Rapid and stable production of 2,3-butanediol by an engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain in a continuous airlift bioreactor

Utilization of renewable feedstocks for the production of bio-based bulk chemicals, such as 2,3-butanediol (2,3-BDO), by engineered strains of the non-pathogenic yeast, Saccharomyces cerevisiae, has recently become an attractive option. In this study, to realize rapid production of 2,3-BDO, a flocculent, 2,3-BDO-producing S. cerevisiae strain YPH499/dPdAdG/BDN6-10/FLO1 was constructed from a previously developed 2,3-BDO-producing strain. Continuous 2,3-BDO fermentation was carried out by the flocculent strain in an airlift bioreactor. The strain consumed more than 90 g/L of glucose, which corresponded to 90% of the input, and stably produced more than 30 g/L of 2,3-BDO over 380 h. The maximum 2,3-BDO productivity was 7.64 g/L/h at a dilution rate of 0.200/h, which was higher than the values achieved by continuous fermentation using pathogenic bacteria in the previous reports. These results demonstrate that continuous 2,3-BDO fermentation with flocculent 2,3-BDO-producing S. cerevisiae is a promising strategy for practical 2,3-BDO production.     

Biosynthesis of archaeal membrane lipids: digeranylgeranylglycerophospholipid reductase of the thermoacidophilic archaeon Thermoplasma acidophilum

The basic core structure of archaeal membrane lipids is 2,3-di-O-phytanyl-sn-glyceryl phosphate (archaetidic acid), which is formed by the reduction of 2,3-di-O-geranylgeranylglyceryl phosphate. The reductase activity for the key enzyme in membrane lipid biosynthesis, 2,3-digeranylgeranylglycerophospholipid reductase, was detected in a cell free extract of the thermoacidophilic archaeon Thermoplasma acidophilum. The reduction activity was found in the membrane fraction, and FAD and NADH were required for the activity. The reductase was purified from a cell free extract by ultracentrifugation and four chromatographic steps. The purified enzyme showed a single band at ca. 45 kDa on SDS-PAGE, and catalyzed the formation of archaetidic acid from 2,3-di-O-geranylgeranylglyceryl phosphate. Furthermore, the enzyme also catalyzed the reduction of 2,3-di-O-geranylgeranylglyceryl phosphate analogues such as 2,3-di-O-phytyl-sn-glyceryl phosphate, 3-O-(2,3-di-O-phytyl-sn-glycero-phospho)-sn-glycerol and 2,3-di-O-phytyl-sn-glycero-phosphoethanolamine. The N-terminal 20 amino acid sequence of the purified enzyme was determined and was found to be identical to the sequence encoded by the Ta0516m gene of the T. acidophilum genome. The present study clearly demonstrates that 2,3-digeranylgeranylglycerophospholipid reductase is a membrane associated protein and that the hydrogenation of each double bond of 2,3-digeranylgeranylglycerophospholipids is catalyzed by a single enzyme.     

抗麻痹性贝毒素GTX2,3单克隆抗体的制备及特性分析

制备抗麻痹性贝毒GTX2,3单克隆抗体。利用醛化法将GTX2,3与载体牛血清白蛋白(BSA)偶联,制备完全抗原。免疫小鼠,取小鼠脾细胞与Sp2/0细胞融合。GTX2,3与钥孔血蓝蛋白(KLH)偶联作为检测抗原,用间接ELISA法筛选阳性克隆株。将筛选的阳性细胞株制备腹水。获得三株稳定分泌抗GTX2,3单克隆抗体的杂交瘤细胞株F4、F10、G9。间接ELISA法检测F10细胞株腹水抗体效价为1.4×10^-5。半抗原GTX2,3与载体蛋白偶联后,作为免疫原,可制备高滴度的抗GTX2,3抗血清和单克隆抗体。该抗体对于藻毒素具有高特异性和高亲和力,可用于污染海产品的麻痹性贝毒的检测。     

Synthesis of Some New Nucleoside Analogues as Potential Antiviral Agents

Abstract

A novel series of pyrimidine nucleoside analogues was synthesized. 2,3-Dideoxy-2,3-anhydro-β-D-lyxofuranose was opened by sodium azide to give the corresponding azido compound, which was reduced by lithium aluminium hydride to lead to 2,3-dideoxy-2,3-epimino-β-D-ribofuranose. Pyrimidine bases were glycosylated with this synthon to give potential antiviral molecules: 1-(2,3-dideoxy-2,3-epimino-β-D-ribofuranosyl)pyrimidines.     

Ornithine decarboxylase activity in embryos depends on temperature of development rather than on the stage of development. Molecular adaptation to temperature changes in poikilothermic animals.

An unknown amino sugar, U-7, which had been detected in the hydrolysate of the polysaccharide fraction (F-A) of Pseudomonas aeruginosa P14 lipopolysaccharide, was isolated from the hydrolysate of whole cells of this micro-organism and converted into the N-acetyl derivative (U-7NAc). On the basis of i.r.-absorption spectrometry, 13C-n.m.r. and 1H-n.m.r. spectroscopy and mass spectrometry, the structure of compound U-7NAc was identified as 2-acetamido-3-amino-2,3-dideoxyhexofuranurono-6,3-lactam. The configuration of compound U-7NAc was then unequivocally identified as 2-acetamido-3-amino-2,3-dideoxy-D-glucofuranurono-6,3-lactam by comparing the synthetic and natural compounds. Compound U-7 and synthetic 2,3-diamino-2,3-dideoxy-D-glucofuranurono-6,3-lactam showed the same behaviour on chromatography. G.l.c.--mass-spectral analyses of fraction F-A and synthetic 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid after methanolyses and trimethylsilylations showed the presence of the same derivative. It was concluded that the amino sugar U-7 was produced from the 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid residue present in fraction F-A.     

Enhanced production of 2,3-butanediol from glycerol by forced pH fluctuations

The glycerol fermentation by Klebsiella pneumoniae occurs by receiving more than five liquid products—organic acids, diols, and ethanol. Aiming to direct the glycerol conversion towards predominant production of 2,3-butanediol (2,3-BD), the main influencing parameters (the aeration and the pH) were investigated during fed-batch processes. The regime of intensive aeration (2.2 vvm air supply) was evaluated as most favorable for 2,3-BD synthesis and ensured the decrease of all other metabolites. Thus, without pH control, 52.5 g/l 2,3-BD were produced, as the carbon conversion of glycerol into 2,3-BD reached 60.6%. Additional enhancement in 2,3-BD production (by significant increase of glycerol utilization) was achieved by the development of a new method of “forced pH fluctuations”. It was realized by consecutive raisings of pH using definite ΔpH value, at exact time intervals, allowing multiple variations. Thus, the optimal conditions for maximal glycerol consumption were defined, and 70 g/l 2,3-BD were produced, which is the highest amount obtained from glycerol as a sole carbon source until now. The forced pH fluctuations emphasized pH as a governing factor in microbial conversion processes.     

High production of 2,3-butanediol from glycerol by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> G31

The microbial production of high amounts of 2,3-butanediol (2,3-BD) from glycerol as a sole carbon source by the Bulgarian isolate Klebsiella pneumoniae G31 was studied in a series of fed-batch processes. The following conditions were evaluated as optimal: micro-aerobic cultivation in modified media, without pH control. Beginning at pH 8, 49.2 g/l of 2,3-BD was produced as negligible concentrations of by-products were received. The pH is the most important factor ruling the 2,3-BD production. Spontaneous pH changes and products formation in time were investigated, performing fermentations with non-controlled pH starting at different initial pH. In lack of external maintenance, the microorganism attempted to control the pH using acetate/2,3-BD alternations of the oxidative pathway of glycerol catabolism, which resulted in pH fluctuations. Thus, the culture secreted 2,3-BD at unequal portions, either allowing or detaining the acetate synthesis. More alkaline initial pH led to enhanced 2,3-BD accumulation as a response to the increased amplitudes of the pH variations. When the pH was maintained constant, the yield of 2,3-BD was very poor. These cultures remained viable only 72 h; whereas, the pH self-controlling cells lived and produced 2,3-BD up to 280 h. In conclusion, the formation of 2,3-BD is a result of an adaptive mechanism of pH self-control, responding to spontaneous pH drops during glycerol fermentation.     

Induction of 2,3-bisphosphoglycerate synthase in Friend leukemia cells

Friend leukemia cells (clone 745A) induced to differentiate with dimethylsulfoxide showed at least a 10-fold increase of 2,3-bisphosphoglycerate synthase and concomitant accumulation of 2,3-bisphosphoglycerate. These changes paralelled that of the number of hemoglobin-positive cells. Accumulation of 2,3-bisphosphoglycerate was also induced by dimethylsulfoxide in the other clone C-10-6, but not in C-9-6 which is resistant to differentiation with dimethylsulfoxide. Induced activity of 2,3-bisphosphoglycerate synthase in clone 745A was neutralized by antiserum prepared from a rabbit which was immunized with human erythrocyte 2,30bisphosphoglycerate synthase. By using this antiserum, biosynthesis of 2,3-bisphosphoglycerate synthase was detected in Friend cells only after induction by dimethylsulfoxide.     

Degradation of 3-phenylbutyric acid by Pseudomonas sp.

Pseudomonas sp. isolated by selective culture with 3-phenylbutyrate (3-PB) as the sole carbon source metabolized the compound through two different pathways by initial oxidation of the benzene ring and by initial oxidation of the side chain. During early exponential growth, a catechol substance identified as 3-(2,3-dihydroxyphenyl)butyrate (2,3-DHPB) and its meta-cleavage product 2-hydroxy-7-methyl-6-oxononadioic-2,4-dienoic acid were produced. These products disappeared during late exponential growth, and considerable amounts of 2,3-DHPB reacted to form brownish polymeric substances. The catechol intermediate 2,3-DHPB could not be isolated, but cell-free extracts were able only to oxidize 3-(2,3-dihydroxyphenyl)propionate of all dihydroxy aromatic acids tested. Moreover, a reaction product caused by dehydration of 2,3-DHPB on silica gel was isolated and identified by spectral analysis as (--)-8-hydroxy-4-methyl-3,4-dihydrocoumarin. 3-Phenylpropionate and a hydroxycinnamate were found in supernatants of cultures grown on 3-PB; phenylacetate and benzoate were found in supernatants of cultures grown on 3-phenylpropionate; and phenylacetate was found in cultures grown on cinnamate. Cells grown on 3-PB rapidly oxidized 3-phenylpropionate, cinnamate, catechol, and 3-(2,3-dihydroxyphenyl)propionate, whereas 2-phenylpropionate, 2,3-dihydroxycinnamate, benzoate, phenylacetate, and salicylate were oxidized at much slower rates. Phenylsuccinate was not utilized for growth nor was it oxidized by washed cell suspensions grown on 3-PB. However, dual axenic cultures of Pseudomonas acidovorans and Klebsiella pneumoniae, which could not grow on phenylsuccinate alone, could grow syntrophically and produced the same metabolites found during catabolism of 3-PB by Pseudomonas sp. Washed cell suspensions of dual axenic cultures also immediately oxidized phenylsuccinate, 3-phenylpropionate, cinnamate, phenylacetate, and benzoate.     

Multifunctional enzyme, bisphosphoglyceromutase/2,3-bisphosphoglycerate phosphatase/phosphoglyceromutase, from human erythrocytes. Evidence for a common active site.

Bisphosphoglyceromutase and 2,3-bisphosphoglycerate phosphatase activities responsible for 2,3-bisphosphoglycerate metabolsim in human red cells are displayed by the same enzyme protein which has phosphoglyceromutase activity [Sasaki, R., et al. (1975) Eur J. Biochem. 50, 581-593]. This enzyme was subjected to chemical modification by trinitrobenzenesulfonate. The three enzyme activities were inactivated by trinitrobenzenesulfonate at the same rate. The sulfhydryl content of the enzyme was unchanged during trinitrophenylation, indicating that derivatization was through the amino group. Trinitrophenylation of about one amino group per mole of the enzyme resulted in complete loss of the three activities. Both 2,3-bisphosphoglycerate and 1,3-bisphosphoglycerate inhibited trinitrophenylation and effectively protected the enzyme from inactivation. Although monophosphoglycerates did not show any protective effect at concentrations which should be adequate based upon their kinetic constants, they were protective at higher concentrations. Inactivation by trinitrophenylation was an apparent first-order reaction. The dissociation constant of the enzyme - 2,3-bisphosphoglycerate complex was determined by analyzing the first-order reaction on the assumption that the protective effect of 2,3-bisphosphoglycerate was due to competition with trinitrobenzenesulfonate. The dissociation constant was in good agreement with kinetic constants of 2,3-bisphosphoglycerate in the enzyme reactions, which indicated that 2,3-bisphosphoglycerate did indeed exert its protective effect through competition with trinitrobenzenesulfonate for an amino group of the enzyme. The protective effect of monophosphoglycerates could be rationalized with kinetic evidence that 2-phosphoglycerate at high concentrations interacts with the 2,3-bisphosphoglycerate binding site. These results indicate that the enzyme exhibits the three enzyme activities at a common active site at which one amino group essential for binding of bisphosphoglycerates is located. Based on the multifunctional properties of this enzyme, a possible mechanism was discussed for regulation of 2,3-bisphosphoglycerate metabolism in human red cells.     

Isolation and identification of an acetoin high production bacterium that can reverse transform 2,3-butanediol to acetoin at the decline phase of fermentation

Acetoin (3-hydroxy-2-butanone), a very popular food spice is now used in many industries (pharmaceuticals, chemicals, paint, etc.). In this study, an acetoin high producing strain, numbered as JNA-310, was newly isolated and identified as Bacillus subtilis which is safe on food industry, based on its physiological, biological tests and 16S rDNA sequence analysis. When glucose was used as carbon source in fermentation, the fermentation characterizations of this strain were analyzed, and a new phenomenon of reverse transforming 2,3-butanediol which was synthesized from glucose in the fermentation broth to acetoin was detected. Before 96 h, glucose which was mainly transformed to 2,3-butanediol and acetoin was totally consumed, and the yield of the two products were 41.7 and 21.0 g/l respectively. Acetoin was only a by product in the fermentation broth at prophase of fermentation. At the end of fermentation, the yield of acetoin was greatly improved and the yield of 2,3-butanediol was declined and the yield of them were about 42.2 and 15.8 g/l, respectively. The results indicated that 2,3-butanediol was reversely transformed to acetoin.     

The absence of 2,3-diphosphoglycerate from myocytes,hepatocytes and adipocytes

Myocytes, hepatocytes and adipocytes were prepared from heart, liver and epididymal fat pad of the rat. No detectable level of 2,3-diphosphoglycerate was found. Evidence is also present which indicates the absence from these cells of 2,3-diphosphoglycerate mutase and 2,3-diphosphoglycerate phosphatase. Previous findings by others of the presence of 2,3-diphosphoglycerate and 2,3-diphosphoglycerate mutase probably resulted from erythrocytes sequestered in the tissue.     

Microbial transformation of quinoline by a Pseudomonas sp.

A Pseudomonas sp. isolated from sewage by enrichment culture on quinoline metabolized this substrate by a novel pathway involving 8-hydroxycoumarin. During early growth of the organism on quinoline, 2-hydroxyquinoline accumulated as the intermediate; 8-hydroxycoumarin accumulated as the major metabolite on further incubation. 2,8-Dihydroxyquinoline and 2,3-dihydroxyphenylpropionic acid were identified as the other intermediates. Inhibition of quinoline metabolism by 1 mM sodium arsenite led to the accumulation of pyruvate, whereas inhibition by 5 mM arsenite resulted in the accumulation of 2-hydroxyquinoline as the major metabolite and 2,8-dihydroxyquinoline as the minor metabolite. Coumarin was not utilized as a growth substrate by this bacterium, but quinoline-grown cells converted it to 2-hydroxyphenylpropionic acid, which was not further metabolized. Quinoline, 2-hydroxyquinoline, 8-hydroxycoumarin, and 2,3-dihydroxyphenylpropionic acid were rapidly oxidized by quinoline-adapted cells, whereas 2,8-dihydroxyquinoline was oxidized very slowly. Quinoline catabolism in this Pseudomonas sp. is therefore initiated by hydroxylation(s) of the molecule followed by cleavage of the pyridine ring to yield 8-hydroxycoumarin, which is further metabolized via 2,3-dihydroxyphenylpropionic acid.     

A manganese-dependent dioxygenase from Arthrobacter globiformis CM-2 belongs to the major extradiol dioxygenase family.

Almost all bacterial ring cleavage dioxygenases contain iron as the catalytic metal center. We report here the first available sequence for a manganese-dependent 3,4-dihydroxyphenylacetate (3,4-DHPA) 2,3-dioxygenase and its further characterization. This manganese-dependent extradiol dioxygenase from Arthrobacter globiformis CM-2, unlike iron-dependent extradiol dioxygenases, is not inactivated by hydrogen peroxide. Also, ferrous ions, which activate iron extradiol dioxygenases, inhibit 3,4-DHPA 2,3-dioxygenase. The gene encoding 3,4-DHPA 2,3-dioxygenase, mndD, was identified from an A. globiformis CM-2 cosmid library. mndD was subcloned as a 2.0-kb SmaI fragment in pUC18, from which manganese-dependent extradiol dioxygenase activity was expressed at high levels in Escherichia coli. The mndD open reading frame was identified by comparison with the known N-terminal amino acid sequence of purified manganese-dependent 3,4-DHPA 2,3-dioxygenase. Fourteen of 18 amino acids conserved in members of the iron-dependent extradiol dioxygenase family are also conserved in the manganese-dependent 3,4-DHPA 2,3-dioxygenase (MndD). Thus, MndD belongs to the extradiol family of dioxygenases and may share a common ancestry with the iron-dependent extradiol dioxygenases. We propose the revised consensus primary sequence (G,T,N,R)X(H,A)XXXXXXX(L,I,V,M,F)YXX(D,E,T,N,A)PX(G,P) X(2,3)E for this family. (Numbers in brackets indicate a gap of two or three residues at this point in the sequence.) The suggested common ancestry is also supported by sequence obtained from genes flanking mndD, which share significant sequence identity with xylJ and xylG from Pseudomonas putida.     

Inhibition of barnacle larval settlement and crustacean toxicity of some hoplonemertine pyridyl alkaloids

Hoplonemertines are carnivorous marine worms, which prey upon crustaceans and annelids. They paralyze their prey by injecting alkaloids with a stylet-bearing proboscis. The dermis of these animals also secretes alkaloids to repel predators. Besides affecting central and peripheral nervous system nicotinic receptors, some pyridyl alkaloids also activate certain chemoreceptor neurons in crustacean walking legs, which sense environmental chemicals. Anabaseine (2-[3-pyridyl]-3,4,5,6-tetrahydropyridyl) and 2,3'-bipyridyl (2,3'-BP) are two nemertine alkaloids, which potently paralyze crustaceans. Anabaseine is an agonist of vertebrate as well as invertebrate nicotinic receptors. While 2,3'-BP is non-toxic to mice, it is toxic to crustaceans. We tested a variety of nemertine pyridyl alkaloids for inhibition of barnacle (Balanus amphitrite) larval settlement and for crustacean toxicity in order to determine whether toxicity could be dissociated from inhibition of larval settlement. We prepared eight C-methylated 2,3'-BP isomers to determine where substitution is permitted without loss of activity. Anti-settlement and toxicity activities were not always related. For instance, 4'-methyl-2,3'-BP displayed only 3% of the crayfish paralytic activity of 2,3'-BP, but inhibited settlement almost 2-fold more effectively. Two other isomers displaying exceptional anti-settlement activity were the 4- and 5-methyl-2,3'-BPs; these also displayed high crustacean toxicity. Nemertelline inhibited barnacle settlement at concentrations similar to 2,3'-BP but was 136-fold less toxic when injected into crayfish. Thus, certain bipyridyls and tetrapyridyls may be useful anti-fouling additives.     

Enantiomeric forms of 9-(5-deoxy-beta-erythro-pent-4-enofuranosyl)adenine and a new preparation of 5-deoxy-D-lyxose.

Methyl 5-deoxy-5-iodo-2,3-O-isopropylidene-beta-D-ribofuranoside (3) was obtained in three steps from D-ribose. Exchange of the isopropylidene group for benzoate groups and acetolysis gave 1-O-acetyl-2,3-di-O-benzoyl-5-deoxy-5-iodo-D-ribofuranose which was coupled with 6-benzamidochloromercuripurine by the titanium tetrachloride method to afford the blocked nucleoside. Treatment with 1,5-diazabicyclo[5.4.0]undec-5-ene in N,N-dimethylformamide and removal of the blocking groups have 9-(5-deoxy-beta-D-erythro-pent-4-enofuranosyl)adenine (9). A similar route starting from methyl 5-deoxy-5-iodo-2,3-O-isopropylidene-alpha-D-lyxofuranoside (14) afforded the enantiomeric nucleoside, 9-(5-deoxy-beta-L-erythro-pent-4-enofuranosyl)adenine (20). Methyl 2,3-O-isopropylidene-alpha-D-mannofuranoside was treated with sodium periodate and then with sodium borohydride to give methyl 2,3-O-isopropylidene-alpha-D-lyxofuranoside (11). Acid hydrolysis afforded D-lyxose. Tosylation of 11 gave methyl 2,3-O-isopropylidene-5-O-p-tolylsulfonyl-alpha dp-lyxofuranoside (12) which was converted into 14 with sodium iodide in acetone. Reduction of 12 gave methyl 5-deoxy-2,3-O-isopropylidene-alpha-D-lyxofuranoside which was hydrolyzed to give 5-deoxy-D-lyxose.     

A new 6-C-alkylation from an alkyl mannofuranoside 5,6-cyclic sulfate

Methyl 6-C-alkyl-6-deoxy-alpha-D-mannofuranoside derivatives have been synthesized from methyl 2,3-O-isopropylidene-5,6-O-sulfuryl-alpha-D-mannofuranoside (1). In a Path A, reaction of the 5,6-cyclic sulfate 1 with 2-lithio-1,3-dithiane afforded 2-(methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-mannofuranosid-6-yl)-1,3-dith iane (2). Treatment of 2 with n-butyllithium then alkyl iodide gave the corresponding 2-(methyl 5-O-alkyl-6-deoxy-2,3-O-isopropylidene-alpha-D-mannofuranosid-6-yl )-1,3- dithiane. Reaction of 2 with n-butyllithium and 5,6-cyclic sulfate 1 furnished 2-[methyl 6-deoxy-2,3-O-isopropylidene-5-O-(methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-manno-furanosid-6-yl)-alpha-D - mannofuranosid-6-yl]-1,3-dithiane. 2-(Methyl 6-deoxy-2,3-O-isopropylidene-5-O-methyl-alpha-D-mannofuranosid- 6-yl)-1,3-dithiane was converted into the lithiated anion, which after treatment with alkyl halide afforded the corresponding 2-alkyl-C-(methyl 6-deoxy-2,3-O-isopropylidene-5-O-methyl-alpha-D-mannofuranosid-6-y l)-1,3- dithiane. In a Path B, 5,6-cyclic sulfate 1 reacted with 2-alkyl-2-lithio-1,3-dithiane derivatives, which led after acidic hydrolysis to 2-alkyl-2-(methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-mannofuranosid-6-yl)-1,3-dith iane accompanied by methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranos-5-u loside as the by-product. This methodology was applied to synthesize 2-(methyl 6-deoxy-2,3-O-isopropylidene-5-O-methyl-alpha-D-mannofuranosid-6-y l)-2- (methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-mannofuranosid-6-yl)-1,3-dith iane.     

Occurrence of multiple forms of alcohol dehydrogenase in Penicillium supplemented with 2,3-butanediol

The NAD-dependent oxidation of ethanol, 2,3-butanediol, and other primary and secondary alcohols, catalyzed by alcohol dehydrogenases derived from Penicillium charlesii, was investigated. Alcohol dehydrogenase, ADH-I, was purified to homogeneity in a yield of 54%. The enzyme utilizes several primary alcohols as substrates, with K_m values of the order of 10^?4m. A K_m value of 60 mm was obtained for R,R,-2,3-butanediol. The stereospecificity of the oxidation of 2-butanol was investigated, and S-(+)-2-butanol was found to be oxidized 2.4 times faster than was R-(?)-2-butanol. The reduction of 2-butanone was shown to produce S-(+)-2-butanol and R-(?)-butanol in a ratio of 7:3. ADH-I is the primary isozyme of alcohol dehydrogenase present in cultures utilizing glucose as the sole carbon source. The level of alcohol dehydrogenase activity increased 7.6-fold in mycelia from cultures grown with glucose and 2,3-butanediol (0.5%) as carbon sources compared with the activity in cultures grown on only glucose. Two additional forms of alcohol dehydrogenase, ADH-II and ADH-III, were present in the cultures supplemented with 2,3-butanediol. These forms of alcohol dehydrogenase catalyze the oxidation of ethanol and 2,3-butanediol. These data suggest that P. charlesii carries out an oxidation of 2,3-butanediol which may constitute the first reaction in the degradation of 2,3-butanediol as well as the last reaction in the mixed-acid fermentation. Alcohol dehydrogenase activities in P. charlesii may be encoded by multiple genes, one which is expressed constitutively and others whose expression is inducible by 2,3-butanediol.