(157ay) Modifying the Operator for More Dynamic Tunable Gene Expression 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. 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. In its native form, P. sacchari cannot efficiently produce enough PHB for the process to be economically feasible and a metabolically engineered strain would be needed to be industrially useful. However, there are limited synthetic biology tools available to attempt metabolic engineering strategies. Here we share our development of an inducible system for gene control in P. sacchari. It is commonly recognized that development of promoters, specifically inducible promoters that can be turned on and off, is one of the easiest and most effective ways to control gene expression. Therefore, we 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 most strongly bind the repressor protein. This resulted in a tighter off state when used to express green fluorescent protein (GFP). Growth in the presence of varying concentrations of the inducer molecule exhibited a graded response, therefore providing a tunable and useful tool for future metabolic engineering of P. sacchari. In addition to the inducible system, we also tested the Anderson promoter library for constitutive (always on) expression of GFP. We saw a range of expression which provides options for constitutive control of gene expression. Current and future work within our lab is focusing on incorporating these regulatory elements into explorations of engineering P. sacchari for more efficient PHB production on complex feedstocks. 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.