homologous recombination-based gene disruption. In addition, rapid sequencing of breeding populations of energy crops can enable marker-assisted selection to accelerate the breeding of energy crops in ways previously not possible. And the rapid and inexpensive sequencing of fermentative and photosynthetic microorganisms in particular is redefining and shortening the timelines associated with strain-development programs for converting sugars, lignocellulosic materials, and CO2 into alternative liquid fuels.

Strains generated through classical mutagenesis that have improved biocatalytic properties can now be analyzed at the molecular level to determine the specific genetic changes that result in the improved phenotype, allowing those changes to be implemented in other strains. In addition, “metagenome” sequence data, obtained by randomly sequencing DNA isolated from environmental samples, are providing vast numbers of new gene sequences that can be used to genetically engineer improved crops and microorganisms.

Synthetic Biology

Improved technologies for synthesizing megabase DNA molecules are being developed that will allow the introduction of entirely new biochemical pathways into energy crops and biofuel-producing microorganisms. These technologies could have a great impact on scientists’ ability to generate plants and microorganisms with desired traits. For example, it is becoming conceivable that large portions of microorganisms’ chromosomes, or even their complete chromosomes, can be replaced in ways that focus most of the cells’ biochemical machinery on producing “next-generation” biofuel molecules boasting both cost and product advantages. Significant hurdles, however, could occur in maintaining the purity of such cultures and in dealing with mutants that gain competitive advantage by producing less of the desired chemicals.

Metabolic and Bioprocess Engineering

In addition to genetic manipulation, new bioengineering technologies are coming on line that will lower the cost of biofuel formation and recovery. While synthetic biology can now provide synthetic DNA for transferring heterologous genes into suitable host cells, metabolic engineering is the enabling technology for constructing functional and even optimal pathways for microbial fuel biosynthesis. This field has matured in only a few years and has an impressive record of accomplishments, many already in industrial practice (for example, biopolymers, alcohols,



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