Heterologous expression of critical pathway genes leads to complex pattern of increased yield of bioplastic precursors inParaburkholderia sacchariITP 101
Authors: Morris Dianna , Chowdhury Niaz Bahar , Immethun Cheryl , Saha Rajib 
Published: November 2024

AbstractRecent research endeavors have turned to sustainably generating useful chemicals from biological platforms. However, conventional model organisms, such asEscherichia coliandSaccharomyces cerevisiae, face limitations, particularly in terms of substrate range and yield for certain metabolites. In this study, we share our work toward the development of the non-model bacterium,Paraburkholderia sacchari(hereafterP. sacchari), as a microbial factory for the production of polyhydroxyalkanoates (PHAs), which are precursors for biodegradable plastic. The particular PHAs of interest produced byP. sacchariinclude poly(3-hydroxybutyrate) (PHB) and the co-polymer produced by the combination of PHB and 3-hydroxyvalerate (3HV) called poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV).P. sacchariproduces PHB from mixtures of hexose and pentose sugars commonly found in lignocellulosic biomass, however PHBV requires co-feeding with propionate. Both plastic precursors have industrial interest, so both PHB and 3HV were chosen as production targets. Due to studies in other bacteria demonstrating PHB yield can be improved by overexpressing genes for critical pathway enzymes, we hypothesized there is a bottleneck in the production pathway leading to PHB inP. saccharias well. To explore this, heterologous genes coding for the three critical enzymes were taken fromCupriavidus necatorH16 (hereafterC. necator) and inserted via plasmid;phaAandbktb(homologous genes for β-ketothiolase),phaB(acetyl-CoA reductase), andphaC(PHA polymerase). PHB production increased following overexpression ofphaB, indicating acetoacetyl-CoA as the limiting enzyme. In fact, overexpression ofphaBwith the synthetic Anderson promoter, BBa_J23 104, increased titer by 162% over wildtype. On the other hand, strategies to improve 3HV had mixed results. Heterologous overexpression of propionyl-CoA transferase (pctfromC. necator), which converts propionate into propionyl-CoA-the starting substrate for the 3HVproduction, showed a 145% increase in 3HV. Yet, internal sourcing of propionyl-CoA from succinyl-CoA following introduction of the sleeping beauty mutase (sbm) operon fromE. colishowed no 3HV production. To this end, Max/Min Driving Force (MDF) thermodynamic analysis of critical PHBV pathways revealed two major limitations of 3HV production: 1) internal sourcing is not thermodynamically favorable; and 2) recycling of propionyl-CoA through the methyl citrate cycle (MCC) is more favorable than 3HV formation. Overall, we have shown promising progress and suggest future directions toward an industrially useful strain ofP. saccharifor PHB and PHBV production.Graphical abstractHighlightsParaburkholderia saccharihas interest as a bioproduction platform for PHAs from complex feedstocks. Removal of PHA pathway bottleneck increases PHB yield by 162%. Improved conversion of fed-in propionate increases 3HV yield by 145%. Internal sourcing of propionyl-CoA does not successfully yield 3HV. Thermodynamic analysis provides insight into difficult conversion of propionyl-CoA to 3HV.