DEVELOPMENT OF PROTEIN DISPLAY SYSTEMS AND GENETIC TOOLS FOR SPORE-FORMING BACTERIA

One major area of synthetic biology is to engineer microbial cells and subcellular systems for diverse applications including biosynthesis, biocatalysis, therapeutics, drug delivery, and bioremediation. For most applications, robust cellular systems are preferred for longer activity half-life and resistance to harsh environments. Two projects related to robust cellular systems involving Gram-positive bacteria are presented in this work. One is to develop thermostable genetic reporters for Geobacilli species and the other is to display an enzyme on the Bacillus subtilis spore surface to enhance its robustness and present an alternative to purified enzymes for industrial applications. Bacillus subtilis and Geobacillus thermoglucosidans are gram-positive, spore-forming bacteria. They secrete many proteins used industrially for the production of paper, food, textiles, chemicals, medicine, and cosmetics. Since G. thermoglucosidans is thermostable with an optimal growth temperature of 60ºC, its secreted proteins are also thermostable which proves advantageous for a variety of industrial applications. Additionally, a strain of G. thermoglucosidans has been used for the production of ethanol from biomass. Unfortunately the inner workings of G. thermoglucosidans are still poorly understood and a genetic toolkit is necessary to better discover how to improve them via genetic engineering for industrial use. Important components of this toolkit are genetic reporters which allow for the analysis of gene expression in G. thermoglucosidans. Fluorescent proteins are commonly used reporters for other bacterial species due to their easily observed and readily measured signal, however no thermostable fluorescent proteins have been shown to be functional in Geobacillus. Seven different fluorescent proteins including mCherry, Venus, GFP, sfGFP, GFPmut3, mCherry (Gt), and Venus (Gt) were tested for stability and functionality in Geobacillus thermoglucosidans. Venus (Gt) and mCherry (Gt) were codon optimized for this bacterium with the goal of increasing expression level and thus improving the fluorescence signal. The fluorescence intensity of each fluorescent protein expressed in G. thermoglucosidans was measured after several hours of bacterial growth at 50ºC and 60ºC. Venus, mCherry, Venus (Gt), mCherry (Gt), and sfGFP all had signal when expressed in G. thermoglucosidans at 50ºC and sfGFP had signal at 60ºC. Therefore, fluorescent reporter proteins in three different colors were found to be functional in G. thermoglucosidans. This will further genetic engineering of the species for thermostable protein production, bioremediation, and biofuel production. Bacillus subtilis is Generally Regarded as Safe (GRAS) by the FDA and amenable toward genetic manipulation. Thus it has been engineered for the production of many heterologous proteins. Oftentimes, proteins secreted by bacteria are purified for industrial use. However, protein purification is expensive and time-consuming and long-term storage of purified proteins requires extremely low temperatures (-20ºC). B. subtilis spores have been used to immobilize a variety of proteins for vaccines, biosensors, and bioremediation applications. Spore surface display eliminates the need for purification and provides a way to easily separate proteins from the final product if necessary. A novel and thermostable laccase, a copper-containing oxidase, was isolated and purified from G. thermoglucosidans. It can be used to degrade lignin and a variety of phenolic compounds and thus has applications for the production of paper, textiles, food, and biofuel. This laccase was isolated, characterized, and immobilized on the surface of B. subtilis spores. The purified and spore displayed laccase were tested for heat stability and catalytic function. The purified laccase showed high activity toward 2,6-dimethoxyphenol (2,6-DMP) and moderate activity toward veratryl alcohol and 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) while the spore displayed laccase showed high activity toward 2,6-DMP. The purified laccase was considerably more heat stable than a commonly used fungal laccase. The spore displayed laccase was also found to be heat stable with a half-life of about 6 hours at 80ºC. The binding affinity of the immobilized laccase for the substrate 2,6-DMP was virtually the same as that of the purified laccase, plus the immobilized laccase showed solid activity. These results show that spore surface display of proteins is a promising, more inexpensive alternative to purifying proteins for industrial use