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Metabolic engineering of Pseudomonas putida for production of vanillylamine from lignin-derived substrates

Heitor Colombelli Manfrão-Netto, João ; Lund, Fredrik LU ; Muratovska, Nina LU ; Larsson, Elin M. ; Skorupa Parachin, Nádia and Carlquist, Magnus LU (2021) In Microbial Biotechnology 14(6). p.2448-2462
Abstract
Whole-cell bioconversion of technical lignins using Pseudomonas putida strains
overexpressing amine transaminases (ATAs) has the potential to become
an eco-efficient route to produce phenolic amines. Here, a novel cell
growth-based screening method to evaluate the in vivo activity of recombinant ATAs towards vanillylamine in P. putida KT2440 was developed. It allowed the identification of the native enzyme Pp-SpuC-II and ATA from Chromobacterium violaceum (Cv-ATA) as highly active towards vanillylamine in vivo. Overexpression of Pp-SpuC-II and Cv-ATA
in the strain GN442ΔPP_2426, previously engineered for reduced vanillin
assimilation, resulted in 94- and... (More)
Whole-cell bioconversion of technical lignins using Pseudomonas putida strains
overexpressing amine transaminases (ATAs) has the potential to become
an eco-efficient route to produce phenolic amines. Here, a novel cell
growth-based screening method to evaluate the in vivo activity of recombinant ATAs towards vanillylamine in P. putida KT2440 was developed. It allowed the identification of the native enzyme Pp-SpuC-II and ATA from Chromobacterium violaceum (Cv-ATA) as highly active towards vanillylamine in vivo. Overexpression of Pp-SpuC-II and Cv-ATA
in the strain GN442ΔPP_2426, previously engineered for reduced vanillin
assimilation, resulted in 94- and 92-fold increased specific
transaminase activity, respectively. Whole-cell bioconversion of
vanillin yielded 0.70 ± 0.20 mM and 0.92 ± 0.30 mM vanillylamine, for Pp-SpuC-II and Cv-ATA,
respectively. Still, amine production was limited by a substantial
re-assimilation of the product and formation of the by-products vanillic
acid and vanillyl alcohol. Concomitant overexpression of Cv-ATA and alanine dehydrogenase from Bacillus subtilis
increased the production of vanillylamine with ammonium as the only
nitrogen source and a reduction in the amount of amine product
re-assimilation. Identification and deletion of additional native genes
encoding oxidoreductases acting on vanillin are crucial engineering
targets for further improvement. (Less)
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author
; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Microbial Biotechnology
volume
14
issue
6
pages
15 pages
publisher
John Wiley & Sons Inc.
external identifiers
  • pmid:33533574
  • scopus:85100356086
ISSN
1751-7907
DOI
10.1111/1751-7915.13764
project
Exploration of Brazilian biodiversity for new biosynthetic pathways applied to lignin valorization
language
English
LU publication?
yes
id
f4493d8f-b033-4c4b-b0fa-f457de126ab4
date added to LUP
2022-02-07 09:18:05
date last changed
2023-01-24 23:06:13
@article{f4493d8f-b033-4c4b-b0fa-f457de126ab4,
  abstract     = {{Whole-cell bioconversion of technical lignins using <i>Pseudomonas putida strains</i><br>
 overexpressing amine transaminases (ATAs) has the potential to become <br>
an eco-efficient route to produce phenolic amines. Here, a novel cell <br>
growth-based screening method to evaluate the <i>in vivo</i> activity of recombinant ATAs towards vanillylamine in <i>P. putida</i> KT2440 was developed. It allowed the identification of the native enzyme Pp-SpuC-II and ATA from <i>Chromobacterium violaceum</i> (<i>Cv-ATA</i>) as highly active towards vanillylamine <i>in vivo</i>. Overexpression of <i>Pp-SpuC-II</i> and <i>Cv-ATA</i><br>
 in the strain GN442ΔPP_2426, previously engineered for reduced vanillin<br>
 assimilation, resulted in 94- and 92-fold increased specific <br>
transaminase activity, respectively. Whole-cell bioconversion of <br>
vanillin yielded 0.70 ± 0.20 mM and 0.92 ± 0.30 mM vanillylamine, for <i>Pp-SpuC-II</i> and <i>Cv-ATA</i>,<br>
 respectively. Still, amine production was limited by a substantial <br>
re-assimilation of the product and formation of the by-products vanillic<br>
 acid and vanillyl alcohol. Concomitant overexpression of <i>Cv-ATA</i> and alanine dehydrogenase from <i>Bacillus subtilis</i><br>
 increased the production of vanillylamine with ammonium as the only <br>
nitrogen source and a reduction in the amount of amine product <br>
re-assimilation. Identification and deletion of additional native genes <br>
encoding oxidoreductases acting on vanillin are crucial engineering <br>
targets for further improvement.}},
  author       = {{Heitor Colombelli Manfrão-Netto, João and Lund, Fredrik and Muratovska, Nina and Larsson, Elin M. and Skorupa Parachin, Nádia and Carlquist, Magnus}},
  issn         = {{1751-7907}},
  language     = {{eng}},
  month        = {{02}},
  number       = {{6}},
  pages        = {{2448--2462}},
  publisher    = {{John Wiley & Sons Inc.}},
  series       = {{Microbial Biotechnology}},
  title        = {{Metabolic engineering of <i>Pseudomonas putida</i> for production of vanillylamine from lignin-derived substrates}},
  url          = {{http://dx.doi.org/10.1111/1751-7915.13764}},
  doi          = {{10.1111/1751-7915.13764}},
  volume       = {{14}},
  year         = {{2021}},
}