The ability to grow at high temperatures makes thermophiles attractive for many fermentation processes. In this work, we used evolutionary engineering to increase ethanol production in the thermophile Geobacillus thermoglucosidasius. This bacterium is a facultative anaerobe, grows at an optimal temperature of 60°C, and can ferment diverse carbohydrates. However, it natively performs mixed-acid fermentation. To improve ethanol productivity, we first eliminated lactate and formate production in two strains of G. thermoglucosidasius, 95A1 and C56-YS93. These deletion strains were generated by selection on spectinomycin, which represents, to the best of our knowledge, the first time this antibiotic has been shown to work with thermophiles. Both knockout strains, however, were unable to grow under microaerobic conditions. We were able to recover growth in G. thermoglucosidasius 95A1 by serial adaptation in the presence of acetic acid. The evolved 95A1 strain 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). Disruption of both genes improved ethanol production in the unadapted strains: however, the increase was significant only when aprt was deleted. In conclusion, we were able to engineer a strain of G. thermoglucosidasius to efficiently produce ethanol from glucose and cellobiose using a combination of metabolic engineering and evolutionary strategies. This work further establishes this thermophile as a platform organism for fuel and chemical production. Biotechnol. Bioeng. 2016;113: 2156-2167. © 2016 Wiley Periodicals, Inc.

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A functional glucokinase (YqgR), N-acetylglucosamine-phosphate mutase (Agm1) and N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) were synthesized simultaneously in an Escherichia coli cell-free system. These three enzymes were coupled to reconstruct a biosynthetic pathway which could produce UDP-N-acetylglucosamine with N-acetylglucosamine as the substrate.

Uridine 5'-diphospho N-acetylglucosamine (UDP-GlcNAc) is an important nucleotide sugar in the biochemistry of all living organisms, and it is an important substrate in the synthesis of oligosaccharides. In the present work, three bioactive enzymes, namely, glucokinase (YqgR), GlcNAc-phosphate mutase (Agm1), and N-acetylglucosamine-1-phosphate uridyltransferase (GlmU), were produced effectively as soluble form in recombinant Escherichia coli. These three enzymes and dried yeast together were used to construct a multistep enzymatic system, which could produce UDP-GlcNAc efficiently with N-acetylglucosamine (GlcNAc) as the substrate. After the optimization of various reaction conditions, 31.5 mMUDP-GlcNAc was produced from 50 mMGlcNAc and 50 mMUMP.