A novel pathway to produce butanol and isobutanol in Saccharomyces cerevisiae

Abstract

Background: The sustainable production of biofuels remains one of the major issues of the upcoming years. Among the number of most desirable molecules to be produced, butanol and isobutanol deserve a prominent place. They have superior liquid-fuel features in respect to ethanol. Particularly, butanol has similar properties to gasoline and thus it has the potential to be used as a substitute for gasoline in currently running engines. Clostridia are recognized as natural and good butanol producers and are employed in the industrial-scale production of solvents. Due to their complex metabolic characteristics and to the difficulty of performing genetic manipulations, in recent years the Clostridia butanol pathway was expressed in other microorganisms such as Escherichia coli and Saccharomyces cerevisiae, but in yeast the obtained results were not so promising. An alternative way for producing fusel alcohol is to exploit the degradation pathway of aminoacids released from protein hydrolysis, where proteins derive from exhausted microbial biomasses at the end of the fermentation processes.

Results: It is known that wine yeasts can, at the end of the fermentation process, accumulate fusel alcohols, and butanol is among them. Despite it was quite obvious to correlate said production with aminoacid degradation, a putative native pathway was never proposed. Starting from literature data and combining information about different organisms, here we demonstrate how glycine can be the substrate for butanol and isobutanol production, individuating at least one gene encoding for the necessary activities leading to butanol accumulation. During a kinetic of growth using glycine as substrate, butanol and isobutanol accumulate in the medium up to 92 and 58 mg/L, respectively.

Conclusions: Here for the first time we demonstrate an alternative metabolic pathway for butanol and isobutanol production in the yeast S. cerevisiae, using glycine as a substrate. Doors are now opened for a number of optimizations, also considering that starting from an aminoacid mixture as a side stream process, a fusel alcohol blend can be generated.

Figure 1

A novel pathway for butanol and isobutanol production. Metabolic pathway for butanol and isobutanol production from glycine in S. cerevisiae through the glyoxylate, β-ethylmalate and α-ketoacids intermediates (grey background arrows). The enzymatic activities involved and the associated gene(s) are also represented. Numbers inside circles indicates the steps of the pathway discussed in this study. The hypothesized isomerisation of α-ketovalerate into α-isoketovalerate is indicated by black arrow.

Figure 2

Butanol and isobutanol accumulation from glycine. Yeast cells were grown in Verduyn medium with ammonium sulphate (5 g/L, left panel) or glycine (15 g/L, right panel) as nitrogen source. Biomass accumulation (square), glycine consumption (triangle), butanol (circle) and isobutanol (diamond) productions are shown. The data presented here are representative of three independent experiments.

Figure 3

Glycine oxidase: reaction and enzymatic activity assay. (A) Reaction catalysed by glycine oxidase. The carbons of the molecules were numbered. (B) Glycine oxidase activity (upper bars), butanol (dark gray columns) and isobutanol (pale gray columns) for the wild type and modified yeast strains overexpressing the bacterial goxB gene optimized for the yeast codon usage. The data presented here are representative of three independent experiments.

Figure 4

Malate synthase activity and glyoxylate bioconversion into butanol and isobutanol. (A) Glyoxylate conversion reaction performed by malate synthase enzyme in the presence of butyryl-CoA. The carbons of the molecules were numbered. (B) Malate synthase activity for the glyoxylate conversion into β-ethylmalate. Left panel: malate synthase activity was assayed using acetyl-CoA or butyryl-CoA as donor group. Right panel: effect of MLS1 deletion on the enzymatic activity. The data presented here are representative of three independent experiments. p ≤ 0,05 = *; p ≤ 0,01 = **; p ≤ 0,001 = ***; p > 0,05 = n.s. (C) Glyoxylate bioconversion. Butanol (full columns) and isobutanol (empty columns) production as well as glyoxylate consumption (triangle) measured at different time point. The data presented here are representative of three independent experiments.

Figure 5

The β-isopropylmalate dehydrogenase involvement in the glycine degradation pathway. The β-isopropylmalate dehydrogenase activity was tested in a coupled reaction using glyoxylate as substrate instead of β-ethylmalate (not commercial available). The carbons of the molecules were numbered. (A) Reaction catalysed by the β-isopropylmalate dehydrogenase enzyme. (B) Activity of β-isopropylmalate dehydrogenase measured in yeast strain ΔLEU2 (empty column) and overexpressing LEU2 gene (grey column). The data presented here are representative of three independent experiments. p ≤ 0,05 = *; p ≤ 0,01 = **; p ≤ 0,001 = ***; p > 0,05 = n.s.

Figure 6

Pyruvate decarboxylase activity and α-ketoacids bioconversion into butanol and isobutanol. (A) α-ketovalerate and α-isoketovalerate conversion reaction performed by pyruvate decarboxylase enzyme. The carbons of the molecules were numbered. (B) Pyruvate decarboxylase deletion effect on butanol and isobutanol accumulation using α-ketovalerate as substrate. Wild type (empty column) and PCD1, 5, 6 deleted strains (full column). The data presented here are representative of three independent experiments. p ≤ 0,05 = *; p ≤ 0,01 = **; p ≤ 0,001 = ***; p > 0,05 = n.s. (C) α-Ketovalerate (left panel) and α-isoketovalerate (right panel) bioconversion. Butanol (dark grey), isobutanol (white) and α-ketovalerate consumption (triangle) were reported. For α-isoketovalerate (triangle), the sole estimated initial concentration is given. The data presented here are representative of three independent experiments.

Figure 7

The evidence of glyoxylate degradation through the Mls, β-IPMD and Pdc(s) activities. The MLS1 deletion effect was evaluated coupled with the LEU2 deletion or overexpression (respectively indicated as ΔMLS1ΔLEU2 and ΔMLS1 LEU2). The data presented here are representative of three independent experiments. p ≤ 0,05 = *; p ≤ 0,01 = **; p ≤ 0,001 = ***; p > 0,05 = n.s.