Increasing energy costs and environmental concerns have emphasized the need to produce sustainable renewable fuels and chemicals. Major efforts to this end are focused on the microbial production of high-energy fuels by cost-effective 'consolidated bioprocesses'. Fatty acids are composed of long alkyl chains and represent nature's 'petroleum', being a primary metabolite used by cells for both chemical and energy storage functions. These energy-rich molecules are today isolated from plant and animal oils for a diverse set of products ranging from fuels to oleochemicals. A more scalable, controllable and economic route to this important class of chemicals would be through the microbial conversion of renewable feedstocks, such as biomass-derived carbohydrates. Here we demonstrate the engineering of Escherichia coli to produce structurally tailored fatty esters (biodiesel), fatty alcohols, and waxes directly from simple sugars. Furthermore, we show engineering of the biodiesel-producing cells to express hemicellulases, a step towards producing these compounds directly from hemicellulose, a major component of plant-derived biomass.

The major regulator controlling the physiological switch between aerobic and anaerobic growth conditions in Escherichia coli is the DNA binding protein FNR. To identify genes controlled by FNR, we used Affymetrix Antisense GeneChips to compare global gene expression profiles from isogenic MG1655 wild-type and ⌬fnr strains grown in glucose minimal media under aerobic or anaerobic conditions. We found that 297 genes contained within 184 operons were regulated by FNR and/or by O 2 levels. The expression of many genes known to be involved in anaerobic respiration and fermentation was increased under anaerobic growth conditions, while that of genes involved in aerobic respiration and the tricarboxylic acid cycle were repressed as expected. The expression of nine operons associated with acid resistance was also increased under anaerobic growth conditions, which may reflect the production of acidic fermentation products. Ninety-one genes with no presently defined function were also altered in expression, including seven of the most highly anaerobically induced genes, six of which we found to be directly regulated by FNR. Classification of the 297 genes into eight groups by k-means clustering analysis indicated that genes with common gene expression patterns also had a strong functional relationship, providing clues for studying the function of unknown genes in each group. Six of the eight groups showed regulation by FNR; while some expression groups represent genes that are simply activated or repressed by FNR, others, such as those encoding functions for chemotaxis and motility, showed a more complex pattern of regulation. A computer search for FNR DNA binding sites within predicted promoter regions identified 63 new sites for 54 genes. We suggest that E. coli MG1655 has a larger metabolic potential under anaerobic conditions than has been previously recognized.

A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome. The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the Red proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn5 transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a lacZ allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn5 complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn5 transposition.

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