248%and 164%,respectively.The values of TTN also increased by 152%,which was in line with the catalytic half-life.Molecular dynamics?MD?simulations,showing that the reasons for the improved catalytic stability were the prolonged incorrect protonation distance?d1??4.9?in Q0 vs.5.5?in Q5?,which increased difficulty of inactivation to occur,and the decreased flexibility of residues in identified flexible regions,thereby enhancing the probability of correct protonation.?3?One-pot production of?-alanine by assembling AspA and PanD in vivo.The BsPanD variant Q5 and EcAspA were co-expressed in one plasmid resulting in strain pBsPA.Production of target product?-alanine was confirmed by whole-cell conversions of pBsPA.However,the imbalance of two reactions was caused by significant difference in in vivo enzyme activity between AspA and PanD?26:1?,resulting in unexpected and excessive accumulation of L-aspartic acid.The in vitro conversions for different activity ratios of AspA to PanD suggested that ratios of 1.0:1–1.5:1 was the optimal.To achieve this optimal ratio,weak RBS was screened and used to decrease the expression of AspA,and the strategy of gene duplication was used to increase the expression of PanD,resulting in strain pBs3PA-1.The whole-cell activity ratio of AspA to PanD decreased to 1.3:1.Furthermore,the conditions for catalysts preparation and conversion system were optimized with the strain pBs3PA-1 in the 5-L fermenter with fumaric acid as the substrate.The optimal catalysts preparation conditions:the inoculation amount of fermentation culture was 5%,5 g·L^-11 lactose was added into the culture when the OD₆₀₀00 of cells reaching 3,and enzymes were induced at 25^oC for 14h.The optimal conversion conditions:37 ^oC,pH 6.5,1 g·L^-11 triton and 20 g·L^-11 whole-cell catalysts were added into the reaction system.Finally,the conversion system was tested in a15-L scale fermenter under optimal conditions,yielding 118.6 g·L^-1?-alanine,with a product/catalyst ratio of 5.9 g·g^-1and a conversion>99%.