Increased availability of NADH in metabolically engineered baker’s yeast improves transaminase-oxidoreductase coupled asymmetric whole-cell bioconversion

Microbial Cell Factories

Research

Cecilia Hägglöf¹  Magnus Carlquist¹  Nora Weber²  Jan Dines Knudsen³ 

[1] Division of Applied Microbiology, Department of Chemistry, Faculty of Engineering, Lund University, PO Box 124, 221 00, Lund, Sweden;Division of Applied Microbiology, Department of Chemistry, Faculty of Engineering, Lund University, PO Box 124, 221 00, Lund, Sweden;Evolva, Duggingerstrasse 23, 4153, Reinach, Switzerland;Division of Applied Microbiology, Department of Chemistry, Faculty of Engineering, Lund University, PO Box 124, 221 00, Lund, Sweden;The Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza 4, 20126, Milano (MI), Italy;

关键词: Whole-cell biocatalysis;    Co-factor regeneration;    Chiral amines;    Chiral alcohols;    Glycerol-3-phosphate dehydrogenase;    Kinetic resolution;    R;    S;   

DOI  :  10.1186/s12934-016-0430-x

 received in 2015-10-29, accepted in 2016-01-25,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundSaccharomyces cerevisiae can be engineered to perform a multitude of different chemical reactions that are not programmed in its original genetic code. It has a large potential to function as whole-cell biocatalyst for one-pot multistep synthesis of various organic molecules, and it may thus serve as a powerful alternative or complement to traditional organic synthetic routes for new chemical entities (NCEs). However, although the selectivity in many cases is high, the catalytic activity is often low which results in low space-time-yields. In the case for NADH-dependent heterologous reductive reactions, a possible constraint is the availability of cytosolic NADH, which may be limited due to competition with native oxidative enzymes that act to maintain redox homeostasis. In this study, the effect of increasing the availability of cytosolic NADH on the catalytic activity of engineered yeast for transamination-reduction coupled asymmetric one-pot conversion was investigated.ResultsA series of active whole-cell biocatalysts were constructed by over-expressing the (S)-selective ω-transaminase (VAMT) from Capsicum chinense together with the NADH-dependent (S)-selective alcohol dehydrogenase (SADH) originating from Rhodococcus erythropolis in strains with or without deletion of glycerol-3-phosphate dehydrogenases 1 and 2 (GPD1 and GPD2). The yeast strains were evaluated as catalysts for simultaneous: (a) kinetic resolution of the racemic mixture to (R)-1-phenylethylamine, and (b) reduction of the produced acetophenone to (S)-1-phenylethanol. For the gpd1Δgpd2Δ strain, cell metabolism was effectively used for the supply of both amine acceptors and the co-factor pyridoxal-5′-phosphate (PLP) for the ω-transaminase, as well as for regenerating NADH for the reduction. In contrast, there was nearly no formation of (S)-1-phenylethanol when using the control strain with intact GPDs and over-expressing the VAMT-SADH coupling. It was found that a gpd1Δgpd2Δ strain over-expressing SADH had a 3-fold higher reduction rate and a 3-fold lower glucose requirement than the strain with intact GPDs over-expressing SADH.ConclusionsOverall the results demonstrate that the deletion of the GPD1 and GPD2 genes significantly increases activity of the whole-cell biocatalyst, and at the same time reduces the co-substrate demand in a process configuration where only yeast and sugar is added to drive the reactions, i.e. without addition of external co-factors or prosthetic groups.

【 授权许可】

CC BY   
© Knudsen et al. 2016