In order to create truly sustainable biofuels, researchers are investigating methods to produce bioethanol from waste plant dry matter, such as wood and straw, known as lignocellulosic feedstocks. The industrial yeast (S. cerevisiae) already used to make bioethanol from glucose-rich crops, such as maize and sugar cane, is not efficient at fermenting the pentose sugars (D-xylose and L-arabinose) found in waste lignocellulosic feedstocks. The woody parts of plants are also resistant to enzymatic degradation and require thermochemical pretreatment to release fermentable sugars. This additional step has the undesirable side effect of producing inhibitors to downstream enzymatic conversions. In a recent study published in Biotechnology for Biofuels, Johan Thevelein from the University of Leuven, Belgium, and colleagues tackled these challenges by creating a transgenic strain of industrial yeast.
Using an evolutionary engineering strategy the researchers were able to improve the yeasts ability to convert D-xylose to ethanol. Transgenic yeast strains have already been created to ferment pentose sugars, by inserting genes for enzymes from fungi and bacteria. However, these haploid laboratory strains do not translate well to an industrial setting, being less robust in large scale batch fermentation than diploid commercial yeast strains.
Thevelein and colleagues engineered novel metabolic properties into the Ethanol red industrial yeast strain, by inserting two expression cassettes containing genes involved in pentose and arabinose metabolism. However this new strain was only able to ferment pentose sugars at a low level. To improve this trait, the parent recombinant strain was encouraged to diversify by chemical mutagenesis and the resulting clones selected for survival on a D-xylose substrate. The genome of the parent strain was then shuffled by mating with a selected pair of the new D-xylose surviving clones. The resulting population, now enriched for desirable mutations, was then serially cultured to further optimise D-xylose conversion and survival, under biofuel production conditions.
Seven generations later and a dominant strain emerged. GS1.11-26 consumes D-xylose over eight fold faster than the parental strain. A major caveat of evolutionary engineering approaches is that beneficial mutations are unlikely to occur. However, the authors demonstrated that overexpression alone of the D-xylose isomerase transgene XI, which is involved in the conversion of D-xylose, was unable to produce the same metabolic advantage as that found in the evolved strain. This suggested that at least one or two novel mutations had occurred in the genome of GS1.11-26.
The evolved strain also showed enhanced survival against inhibitors that were produced specifically in the environment of the chosen selection medium used during fermentation, indicating that the serial culture process highly adapts the organism to the specific fermentation niche used. Deleterious mutations did however also arise; the evolved strain was less able to grow in aerobic conditions than the parent strain. Nonetheless, overall GS1.11-26 presents a robust yeast strain that is capable of complete fermentation of D-xylose from waste softwood. Further development of this strain may prove beneficial to industry.