Metabolic engineering of Pseudomonas putida for increased polyhydroxyalkanoate production from lignin

Abstract

SummaryMicrobial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic‐catabolic pathways are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole‐cell biocatalyst for funnelling lignin‐derived compounds to value‐added products, including its native carbon storage product, medium‐chain‐length polyhydroxyalkanoates (mcl‐PHA). In this work, a series of metabolic engineering targets to improve mcl‐PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p‐coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene phaZ was knocked out, and the genes involved in β‐oxidation (fadBA1 and fadBA2) were deleted. Additionally, to increase carbon flux into mcl‐PHA biosynthesis, phaG, alkK, phaC1 and phaC2 were overexpressed. The best performing strain – which contains all the genetic modifications detailed above – demonstrated a 53% and 200% increase in mcl‐PHA titre (g l−1) and a 20% and 100% increase in yield (g mcl‐PHA per g cell dry weight) from p‐coumaric acid and lignin, respectively, compared with the wild type strain. Overall, these results present a promising strain to be employed in further process development for enhancing mcl‐PHA production from aromatic compounds and lignin.

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

“…In addition, it is known that phaZ (mcl-PHA depolymerase) knockout and phaC (mcl-PHA synthase) knockin enhance mcl-PHA accumulation in P. putida 19 . Therefore, phaZ was deleted and replaced by a copy of phaC1, generating the mutant KTc9n13 (Table 1 and Supplementary Table 3).…”

“…P. putida can catabolize ferulic acid via a coenzyme A-dependent, non-β-oxidative pathway 18 . Moreover, it is a well-known mcl-PHA-producing organism 13,19 . However, the conversion efficiency is low, posing a challenge for the application of this organism in ferulic acid-to-PHA 9,19 .…”

“…Moreover, it is a well-known mcl-PHA-producing organism 13,19 . However, the conversion efficiency is low, posing a challenge for the application of this organism in ferulic acid-to-PHA 9,19 . First, ferulic acid is highly toxic to the bacterial cell, inhibiting cell growth and metabolism 8 .…”

“…Second, high mcl-PHA yields in P. putida are more favored to be produced from fatty acids 20,21 . Employing non-fatty acid feedstock, especially aromatic compounds, greatly limits mcl-PHA accumulation 19,22 . Hence, metabolic engineering of P. putida strains is required to address these two key issues.…”

Strategies for the production of biochemicals in bioenergy crops

“…Several microbial strains have been developed for bio-based production of p-HS using pCA as direct precursor [62,63]. Other engineered microbial strains have been designed to upgrade pCA into resveratrol, an antioxidant with potential benefits for human health [64][65][66], as well as the biofuel precursor bisabolene [18], the platform chemical muconic acid [67], the biodegradable polyester precursor lactic acid [68], and polyhydroxyalkanoate polyesters [20,69,70]. Moreover, pCA can be transformed chemically to deep eutectic solvents potentially useful for biomass pretreatments [40] and converted to different types of high-performance polymers [71,72].…”