Improved Production of Corn Ethanol

The production of ethanol from corn or grain starch is mature technology that was the target of early genetic engineering enzyme production in the early 1980s with the cloning and expression of the key enzymes required for glucose production. For example, α-amylase, glucoamylase and other accessory enzymes such as pullulanase and isomaltase needed for cleavage of α-1,4- and α-1,6-glyceride linkages in starch and elimination of reversion sugars were genetically cloned and made commercially available. Production of many of these enzymes proceeded through classical protein engineering improvement strategies to improve thermostability, pH optimum/stability and specific activity in conjunction with genetic and process improvements to improve the volumetric productivity of the enzyme fermentation. Improvements in characteristics and production levels of these enzymes have expanded applications, but the largest markets for enzymes, such as sweetener and detergent enzymes, has become a commodity business. This has made further research for incremental improvements difficult to support.
The fermentation microorganism Saccharomyces cerevisiae is a highly productive and efficient producer of ethanol from glucose within both the wet and dry mill processes. However, opportunities remain for process improvement using new approaches to analyze the metabolic
limitations that may arise. Fundamental analysis of the S. cerevisiae genome has been investigated for laboratory fermentation. In addition, this technology has been applied in the beer and wine industry with the temporal analysis of the fermentation of simple sugars by S. cerevisiae. Interesting differences have been detected among wine yeast strains that ferment primarily glucose and fructose from wine grapes Vitis vinifera L. For example. microarray analysis of natural vineyard populations of S. cerevisiae found significant differences in amino acid expressions, especially methionine, which showed that the natural population can vary significantly in gene expression due to segregation patterns of relatively few genes. These differences presumably may impact flavor components in the fermentation broth, in this case wine. In addition, transcriptomic analysis has been used to determine the impact of high ethanol levels routinely reached during fermentation, especially with wine and champagne yeast where ethanol tolerance is required to meet product specifications. A more holistic approach has been taken for beer production where the yeast must ferment hydrolyzed starch from added grains along with supplemented sugar. Distinct differences detected in the proteome of ale versus lager beer yeast have allowed researchers to conclude that the yeast involved has undergone significant interspecies hybridization and evolution as a ‘genetic tree’ can be derived with selected yeast. To some extent it is encouraging that very traditional, long-standing industries such as wine and beer manufacturing have already applied the tools of systems biology to their craft, yielding potentially beneficial insights into product improvement.
Among all yeast fermentations, glycerol has long been accepted as an expected co-product of ethanol production, which must be removed during processing, especially for the fuel alcohol industry. Significant progress has been made in understanding why glycerol is produced, providing an avenue to strain improvement. It has been determined that glycerol is produced or induced as an osmotic stress response. Indeed, mutant analysis has made progress in identifying specific mutant changes that impact glycerol production, permitting strategies for reduction of glycerol production including strategies to limit substrate, modify acid/base additions or other known engineering conditions present in commercial fermenters.Further research is needed as additional S. cerevisiae glycerol mutants are produced. Through rapid sampling and processing, the yeast RNA can be acquired and processed for gene expression analysis, for example, providing information on the gene expression under way even from industrial-sized fermenters. This information, coupled with closely monitored fermentation broth ethanol, glycerol and other byproducts (external metabolome), will provide information on the fermentation temporal changes permitting engineering and possible genetic engineering solutions as needed. However,  such investigations must be limited to on-going inefficiencies or difficulties that impose a sufficient economic burden to warrant research time and costs.