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

Abstract

Summary 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.

Renewable Vanillylamine Synthesis from Lignin-Derived Feedstocks

“…33,34 Previously, Manfrao-Netto et al explored the conversion of vanillin and ferulic acid to vanillylamine in P. putida using the combination of transaminase and L-alanine dehydrogenase (AlaDH), which consumes only ammonium and NADH. 35 The byproduct pyruvate was regenerated to L-alanine by AlaDH, thus resulting in the redox self-sufficient cascade possessing high atom efficiency. However, the yield of vanillylamine is extremely low (0.473 mM vanillylamine from 3.5 mM ferulic acid).…”

“…Unlike vanillin, vanillic acid has a more stable structure and often can be manufactured in large quantities. However, the biosynthesis of vanillylamine is mainly based on vanillin to date, , and there has been no report on the bioconversion of vanillic acid to vanillylamine. According to the literature, CAR from S. rotundus DSM44985 has a wide range of substrate specificity and can efficiently convert vanillic acid to vanillin.…”

Valorization of lignin components into gallate by integrated biological hydroxylation, O-demethylation, and aryl side-chain oxidation

“…In previous studies, lignin and lignin-derived aromatics were converted to microbial lipids (24), cis, cis-muconate (25)(26)(27)(28), polyhydroxyalkanoate (19), pyridine-dicarboxylic acids (29,30), vanillin (31), vanillylamine (32), itaconate (33), pyruvate, lactate (34), succinate (35), alkane, wax ester (36), etc. by native or modified microbial metabolic pathways.…”

Recombinant yeast for production of the pain receptor modulator nonivamide from vanillin

“…However, these have only been applied for whole-cell production of chiral amines by kinetic resolution of racemic amines (amine to ketone), since the reverse direction (ketone to amine) has been proven to be exceptionally difficult to achieve in vivo (Weber et al, 2014a;Weber et al, 2014b;Knudsen et al, 2016;Weber et al, 2017). This was though recently achieved for vanillylamine production in engineered Escherichia coli (Fu et al, 2021) and Pseudomonas putida (Manfrao-Netto et al, 2021), both expressing CvTA and alanine dehydrogenase from Bacillus subtilis (BsAlaDH) (Koszelewski et al, 2008). Key to success was the coexpression of CvTA and BsAlaDH in combination with appropriate reaction conditions in favour of amine formation.…”