Successful design and development of genetically engineered Saccharomyces yeasts for effective cofermentation of glucose and xylose from cellulosic biomass to fuel ethanol.

Ethanol is an effective, environmentally friendly, nonfossil, transportation biofuel that produces far less pollution than gasoline. Furthermore, ethanol can be produced from plentiful, domestically available, renewable, cellulosic biomass. However, cellulosic biomass contains two major sugars, glucose and xylose, and a major obstacle in this process is that Saccharomyces yeasts, traditionally used and still the only microorganisms currently used for large scale industrial production of ethanol from glucose, are unable to ferment xylose to ethanol. This makes the use of these safest, most effective Saccharomyces yeasts for conversion of biomass to ethanol economically unfeasible. Since 1980, scientists worldwide have actively been trying to develop genetically engineered Saccharomyces yeasts to ferment xylose. In 1993, we achieved a historic breakthrough to succeed in the development of the first genetically engineered Saccharomyces yeasts that can effectively ferment both glucose and xylose to ethanol. This was accomplished by carefully redesigning the yeast metabolic pathway for fermenting xylose to ethanol, including cloning three xylose-metabolizing genes, modifying the genetic systems controlling gene expression, changing the dynamics of the carbon flow, etc. As a result, our recombinant yeasts not only can effectively ferment both glucose and xylose to ethanol when these sugars are present separately in the medium, but also can effectively coferment both glucose and xylose present in the same medium simultaneously to ethanol. This has made it possible because we have genetically engineered the Saccharomyces yeasts as such that they are able to overcome some of the natural barrier present in all microorganisms, such as the synthesis of the xylose metabolizing enzymes not to be affected by the presence of glucose and by the absence of xylose in the medium. This first generation of genetically engineered glucose-xylose-cofermenting Saccharomyces yeasts relies on the presence of a high-copy-number 2 mu-based plasmid that contains the three cloned genetically modified xylose-metabolizing genes to provide the xylose-metabolizing capability. In 1995, we achieved another breakthrough by creating the super-stable genetically engineered glucose-xylose-cofermenting Saccharomyces yeasts which contain multiple copies of the same three xylose-metabolizing genes stably integrated on the yeast chromosome. This is another critical development which has made it possible for the genetically engineered yeasts to be effective for cofermenting glucose and xylose by continuous fermentation. It is widely believed that the successful development of the stable glucose-xylose-cofermenting Saccharomyces yeasts has made the biomass-to-ethanol technology a step much closer to commercialization. In this paper, we present an overview of our rationales and strategies as well as our methods and approaches that led to the ingenious design and successful development of our genetically engineered Saccharomyces yeasts for effective cofermentation of glucose and xylose to biofuel ethanol.