The ethanol-producing bacterium Zymomonas mobilis was metabolically engineered to broaden its range of fermentable substrates to include the pentose sugar xylose. Two operons encoding xylose assimilation and pentose phosphate pathway enzymes were constructed and transformed into Z. mobilis in order to generate a strain that grew on xylose and efficiently fermented it to ethanol. Thus, anaerobic fermentation of a pentose sugar to ethanol was achieved through a combination of the pentose phosphate and Entner-Doudoroff pathways. Furthermore, this strain efficiently fermented both glucose and xylose, which is essential for economical conversion of lignocellulosic biomass to ethanol.

Efficient conversion of lignocellulosic biomass requires biocatalysts able to tolerate inhibitors produced by many pretreatment processes. Recombinant Zymomonas mobilis 8b, a recently developed integrant of Zymomonas mobilis 31821(pZB5), tolerated acetic acid up to 16 g l(-1) and achieved 82%-87% (w/w) ethanol yields from pure glucose/xylose solutions at pH 6 and temperatures of 30 degrees C and 37 degrees C. An ethanol yield of 85% (w/w) was achieved on glucose/xylose from hydrolysate produced by dilute sulfuric acid pretreatment of corn stover after an overliming' process was used to improve hydrolysate fermentability.

The substrate fermentation range of the ethanologenic bacterium Zymomonas mobilis was expanded to include the pentose sugar, L-arabinose, which is commonly found in agricultural residues and other lignocellulosic biomass. Five genes, encoding L-arabinose isomerase (araA), L-ribulokinase (araB), L-ribulose-5-phosphate-4-epimerase (araD), transaldolase (talB), and transketolase (tktA), were isolated from Escherichia coli and introduced into Z. mobilis under the control of constitutive promoters that permitted their expression even in the presence of glucose. The engineered strain grew on and produced ethanol from L-arabinose as a sole C source at 98% of the maximum theoretical ethanol yield, based on the amount of consumed sugar. This indicates that arabinose was metabolized almost exclusively to ethanol as the sole fermentation product, with little by-product formation. Although no diauxic growth pattern was evident, the microorganism preferentially utilized glucose before arabinose, apparently reflecting the specificity of the indigenous facilitated diffusion transport system. This microorganism may be useful, along with the previously developed xylose-fermenting Z. mobilis (M. Zhang, C. Eddy, K. Deanda, M. Finkelstein, and S. Picataggio, Science 267:240-243, 1995), in a mixed culture for efficient fermentation of the predominant hexose and pentose sugars in agricultural residues and other lignocellulosic feedstocks to ethanol.

The Zymomonas mobilis gene encoding acid phosphatase, phoC, has been cloned and sequenced. The gene spans 792 base pairs and encodes an Mr 28,988 polypeptide. This protein was identified as the principal acid phosphatase activity in Z. mobilis by using zymograms and was more active with magnesium ions than with zinc ions. Its promoter region was similar to the -35 "pho box" region of the Escherichia coli pho genes as well as the regulatory sequences for Saccharomyces cerevisiae acid phosphatase (PHO5). A comparison of the gene structure of phoC with that of highly expressed Z. mobilis genes revealed that promoters for all genes were similar in degree of conservation of spacing and identity with the proposed Z. mobilis consensus sequence in the -10 region. The phoC gene contained a 5' transcribed terminus which was AT rich, a weak ribosome-binding site, and less biased codon usage than the highly expressed Z. mobilis genes.

Zymomonas mobilis is an unusual microorganism which utilizes both iron-containing alcohol dehydrogenase (ADHII) and zinc-containing alcohol dehydrogenase (ADHI) isoenzymes during fermentative growth. This organism is obligately ethanologenic, and alcohol dehydrogenase activity is essential. The activities of ADHI and ADHII were altered by supplementing growth medium with iron or zinc salts and by iron starvation. Growth under iron-limiting conditions (chelators, minimal medium) reduced ADHII activity but did not prevent the synthesis of the ADHII protein. The inactive form of this enzyme appeared quite stable, was not renatured by iron addition, and persisted in the cell. The iron-induced increase in ADHII activity required de novo synthesis which was blocked by antibiotic additions. The ability of Z. mobilis to synthesize ADHII and ADHI may be advantageous in nature.Zymomonas mobilis is an obligately fermentative, gramnegative bacterium in which substrate-level phosphorylation from glycolysis provides the sole source of energy (16). In this organism, ethanol production from pyruvate is the dominant route for NADH oxidation, leading to the conversion of 95% of the metabolized sugar to ethanol and carbon dioxide. The ethanologenic pathway in Z. mobilis is identical to that in Saccharomyces cerevisiae and consists of two essential activities, pyruvate decarboxylase and alcohol dehydrogenase (ADH).ADH activity is typically present as multiple isoenzymes in procaryotes (1,24) and eucaryotes (3, 9). Two isoenzymes of ADH have been identified in Z. mobilis (22). ADHI is a zinc enzyme (5) and appears similar in many respects to the alcohol dehydrogenases of S. cerevisiae (15, 23), fungi (10), and plants (2, 9). ADHII is very unusual and does not contain zinc (17)(18)(19). This enzyme has been variously reported as either a dimer (8) or a tetramer (17) of identical subunits with either iron (17)(18)(19) or cobalt (8) as the bound transition metal.The gene encoding Z. mobilis ADHII has been cloned, sequenced, and expressed in Escherichia coli as a part of a recombinant pathway for alcohol production (6,7). On the basis of amino acid sequence similarities, this gene (4) In this study, we have investigated the effects of added metals (primarily zinc and iron) and iron depletion by chelators on the abundance of ADH isoenzymes in Z.mobilis. MATERIALS AND METHODSOrganism and growth conditions. Z. mobilis CP4 (4) was maintained on complex solid medium containing 1.5% agar, 2% glucose, 1.0% yeast extract, and 0.03% monobasic potassium phosphate. Broth cultures were grown in a similar complex medium containing 10% glucose and 0.5% yeast extract or in minimal medium containing 10% glucose, 0.05% sodium chloride, 0.05% magnesium sulfate, and 0.1% each ammonium sulfate, dibasic potassium phosphate, and monobasic potassium phosphate. A mixture of filter-sterilized vitamins was also added to produce a final concentration of 5 mg of calcium pantothenate and 1 mg each of thiamine hydrochloride, pyridoxine hydrochloride, biotin, and nic...