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.

We have engineered an industrial strain of the yeast, Candida tropicalis, for the efficient production of long-chain dicarboxylic acids, which are important raw materials for the chemical industry. By sequential disruption of the four genes encoding both isozymes of the acyl-CoA oxidase which catalyzes the first reaction in the beta-oxidation pathway, alkane and fatty acid substrates have been successfully redirected to the omega-oxidation pathway. Consequently, the conversion efficiency and chemical selectivity of their terminal oxidation to the corresponding dicarboxylic acids has been improved to 100 percent. The specific productivity of the bioconversion has been increased further by amplification of the cytochrome P450 monooxygenase and NADPH-cytochrome reductase genes encoding the rate-limiting omega-hydroxylase in the omega-oxidation pathway. The amplified strains demonstrated increased omega-hydroxylase activity and a 30% increase in productivity compared to the beta-oxidation-blocked strain in fermentations. The bioconversion is effective for the selective terminal oxidation of both saturated and unsaturated linear aliphatic substrates with chain-lengths ranging from 12 carbons to 22 carbons and also avoids the undesirable chain modifications associated with passage through the beta-oxidation pathway, such as unsaturation, hydroxylation, or chain shortening. It is now possible to efficiently produce a wide range of previously unavailable saturated and unsaturated dicarboxylic acids with a high degree of purity.

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.

A recently developed transformation system has been used to facilitate the sequential disruption of the Candida tropicalis chromosomal POX4 and POXS genes, encoding distinct isozymes of the acyl coenzyme A (acyl-CoA) oxidase which catalyzes the first reaction in the ,8-oxidation pathway. The URA3-based transformation system was repeatedly regenerated by restoring the uracil requirement to transformed strains and POXS genes had been disrupted confirmed that all functional acyl-CoA oxidase genes had been inactivated. This strain cannot utilize alkanes or fatty acids for growth, indicating that the "-oxidation pathway has been functionally blocked.Peroxisomes represent a class of organelles that are ubiquitous among eukaryotic organisms and are involved in a variety of metabolic processes such as ,8-oxidation and the degradation of hydrogen peroxide. The importance of these organelles to cellular metabolism has been made clear by the recent discovery of a new class of inherited peroxisomal disorders such as Zellweger syndrome, which has been reported to be due to a recessive mutation that abolishes the import of peroxisomal matrix proteins (11). However, the molecular events leading to compartmentalization in eukaryotic cells and to the localization of peroxisomal proteins are still largely unknown and are the subject of intense study.The yeast Candida tropicalis is an important organism for the study of peroxisome biogenesis and the localization of peroxisomal proteins, since these organelles are rapidly and abundantly induced following growth on either alkane or fatty acid substrates (10). At least 18 different peroxisomal proteins have been detected in oleic acid-induced cells, and several of these have been identified as enzymes associated with the 1-oxidation pathway (4). Localization of acyl coenzyme A (acyl-CoA) oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase activities has shown that 13-oxidation takes place solely in the peroxisomes of this yeast (14). Peroxisomal acyl-CoA oxidase is an octomeric flavoprotein (molecular weight 600,000) which catalyzes the first reaction in the 1-oxidation pathway by the stoichiometric conversion of acyl-CoA to enoyl-CoA for substrates with chain lengths from 4 to 20 carbons (12). The genes encoding the subunits of distinct acyl-CoA oxidase isozymes (7-9), designated POX4 and POX5, have been cloned and sequenced. These genes encode polypeptides (designated PXP4 and PXP5) composed of 708 and 661 amino acids, respectively, following the * Corresponding author.removal of the initiating methionine during maturation (8). Since these polypeptides were found to share 63% homology, it was suggested that the two acyl-CoA oxidase isozymes arose by a gene duplication event followed by a localized exchange or replacement of functional domains. However, the functional differences between these isozymes, if any, have not been determined. A third member of the acyl-CoA oxidase gene family has been identified on the basis of sequence homology as P...

A recently developed transformation system has been used to facilitate the sequential disruption of the Candida tropicalis chromosomal POX4 and POX5 genes, encoding distinct isozymes of the acyl coenzyme A (acyl-CoA) oxidase which catalyzes the first reaction in the beta-oxidation pathway. The URA3-based transformation system was repeatedly regenerated by restoring the uracil requirement to transformed strains, either through selection for spontaneous mutations or by directed deletion within the URA 3 coding sequence, to permit sequential gene disruptions within a single strain of C. tropicalis. These gene disruptions revealed the diploid nature of this alkane- and fatty acid-utilizing yeast by showing that it contains two copies of each gene. A comparison of mutants in which both POX4 or both POX5 genes were disrupted revealed that the two isozymes were differentially regulated and displayed unique substrate profiles and kinetic properties. POX4 was constitutively expressed during growth on glucose and was strongly induced by either dodecane or methyl laurate and to a greater extent than POX5, which was induced primarily by dodecane. The POX4-encoded isozyme demonstrated a broad substrate spectrum in comparison with the narrow-spectrum, long-chain oxidase encoded by POX5. The absence of detectable acyl-CoA oxidase activity in the strain in which all POX4 and POX5 genes had been disrupted confirmed that all functional acyl-CoA oxidase genes had been inactivated. This strain cannot utilize alkanes or fatty acids for growth, indicating that the beta-oxidation pathway has been functionally blocked.