Ehanced Production Of Recombinant Formate Dehydrogenase With Pseudo-exponential Fed-batch Cultivation

Formate dehydrogenase(FDH) is known as one of the most promising catalyst for in situ regeneration ofβ-nicotinamide adenine dinucleotide(NADH).FDHs are mainly NAD⁺-dependent in aerobic organisms,which catalyze oxidation of formate to carbon dioxide with commitment reduction of NAD⁺ to NADH.NADH regeneration using FDH can be widely applied in asymmetric reduction of prochiral carbonyl compounds with alcohol dehydrogenases (ADH),a promising route for synthesis of valuable optically active alcohols,amino acids, hydroxyl acids or esters.However,large-scale production of FDH is still a tough task at present, because acetate overflow in aerobically grown cultures of E.coli is a major problem in high cell density cultivation for large-scale production of FDH.The objective of this work is to develop an efficient high cell density cultivation process with pseudo-exponential fed-batch strategy,focused on reducing the formation of acetate and overflow metabolism by optimizing the fed-batch strategy,and finally grew recombinant E.coli to high cell densities for enhanced production of FDH intracellularly,saving time and energy cost.The efficiency of in situ NADH regeneration by FDH was evaluated by reducing 4-Chloro acetoacetate ethyl(COBE) to(R)-4-chloro-3-hydroxy ethyl butyrate(CHBE) when FDH is coupled with ADH from Rhodococcus erythropolis ATCC 17896.Firstly,the glucose feeding rate was stepwisely changed every 2 hours to simulate the exponential feeding strategy,controlling the specific growth rate at the predetermined set point ofμ_set.Then the apparent yield coefficient Y_X/S was investigated and set at 0.3 g DCW/g glucose cultivating E.coli BL21(DE3)/pET 28a(+)-fdh.Based on the preliminary results,induction time and fed-batch strategy in the pre-induction stage were optimized for fermentation of recombinant E.coli in completely defined medium.And pseudo-exponential feeding strategy under three different specific growth rates in post-induction stage was further investigated to reduce acetate accumulation,prolong the FDH production period,and most importantly to maximize FDH production and intracellular FDH activity.This three-step cultivation was operated as follows: Pseudo-exponential fed-batch fermentation controlled at specific growth rate of 0.05 h^-1 was employed in pre-induction stage after batch fermentation.Until the biomass reached 11.0 g DCW/L(with OD₆₀₀ around 25),induction was carded out by adding 0.4 mM IPTG and the temperature was reduced to 28℃.The specific growth rate in the subsequent post-induction stage was set at 0.08 h^-1.pH was maintained at 7.0 during the whole fermentation.The biomass attained was 41.20 g DCW/L(OD₆₀₀ 114.8).The maximum FDH production and intracellular FDH activity,was 26.24×10³ U/L and 636.9 U/g DCW,respectively.The FDH production abtained in this three-step way was enhanced by 1.17 times than the earlier result got from two-step cultivation with batch and pH-stat fed-batch.The results showed that low set-value of specific growth rate led to lower glucose feeding rate and less overflow metabolism,thus delayed the formation of acetate,and finally increased the FDH production significantly.And it also helps to save time and energer greatly.Recombinant E.coli BL21(DE3)/pET 28a(+)-adh was constructed with ADH gene from Rhodococcus erythropolis ATCC 17896,and the ADH expression condition was optimized.Then COBE was reduced to produce(R)-CHBE by ADH coupled with in situ NADH regeneration catalyzed by FDH.15 mM COBE was reduced completely in less than 2 hours.And the productivity of(R)-CHBE was 7.125 mol/(L·h),the enantio excess was 89(±1.4)%,with the total turnover number(TTN) at 150 mol/mol.The successful application proved FDH produced in our lab was capable and applicable in efficient NADH regeneration.FDH is a promising NADH regenerator for synthesis of high value products.