Discovery Of Conjugated Polyketone Reductases For Asymmetricly Catalyzing The Synthesis Of D-(-)-Pantolactone

D-?-?-pantolactone?D-PL?is a key chiral intermediate for the synthesis of D-pantothenate calcium?vitamin B₅?,D-pantothenic acid and?R?-catechol.D-calcium pantothenate calcium and D-pantothenic acid are mainly used in the fields of medicine,food,feed and daily chemicals,and its demand is increasing continuously each year.At present,D-?-?-pantolactone is produced by hydrolysis kinetic resolution in the industry.First,the racemic panthionyl lactone is further esterified by an aldol condensation reaction of isobutyraldehyde and formaldehyde under an acidic condition with addition of hydrogen cyanide.The racemic pantolactone is further catalyzed by a hydrolase and racemized by a strong base to obtain D-?-?-pantolactone with a high optical purity.The main disadvantages of the traditional method are the use of highly toxic compound hydrogen cyanide and a large amount of strong acid and alkali,which are not consistent with the demands for development of of green chemical engineering.So far,the asymmetric reduction of prochiral carbonyl compounds to corresponding chiral alcohols by oxidoreductase is considered to be the most efficient synthetic method.Conjugated polyketone reductases?CPRs?asymmetrically reduce dihydro-4,4-dimethyl-2,3-furandione to D-?-?-pantolactone.The advantages of this approach compared with the tranditional hydrolysis are as follows.First,CPRs catalyzed the synthesis of D-?-?-pantolactone with a high catalytic stereoselectivity,enantiomeric excess?e.e.?>99.9%.Second,isobutyraldehyde and diethyl oxalate were used to synthesize intermediate,which avoided the use of the highly toxic hydrocyanic acid and strong acid and alkali.However,the reported conjugated polyketone reductase genes are relatively scarce.So far,only CPR-C1 and CPR-2genes from Candida parapsilosis IFO 0708 have been reported to catalyze the synthesis of D-?-?-pantolactone.In addition,the expression levels of CPR-C1 and CPR-C2 were lowin E.coli,which directly lead to the low catalytic efficiency of recombinant cells.Conjugated polyketone reductase?CPR?belongs to nicotinamide adenine dinucleotide phosphate?NADPH?-dependented aldo-keto reductase.In this work,we developed a strategy to discove novel CPR gene from GenBank database by gene miningusing CPR-C1 and CPR-C2 as query sequences.Seven putative CPR genes from different microorganisms were cloned and functionally expressedin Eschericha coli.Then we screened target CPR based on the catalytic activity and stereoselectivity.The putative CPR gene from Candida orthopsilosis Co 90-125?CorCPR?exhibited high catalytic activity and excellent stereoselectivity.Then it was cloned and functionallyexpressedinEscherichiacoliBL21?DE3?.Using Dihydro-4,4-dimethyl-2,3-furandione as substrate,the catalytic activity and enantiomeric excess of recombinant CorCPR were 49 U/mg protein and e.e.>99%,respectively.At the same time,the enzymatic properties of the recombinant CorCPR were systematically characterized,the optimum pH and temperature of recombinant CorCPR were 6.0-7.0 and 40?,respectively.The recombinant Cor CPR exhibited high thermal stability,and the half-lives(?_1/2)of 40,45 and 50°C were 8.0 h,3.7 h and 52 min,respectively.Recombinant CorCPR exhibited the highest activity and stereoselectivity toward KPL.The K_m and V_max toward KPL were 1.3 mM and 227.3?mol/min/mg protein,respectively.Other?-ketoesters were not the optimal substrates of CorCPR due to the relatively low activities and low stereoselectivities.In addition,CorCPR did not catalyze?-ketoesters to corresponding alcohols.Moreover,the synthetic process of D-pantolactone was studied suing recombinant CorCPR.We constructed recombinant E.coli cell harboring expression plasmid pACYCDuet-CorCPR-GDH,and selected glucose dehydrogenase?GDH?from Bacillus subtilis 168 for the regeneration of coenzyme NADPH.When the resting E.coli cells harboring pACYCDuet-CorCPR-GDH were used for the synthesis of D-?-?-pantolactone,the optimum pH and temperature of the catalyticreaction were6.5 and 35°C,respectively.The optimum molar ratio of glucose to substrate was1.25:1,and extra coenzyme was not added in the reaction mixture.Meanwhile,we developed a continuous catalytic process to avoid the spontaneous hydrolysis of substrate ketopantoyl lactone,1 M substrate KPL and 1.25 M glucose were concentrated in 5 mL buffer?pH 2.5?and continuously feeded into the reaction mixture at a flow rate of 11?mol·min^-1 and 14?mol·min^-1,respectively.The final product concentration reached 379 mM in the reaction mixture.Ketopantoyl lactone was reduced to?R?-PL with 95%conversion and>99%enantiomeric excess within7.5 h,and the space time yield was 157 g·L^-1·d^-1.