Multi-functionalizing Pseudomonas putida KT2440 for broad carbon utilization and 3-hydroxy acids production

RWTH Publications (RWTH Aachen)

Abstract

With the increasing concerns of maintaining a beautiful living environment, microbiologists need to consider how to address high-complexity non-biodegradable wastes and global warming driven by greenhouse-gas over-emission. “Open-loop” upcycling of these wastes into biodegradable material building blocks using metabolically flexible microorganisms could be a promising approach. Under this context, Pseudomonas putida KT2440 (P. putida), featuring versatile metabolism and a relatively complete gene editing toolkit, was selected and engineered. To accelerate strain engineering, the widely used pEMG/pSW genome-editing workflow was optimized by re-assembling it into a single plasmid and introducing strict inducible control of I-SceI together with SacB counterselection. This shortened the turnaround time from 9 to 3 days. The resulting vectors (pEMG-RIS, pEMG-AIS, pEMG-GRIS) were shared with the Pseudomonas community and deposited at Addgene. As biodegradable plastic alternatives, polyhydroxyalkanoates (PHA) are attractive, yet intracellular granule recovery is often unfeasible; therefore, the secreted monomers 3-hydroxy fatty acids (3-HFAs) were in focus. A non-degradable 3-HFAs chassis was built by deleting multiple CoA ligases. PhaG was re-identified as a thioesterase and was constitutively expressed for direct synthesis of C8–C14 3-HFAs, with scalability validated in bioreactors. In parallel, an indirect route using RhlA variants generated HAAs, which were converted to free 3-HFAs by endogenous esterase(s), enabling tunable chain-length distribution. To tackle mixed hydrolysable plastics, the P. putida ETAB strain was constructed enabling simultaneous co-consumption of five monomers, ethylene glycol (EG), terephthalic acid (TA), adipic acid (AA), 1,4-butanediol (BDO), and lactic acid (LA), derived from common hydrolysable plastics (PET, PU, PBAT, and PLA). Its robustness was evaluated in a 21-day continuous fermentation, revealing two beneficial mutations; the reverse-engineered strain was further developed to produce R-3-hydroxybutyrate (R-3HB) from real hydrolysates of an enzymatically depolymerized PET/PBAT/TPU blend. In addition, the engineering scope was extended to non-hydrolysable plastics and C2-based bioconversion. For non-hydrolysable plastics, polystyrene-derived styrene was addressed by integrating the sty operon into P. putida. Deletion of paaK enabled near-quantitative accumulation of phenylacetate (PAA) from styrene, while deletion of paaF plus expression of ter allowed production of adipic acid (AA). Besides, to improve C2-based bioconversion, the ATP-intensive acetyl-CoA synthetase route for acetate assimilation was removed and replaced by an ackA-pta pathway (identified by insertion/screening), yielding an efficient platform for R-3HB production from acetate with formate or ethanol. We also integrated a functional soluble hydrogenase (SH) into P. putida, demonstrating vitality with hydrogen as a carbon-free reductant, while its broader metabolic-engineering applications remain to be explored. In summary, these developments highlight the promise of P. putida in addressing sustainability-related challenges.

Authors 1

1. Hao Meng corresponding Aachen

   RWTH Aachen University

   Affiliation as printed

   RWTH Aachen