Like E. coli, Klebsiella oxytoca is able to metabolize a variety of biomass — derived monomeric sugars, but unlike E. coli it also has the native ability to transport and metabolize cellulose subunits cellobiose and cellotriose [50, 51]. The PET operon was expressed in K. oxytoca concurrent with the ori­ginal E. coli work [52] and was later chromosomally integrated, resulting in strain P2 [51]. Ethanol production by K. oxytoca P2 from various substrates has been reported (Table 1) [53-56]. K. oxytoca strain BW21, which was de­rived from strain P2 by elimination of the butanediol pathway, produces over 40 g L-1 ethanol in 48 h in OUM1 medium. OUM1 is a medium designed spe­cifically for K. oxytoca, as described below [57].

When Z. mobilis was selected as the source of the PET operon, Z. mo — bilis and Gram-positive Sarcina ventriculi were the only known bacteria with PDC activity [58-61]. Since that time, PDC activity has been identified in other bacteria, including Gram-negative Acetobacter pasteurianus [62] and Zymobacter palmae [3]. The Z. palmae PDC has a higher specificity and lower pyruvate Km than Z. mobilis, S. ventriculi, and A. pasteurianus [63]. The S. ventriculi PDC is different from the Z. mobilis enzyme but is highly related to the PDC found in fungi; its expression in E. coli requires the presence of accessory tRNA due to differences in codon usage [63]. In A. pasteurianus, PDC seems to have the unusual role of functioning in an aerobic pathway, contributing primarily to the conversion of pyruvate to acetaldehyde [62].

The robustness of Gram-positive organisms is appealing for industrial ap­plications, but initial attempts to express the Z. mobilis homoethanol pathway in Bacillus and lactic acid bacteria had limited success [64-67]. However, the discovery of new PDC forms has enabled renewed engineering attempts. The PDCs from S. ventriculi, A. pasteurianus, and Z. mobilis, as well as S. cerevisiae, were each expressed in Bacillus megaterium, with the S. ven — triculi PDC showing the highest activity. When coupled with ADH from Geobacillus stearothermophilus, the S. ventriculi PDC enabled B. megaterium to convert 13.2 gL-1 pyruvate to 3.3 gL-1 ethanol, a tenfold increase rela­tive to strains lacking PDC [68]. The S. ventriculi PDC was also expressed in Lactobacillus plantarum, with production of up to 6 g L-1 ethanol from 40gL-1 glucose [69]. Recent attempts to express the Z. mobilis pathway in Corynebacterium glutamicum have resulted in production of ethanol from glucose [70].

Considerable effort has been extended to engineering of Z. mobilis for im­proved ethanol production, as covered elsewhere in this volume. However, the ethanol titers attained by ethanologenic E. coli LY168 in minimal medium exceed published values for Z. mobilis in rich medium (Table 2).

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