Eliminating the isoleucine biosynthetic pathway to reduce competitive carbon outflow during isobutanol production by Saccharomyces cerevisiae.
BACKGROUND
Isobutanol is an important biorefinery target alcohol that can be used as a fuel, fuel additive, or commodity chemical. Baker's yeast, Saccharomyces cerevisiae, is a promising organism for the industrial manufacture of isobutanol because of its tolerance for low pH and resistance to autolysis. It has been reported that gene deletion of the pyruvate dehydrogenase complex, which is directly involved in pyruvate metabolism, improved isobutanol production by S. cerevisiae. However, the engineering strategies available for S. cerevisiae are immature compared to those available for bacterial hosts such as Escherichia coli, and several pathways in addition to pyruvate metabolism compete with isobutanol production.


RESULTS
The isobutyrate, pantothenate or isoleucine biosynthetic pathways were deleted to reduce the outflow of carbon competing with isobutanol biosynthesis in S. cerevisiae. The judicious elimination of these competing pathways increased isobutanol production. ILV1 encodes threonine ammonia-lyase, the enzyme that converts threonine to 2-ketobutanoate, a precursor for isoleucine biosynthesis. S. cerevisiae mutants in which ILV1 had been deleted displayed 3.5-fold increased isobutanol productivity. The ΔILV1 strategy was further combined with two previously established engineering strategies (activation of two steps of the Ehrlich pathway and the transhydrogenase-like shunt), providing 11-fold higher isobutanol productivity as compared to the parent strain. The titer and yield of this engineered strain was 224 ± 5 mg/L and 12.04 ± 0.23 mg/g glucose, respectively.


CONCLUSIONS
The deletion of competitive pathways to reduce the outflow of carbon, including ILV1 deletion, is an important strategy for increasing isobutanol production by S. cerevisiae.
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan. ida.kengo@gmail.com.
Kengo
K
Ida
Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan. junjun@port.kobe-u.ac.jp.
Jun
J
Ishii
Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan. fmatsuda@bio.eng.osaka-u.ac.jp.|Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Suita, Osaka, 565-0871, Japan. fmatsuda@bio.eng.osaka-u.ac.jp.|RIKEN Biomass Engineering Program, 1-7-22 Suehiro, Tsurumi, Yokohama, 230-0045, Japan. fmatsuda@bio.eng.osaka-u.ac.jp.
Fumio
F
Matsuda
Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan. kondo814@gmail.com.|Present address: Faculty of Engineering, Hokkaido University, N13W8, Sapporo, 060-8628, Japan. kondo814@gmail.com.
Takashi
T
Kondo
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan. akondo@kobe-u.ac.jp.|RIKEN Biomass Engineering Program, 1-7-22 Suehiro, Tsurumi, Yokohama, 230-0045, Japan. akondo@kobe-u.ac.jp.
Akihiko
A
Kondo
D000440:Butanols / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism*; D007532:Isoleucine / Q000096:biosynthesis / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D000440:Butanols; D007532:Isoleucine; C040507:isobutyl alcohol; D002244:Carbon