Redox self-sufficient whole cell biotransformation for amination of alcohols

Klatte, Stephanie; Wendisch, Volker F.

doi:10.1016/j.bmc.2014.05.012

Cited by 50 publications

(45 citation statements)

References 34 publications

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“…2Engineering cofactor or co-substrate balance. a NAD(P)H regeneration systems formed via coupling with a regeneration reaction [56]; b redox self-sufficient amination via coupling with an alcohol dehydrogenase, l -alanine-dependent transaminase and l -alanine dehydrogenase [62, 63]; c redox self-sufficiency via a two-enzyme cascade for the hydrogen-borrowing amination of alcohols [54]; d reconstitution of TCA cycle using a DAOCS-catalysed reaction for 2-OG supply and regeneration [66]; e cofactor self-sufficient system established via a bridging mechanism (enzymes) to enable the simultaneous regeneration of cofactors and redox equivalent …”

Section: Whole-cell Biocatalyst Design Principlesmentioning

confidence: 99%

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Whole-cell biocatalysts by design

2017

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Whole-cell biocatalysts provide unique advantages and have been widely used for the efficient biosynthesis of value-added fine and bulk chemicals, as well as pharmaceutically active ingredients. What is more, advances in synthetic biology and metabolic engineering, together with the rapid development of molecular genetic tools, have brought about a renaissance of whole-cell biocatalysis. These rapid advancements mean that whole-cell biocatalysts can increasingly be rationally designed. Genes of heterologous enzymes or synthetic pathways are increasingly being introduced into microbial hosts, and depending on the complexity of the synthetic pathway or the target products, they can enable the production of value-added chemicals from cheap feedstock. Metabolic engineering and synthetic biology efforts aimed at optimizing the existing microbial cell factories concentrate on improving heterologous pathway flux, precursor supply, and cofactor balance, as well as other aspects of cellular metabolism, to enhance the efficiency of biocatalysts. In the present review, we take a critical look at recent developments in whole-cell biocatalysis, with an emphasis on strategies applied to designing and optimizing the organisms that are increasingly modified for efficient production of chemicals.

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Section: Whole-cell Biocatalyst Design Principlesmentioning

confidence: 99%

“…Recently, a self-sufficient redox system that utilizes the direct coupling of oxidizing and reducing enzymatic reactions has been developed [59, 62–64]. Thus, neither additional substrate nor another regenerating enzyme is required for this type of recycling reaction.…”

Section: Whole-cell Biocatalyst Design Principlesmentioning

confidence: 99%

Whole-cell biocatalysts by design

2017

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“…For the use of l -alanine as amine donor, a multitude of enzymatic in vitro co-product removal systems have previously been developed, e.g. based on the conversion of pyruvate back to l -alanine by alanine dehydrogenase (Aldh) [26, 51], or pyruvate to acetaldehyde and CO 2 by Pdc [52]. Yeast possess a number of enzymes that catalyse the conversion of pyruvate, e.g.…”

Section: Discussionmentioning

confidence: 99%

“…To increase the activity of PLP-dependent reactions, PLP is typically supplemented directly to the reaction solution thereby relieving potential limitations in availability [12, 25, 26]. An alternative to supplementation is the increase of intracellular levels by metabolic engineering of PLP biosynthetic pathways.…”

Section: Introductionmentioning

confidence: 99%

Improvement of whole-cell transamination with Saccharomyces cerevisiae using metabolic engineering and cell pre-adaptation

2017

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BackgroundWhole-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.ResultsHerein, 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.ConclusionsAltogether, 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 materialThe online version of this article (doi:10.1186/s12934-016-0615-3) contains supplementary material, which is available to authorized users.

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“…Thereby,a nA DH from Bacillus stearothermophilus was used for substrate oxidation, at ransaminase from Vibriof luvialis was applied for aldehyde-intermediate amination and an alanine dehydrogenase from B. subtilis was expressed for recycling NADH. [199] Connecting an enzyme to performt he desired reaction to ac ofactor recycling enzyme via al inker-domainr epresents another elegant solution to the problem. This was demonstrated for several BVMOs which were linked to at hermostable variant of phosphite dehydrogenase.…”

Section: Cofactor Recyclingmentioning

confidence: 99%