(278b) Heterologous Gene Expression Yields Higher Polyhydroxybutyrate Production in Paraburkholderia Sacchari

Recent research endeavors have turned to generating useful chemicals from biological platforms as an environmentally responsible alternative to non-sustainable sources. One such chemical is polyhydroxybutyrate (PHB), which is a biodegradable plastic. One bacterium of interest for production of PHB is Paraburkholderia sacchari LMG 19450 LFM101. This non-model bacterium is a relatively high efficiency producer of PHB and can utilize both C5 and C6 sugars, making it an attractive candidate for PHB production from cheap feedstocks that may have a mixture of carbon sources, such as corn kernel fiber (CFK). In its native form, P. sacchari cannot efficiently produce enough PHB for the process to be economically feasible; however, there are limited synthetic biology tools available to attempt metabolic engineering strategies to rectify this shortcoming. Here we share our expansion on synthetic biology tools through characterization of a synthetic promoter set, development of an inducible system for gene control, and use of the RSF origin of replication in P. sacchari. We also expand on metabolic engineering strategies by demonstrating an increase in PHB production following expression of heterologous genes from another PHB producing bacterium, Cupriavidus necator. It is commonly recognized that transcriptional control is one of the easiest and most effective ways to control gene expression. To explore constitutive (always on) synthetic transcriptional control, we tested the Anderson promoter library for expression of GFP. We saw a range of expression which provides options for constitutive control of gene expression. We also sought to develop an inducible system within P. sacchari. The lac system from E. coli with the promoter P_LacUV5 has been shown to achieve gene expression in P. sacchari, however, the off state is extremely leaky. We modified the lac system by changing the operator sites surrounding the promoter region to the Oid sequence shown to bind the repressor protein most strongly, resulting in a tighter off state when used to express green fluorescent protein (GFP). The presence of varying concentrations of the inducer molecule exhibited a graded expression response, therefore providing a tunable and useful tool for future metabolic engineering of P. sacchari. We also demonstrate that the RSF origin of replication is a candidate for plasmid maintenance within P. sacchari. Our findings show that a plasmid containing RSF and red fluorescent protein (RFP) as the reporter can express RFP within P. sacchari. This provides another origin of replication option, in addition to the previously used pBBR1-oriV, for plasmid- based gene expression within P. sacchari, which will be a useful tool for future multi-plasmid expression strategies. Finally, we have been able to demonstrate increased production of PHB from P. sacchari by incorporating heterologous genes from C. necator. A set of genes from C. necator (phaA, phaB, phaC, and bktb) were each individually inserted via plasmid and maintained with kanamycin resistance. These genes were chosen as they code for key enzymes in the PHB pathway which have been shown to work efficiently and we hypothesized that overexpression could increase PHB production. After 48 hours growth in nitrogen limited medium with 15 g/L glucose, there was a 3.5X increase in PHB production with the P. sacchari strain containing bktb under the constitutive control of the promoter P_lac. This gene, bktb, codes for β-ketothiolase, which converts Acetyl-CoA to Acetoacetyl-CoA as part of the PHB production pathway. Current and future work incorporates the synthetic biology tools described here into further exploration of how heterologous genes can improve PHB production within P. sacchari. Our findings lay a foundation for further development of a bioproduction system for PHB production; a direction of research that will provide a sustainable, environmentally responsible, and economically beneficial method for creating biodegradable plastics.