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-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. Yueyue Zhou et al. develop a genetic engineering method that increases the production of polyhydroxyalkanoate from ferulic acid, which is toxic at high concentrations. This study provides insight into the bioconversion of the aromatic compound in Pseudomonas.

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DOI

10.1038/s42003-020-0824-5

Journal

Communications Biology

Year

2020