Enhancement of the NAD(P)(H) Pool in Saccharomyces cerevisiae

Abstract

Asymmetric biosyntheses allow for an efficient production of chiral building blocks. The application of whole cells as biocatalysts for asymmetric syntheses is advantageous because they already contain the essential coenzymes NAD(H) or NADP(H), which additionally can be regenerated in the cells. Unfortunately, reduced catalytic activity compared to the oxidoreductase activity is observed in many cases during whole‐cell biotransformation. This may be caused by low intracellular coenzyme pool sizes and/or a decline in intracellular coenzyme concentrations. To enhance the intracellular coenzyme pool sizes, the effects of the precursor metabolites adenine and nicotinic acid on the intracellular accumulation of NAD(H) and NADP(H) were studied in Saccharomyces cerevisiae. Based on the results of simple batch experiments with different precursor additions, fed‐batch processes for the production of yeast cells with enhanced NAD(H) or enhanced NADP(H) pool sizes were developed. Supplementation of the feed medium with 95 mM adenine and 9.5 mM nicotinic acid resulted in an increase of the intracellular NAD(H) concentration by a factor of 10 at the end of the fed‐batch process compared to the reference process. The final NAD(H) concentration remains unchanged if the feed medium was solely supplemented with 95 mM adenine, but intracellular NADP(H) was increased by a factor of 4. The effects of NADP(H) pool sizes on the asymmetric reduction of ethyl‐4‐chloro acetoacetate (CAAE) to the corresponding (S)‐4‐chloro‐3‐hydroxybutanoate (S‐CHBE) was evaluated with S. cerevisiae FasB His6 as an example. An intracellular threshold concentration above 0.07 mM NADP(H) was sufficient to increase the biocatalytic S‐CHBE productivity by 25 % compared to lower intracellular NADP(H) concentrations.

Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD + -Auxotrophic Mutant

“…It has been demonstrated that about 20% total cellular NAD(H) in wild-type cells is freely accessible and the (4). In several studies, the cellular cofactor concentration has been increased through manipulating the NAD ϩ biosynthetic pathway (12,16) or feeding permeabilized cells (18,36,37). The NAD(H) level was increased up to 6-fold previously (12).…”

Improvement of NADPH-dependent P450-mediated biotransformation of 7α,15α-diOH-DHEA from DHEA by a dual cosubstrate-coupled system

“…Both nicotinic acid and glucose showed beneficial effects on DHEA biotransformation, and the pathways for NADPH synthesis by these two cosubstrates were different [15,23]. Therefore, the dual cosubstrate combination of nicotinic acid and glucose was investigated to further improve the 7α,15α-diOH-DHEA molar conversion.…”

“…Nicotinic acid, nicotinamide and tryptophane can synthesize NADPH through related metabolic pathways in eukaryotic cells [15]. If the intracellular NADPH/NADP ratio and 7α,15α-diOH-DHEA molar conversion directly increased by the addition of coenzyme precursors, it could demonstrate that the limiting factor during DHEA hydroxylation was the low NADPH/NADP ratio from another perspective.…”

Enhanced direct fermentation of cassava to butanol by Clostridium species strain BOH3 in cofactor-mediated medium

“…Consequently, a lack or inefficient regeneration of the cofactor NADH or NADPH would shutdown the butanol dehydrogenase activity. Cofactor manipulations can potentially become a powerful tool for improvement of overall process yield and productivity [ 18 , 19 ]. Therefore, a feasible way to improve the economic efficacy of butanol production is to maximize cassava hydrolysis and the butanol yield in the cassava-based direct ABE fermentation.…”