The rising cost of petroleum along with human dependency on crude-oil imports has opened up an opportunity for chemicals and bio-based fuels to become economically competitive. With new technologies developing on one side, petrochemicals and petroleum-based automotive fuels are effectively replaced with plant and plant-derived materials. This way the supplementation of national energy supply using sustainable resources has become feasible. Today, nearly 65 percent of oil consumed in the United States is imported. Approximately, in 2005, 211 billion gallons were burned as automotive fuel. This has urged the need for the development of an alternative and a renewable transportation fuel (example, ethanol) to reduce the imported-oil dependency of the US thereby contributing to the preservation of natural resources.

Brazil was first country to successfully implement the use of sugar-derived ethanol as the primary component of automotive fuel nearly 3 decades ago. In 2006, the United States produced around 4.9 billion gallons of ethanol fuel. Lignocellulosic materials provided the opportunity to expand ethanol production. Lignocellulosic is a complex substance accounting for 90 percent of dry weight of plant material. It is considered the most abundant renewable-energy source in the world today and is made up of cell-wall structural polymers (lignin, hemicellulose, pectin, and cellulose).
Due to the complex nature of lignocellulose and the biological limitations of catalysts existing today, the process designs (conceptual) for lignocellulose-based ethanol production is complicated than starch-based process. A microbial catalyst capable of metabolizing lignocellulose and the constitutive sugars is expected to simplify the production process and reduce the ethanol production cost.

For years, Saccharomyces has acted as the primary biocatalyst for ethanol production on commercial level. Since both Z.mobilis and Saccharomyces are naturally ethanologenic, these organisms are clear candidates for ethanol production. However, both these organisms do not have the ability to utilize pentose sugars, the main component the biomass hemicellulose fraction. E.coli also does not have the ability to produce ethanol as the main formation product but can utilize both pentose and hexose sugars and pectin's uronic acid constituents.

Performance and Engineering of Ethanologenic E.coli
Ethanologenic E.coli includes a combination of metabolic evolution and directed engineering. The Z.mobilis homoethanol pathway, also called PET Operon, was introduced into E.coli in the plasmids and the derivatives produced ethanol as the cardinal fermentation product. The PET Operon is integrated into the chromosome in a stable way at pfl locus with an antibiotic-resistance marker. Spontaneous high antibiotic resistance and mutants exhibiting high-ADH activity were specifically selected ensuring high PET activity. Side reactions, which tend to drain carbon from ethanol, are eliminated either physiologically or by mutation.

Ethanologenic Biocatalyst KO11
The resulting strain called KO11 produces ethanol at 95 percent yield in the complex media. Though, it was initially reported that strain KO11 was derived from E.coli B, it was recently found out that the real parental strain is E.coli W. While, ethanol production by KO11 is high as in yeast, the tolerance level of ethanol is comparatively low in commercially employed yeast strains. In a complex media, strain KO11 exhibits complete absence of growth in the presence of 35gL-1 ethanol and only 30 percent survival chance from exposure 30 s to 100gL-1 ethanol.

Engineering Scheme
Strain KO11 is used as the starting point, mutant strains that significantly increased the tolerance level of ethanol were isolated. This is 3-month metabolic evolution process and consists of alternating selection periods in a liquid media for increased ethanol tolerance and solid media selection for increased ethanol production. The final product yielded at the end this evolution is the strain LY01 that grows in the presence of 50gL-1 ethanol with 80 percent survival chance from exposure 30 s to 100gL-1 ethanol.

Utilizing Substrates
The utilization of KO11 for ethanol production from biomass has had multiple demonstrations with numerous substrates including agricultural residues, rice hulls, hulls, sugarcane bagasse, corn-cobs, AFEX-pretreated fibers, willow, sweet whey, cotton gin waste, brewery waste, orange peel, and Pinus sp. hydrosylate. Strain KO11 is robust to changes in pH and temperature.
While ethanol yields obtained from xylose and glucose differ in transport mechanisms resulting in lower ATP yield for xylose. Both LY01 and KO11 grow 50 percent faster and produce about three times much ATP from glucose to xylose.

The metabolic evolution of LY01 and KO11 has proved to be great success and have been applied to other ethanologenic biocatalyst designs and in the production of commercial commodities.

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