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Researchers significantly boost yield of isobutanol from engineered yeast using new synthesis pathway located in the cytosol
6 November 2012
A team at the Institute of Molecular Biosciences, Goethe-University Frankfurt led by Prof. Dr. Eckhard Boles, has developed a new synthesis pathway for engineering the industrial yeast Saccharomyces cerevisiae to improve the production of isobutanol via fermentation. The work, noted Boles, is being done for the Swiss biofuels and biochemical company Butalco, of which he is a co-founder. (Earlier post.)
In an open access paper published in the journal Biotechnology for Biofuels, the team reported achieving a titer of more than 630 mg/L isobutanol with a yield of nearly 15 mg/g glucose. The highest values reported before for recombinant S. cerevisiae were about 150 mg/L isobutanol and a yield of 6.6 mg/g glucose, they noted. Additional engineering should lead to even higher isobutanol production, they suggested.
Yeast naturally produces low amounts of isobutanol as a by-product of fermentation. Yeast can synthesize isobutanol via a three-step catalytic breakdown of valine (the Ehrlich pathway) through which valine undergoes transamination to 2-ketoisovalerate (KIV) catalyzed by branched-chain amino acid aminotransferase (Bat2). The subsequent decarboxylation and reduction of KIV to isobutanol is catalyzed by ketoacid decarboxylase (KDC) and alcohol dehydrogenase (ADH) with isobutyraldehyde as an intermediate.
The coupling of valine biosynthetic enzymes with valine degrading enzymes via the common intermediate KIV would result in a direct isobutanol synthesis pathway. Such a strategy could be successfully transferred into different bacterial microorganisms. In various recent publications, the metabolic flux towards isobutanol production was improved by overexpressing endogenous or heterologous genes of valine synthesis and degradation. E.g., engineered recombinant E. coli strains were able to produce more than 20 g/L isobutanol, whereby isobutanol amounts could be further enhanced up to 50 g/L by using a 1 L bioreactor connected to a gas-stripping system. Production of isobutanol with Bacillus subtilis and Corynebacterium glutamicum could be achieved up to 2.62 g/L and 4.9 g/L, respectively.
One of the major problems of most bacterial host organisms in large production processes is their low tolerance towards fermentation inhibitors and to isobutanol. The yeast S. cerevisiae seems to be more promising as a host for isobutanol production.—Brat et al.
Unlike in bacteria, the researchers noted, in S. cerevisiae the anabolic reactions providing KIV are separated from the catabolic reactions producing isobutanol. The anabolic reactions—part of valine biosynthesis—are located in the mitochondrial matrix; the Ehrlich pathway reactions take place in the cytosol—the fluid component of cytoplasm, excluding organelles and the insoluble, usually suspended, cytoplasmic components.
Brat et al. hypothesized that putting all the enzymes in the same compartment would increase the production of isobutanol—using the cytosol as the location of the new isobutanol synthesis pathway seemed promising. This would also avoid any transport of intermediates across intracellular membranes.
Accordingly, they relocated the enzymes of valine biosynthesis from the mitochondrial matrix into the cytosol.
They found that overexpression of cytosolically located enzymes did not significantly increase isobutanol production. However, eliminating the competing mitochondrial valine pathway together with omitting valine from the fermentation medium resulted in strongly increased isobutanol production.
In this work, we expressed a valine biosynthetic pathway from pyruvate to KIV in the cytosol of yeast cells. Simultaneous blocking of the mitochondrial pathway and omission of valine from the fermentation medium pushed and pulled pyruvate into and through the new pathway. Changing the ‘anabolic’ codon usage of valine synthesis genes into a ‘catabolic’ codon usage further improved flux through the new pathway. Overexpression of KDC and ADH activities increased the conversion of KIV to isobutanol. The highest measured isobutanol titer of 0.6 g/L represents the highest titer ever reported for recombinant S. cerevisiae.—Brat et al.
Dawid Brat, Christian Weber, Wolfram Lorenzen, Helge B Bode and Eckhard Boles (2012) Cytosolic re-localization and optimization of valine synthesis and catabolism enables increased isobutanol production with the yeast Saccharomyces cerevisiae Biotechnology for Biofuels 5:65 doi: 10.1186/1754-6834-5-65
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