——Applied Microbiol Biotechnol (2017)101：2189-2199

Chunkai Gu^1,2， Genyu Wang¹，Shuai Mai¹，Pengfei Wu¹，Jianrong Wu²，Gehua Wang¹，Hongjuan Liu¹，Jianan Zhang¹

（1.Institute of Nuclear and New Energy Technology, TsinghuaUniversity; 2.The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University）

Abstract： Butanol is an ideal renewable biofuel which possesses superior fuel properties. Previously, butanol-producing symbiotic system TSH06 was isolated in our lab, with microoxygen tolerance ability. To boost butanol yield for large-scale industrial production, TSH06 was used as parental, strain and subjected to atmospheric and room temperature plasma (ARTP) and four rounds of genome shuffling (GS). ARTP mutant and GS strain were co-cultured with facultative anaerobic Bacillus cereus TSH2 to form a symbiotic system with microoxygen tolerance, which was then subjected to fermentation. Relative messenger RNA (mRNA) level of key enzyme gene was measured by real-time PCR. The highest butanol titer of TS4-30 reached 15.63 g/L, which was 34% higher than TSH06 (12.19 g/L). Compared with parental strain, mRNA of acid-forming gene in TS4-30 decreased in acidogenesis phase, while solvent-forming gene increased in solventogenesis phase. This gene expression pattern was consistent with high butanol yield and low acid level in TS4-30. In summary, symbiotic system TS4-30 was obtained with butanol titer improvement and microoxygen tolerance.

Fig. 6 Relative transcription ratio of key genes at different fermentation phases. a Acidproducing phase. b Butanolproducing phase. c Metabolic pathway of acid-producing phase in the ABE fermentation. Ak acetate kinase, Pta phosphate acetyltransferase, Bk butyrate kinase, Ptb phosphate butyryltransferase, CoAT CoA transferase (encoded by ctf α and β), ADC acetoacetate decarboxylase, Thl thiolase, Hbd 3-hydroxybutyryl-CoA dehydrogenase, Cro crotonase,Bdc butyryl-CoA dehydrogenase, Aad acetaldehyde-CoA/alcohol dehydrogenase, BdhA NADHdependent butanol dehydrogenase A, BdhB NADHdependent alcohol dehydrogenase B.