Metabolic Engineering Of Escherichia Coli,Clostridium Tyrobutyricum For Phenyllactic Acid And N-butanol Production

Metabolic engineering is the practice of optimizing genetic and regulatory processes within cells to increase the cells' production of a certain substance.In this study ? constructed new biosynthesis pathways in E.coli and Clostridium tyrobutyricum for phenyllactic acid and n-butanol production,respectively.1.Phenyllactic acid(2-hydroxy-3-phenylpropanonic,PLA)is naturally distributed in honey and fermented foods.PLA exhibits broad spectrum inhibitor activity towards bacteria and fungi including food quality deterioration inducing pathogenic species.Lactic acid bacteria can produce PLA at very low efficiency from phenylalanine and tyrosine.The biosynthesis of PLA could be further improved.Characterization of a novel L-phenylalanine oxidase from Coprinopsis cinereusA novel L-phenylalanine oxidase gene from a species of mushroom Coprinopsis cinereus was cloned.With L-amino acid oxidase from Hebeloma cylindrosporum,which is the closest one,it shared only 30.6%sequence identity.This recombinant protein was expressed in Escherichia coli,purified and biochemically characterized.It contained 778 amino acids and was quite different compared with all previously studied enzymes.This enzyme exhibited highest specific activity of 6.04 U/mg towards L-phenylalanine and the optimal pH,temperature of the enzyme catalyzed reaction were 8.5 and 45?.The enzyme was stable up to 55? within pH range 7.0-9.5.It could oxidize L-phenylalanine to phenylpyruvic acid at high titer(8.1 g/L),conversion ratio(97.4%)and productivity(1.02 g/L h)within 8 h.The product phenylpyruvic acid could subsequently be catalyzed to produce phenyllactic acid.Coexpression of L-phenylalanine oxidase and L-lactate dehydrogenase in E.coli for phenyllactic acid biosynthesis from phenylalanineThe production of phenyllactic acid from phenylalanine by recombinant E.coli(ldh-lpox)overexpressing L-phenylalanine oxidase and L-lactate dehydrogenase was studied.Aromatic aminotransferase naturally in phenylalanine mechanism of lactic acid bacteria was replaced by L-phenylalanine oxidase avoiding the participation amino acceptor.The novel phenyllactic acid synthesis pathway was confirmed to be efficient and two different biocatalyst processes were carried out and optimized for phenyllactic acid production.In whole cell biosynthesis process at high cell density,E.coli(ldh-lpox)could produce 9.76 mM of phenyllactic with conversion ratio of 28.1%.Similarly,in the two-temperature-stage,comparable phenyllactic acid production of 8.85 mM was obtained under the optimal condition.2.n-Butanol is a promising advanced biofuel with superior fuel properties,including a 30%higher energy content and lower hygroscopicity and volatility compared to ethanol.In addition,as the conventional ABE fermentation feedstock usually accounts for more than 50%of the product cost,alternative low-cost feedstocks have been a focus of recent studies of ABE fermentation.Metabolic engineering of Clostridium tyrobutyricum expressing sucrose catabolic genes and adhE2 for n-butanol production from sugarcane juiceC.tyrobutyricum is a promising organism for butyrate and n-butanol production,but cannot grow on sucrose.Three genes(scrA,scrB,and scrK)involved in the sucrose catabolic pathway,along with an aldehyde/alcohol dehydrogenase gene(adhE2)were cloned from Clostridium acetobutylicum and introduced into C.tyrobutyricum(?ack)with acetate kinase(ack)knockout.In batch fermentation,the engineered strain Ct([ack)-pscrBAK produced 14.8-18.8 g/L butanol,with a high butanol/total solvent ratio of 0.94(w/w),from sucrose and sugarcane juice.Moreover,stable high butanol production with a high butanol yield of 0.25 g/g and productivity of 0.28 g/L·h was obtained in batch fermentation without using antibiotics for selection pressure,suggesting that Ct(Aack)-pscrBAK is genetically stable.Furthermore,sucrose utilization by Ct(?ack)-pscrBAK was not inhibited by glucose,which would usually cause carbon catabolite repression on solventogenic clostridia.Ct(Aack)-pscrBAK is thus advantageous for use in biobutanol production from sugarcane juice and other sucrose-rich feedstock.Butanol production from sucrose and sugarcane juice by Ct(Aack)-pscrBAK in a fibrous bed bioreactor using corn steep liquor as nitrogen sourceIn free cell fermentation,butanol production of 16 g/L at a yield of 0.31 g/g and productivity of 0.33 g/L·h was obtained from sucrose and yield of 0.24 g/g and productivity of 0.30 g/L·h from sugarcane juice containing sucrose,glucose and fructose.The fermentation was also studied in a fibrous bed bioreactor(FBB)operated in a repeated batch mode for 10 consecutive cycles in 10 days,achieving an average butanol yield of 0.21 g/g and productivity of 0.53 g/L h from sugarcane juice,demonstrating its long-term stability without applying the antibiotic selection pressure.