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

Yueyue Zhou,Jizhong Zhou,Nianzhi Jiao,Lu Lin,Zhichao Zhang,Heng Wang

doi:10.1038/s42003-020-0824-5

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.

Highlights

Ferulic acid is a ubiquitous phenolic compound in lignocellulose, which is recognized for its role in the microbial carbon catabolism and industrial value
The results suggested pyrF was knockedout, validating this CRISPR/Cas9-λ-Red system works in KT2440, consistent with the previous study[28] (Supplementary Notes)
Several genome-editing approaches have been developed for P. putida KT244026,28,33, our CRISPR/Cas9n-λ-Red genome-editing strategy (CRP) method proved to be an efficient, fast and convenient tool (Table 3)

Summary

Introduction

Ferulic acid is a ubiquitous phenolic compound in lignocellulose, which is recognized for its role in the microbial carbon catabolism and industrial value. We develop a genome editing strategy for Pseudomonas putida KT2440 using an integrated CRISPR/Cas9nλ-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. A wide variety of genome-editing tools have been developed for metabolic engineering in Pseudomonas, the majority of which are based on homologous recombination[23] Such methods have very low mutation efficiency and are time consuming[23,24,25].

Methods

Results

Conclusion