Improving bioplastic production by Rhodopseudomonas palustris TIE-1 using synthetic biology and metabolic engineering

Abstract With the increasing demand for sustainably produced renewable resources, it is important to look towards microorganisms capable of producing bioproducts such as biofuels and bioplastics. Though many systems for bioproduct production are well documented and tested in model organisms, it is essential to look beyond to non-model organisms to expand the field and take advantage of metabolically versatile strains. This investigation centers on Rhodopseudomonas palustris TIE-1, a purple, non-sulfur autotrophic, and anaerobic bacterium capable of producing bioproducts that are comparable to their petroleum-based counterparts. To induce bioplastic overproduction, genes that might have a potential role in the PHB biosynthesis such as the regulator, phaR, and phaZ known for its ability to degrade PHB granules were deleted using markerless deletion. Mutants in pathways that might compete with polyhydroxybutyrate (PHB) production such as glycogen and nitrogen fixation previously created to increase n -butanol production by TIE-1 were also tested. In addition, a phage integration system was developed to insert RuBisCO (RuBisCO form I and II genes) driven by a constitutive promoter P aphII into TIE- 1 genome. Our results show that deletion of the phaR gene of the PHB pathway increases PHB productivity when TIE-1 was grown photoheterotrophically with butyrate and ammonium chloride (NH 4 Cl). Mutants unable to make glycogen or fix dinitrogen gas show an increase in PHB productivity under photoautotrophic growth conditions with hydrogen. In addition, the engineered TIE-1 overexpressing RuBisCO form I and form II produces significantly more polyhydroxybutyrate than the wild type under photoheterotrophy with butyrate and photoautotrophy with hydrogen. Inserting RuBisCO genes into TIE-1 genome is a more effective strategy than deleting competitive pathways to increase PHB production in TIE-1. The phage integration system developed for TIE-1 thus creates numerous opportunities for synthetic biology in TIE-1.