Engineering geobacillus thermoglucosidasius for efficient production of fuels and chemicals

Geobacillus;Metabolic engineering;Evolutionary engineering;Ethanol;2R, 3R-butandiol (R-BDO);Protein secretion

Zhou, Jiewen

关键词: Geobacillus;    Metabolic engineering;    Evolutionary engineering;    Ethanol;    2R, 3R-butandiol (R-BDO);    Protein secretion;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/99510/ZHOU-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The ability to grow at high temperature makes thermophiles attractive for many fermentation processes. In this thesis, we aimed to develop the thermophile Geobacillus thermoglucosidasius as a cell factory for fuels and chemicals production from renewable biomass. G. thermoglucosidasius is a facultatively anaerobic thermophilic bacterium growing between 37°C to 70°C, and can ferment diverse carbohydrates. Firstly, we engineered two strains of G. thermoglucosidasius 95A1 (95A1) and G. thermoglucosidasius C56-YS93 (C56) for improved ethanol production by employing an evolutionary engineering strategy. We eliminated lactate and formate formation in both of the strains to divert carbon source to ethanol production. However, strains were unable to grow under microaerobic conditions. Growth of 95A1 was then recovered by serial adaptation of the strain in the presence of acetate. The evolved strain of 95A1 was able to efficiently produce ethanol during growth on glucose or cellobiose. Genome sequencing identified loss-of-function mutations in adenine phosphoribosyltransferase (aprt) and the stage III sporulation protein AA (spoIIIAA). Their effect on improving ethanol production was verified by disruption of both genes. By comparison of two strains, 95A1 was a good ethanol producer and easily genetic engineered.G. thermoglucosidasius 95A1 was furtherly developed to produce a valuable biochemical of 2R, 3R-butandiol (R-BDO). Strong promoter from lactate dehydrogenase (ldh)_from G. thermodenitrificans was selected from different promoters for efficient pathway construction. The new R-BDO biosynthetic pathway was constructed in 95A1 through testing different combination of enzymes. As a result, an efficient pathway was obtained, which was composed of heterologous acetolactate synthase (alsS) from Bacillus. subtilis and acetolactate decarboxylase (alsD) from Streptococcus thermophilus. In order to enhance R-BDO production, different fermentation conditions were optimized, including oxygen supply, temperature, inoculation time, different media and addition of yeast extract. With optimal conditions, 7.2 g/L R-BDO was achieved after 48 h at 55°C. Different alcohol dehydrogenase (adh) was also deleted separately to divert more carbon source to R-BDO, but it only increased the yield but not production.Secondly, we engineered G. thermoglucosidasius 95A1 to secrete heterologous protein secretion since it was found to have capability of secreting proteins at high titers in our lab. To improve heterologous protein secretion, 25 signal peptides from G. thermoglucosidasius C56-YS93, predicted by SignalP 4.1 server, were screened and characterized for different secretory target proteins. Three thermostable hydrolase for biofuel production were selected: α-amylase (amyE) from G. stearothermophilus, endoglucanase (eglS) from B. subtilis and cellulase (celA) from Caldicellulosiruptor bescii. The optimal signal peptides for different enzymes were determined by measuring enzyme activity in supernatant of culture compared with that of their native signal peptides. One signal peptide was found to have efficient secretion with all of three enzymes. Lastly, G. thermoglucosidasius 95A1 was engineered for bioethanol production by consolidated bioprocess. Efficient ethanol production from starch was achieved by transforming α-amylase secretion plasmid into the evolved strain for ethanol production. Ethanol production from cellulose was also tried in the evolved strains harboring the cellulase secretion plasmids. However, the production was inefficient, which needed more effort to be improved. In summary, the work in this thesis established this thermophile as a platform organism for fuel and chemical production from biomass.