Dataset Information

Vanillin Production in Pseudomonas: Whole-Genome Sequencing of Pseudomonas sp. Strain 9.1 and Reannotation of Pseudomonas putida CalA as a Vanillin Reductase.

ABSTRACT: Microbial degradation of lignin and its related aromatic compounds has great potential for the sustainable production of chemicals and bioremediation of contaminated soils. We previously isolated Pseudomonas sp. strain 9.1 from historical waste deposits (forming so-called fiber banks) released from pulp and paper mills along the Baltic Sea coast. The strain accumulated vanillyl alcohol during growth on vanillin, and while reported in other microbes, this phenotype is less common in wild-type pseudomonads. As the reduction of vanillin to vanillyl alcohol is an undesired trait in Pseudomonas strains engineered to accumulate vanillin, connecting the strain 9.1 phenotype with a genotype would increase the fundamental understanding and genetic engineering potential of microbial vanillin metabolism. The genome of Pseudomonas sp. 9.1 was sequenced and assembled. Annotation identified oxidoreductases with homology to Saccharomyces cerevisiae alcohol dehydrogenase ScADH6p, known to reduce vanillin to vanillyl alcohol, in both the 9.1 genome and the model strain Pseudomonas putida KT2440. Recombinant expression of the Pseudomonas sp. 9.1 FEZ21_09870 and P. putida KT2440 PP_2426 (calA) genes in Escherichia coli revealed that these open reading frames encode aldehyde reductases that convert vanillin to vanillyl alcohol, and that P. putida KT2440 PP_3839 encodes a coniferyl alcohol dehydrogenase that oxidizes coniferyl alcohol to coniferyl aldehyde (i.e., the function previously assigned to calA). The deletion of PP_2426 in P. putida GN442 engineered to accumulate vanillin resulted in a decrease in by-product (vanillyl alcohol) yield from 17% to ?1%. Based on these results, we propose the reannotation of PP_2426 and FEZ21_09870 as areA and PP_3839 as calA-II IMPORTANCE Valorization of lignocellulose (nonedible plant matter) is of key interest for the sustainable production of chemicals from renewable resources. Lignin, one of the main constituents of lignocellulose, is a heterogeneous aromatic biopolymer that can be chemically depolymerized into a heterogeneous mixture of aromatic building blocks; those can be further converted by certain microbes into value-added aromatic chemicals, e.g., the flavoring agent vanillin. We previously isolated a Pseudomonas sp. strain with the (for the genus) unusual trait of vanillyl alcohol production during growth on vanillin. Whole-genome sequencing of the isolate led to the identification of a vanillin reductase candidate gene whose deletion in a recombinant vanillin-accumulating P. putida strain almost completely alleviated the undesired vanillyl alcohol by-product yield. These results represent an important step toward biotechnological production of vanillin from lignin using bacterial cell factories.

SUBMITTER: Garcia-Hidalgo J

PROVIDER: S-EPMC7054097 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Vanillin Production in Pseudomonas: Whole-Genome Sequencing of Pseudomonas sp. Strain 9.1 and Reannotation of Pseudomonas putida CalA as a Vanillin Reductase.

García-Hidalgo Javier J Brink Daniel P DP Ravi Krithika K Paul Catherine J CJ Lidén Gunnar G Gorwa-Grauslund Marie F MF

Applied and environmental microbiology 20200302 6

Microbial degradation of lignin and its related aromatic compounds has great potential for the sustainable production of chemicals and bioremediation of contaminated soils. We previously isolated Pseudomonas sp. strain 9.1 from historical waste deposits (forming so-called fiber banks) released from pulp and paper mills along the Baltic Sea coast. The strain accumulated vanillyl alcohol during growth on vanillin, and while reported in other microbes, this phenotype is less common in wild-t ...[more]

PMID: 31924622

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Project description:The gene loci vdh, vanA, and vanB, which are involved in the bioconversion of vanillin to protocatechuate by Pseudomonas sp. strain HR199 (DSM 7063), were identified as the structural genes of a novel vanillin dehydrogenase (vdh) and the two subunits of a vanillate demethylase (vanA and vanB), respectively. These genes were localized on an EcoRI fragment (E230), which was cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The vdh gene was identified on a subfragment (HE35) of E230, and the vanA and vanB genes were localized on a different subfragment (H110) of E230. The nucleotide sequences of fragment HE35 and part of fragment H110 were determined, revealing open reading frames of 1062, 951, and 1446 bp, representing vanA, vanB, and vdh, respectively. The vdh gene was organized in one operon together with a fourth open reading frame (ORF2), of 735 bp, which was located upstream of vdh. The deduced amino acid sequences of vanA and vanB exhibited 78.8 and 62.1% amino acid identity, respectively, to the corresponding gene products from Pseudomonas sp. strain ATCC 19151 (F. Brunel and J. Davison, J. Bacteriol. 170:4924-4930, 1988). The deduced amino acid sequence of the vdh gene exhibited up to 35.3% amino acid identity to aldehyde dehydrogenases from different sources. The deduced amino acid sequence of ORF2 exhibited up to 28.4% amino acid identity to those of enoyl coenzyme A hydratases. Escherichia coli strains harboring fragment E230 cloned in pBluescript SK- converted vanillin to protocatechuate via vanillate, indicating the functional expression of vdh, vanA, and vanB in E. coli. High expression of vdh in E. coli was achieved with HE35 cloned in pBluescript SK-. The resulting recombinant strains converted vanillin to vanillate at a rate of up to 0.3 micromol per min per ml of culture. Transfer of vanA, vanB, and vdh to Alcaligenes eutrophus and to different Pseudomonas strains, which were unable to utilize vanillin or vanillate as carbon sources, respectively, conferred the ability to grow on these substrates to these bacteria.

Project description:BackgroundAlthough a transition toward sustainable production of chemicals is needed, the physiochemical properties of certain biochemicals such as biosurfactants make them challenging to produce in conventional bioreactor systems. Alternative production platforms such as surface-attached biofilm populations could potentially overcome these challenges. Rhamnolipids are a group of biosurfactants highly relevant for industrial applications. However, they are mainly produced by the opportunistic pathogen Pseudomonas aeruginosa using hydrophobic substrates such as plant oils. As the biosynthesis is tightly regulated in P. aeruginosa a heterologous production of rhamnolipids in a safe organism can relive the production from many of these limitations and alternative substrates could be used.ResultsIn the present study, heterologous production of biosurfactants was investigated using rhamnolipids as the model compound in biofilm encased Pseudomonas putida KT2440. The rhlAB operon from P. aeruginosa was introduced into P. putida to produce mono-rhamnolipids. A synthetic promoter library was used in order to bypass the normal regulation of rhamnolipid synthesis and to provide varying expression levels of the rhlAB operon resulting in different levels of rhamnolipid production. Biosynthesis of rhamnolipids in P. putida decreased bacterial growth rate but stimulated biofilm formation by enhancing cell motility. Continuous rhamnolipid production in a biofilm was achieved using flow cell technology. Quantitative and structural investigations of the produced rhamnolipids were made by ultra performance liquid chromatography combined with high resolution mass spectrometry (HRMS) and tandem HRMS. The predominant rhamnolipid congener produced by the heterologous P. putida biofilm was mono-rhamnolipid with two C10 fatty acids.ConclusionThis study shows a successful application of synthetic promoter library in P. putida KT2440 and a heterologous biosynthesis of rhamnolipids in biofilm encased cells without hampering biofilm capabilities. These findings expands the possibilities of cultivation setups and paves the way for employing biofilm flow systems as production platforms for biochemicals, which as a consequence of physiochemical properties are troublesome to produce in conventional fermenter setups, or for production of compounds which are inhibitory or toxic to the production organisms.

Dataset Information

Vanillin Production in Pseudomonas: Whole-Genome Sequencing of Pseudomonas sp. Strain 9.1 and Reannotation of Pseudomonas putida CalA as a Vanillin Reductase.

Publications

Vanillin Production in <i>Pseudomonas</i>: Whole-Genome Sequencing of <i>Pseudomonas</i> sp. Strain 9.1 and Reannotation of Pseudomonas putida CalA as a Vanillin Reductase.

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