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Increased availability of NADH in metabolically engineered baker's yeast improves transaminase-oxidoreductase coupled asymmetric whole-cell bioconversion
- Source :
- Microbial Cell Factories
- Publication Year :
- 2015
-
Abstract
- 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.
- Subjects :
- 0106 biological sciences
0301 basic medicine
Saccharomyces cerevisiae
Bioengineering
Glycerolphosphate Dehydrogenase
01 natural sciences
Applied Microbiology and Biotechnology
Cofactor
Kinetic resolution
Metabolic engineering
03 medical and health sciences
Chiral alcohols
Oxidoreductase
010608 biotechnology
Phenethylamines
(S)-1-phenylethanol
Chiral amines
(R)-1-phenylethylamine
Benzyl Alcohols
Transaminases
Alcohol dehydrogenase
chemistry.chemical_classification
Co-factor regeneration
Whole-cell biocatalysis
biology
Research
Alcohol Dehydrogenase
Acetophenones
Stereoisomerism
biology.organism_classification
NAD
Yeast
030104 developmental biology
Glycerol-3-phosphate dehydrogenase
Glucose
chemistry
Biochemistry
Metabolic Engineering
Benzaldehydes
biology.protein
Biocatalysis
Metabolome
Oxidoreductases
Biotechnology
Subjects
Details
- ISSN :
- 14752859
- Volume :
- 15
- Database :
- OpenAIRE
- Journal :
- Microbial cell factories
- Accession number :
- edsair.doi.dedup.....a0edf2bca914ff3c7603356fb5c06394