Enhancing Biosynthesis Of L-DOPA By Metabolic Engineering And Enzyme Engineering Strategies

Levodopa（L-DOPA）is mainly used for the treatment of Parkinson’s disease.While chemical synthesis of L-DOPA has developed maturely,it is necessary for heavy metal.Biosynthesis of L-DOPA displayed attractive advantages,such as mild reaction condition,non-pollution,and high enantioselectivity.Presently,biosynthesis of L-DOPA is mainly catalyzed by tyrosine phenol lyase（TPL）with pyruvate,catechol as substrates.Pyruvate produced by fermentation technology has gained rapid development.Therefore,biosynthesis of L-DOPA bound with pyruvate fermentation is new green mode,which accords with sustainable development.This study mainly includes:expression optimization of TPL and the improvement of enzyme properties,optimization of the whole cells biotransformation,construction of the cofactor regeneration system and construction of the reaction kinetics at fermenter level.The main results are as follows:（1）Optimizing the expression of TPL and the whole cells biotransformation in shaking flask level.TPL that was derived from Citrobacter freundii,Erwinia herbicola,and Rhodobacter capsulatus,was expressed in Eescherichia coli,respectively.The enzyme activity of TPL from C.freundii showed the highest.Expression of TPL and whole cells biotransformation was optimized at flask level.And,the optimal initial concentration of substrates and the optimal feeding-batch strategy was determined.（2）Improving thermostability of TPL by site-directed mutation.Residues in mutation group were virtually mutated by Discovery Studio to predict key amino acids.Result showed that GLU313 was not only located near the activity pocket but was also associated with the N-terminal arm through aαhelix.E313W and E313M with obvious advantages were achieved through analysis and screening,which showed extended half-life,enhanced dissolving temperature and improved thermostability.Therefore,L-DOPA yield from E313W and E313M was up to 47.5 g/L and 62.1 g/L,exhibiting improvements of 107.3%and 174.8%relative to that of TPL,respectively.（3）Inducing the formation of active TPL aggregates by terminally attached functional peptides.The inclusion bodies of TPL almostly show no enzyme activity.By fusing functional peptides at the carboxyl end of TPL,the inclusion bodies of TPL-DLK6,TPL-ELP10,TPL-EAK16,TPL-18A and TPL-GFIL16 showed enzyme activity.TPL-DKL6,TPL-EAK16,TPL-18A,and TPL-GFIL16 displayed obviously improved thermostability with extended half-life.The strain expressing TPL-EAK16 displayed the highest L-DOPA yield,53.8 g/L,by whole cells biotransformation.（4）Constructing metabolic pathway of pyridoxal 5-phosphate（PLP）.Metabolic pathway of PLP from Bacillus subtilis 168 was introduced by co-expressing TPL and Pdx S/T.The ribose 5-phosphate was the precursor,and Pdx S/T was the key enzyme in the metabolic pathway of PLP.Based on the binding of PLP with riboswitch RNA,PLP metabolism level was regulated at translation level.As a result,L-DOPA yield of BL21-TPLST Ribo1,BL21 TPLST-Ribo2 was increased to 69.8 g/L,66.9 g/L by whole cells biotransformation,respectively.（5）Optimizing whole cells biotransformation at fermenter level and constructing kinetic model.Sodium pyruvate and catechol was maintained at optimum concentration range by continuous flow strategy.As a result,L-DOPA yield was up to 75.3 g/L.The key constants of the synthesis kinetics of L-DOPA were determined with double reciprocal treatment for substrate-initial reaction rate,such as,""^#=4.52 g/L,_%"^#=20.18 g/L,_%"^&=39.79 g/L,V_（）*=0.53 g/L/min.Pyruvate was produced by Candida glabrata 4H2 strain,which was used as substrate in whole cells biotransformation.As a result,the actual value of L-DOPA synthesis rate was 0.233g/L/min,0.251 g/L/min,respectively.The theoretical value was 0.275 g/L/min,0.256g/L/min,respectively.