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Background Saccharomyces 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. Results A 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. Conclusions Overall 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. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0430-x) contains supplementary material, which is available to authorized users.

Background One-pot multi-step biocatalysis is advantageous over step-by-step synthesis as it reduces the number of process operation units, leading to significant process intensification. Whole-cell biocatalysis with metabolically active cells is especially valuable since all enzymes can be co-expressed in the cell whose metabolism can be exploited for supply of co-substrates and co-factors. Results In this study, a heterologous enzymatic system consisting of ω-transaminase and ketone reductase was introduced in Saccharomyces cerevisiae, and evaluated for one-pot stereo-selective conversion of amines to alcohols. The system was applied for simultaneous kinetic resolution of racemic 1-phenylethylamine to (R)-1-phenylethylamine and reduction of the ketone intermediate to (R)-1-phenylethanol. Glucose was used as sole co-substrate for both the supply of amine acceptor and the regeneration of NADPH in the reduction step. Conclusions The whole-cell biocatalyst was shown to sustain transaminase-reductase-catalyzed enantioselective conversion of amines to alcohols with glucose as co-substrate. The transamination catalyzed by recombinant vanillin aminotransferase from Capsicum chinense proved to be the rate-limiting step as a three-fold increase in transaminase gene copy number led to a two-fold increased conversion. The (R)-selective NADPH-dependent alcohol dehydrogenase from Lactobacillus kefir proved to be efficient in catalyzing the reduction of the acetophenone generated in the transamination reaction.

Background Whole-cell biocatalysis based on metabolically active baker’s yeast with engineered transamination activity can be used to generate molecules carrying a chiral amine moiety. A prerequisite is though to express efficient ω-transaminases and to reach sufficient intracellular precursor levels. Results Herein, the efficiency of three different ω-transaminases originating from Capsicum chinense, Chromobacterium violaceum, and Ochrobactrum anthropi was compared for whole-cell catalyzed kinetic resolution of racemic 1-phenylethylamine to (R)-1-phenylethylamine. The gene from the most promising candidate, C. violaceum ω-transaminase (CV-TA), was expressed in a strain lacking pyruvate decarboxylase activity, which thereby accumulate the co-substrate pyruvate during glucose assimilation. However, the conversion increased only slightly under the applied reaction conditions. In parallel, the effect of increasing the intracellular pyridoxal-5′-phosphate (PLP) level by omission of thiamine during cultivation was investigated. It was found that without thiamine, PLP supplementation was redundant to keep high in vivo transamination activity. Furthermore, higher reaction rates were achieved using a strain containing several copies of CV-TA gene, highlighting the necessity to also increase the intracellular transaminase level. At last, this strain was also investigated for asymmetric whole-cell bioconversion of acetophenone to (S)-1-phenylethylamine using l-alanine as amine donor. Although functionality could be demonstrated, the activity was extremely low indicating that the native co-product removal system was unable to drive the reaction towards the amine under the applied reaction conditions. Conclusions Altogether, our results demonstrate that (R)-1-phenylethylamine with >99% ee can be obtained via kinetic resolution at concentrations above 25 mM racemic substrate with glucose as sole co-substrate when combining appropriate genetic and process engineering approaches. Furthermore, the engineered yeast strain with highest transaminase activity was also shown to be operational as whole-cell catalyst for the production of (S)-1-phenylethylamine via asymmetric transamination of acetophenone, albeit with very low conversion. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0615-3) contains supplementary material, which is available to authorized users.