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

Manfrão‐Netto, João Heitor Colombelli and Lund, Fredrik and Muratovska, Nina and Larsson, Elin M. and Parachin, Nádia Skorupa and Carlquist, Magnus (2021) Metabolic engineering of Pseudomonas putida for production of vanillylamine from lignin‐derived substrates. Microbial Biotechnology . ISSN 1751-7915. (In Press) https://resolver.caltech.edu/CaltechAUTHORS:20210208-103919031

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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 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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1111/1751-7915.13764DOIArticle
ORCID:
AuthorORCID
Manfrão‐Netto, João Heitor Colombelli0000-0001-5600-0219
Larsson, Elin M.0000-0003-1341-5937
Additional Information:© 2021 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Version of Record online: 03 February 2021; Manuscript accepted: 20 January 2021; Manuscript received: 14 October 2020. The authors would like to thank Javier García‐Hidalgo (Applied Microbiology, Lund university) for kindly providing the P. putida strain GN442ΔPP_2426, and Esteban Martínez‐García and Tomás Aparicio from the National Center for Biotechnology (CSIC, Madrid, Spain) for kindly providing the plasmid pSEVA424. This study was financially supported by the Program for Institutional Internationalization from the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES/PrInt) – Project number: 88887.364008/2019-00, and Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES/MEC) – Grant Number: 88881.155712/2017-01 and STINT (The Swedish Foundation for International Cooperation in Research and Higher Education) Grant number: BR2017-7077. The authors declare that they have no conflict of interest. Author contributions: J.H.C.M.N. contributed to the design of the study, performed the bioinformatics analysis, design and performed the experiments, analysed the data and drafted the manuscript. F.L. and N.M. helped to perform the bioconversions experiments, the growth curves and helped with the data interpretation and write the paper. N.M. developed the HPLC setup used and helped with the analytical analyses, the data interpretation and to write the Experimental Procedures section. E.M.L. performed the growth curves in microplates and prepared the parameter fitting for the growth curves, helped to perform the bioconversions experiments and write the paper. N.S.P. and MC revised and helped to write the manuscript. MC conceived and designed the study and participated in the experimental design and data interpretation. All authors read and approved the final manuscript.
Funders:
Funding AgencyGrant Number
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)88887.364008/2019‐00
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)88881.155712/2017‐01
Swedish Foundation for International Cooperation in Research and Higher EducationBR2017‐7077
Record Number:CaltechAUTHORS:20210208-103919031
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210208-103919031
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:107947
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:08 Feb 2021 19:05
Last Modified:08 Feb 2021 19:05

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