Metabolic engineering of the 2-ketobutyrate biosynthetic pathway for 1-propanol production in Saccharomyces cerevisiae

Abstract

Background: To produce 1-propanol as a potential biofuel, metabolic engineering of microorganisms, such as E. coli, has been studied. However, 1-propanol production using metabolically engineered Saccharomyces cerevisiae, which has an amazing ability to produce ethanol and is thus alcohol-tolerant, has infrequently been reported. Therefore, in this study, we aimed to engineer S. cerevisiae strains capable of producing 1-propanol at high levels.

Results: We found that the activity of endogenous 2-keto acid decarboxylase and alcohol/aldehyde dehydrogenase is sufficient to convert 2-ketobutyrate (2 KB) to 500 mg/L 1-propanol in yeast. Production of 1-propanol could be increased by: (i) the construction of an artificial 2 KB biosynthetic pathway from pyruvate via citramalate (cimA); (ii) overexpression of threonine dehydratase (tdcB); (iii) enhancement of threonine biosynthesis from aspartate (thrA, thrB and thrC); and (iv) deletion of the GLY1 gene that regulates a competing pathway converting threonine to glycine. With high-density anaerobic fermentation of the engineered S. cerevisiae strain YG5C4231, we succeeded in producing 180 mg/L 1-propanol from glucose.

Conclusion: These results indicate that the engineering of a citramalate-mediated pathway as a production method for 1-propanol in S. cerevisiae is effective. Although optimization of the carbon flux in S. cerevisiae is necessary to harness this pathway, it is a promising candidate for the large-scale production of 1-propanol.

Keywords: 1-Propanol; 2-Ketobutyrate; Fermentation; S. cerevisiae; Yeast.

Fig. 1

Pathways for 1-propanol production in S. cerevisiae. a–d show different theoretical methods to achieve production of this metabolite. Red letters indicate genes that are overexpressed. Blue and green letters indicate genes that are deleted. ARO3 and ARO4 encode 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase. ALT1 and ALT2 encode alanine transaminase. CIT1, CIT2 and CIT3 encode citrate synthase. MET2 encodes l-homoserine-O-acetyltransferase. GLY1 encodes threonine aldolase. ILV2 and ILV6 encode acetolactate synthase. ILV3 encodes dihydroxyacid dehydratase. ILV5 encodes acetohydroxyacid reductoisomerase. BAT1 and BAT2 encode branched-chain amino acid transaminase. cimA encodes citramalate synthase. leuC and leuD encode citramalate hydrolyase. LEU2 encodes 3-isopropylmalate dehydrogenase. asd encodes aspartate-semialdehyde dehydrogenase. thrA encodes aspartokinase and homoserine dehydrogenase I. thrB encodes homoserine kinase. thrC encodes threonine synthase. tdcB encodes threonine dehydratase

Fig. 2

Production of 1-propanol from added 2 KB in various KDC- and ADH-overexpressing S. cerevisiae YPH499 strains

Fig. 3

Production of 1-propanol in S. cerevisiae YPH499 strains expressing an artificially engineered pathway from pyruvate to 2 KB. (Mj: M. jannaschii, Ec: E. coli, Cb: Clostridium beijerinckii)

Fig. 4

Production of 1-propanol in S. cerevisiae YPH499 strains expressing an artificially engineered pathway from pyruvate to 2 KB and overexpression of genes encoding the enzyme threonine dehydratase. (Mj: M. jannaschii, Ec: E. coli, Cb: Clostridium beijerinckii)

Fig. 5

Deletion of metabolic pathways competing with 1-propanol production in yeast. a 1-Propanol production in strains from a single gene deletion library of BY4741. b Comparison of BY4741 with BY4741ΔGLY1. c Comparison of YPH499 with YPH499ΔGLY1. (Mj: M. jannaschii, Ec: E. coli, Cb: Clostridium beijerinckii)

Fig. 6

Production of 1-propanol in S. cerevisiae YPH499ΔGLY1 strains with an artificially engineered pathway from pyruvate to 2 KB and overexpression of tdcB and genes encoding for threonine synthase. (Cb: Clostridium beijerinckii)

Fig. 7

Oxygen-limited fermentation of engineered YPH499ΔGLY1 strains. (Cb: Clostridium beijerinckii)