Dataset Information

Development of a CRISPR/Cas9n-based tool for metabolic engineering of Pseudomonas putida for ferulic acid-to-polyhydroxyalkanoate bioconversion.

ABSTRACT: Ferulic acid is a ubiquitous phenolic compound in lignocellulose, which is recognized for its role in the microbial carbon catabolism and industrial value. However, its recalcitrance and toxicity poses a challenge for ferulic acid-to-bioproducts bioconversion. Here, we develop a genome editing strategy for Pseudomonas putida KT2440 using an integrated CRISPR/Cas9n-?-Red system with pyrF as a selection marker, which maintains cell viability and genetic stability, increases mutation efficiency, and simplifies genetic manipulation. Via this method, four functional modules, comprised of nine genes involved in ferulic acid catabolism and polyhydroxyalkanoate biosynthesis, were integrated into the genome, generating the KTc9n20 strain. After metabolic engineering and optimization of C/N ratio, polyhydroxyalkanoate production was increased to ~270?mg/L, coupled with ~20?mM ferulic acid consumption. This study not only establishes a simple and efficient genome editing strategy, but also offers an encouraging example of how to apply this method to improve microbial aromatic compound bioconversion.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC7058019 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Development of a CRISPR/Cas9n-based tool for metabolic engineering of Pseudomonas putida for ferulic acid-to-polyhydroxyalkanoate bioconversion.

Zhou Yueyue Y Lin Lu L Wang Heng H Zhang Zhichao Z Zhou Jizhong J Jiao Nianzhi N

Communications biology 20200305 1

Ferulic acid is a ubiquitous phenolic compound in lignocellulose, which is recognized for its role in the microbial carbon catabolism and industrial value. However, its recalcitrance and toxicity poses a challenge for ferulic acid-to-bioproducts bioconversion. Here, we develop a genome editing strategy for Pseudomonas putida KT2440 using an integrated CRISPR/Cas9n-λ-Red system with pyrF as a selection marker, which maintains cell viability and genetic stability, increases mutation efficiency, an ...[more]

PMID: 32139868

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Project description:Pseudomonas putida CSV86 degrades lignin-derived metabolic intermediates, viz, veratryl alcohol, ferulic acid, vanillin, and vanillic acid, as the sole sources of carbon and energy. Strain CSV86 also degraded lignin sulfonate. Cell respiration, enzyme activity, biotransformation, and high-pressure liquid chromatography (HPLC) analyses suggest that veratryl alcohol and ferulic acid are metabolized to vanillic acid by two distinct carbon source-dependent inducible pathways. Vanillic acid was further metabolized to protocatechuic acid and entered the central carbon pathway via the β-ketoadipate route after ortho ring cleavage. Genes encoding putative enzymes involved in the degradation were found to be present at fer, ver, and van loci. The transcriptional analysis suggests a carbon source-dependent cotranscription of these loci, substantiating the metabolic studies. Biochemical and quantitative real-time (qRT)-PCR studies revealed the presence of two distinct O-demethylases, viz, VerAB and VanAB, involved in the oxidative demethylation of veratric acid and vanillic acid, respectively. This report describes the various steps involved in metabolizing lignin-derived aromatic compounds at the biochemical level and identifies the genes involved in degrading veratric acid and the arrangement of phenylpropanoid metabolic genes as three distinct inducible transcription units/operons. This study provides insight into the bacterial degradation of lignin-derived aromatics and the potential of P. putida CSV86 as a suitable candidate for producing valuable products.IMPORTANCEPseudomonas putida CSV86 metabolizes lignin and its metabolic intermediates as a carbon source. Strain CSV86 displays a unique property of preferential utilization of aromatics, including for phenylpropanoids over glucose. This report unravels veratryl alcohol metabolism and genes encoding veratric acid O-demethylase, hitherto unknown in pseudomonads, thereby providing new insight into the metabolic pathway and gene pool for lignin degradation in bacteria. The biochemical and genetic characterization of phenylpropanoid metabolism makes it a prospective system for its application in producing valuable products, such as vanillin and vanillic acid, from lignocellulose. This study supports the immense potential of P. putida CSV86 as a suitable candidate for bioremediation and biorefinery.

Project description:From a ferulic-acid-degrading Pseudomonas fluorescens strain (BF13), we have isolated a transposon mutant, which retained the ability to bioconvert ferulic acid into vanillic acid but lost the ability to further degrade the latter acid. The mutant, BF13-97, was very stable, and therefore it was suitable to be used as a biocatalyst for the preparative synthesis of vanillic acid from ferulic acid. By use of resting cells we determined the effect on the bioconversion rate of several parameters, such as the addition of nutritional factors, the concentration of the biomass, and the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with cells pregrown on M9 medium containing p-coumaric acid (0.1% [wt/vol]) as a sole carbon source and yeast extract (0.001% [wt/vol]) as a source of nutritional factors. Under these conditions, 1 mg (wet weight) of biomass produced 0.23 mg of vanillic acid per h. The genomic region of BF13-97 flanking the transposon's site of insertion was cloned and sequenced revealing two open reading frames of 1,062 (vanA) and 954 (vanB) bp, respectively. The van genes are organized in a cluster and encode the subunits of the vanillate-O-demethylase, which catalyzes the first step of the vanillate catabolism. Amino acid sequences deduced from vanA and vanB genes were shown to have high identity with known VanAs and VanBs from Pseudomonas and Acinetobacter spp. Highly conserved regions known to exist in class IA oxygenases were also found in the vanillate-O-demethylase components from P. fluorescens BF13. The terminal oxygenase VanA is characterized by a conserved Rieske-type [2Fe-2S](R) ligand center. The reductase VanB contains a plant-type ferredoxin [2Fe-2S](Fd), flavin mononucleotide, and NAD-ribose binding domains which are located in its C-terminal and N-terminal halves, respectively. Transfer of wild-type vanAB genes to BF13-97 complemented this mutant, which recovered its ability to grow on either vanillic or ferulic acid.

Dataset Information

Development of a CRISPR/Cas9n-based tool for metabolic engineering of Pseudomonas putida for ferulic acid-to-polyhydroxyalkanoate bioconversion.

Publications

Development of a CRISPR/Cas9n-based tool for metabolic engineering of Pseudomonas putida for ferulic acid-to-polyhydroxyalkanoate bioconversion.

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