Engineering the leucine biosynthetic pathway for isoamyl alcohol overproduction in Saccharomyces cerevisiae.
Isoamyl alcohol can be used not only as a biofuel, but also as a precursor for various chemicals. Saccharomyces cerevisiae inherently produces a small amount of isoamyl alcohol via the leucine degradation pathway, but the yield is very low. In the current study, several strategies were devised to overproduce isoamyl alcohol in budding yeast. The engineered yeast cells with the cytosolic isoamyl alcohol biosynthetic pathway produced significantly higher amounts of isobutanol over isoamyl alcohol, suggesting that the majority of the metabolic flux was diverted to the isobutanol biosynthesis due to the broad substrate specificity of Ehrlich pathway enzymes. To channel the key intermediate 2-ketosiovalerate (KIV) towards α-IPM biosynthesis, we introduced an artificial protein scaffold to pull dihydroxyacid dehydratase and α-IPM synthase into the close proximity, and the resulting strain yielded more than twofold improvement of isoamyl alcohol. The best isoamyl alcohol producer yielded 522.76 ± 38.88 mg/L isoamyl alcohol, together with 540.30 ± 48.26 mg/L isobutanol and 82.56 ± 8.22 mg/L 2-methyl-1-butanol. To our best knowledge, our work represents the first study to bypass the native compartmentalized α-IPM biosynthesis pathway for the isoamyl alcohol overproduction in budding yeast. More importantly, artificial protein scaffold based on the feature of quaternary structure of enzymes would be useful in improving the catalytic efficiency and the product specificity of other enzymatic reactions.
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore. jifeng_yuan@biotrans.a-star.edu.sg.|Temasek Laboratories, National University of Singapore, T-Lab Building 5A, Engineering Drive 1, Singapore, 117411, Singapore. jifeng_yuan@biotrans.a-star.edu.sg.|Biotransformation Innovation Platform, Agency for Science, Technology and Research (A*STAR), Singapore, 138673, Singapore. jifeng_yuan@biotrans.a-star.edu.sg.
Jifeng
J
Yuan
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore.
Pranjul
P
Mishra
Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore.|Temasek Laboratories, National University of Singapore, T-Lab Building 5A, Engineering Drive 1, Singapore, 117411, Singapore.|Singapore Institute of Technology, 10 Dover Drive, Singapore, 138683, Singapore.
Chi Bun
CB
Ching
D020001:1-Butanol / Q000378:metabolism; D053898:Biosynthetic Pathways; D000440:Butanols; D003600:Cytosol / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D045782:Hemiterpenes; D006836:Hydro-Lyases; D007930:Leucine / Q000737:chemistry*; D053858:Metabolic Networks and Pathways; D010421:Pentanoic Acids / Q000737:chemistry; D000439:Pentanols / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000378:metabolism; D012313:RNA / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D000440:Butanols; D045782:Hemiterpenes; D010421:Pentanoic Acids; D000439:Pentanols; D011506:Proteins; C008216:isovaleric acid; C040507:isobutyl alcohol; D012313:RNA; C032765:tert-amyl alcohol; D020001:1-Butanol; C029683:isopentyl alcohol; D006836:Hydro-Lyases; C020052:dihydroxyacid dehydratase; D007930:Leucine
Artificial protein scaffold; Ehrlich degradation pathway; Enzyme co-localization; Isoamyl alcohol; Isobutanol; α-IPM biosynthetic pathway