Construction Of Genetically Engineered Strain For Biosynthesis Of Beta-Alanine

?-alanine is an important pharmaceutical and chemical raw material,which has been widely applied to food,medical,environmental and many other industrial fields.Previous research has been verified that L-aspartate-?-decarboxylase(PanD)could convert L-aspartate to ?-alanine by specifically removing ?-carboxyl group from L-aspartate.In order to producing ?-alanine from L-aspartate,panD gene from Bacillus subtilis was cloned and heterologously expressed in E.coli BL21(DE3)to obtain transformed PanD-expressing E.coli strains in this work.Then,several optimizing strategies were carried out for increasing the expression level of PanD and cell density,which led to improved production of ?-alanine.For vector constructions,p ET-22b(+),p ET-29a(+)and p ET-30a(+)were used as backbones for producing panD-expressing vectors,p ET-22b(+)-panD,p ET-29a(+)-panD,and p ET-30a(+)-panD.The obtained three panD-expressing vectors were then transformed to E.coli BL21(DE3)for examining the efficiency of PanD productivity.Results showed that all three transformants could produce active PanD and E.coli strains transformed with p ET-22b(+)-panD and p ET-30a(+)-panD were selected for further investigation according to their better PanD catalytic activity.In order to increase the expression level of PanD in E.coli,codon optimization of B.subtilis panD sequence was performed to produce two new E.coli transformants E.coli BL21(DE3)/p ET-22b(+)-panDo(22b-1)and E.coli BL21(DE3)/p ET-30a(+)-panDo(30a-1).After testing PanD expression levels and catalytic efficiencies with these two E.coli strains,it's found that both PanD expression level and catalytic efficiency in strain 30a-1 were significantly increased,which was leading to 2-fold promotion of ?-alanine productivity in comparison with that in E.coli transformant without panD codon optimization.Meanwhile,no obvious variation of ?-alanine productivity was observed with or without panD codon optimization in strain 22b-1.Strain 30a-1 was then selected for optimizing ?-alanine production by applying auto induction medium for increasing cell density and ?-alanine productivity.Results showed that cell density was increased about 2.2 times higher than that growing in IPTG-inducing medium,while ?-alanine production was increased to 140.82 mmol/L,which was 2.5 times higher than that by using IPTG-inducing medium.By using auto induction medium,the enzyme activity of PanD could reach 20.57 U/m L in strain 30a-1.In summary,three different expression vectors were used as backbones for constructing panD-expressing vectors to produce transformed E.coli BL21(DE3)strains.Based on B.subtilis panD codon optimization followed by examination of enzyme activity and ?-alanine productivity,strain 30a-1 was selected for further possible industrial application of producing?-alanine.