Levels of enzymes of the pentose phosphate pathway in Pachysolen tannophilus Y-2460 and selected mutants Anil H. Lachke Division of Biochemical Sciences, National Chemical Laboratory, Poona 411008, India and Thomas W. Jeffries US Department of Agriculture, Forest Service, Forest Products Laboratory, One Gifford Pinchot Drive, Madison, WI 53705-2398, USA (Received 24 September 1985; revised 6 January 1986) The compositions of intracellular pentose phosphate pathway enzymes have been examined in mutants of Pachysolen tannophilus NRRL Y-2460 which possessed enhanced D-xylose fermentation rates. The levels of oxidoreductive enzymes involved in converting D-xylose to D-xylulose via xylitol were 1.5-14.7-fold higher in mutants than in the parent. These enzymes were still under inductive control by D-xylose in the mutants. The D-xylose reductase activity (EC 1.1.1.21) which catalyses the conversion of D-xylose to xylitol was supported with either NADPH or NADH as coenzyme in all the mutant strains. Other enzyme specific activities that generally increased were: xylitol dehydro- genase (EC 1.1.1.9), 1.2-1.6-fold; glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 1.9-2.6-fold; D-xylulose-5-phosphate phosphoketolase (EC 4.1.2.9), 1.2-2.61fold; and alcohol dehydrogenase (EC 1.1.1.1). 1.5-2.7-fold. The increase of enzymatic activities, 5.3-10.3-fold, occurring in D- xylulokinase (EC 2.7.1.17), suggested a pivotal role for this enzyme in utilization of D-xylose by these mutants. The best ethanol-producing mutant showed the highest ratio of NADH- to NADPH- linked D-xyloSe reductase activity and high levels of all other pentose phosphate pathway enzymes assayed. Keywords: Pentose phosphate pathway; Pachysolen tannophilus; D-xylose metabolism Introduction D-xylose is first reduced by an NADPH-linked D-xylose reductase (alditol:NADP+/NAD+ 1 -oxidoreductase, EC Xylans, the principal constituents of hemicelluloses, can be 1.1.1.21) to form xylitol. The reoxidation of xylitol to obtained from woody angiosperms (hardwoods) and agri- D-xylulose is then effected by another NAD-linked dehyd- cultural residues. Sugars derived from xylans offer potential rogenase (xylitol:NAD+ 2-oxidoreductase, D-xylulose feedstock for generating food and fuel. The hemicellulosic reductase, EC 1.1.19). D-Xylulose thus formed is then sugars can be recovered from lignocellulose more readily phosphorylated to form a key intermediate, D-xylulose-5- than glucose but are fermented with greater difficulty than phosphate. Beyond this point, much of the pathway is glucose or pure sugars.1 assumed from biochemical studies with other organisms. In recent years, numerous yeasts, particularly Pachy- In one route, phosphate acetylating enzyme, phospho- solen tannophilus,2 Candida shehatae,3 and Pichia stipitis4 ketolase (D-xylulose-5-phosphate D-glyceraldehyde-3- have been found to produce ethanol from D-xylose. Of phosphate-lyase, EC 4.1.2.9) converts D-xylulose-5-phos- these, P. tannophilus has been studied most extensively,5 phate to glyceraldehyde-3-phosphate and acetyl phosphate and a number of mutants exhibiting improved rates of D- in the presence of cocarboxylase and Mg2+. In another xylose fermentation have been obtained from the type route, D-xylulose-5-phosphate proceeds by way of epi- culture, P. tannophilus NRRL Y-2460.6 This research merase, isomerase, transketolase and transaldolase to form sought to characterize the parent strain and the mutants glyceraldehyde-3-phosphate and D-fructose-6-phosphate. biochemically in order to determine if altered levels of key Glyceraldehyde-3-phosphate is an intermediate in both enzymes could account for the improved performance of the pentose phosphate pathway (PPP) and Embden- the mutants. Meyerhof-Parnas (EMP) pathway.8-10 The net result of a In yeasts and moulds, D-xylose is converted to D-xylulose homoethanolic fermentation through this latter route in two enzymatic steps.7 In the oxidoreductive pathway, involving transketolase is that 3 mol of xylose yield 5 mol 1986 Butterworth & Co. (Publishers) Ltd Enzyme Microb. Technol., 1986, vol. 8, June 353 Papers of ethanol and 5 mol of carbon dioxide, i.e. 1.67 mol C2 filter sterilized in 10 × strength and either 4.5 or 6.0% per mol C5 utilized. Both the ATP and the ethanol yields D-xylose or D-glucose as sole carbon sources. An inoculum of this homoethanolic route are identical to those obtained was grown by transferring each strain from an agar slant with glucose via the Embden-Meyerhofpathway. into a 125 ml Erlenmeyer flask containing 50 ml of the The pathway for fermenting D-xylose by P. tannophilus medium and agitating at 100 rev min-1, 32°C, for 48 h. is poorly characterized. P. tannophilus unambiguously Inoculum cells were washed with sterile water and diluted exhibits an anaerobic fermentation of D-xylose; however it so that cells grown under different conditions would all forms significant quantities of acetic acid under anaerobic give the same initial absorbance at 525 nm.14 Five ml of conditions or when cells are grown aerobically on nitrate.11 each cell suspension containing on average 45 mg (±2) dry It is possible that this product could arise from phospho- weight (0.8g wet wt) cells was used as inoculum. The ketolase activity. We decided therefore to determine the strains were grown on a rotatory shaker (100 rev min-1 ) at enzymic activities of various PPP enzymes in parent and 32°C in 125ml Erlenmeyer flasks containing 50ml of previously obtained mutant yeast strains6 in order to better medium. The cells were harvested by centrifugation (7000 characterize the pathway and elucidate the rate-limiting rev min-1 , 20 min, 44°C) after 48 h and washed with step(s) in the overall process. potassium phosphate buffer (0.1 M, pH 7.1) containing The mutant strains examined here were enriched and 10 mM cysteine-HCl, 1 mM dithiothreitol, 1 mM EDTA, selected on the basis of their abilities to grow preferentially and 5 mM MgCl2. in broth and to form large colonies on agar with either nitrate or ammonia as nitrogen sources and with xylitol as a carbon source. Nitrate was chosen as the sole nitrogen Preparation of cell homogenates source for enrichment and selection because of its recog- The harvested yeast cells (4-6g), usually from six nized ability to induce higher levels of pentose phosphate Erlenmeyer flasks, were suspended in 15 ml 100 mM pathway enzymes in yeasts, fungi and plant cells.12,13 potassium phosphate buffer, pH 7.1, and placed in pre- Xylitol was chosen as the sole carbon source because it cooled thick-walled bottles containing 35 g (HCl-washed was restrictive for these cells, and it could be used to and rinsed in water) glass beads (0.5 mm diameter). This distinguish between fast- and slow-growing colonies on a mixture was broken in a Braun model cell homogenizer plate. The mutant strains examined have produced ethanol by agitating for 3 min while cooling to 1-5°Cwith liquid approximately twice as fast and in nearly 30% better yield CO2. The supernatant solution was separated from glass than the parent strain under the conditions employed? beads and cell debris by centrifugation (12 000 rev min-1 , The objective of the present work was to determine 30 min, 4-6°C) and used immediately for enzyme assays. intracellular levels of some of the enzymes of the PPP in the parent and isolated mutant strains, when grown under welldefined aerobic conditions. The results of this research Enzyme assays should be useful in future comparative biochemical studies NAD(H) and NADP(H)-dependent reduction or oxidation with C. shehatae, P. stipitis and other yeasts. of the polyol, ketose or aldose substrates by cell-free extracts were followed on a Gilford model 250 spectro- Materials and methods photometer at 340 nm using 1 cm light path quartz cuvettes thermostatically controlled at 30°C. The enzyme sample in Chemicals the assay was diluted appropriately to give a linear reaction D-Xylose, xylitol, D-xylulose; NAD; NADH; NADP; velocity for at least 5 min. In every assay, control recordings NADPH, type 1; lactic dehydrogenase, type 1 (LDH); were made without added cell homogenate, coenzyme or adenosine 5'-triphosphate, phosphoenol pyruvate, D- substrate, respectively. ε340 nm values were converted to xylulose, thiamin pyrophosphate, phosphoriboisomerase, NAD(P)H concentrations using ε (mM) = 6.22. pyruvate kinase, D-ribulose-5-phosphate 3-epimerase, and Activities of all enzymes are expressed as international D-ribose-5-phosphate were obtained from sigma Chemical units (U), where one unit is the amount of enzyme necessary Company. Bacto Yeast Nitrogen Base was obtained from to catalyse the oxidation or reduction of one micromole Difco. Other chemicals used were of reagent grade. of coenzyme (NAD/NADP) per minute at 30°C under the given experimental conditions. Specific activities are Microorganisms expressed as units mg protein-1. The reaction mixtures and Pachysolen tannophilus Boidin et Adzet NRRL Y-2460 other conditions for the individual enzymes are as follows. (ATCC 32691) was obtained from the yeast collection at the USDA Northern Regional Research Center, Peoria, IL, D-Xylose reductase (EC 1.1.1.21) (alditol:NADP+/ and maintained on yeast malt agar (YMA, Difco). Muta- NAD+ 1-axidoreductase). The activity of D-xylose reductase genesis, enrichment, isolation and selection of mutant was determined using NADPH and NADH as coenzyme 7 strains of P. tannophilus Y-2460, viz. NO3→NO3-7,NO3→ according to the method described by Chiang and Knight. U-4, NO3→NO3-4, U→U-27 and U→NO3-6, have been The reaction mixture contained 0.1 M Tris-HCl buffer, 6 -3 described previously.