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

Communications Biology
Yueyue ZhouNianzhi Jiao

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-λ-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.

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Citations

Aug 14, 2020·Applied Microbiology and Biotechnology·Anna WeimerChristoph Wittmann
Sep 27, 2020·Biotechnology Journal·Tobias SchwanemannBenedikt Wynands
Apr 1, 2021·Biotechnology Advances·Maria Martin-PascualVitor A P Martins Dos Santos
Jul 30, 2021·International Journal of Biological Macromolecules·Rajendran MuthurajTizazu H Mekonnen
Sep 14, 2021·Frontiers in Bioengineering and Biotechnology·Yidong LiuCuiqing Ma

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Methods Mentioned

BETA
PCR
reverse transcription-PCR
Assay

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