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

WEBER, NORA LU ; Gorwa-Grauslund, Marie-Francoise LU and Carlquist, Magnus LU (2017) In Microbial Cell Factories 16(1).
Abstract

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... (More)

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.

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author
organization
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type
Contribution to journal
publication status
published
subject
keywords
Amine transaminase, Chiral amine, Co-substrate, Pyridoxal-5'-phosphate, Pyruvate decarboxylase, Whole-cell bioconversion, Yeast
in
Microbial Cell Factories
volume
16
issue
1
pages
12 pages
publisher
BioMed Central
external identifiers
  • scopus:85010214325
  • wos:000391322400003
ISSN
1475-2859
DOI
10.1186/s12934-016-0615-3
language
English
LU publication?
yes
id
62927119-5065-4936-8645-eb904cbf35d7
date added to LUP
2017-02-02 12:40:37
date last changed
2018-07-01 04:42:34
@article{62927119-5065-4936-8645-eb904cbf35d7,
  abstract     = {<p>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 &gt;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.</p>},
  articleno    = {3},
  author       = {WEBER, NORA and Gorwa-Grauslund, Marie-Francoise and Carlquist, Magnus},
  issn         = {1475-2859},
  keyword      = {Amine transaminase,Chiral amine,Co-substrate,Pyridoxal-5'-phosphate,Pyruvate decarboxylase,Whole-cell bioconversion,Yeast},
  language     = {eng},
  month        = {01},
  number       = {1},
  pages        = {12},
  publisher    = {BioMed Central},
  series       = {Microbial Cell Factories},
  title        = {Improvement of whole-cell transamination with Saccharomyces cerevisiae using metabolic engineering and cell pre-adaptation},
  url          = {http://dx.doi.org/10.1186/s12934-016-0615-3},
  volume       = {16},
  year         = {2017},
}