UC Berkeley

The demand for thermophilic microbial host systems for the production of bio-products has increased over the years due to the inadequacy of the currently available mesophilic systems. However, genetic tools for studying thermophilic organisms are sparingly available. This study evaluated the potential of S. acidocaldarius, a hyperthermophilic archaea, as a platform for metabolic engineering, especially cellulosic biofuels. Bioconversion of ligno-cellulosic biomass into biofuels involves the use of a wide array of thermophilic enzymes such as endo-glucanases and β-glycosidase, most of which are not properly expressed in E. coli and S. cerevisiae. S. acidocaldarius grows optimally at 75 - 80oC and pH 2-3. This organism utilizes most cellulosic sugars with the exception of cellobiose. The dissertation reports the absence of glucose-induced diauxie during consumption of multiple cellulosic sugars such as arabinose and xylose as co-carbon sources. The organism utilized combinations of 1 g/L each of glucose and xylose simultaneously with a specific growth rate of 0.079 h^-1. The organism did not show preference to glucose or any of the sugars tested. However, the organism grew faster on 2 g/L xylose (0.074 h^-1) than on equal amount of glucose (0.022 h^-1). The consumption of most cellulosic sugars by this organism makes it a potential candidate for cellulosic bio-products engineering. During growth on multiple sugars, the organism consumed each sugar at a rate that was roughly proportional to its concentration in the growth medium. The mechanism of this novel regulation is not fully understood and is currently being investigated. This study also focused on developing reliable genetic tools such as recipient strains with selectable traits and effective plasmid system to achieve the goal of making this organism a potent platform for metabolic engineering. A number of pRN1-based shuttle vectors were developed for heterologous expression in S. acidocaldarius. The role of a 241-bp region downstream of the orf904 as the putative origin of replication of pRN1 was also investigated. The results indicated that orf56, orf904 and the putative origin were the minimum replicon of pRN1. A 100-bp stem-loop structure within this putative origin was proposed to be the double-strand origin of replication of the plasmid. The impact of a functional β-glycosidase on cellobiose metabolism in S. acidocaldarius was evaluated by inserting lacS from S. solfataricus into the chromosome of the organism. The gene (lacS) established β-glycosidase activity in vivo but did not establish cellobiose metabolism, suggesting that S. acidocaldarius lacks a transporter for cellobiose. Overall, this study enhanced our knowledge of sugar metabolism in S. acidocaldarius and highlighted the development of genetic tools and strategy for utilizing the organism as a platform for metabolic engineering.