Heterologous constitutive production of short-chain-length polyhydroxyalkanoates in Pseudomonas putida KT2440: the involvement of IbpA inclusion body protein

Front Bioeng Biotechnol. 2023 Nov 1:11:1275036. doi: 10.3389/fbioe.2023.1275036. eCollection 2023.

Abstract

Designing cell factories for the production of novel polyhydroxyalkanoates (PHAs) via smart metabolic engineering is key to obtain à la carte materials with tailored physicochemical properties. To this end, we used the model medium-chain-length-PHA producing bacterium, P. putida KT2440 as a chassis, which is characterized by its metabolic versatility and stress tolerance. Different PHA biosynthetic modules were assembled in expression plasmids using the Golden gate/MoClo modular assembly technique to implement an orthogonal short-chain-lengh-PHA (scl-PHA) switch in a "deaf" PHA mutant. This was specifically constructed to override endogenous multilevel regulation of PHA synthesis in the native strain. We generated a panel of engineered approaches carrying the genes from Rhodospirillum rubrum, Cupriavidus necator and Pseudomonas pseudoalcaligenes, demonstrating that diverse scl-PHAs can be constitutively produced in the chassis strain to varying yields from 23% to 84% PHA/CDW. Co-feeding assays of the most promising engineered strain harboring the PHA machinery from C. necator resulted to a panel of PHBV from 0.6% to 19% C5 monomeric incorporation. Chromosomally integrated PHA machineries with high PhaCCn synthase dosage successfully resulted in 68% PHA/CDW production. Interestingly, an inverse relationship between PhaC synthase dosage and granule size distribution was demonstrated in the heterologous host. In this vein, it is proposed the key involvement of inclusion body protein IbpA to the heterologous production of tailored PHA in P. putida KT2440.

Keywords: IbpA inclusion body protein; Pseudomonas putida; modular cloning assembly; polyhydroxyalkanoates; synthetic biology.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement number 633962 (P4SB), 814418 (SinFonia) and 870294 (MIX-up). This work was supported by the CSIC Interdisciplinary Thematic Platform (PTI+) Sustainable Plastics towards a Circular Economy (PTI-Susplast+), the Community of Madrid (P2018/NMT4389) and the Spanish Ministry of Science and Innovation under the research grant BIOCIR (PID 2020-112766RB-C21).