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/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. 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.

Pseudomonas as Versatile Aromatics Cell Factory

“…However, through inactivation of the protocatechuate degradation pathway and simultaneous integration of protocatechuate decarboxylase, many other aromatic substrates present in lignin hydrolysate (e.g., 4-coumarate, ferulate, 4-hydroxybenzoate) can be funneled to catechol and subsequently to cis,cis-muconate. Thus, metabolic funneling of heterogonous mixtures of many different lignin-derivable compounds to a common product is attractive as this facilitates product isolation and purification that is also applicable to non-aromatic products including polyhydroxyalkanoate (PHA) [22,211,212] or organic acids including PYR and lactate. [213]…”

Challenges and Advances in Genome Editing Technologies in Streptomyces

“…Cas9n only causes single-stranded DNA breaks, which can mitigate the lethal effects of Cas9. Currently, Cas9n has been adopted for the development of efficient and precise genome editing tools in several bacteria, such as Clostridium cellulolyticum [65], Pseudomonas putida [66], Escherichia coli [67], Lactobacillus casei [68], and Bacillus licheniformis [69]. However, Cas9n has not been adopted for Streptomyces genome editing.…”

Bioconversion of biphenyl to a polyhydroxyalkanoate copolymer by Alcaligenes denitrificans A41

“…In conclusion, the native strain A41 provided here the prototype of a microbial platform for the production of copolymer P(3HB- co -3HV) by supplying a cytotoxic biphenyl. In the near future, a recombinant technology (Zhou et al 2020 ) will be applied for the strain A41 platform, in order to improve PHA productivity and alter properties related to molecular weight and monomeric composition.…”