Aureobasidium Pullulans Var Research Articles

Biotechnological application of Aureobasidium spp. as a promising chassis for biosynthesis of ornithine-urea cycle-derived bioproducts.

The ornithine-urea cycle (OUC) in fungal cells has biotechnological importance and many physiological functions and is closely related to the acetyl glutamate cycle (AGC). Fumarate can be released from argininosuccinate under the catalysis of argininosuccinate lyase in OUC which is regulated by the Ca2+ signaling pathway and over 93.9 ± 0.8 g/L fumarate can be yielded by the engineered strain of Aureobasidium pullulans var. aubasidani in the presence of CaCO3. Furthermore, 2.1 ± 0.02 mg of L-ornithine (L-Orn)/mg of the protein also can be synthesized via OUC by the engineered strains of Aureobasidum melanogenum. Fumarate can be transformed into many drugs and amino acids and L-Orn can be converted into siderophores (1.7 g/L), putrescine (33.4 g/L) and L-piperazic acid (L-Piz) (3.0 g/L), by different recombinant strains of A. melanogenum. All the fumarate, L-Orn, siderophore, putrescine and L-Piz have many applications. As the yeast-like fungi and the promising chassis, Aureobasidium spp, have many advantages over any other fungal strains. Further genetic manipulation and bioengineering will enhance the biosynthesis of fumarate and L-Orn and their derivates.

The role of the acetyl-glutamate cycle in fumarate biosynthesis through L-ornithine supply via the ornithine-urea cycle in Aureobasidium pullulans var. aubasidani

In Aureobasidium pullulans var. Aubasidani, the ornithine-urea cycle (OUC) plays a role in fumarate biosynthesis with L-ornithine serving as a significant intermediate. The acetyl-glutamate cycle (AGC) is also responsible for synthesizing L-ornithine. But it remains unclear whether AGC can assist OUC in fumarate biosynthesis. N-acetyl-glutamate kinase and N-acetyl-gamma-glutamyl-phosphate reductase present in AGC were both encoded by the ARGBC gene in A. pullulans var. Aubasidani and N-acetyl-glutamate kinase was observed to be insensitive to feedback inhibition by L-arginine from OUC. This observation could be attributed to the presence of conserved residues, specifically L-threonine (T340) and L-arginine (R336), in the N-acetyl-glutamate kinase as opposed to the conserved L-threonine (T340) and L-lysine (K336) residues that could interact with L-arginine. Hence, AGC remained active and consistently produced L-ornithine for OUC. The deletion of the ARGBC gene in the parent glucose oxidase-deficient mutant (Δgox) led to decreased L-ornithine accumulation and reduced fumarate production in the disruptant Δgox Δargbc while complementation of the ARGBC gene enhanced accumulation of L-ornithine and restored fumarate production by the complementing strain ARGBC-H. The introduction of exogenous L-arginine clearly impacted fumarate production and cell growth in the disruptant strain Δgox Δargbc. It was concluded that achieving elevated fumarate levels through OUC, maintaining the activity of AGC for high L-ornithine accumulation was crucial. The produced fumarate had many applications.

The ornithine-urea cycle involves fumaric acid biosynthesis in Aureobasidium pullulans var. aubasidani, a green and eco-friendly process for fumaric acid production

The current petroleum chemical methods for fumaric acid production can cause heavy pollution and global warming. In this study, the engineered strains of A. pullulans var. aubasidani were found to be suitable for green fumaric acid producer. Removal and complementation of the relevant genes showed only the ornithine-urea cycle (OUC) was involved in high level fumarate biosynthesis which was controlled by the Ca2+ signaling pathway. Removal of both the GOX gene encoding glucose oxidase and the PKS1 gene encoding the polyketide synthase for 3,5-dihydroxydecanoic acid biosynthesis and overexpression of the PYC gene encoding pyruvate carboxylase made the strain e-PYC produce 88.1 ± 4.3 g/L of fumarate at flask level and 93.9 ± 0.8 g/L of fumarate during the fed-batch fermentation. As a yeast-like fungal strain, it was very easy to cultivate A. pullulans var. aubasidani DH177 and their mutants in the bioreactor and to edit its genomic DNAs to enhance fumarate production. It was found that 2 mol of CO2 could be fixed during a maximal theoretical yield of 2 mol of fumarate per mole of glucose consumed in the OUC. Therefore, the OUC-mediated fumarate biosynthesis pathway in A. pullulans var. aubasidani was a green and eco-friendly process for the global sustainable development and carbon neutrality.

First Report of Aureobasidium pullulans var. pullulans Causing Spot Blight of Pear (Pyrus pyrifolia) in Zhejiang Province of China.

Asian pear (Pyrus pyrifolia Nakai cv. cuiguan), is widely grown in Zhejiang province of China. In April 2019, symptoms consisting of small black, round leaf spots and blight flower petals were observed on over 30 % of 'Cuiguan' pear trees in an orchard (ca. 0.8 ha) near Cixi city, Zhejiang province, China. Initially, leaf spots were observed on leaf petioles, which, with time, enlarged and coalesced into necrotic streaks (1-2 cm) along the length of the petioles. Irregularly, reddish brown spots developed on flower petals, which hastened their senescence. Additional symptoms included round, black spots on leaves (2-3 mm in diameter) and necrosis of shoot tips. Symptomatic tissues from petals, petioles and leaves were plated onto potato dextrose agar (PDA). After five days of incubation at 26 °C, slimy fungal colonies (48×48 mm) with pinkish to orange-colored mycelia and with regular annulations were isolated from all tissues. After 10 days, cultured were shiny and dark brown in the center. The color of conidia ranged from hyaline to dark brown. Hyaline conidia were blastic, unicellular, ellipsoidal, smooth, with lengths that ranged from 11.03 to 27.14 (avg. 18.38) μm, and widths that ranged from 3.45 to 8.86 (avg. 6.04) μm (n = 50). Dark brown conidia were 1 to 2 celled, 10.89 to 26.03 (avg. 17.41) μm in length and 4.26 to 12.15 (avg. 6.94) μm in width (n = 50), and a slight septal constriction. Conidiogenous cells were clavate, hyaline, eseptate and top smoothly with 3-11 spores. Indistinct scars remained when the conidia dislodge from the conidiogenous cells. Single spore isolation was used to obtain pure cultures. Mycelia and conidia were scraped from cultures and DNA was extracted using Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech). Amplified PCR products from the internal transcribed spacer (ITS) region ITS1F/ITS4 (White et al. 1990), the partial 28S rDNA (LSU) NL1/NL4 (Boekhout et al. 1995), the β-tubulin (TUB) gene Bt2a/Bt2b (Glass & Donaldson 1995) and the partial elongase gene (ELO) ELO2-F/ELO2-R (Zalar et al. 2008) were sequenced (Tsingke Biotechnology Co., Hangzhou, Zhejiang). A blast search (GenBank Accession No. MT107050, OK485685, OK631951, OK631950) showed 99% Aureobasidium pullulans reference isolate CBS584.75 and EXF-150, which was consistent with the morphological data (Cene et al. 2014). Three-yr-old seedlings from 'Cuiguan' pears were spray with conidial suspension (106 conidia/ml) on the both sides of leaves without wounding. In a greenhouse (26 °C, natural light), six inoculated plants and three noninoculated plants (sprayed with sterile distilled water) enclosed in plastic bags to maintain humidity for 72 h. At 5 days after inoculation, shoot tips blackened and began to wilt. At 15 days after inoculation, symptoms similar to those on the original sample developed on inoculated petioles and leaves, whereas the control plants remained healthy. No symptoms were observed on leaves that were mature at the time of inoculation. Aureobasidium pullulans var. pullulans was reisolated from all inoculated plant. Overall, this disease shortened the life of pear flowers and reduced fruit set. To our knowledge, A. pullulans var. pullulans has not previously been reported as a pathogen of P. pyrifolia.

Genome Sequence of the Black Yeast-Like Strain Aureobasidium pullulans var. aubasidani CBS 100524.

In this work, we present the whole-genome sequence and the complete mitochondrial sequence of the black yeast-like strain Aureobasidium pullulans var. aubasidani CBS 100524, which produces the exopolysaccharide aubasidan and was previously isolated from Betula sp. slime flux from the Leningrad Region of Russia.

Simultaneous production of single cell oil and fumaric acid by a newly isolated yeast Aureobasidium pullulans var. aubasidani DH177.

Microbial oils can be used for biodiesel production and fumaric acid (FA) is widely used in the food and chemical industries. In this study, the production of lipids and FA by Aureobasidium pullulans var. aubasidani DH177 was investigated. A high initial carbon/nitrogen ratio in the medium promoted the accumulation of lipids and FA. When the medium contained 12.0% glucose and 0.2% NH4NO3, the yeast strain DH177 accumulated 64.7% (w/w) oil in its cells, 22.4g/l cell biomass and 32.3g/l FA in a 5-L batch fermentation. The maximum yields of oil and FA were 0.12g/g and 0.27g/g of consumed sugar, respectively. The compositions of the produced fatty acids were C14:0 (0.6%), C16:0 (24.9%), C16:1 (4.4%), C18:0 (2.1%), C18:1 (57.6%), and C18:2 (10.2%). Biodiesel obtained from the extracted oil burned well. This study provides the pioneering utilization of the yeast strain DH177 for the integrated production of oil and FA.

Efficient Production of Polymalic Acid by a Novel Isolated Aureobasidium pullulans Using Metabolic Intermediates and Inhibitors.

Polymalic acid (PMA) is a linear anionic polyester composed of L-malic acid monomers, which have potential applications as drug carriers, surgical suture, and biodegradable plastics. In this study, a novel strain of Aureobasidium pullulans var. melanogenum GXZ-6 was isolated and identified according to the morphological observation and deoxyribonucleic acid internal-transcribed spacer sequence analysis, and the product of PMA was characterized by FT-IR, 13C-NMR, and 1H-NMR spectra. The PMA titer of GXZ-6 reached 62.56 ± 1.18gL-1 with productivity of 0.35gL-1h-1 using optimized medium with addition of metabolic intermediates (citrate and malate) and inhibitor (malonate) by batch fermentation in a 10-L fermentor. Besides that the malate for PMA synthesis in GXZ-6 might mainly come from the glyoxylate cycle, based on results, citrate, malate, malonate, and maleate increased while succinate and fumarate inhibited the production of PMA, which was different from that of other A. pullulans. This study provided a potential strain and a simple metabolic control strategy for high-titer production of PMA and shared novel information on the biosynthesis pathway of PMA in A. pullulans.

Biofilm of Aureobasidium pullulans var. pullulans on winter wheat kernels and its effect on other microorganisms

Winter wheat, grown under greenhouse conditions, was protected four times with a cell suspension of Aureobasidium pullulans var. pullulans during the growing season. After harvest, the distribution and survival rates of the studied biocontrol agent were analyzed under a scanning electron microscope. The abundance of filamentous fungi, yeasts, pseudomonads and Azotobacter bacteria was determined by inoculation onto selective agar media. A. pullulans produced mostly unicellular chlamydospores on the surface and in the brush of kernels. Multicellular blastospore conglomerates secreted extracellular polymeric substances (EPS), and their biofilms were found in the brush and crease of kernels. The application of a cell suspension of A. pullulans with the density of 104 CFU to winter wheat spikes, repeated four times, inhibited the growth of pseudomonads, Azotobacter bacteria and filamentous fungi.

Enhanced production of Ca²⁺-polymalate (PMA) with high molecular mass by Aureobasidium pullulans var. pullulans MCW.

BackgroundPolymalic acid (PMA) has many applications in food and medical industries. However, so far it has not been commercially produced by fermentation. Therefore, it is very important how to develop an economical process for a large scale production of PMA by one step fermentation.ResultsAfter over 200 strains of Aureobasidium spp. isolated from the mangrove systems in the South of China were screened for their ability to produce Ca2+-polymalate (PMA), it was found that Aureobasidium pullulans var. pullulans MCW strain among them could produce high level of Ca2+-PMA. The medium containing only 140.0 g/L glucose, 65.0 g/L CaCO3 and 7.5 g/L corn steep liquor was found to be the most suitable for Ca2+-PMA production. Then, 121.3 g/L of Ca2+-PMA was produced by A. pullulans var. pullulans MCW strain within 120 h at flask level. During 10-L batch fermentation, 152.52 g/L of Ca2+-PMA in the culture and 8.6 g/L of cell dry weight were obtained within 96 h, leaving 4.5 g/L of reducing sugar in the fermented medium. After purification of the Ca2+-PMA from the culture and acid hydrolysis of the purified Ca2+-PMA, HPLC analysis showed that A.pullulans var. pullulans MCW strain produced only one main component of Ca2+-PMA and the hydrolysate of the purified Ca2+-PMA was mainly composed of l-malic acid. Mw (the apparent molecular weight) of the purified PMA was 2.054 × 105 (g/moL) and the purified PMA was estimated to be composed of 1784 l-malic acids.ConclusionsIt was found that A. pullulans var. pullulans MCW strain obtained in this study could yield 152.52 g/L of Ca2+-PMA within the short time, the produced PMA had the highest molecular weight and the medium for production of Ca2+- PMA by this yeast was very simple.

Hydrocarbons, the advanced biofuels produced by different organisms, the evidence that alkanes in petroleum can be renewable.

It is generally regarded that the petroleum cannot be renewable. However, in recent years, it has been found that many marine cyanobacteria, some eubacteria, engineered Escherichia coli, some endophytic fungi, engineered yeasts, some marine yeasts, plants, and insects can synthesize hydrocarbons with different carbon lengths. If the organisms, especially some native microorganisms and engineered bacteria and yeasts, can synthesize and secret a large amount of hydrocarbons within a short period, alkanes in the petroleum can be renewable. It has been documented that there are eight pathways for hydrocarbon biosynthesis in different organisms. Unfortunately, most of native microorganisms, engineered E. coli and engineered yeasts, only synthesize a small amount of intracellular and extracellular hydrocarbons. Recently, Aureobasidium pullulans var. melanogenum isolated from a mangrove ecosystem has been found to be able to synthesize and secret over 21.5 g/l long-chain hydrocarbons with a yield of 0.275 g/g glucose and a productivity of 0.193 g/l/h within 5 days. The yeast may have highly potential applications in alkane production.

Deterioration of expanded polystyrene caused by Aureobasidium pullulans var. melanogenum

An expanded-polystyrene factory located in northern Buenos Aires reported unusual dark spots causing esthetic damage in their production. A fungal strain forming black-olive colonies on extract malt agar medium was isolated from the damaged material and identified as Aureobasidium pullullans var. melanogenum. This fungus is particularly known for its capacity to produce hydrolytic enzymes and a biodegradable extracellular polysaccharide known as pullulan, which is used in the manufacture of packaging material for food and medicine. Laboratory tests were conducted to characterize its growth parameters. It was found that the organism was resistant to a wide range of pHs but did not survive at temperatures over 65°C. The proposed action plan includes drying of the material prior to packaging and disinfection of the machinery used in the manufacturing process and of the silos used for raw material storage.

Antagonistic yeasts competes for iron with winter wheat stem base pathogens

Aureobasidium pullulans and Sporobolomyces roseus are a saprotrophic yeasts fungi commonly found on the leaves of winter wheat and on wheat kernels. The objective of this study was to compare the inhibitory effects of two species of yeasts fungi, Aureobasidium pullulans var. pullulans (de Bary) G. Arnaud and Sporobolomyces roseus Kluyver & van Niel, on the causal agents of stem base diseases, Rhizoctonia cerealis v. d. Hoeven, Gaeumannomyces graminis (Sacc.) Arx & D. Olivier, Helgardia herpotrichoides (Fron) Crous & W. Gams, Fusarium oxysporum (Schlecht) Snyd. et Hans.) and Fusarium culmorum (W. G. Smith). A. pullulans showed stronger inhibitory activity than S. roseus. Among the 70 A. pullulans isolates tested in the study, 25 were capable of suppressing the colony growth of R. cerealis under in vitro conditions. This is the first study to show that A. pullulans competes for iron with stem base pathogens, in particular with fast-growing R. cerealis and F. culmorum. Under greenhouse conditions, A. pullulans protected winter wheat seedlings against infection caused by F. culmorum, from two to four times compared with the control, and its protective effect was determined by the infection susceptibility of wheat cultivars and the time interval between the application of A. pullulans and inoculation with F. culmorum.

Heavy oils, principally long-chain n-alkanes secreted by Aureobasidium pullulans var. melanogenum strain P5 isolated from mangrove system.

In this study, the yeast strain P5 isolated from a mangrove system was identified to be a strain of Aureobasidium pullulans var. melanogenum and was found to be able to secrete a large amount of heavy oil into medium. After optimization of the medium for heavy oil production and cell growth by the yeast strain P5, it was found that 120.0g/l of glucose and 0.1% corn steep liquor were the most suitable for heavy oil production. During 10-l fermentation, the yeast strain P5 produced 32.5g/l of heavy oil and cell mass was 23.0g/l within 168h. The secreted heavy oils contained 66.15% of the long-chain n-alkanes and 26.4% of the fatty acids, whereas the compositions of the fatty acids in the yeast cells were only C16:0 (21.2%), C16:1(2.8%), C18:0 (2.9%), C18:1 (39.8%), and C18:2 (33.3%). We think that the secreted heavy oils may be used as a new source of petroleum in marine environments. This is the first report of yeast cells which can secrete the long-chain n-alkanes.

In vitro activities of eight antifungal drugs against 104 environmental and clinical isolates of Aureobasidium pullulans.

Aureobasidium pullulans is an unusual agent of phaeohyphomycosis. The in vitro activities of antifungals against 104 isolates of Aureobasidium pullulans var. pullulans and A. pullulans var. melanigenum revealed low MIC90s of amphotericin B, posaconazole, and itraconazole. However, they were resistant to fluconazole (≥64 μg/ml) and had high MICs of voriconazole, isavuconazole, caspofungin, and micafungin.

High-level pullulan production by Aureobasidium pullulans var. melanogenium P16 isolated from mangrove system

After over 100 strains of Aureobasidium spp. isolated from mangrove system were screened for their ability to produce exopolysaccharide (EPS), it was found that Aureobasidium pullulans var. melanogenium P16 strain among them could produce high level of EPS. Under the optimal conditions, 65.3g/L EPS was produced by the P16 strain within 120h at flask level. During 10-L batch fermentation, when the medium contained 120.0g/L sucrose, 67.4g/L of EPS and 23.1g/L of cell dry weight in the culture were obtained within 120h, leaving 0.78g/L of reducing sugar and 11.4g/L of total sugar in the fermented medium. It should be stressed that during the fermentation, no melanin was observed. After purification, the purified EPS was confirmed to be pullulan. This is the first time to report that A. pullulans var. melanogenium P16 strain isolated from the mangrove system can produce high level of pullulan.

Evaluation of single cell oil from Aureobasidium pullulans var. melanogenum P10 isolated from mangrove ecosystems for biodiesel production

In this study, the yeast strain P10 which was identified to be a member of Aureobasidium pullulans var. melanogenum isolated from the mangrove ecosystems was found to be able to accumulate high content of oil in its cells. After optimization of the medium for lipid production and cell growth by the yeast strain P10, it was found that 8.0g of glucose per 100ml, 0.02g of yeast extract per 100ml, 0.02g of ammonium sulfate per 100ml, pH 6.0 in the medium were the most suitable for lipid production. During 10-l fermentation, a titer was 66.3g oil per 100g of cell dry weight, cell mass was 1.3g per 100ml, a yield was 0.11g of oil per g of consumed sugar and a productivity was 0.0009g of oil per g of consumed sugar per h within 120h. At the same time, only 0.07g of reducing sugar per 100ml was left in the fermented medium. The compositions of the fatty acids produced were C16:0 (26.7%), C16:1(1.7%), C18:0 (6.1%), C18:1 (44.5%), and C18:2 (21.0%). The biodiesel produced from the extracted lipid could be burnt well.

POTENTIAL OF LACCASE PRODUCED FROM MICROFUNGUS, AUREOBASIDIUM PULLULANS VAR. MELANOGENUM, TO DEGRADE POLY-AROMATIC HYDROCARBONS

Poly-aromatic hydrocarbons (PAHs) are a group of persistent organic pollutants that are resistant to degradation and can remain in the environment for a long time. PAHs are mainly produced as by-products of incomplete combustion, such as from fossil fuel burning, forest fires, coal fires or vehicle emissions. Currently, PAHs increasingly cause major environmental problems because of continuing industrial development. Biodegradation by microbes is the major natural mechanism for PAHs removal from the environment. The main objective of this research was to study the biodegradation of four types of PAHs (naphthalene, anthracene, pyrene and benzo[a]pyrene) by laccase produced from the lipolytic yeast, Aureobasidium pullulans var. melanogenum , screened from Sichang Island (Thailand). The optimal condition for growing A. pullulans var. melanogenum and its laccase production was first evaluated, and revealed that laccase production could be induced with 2 mM guaiacol and 0.5 M CuSO 4 in the yeast malt (YM) growth medium. Laccase was then lyophilized and stored in capsule form at +4oC to maintain enzymatic activity. The subsequent ability to degrade naphthalene, anthracene, pyrene and benzo[a]pyrenewas determined using high performance liquid chromatography. The results revealed that the laccase activity of one capsule (0.16 g) of lyophilized laccase was 711 U mL -1 (4.4 U mg -1 ) and could degrade a mixture of benzo[a]pyrene (37 µg mL -1 ), anthracene (7.5 µg mL -1 ), pyrene (99 µg mL -1 ) and naphthalene (90 µg mL -1 ) by 45.33, 38.16, 25.38 and 24.35% after 48 hours incubation respectively. Furthermore, the result also revealed that laccase (5 g) from A. pullulans can degrade naphthalene and anthracene in soil samples by 51.34 and 85.06% respectively, after incubation for 9 days.

New data on the distribution of certain psychrophilic yeasts in Moscow oblast

Several rare species and varieties of psychrophilic yeasts were isolated from the Sphagnum mosses and paludal vascular plants of Moscow oblast. Based on their 26S rDNA D1/D2 nucleotide sequences, they were assigned to the species Sterigmatosporidium polymorphum, Rhodotorula psychrophenolica, and Aureobasidium pullulans var. subglaciale. Thus, a new habitat of S. polymorphum was found and yeasts known previously only for the Alpine environments and the Arctic region have been isolated in the central regions of Russia for the first time.

Yeasts and Yeast-Like Fungi Associated with Tree Bark: Diversity and Identification of Yeasts Producing Extracellular Endoxylanases

A total of 239 yeast strains was isolated from 52 tree bark samples of the Medaram and Srisailam forest areas of Andhra Pradesh, India. Based on analysis of D1/D2 domain sequence of 26S rRNA gene, 114 strains were identified as ascomycetous; 107 strains were identified as basidiomycetous yeasts; and 18 strains were identified as yeast-like fungi. Among the ascomycetous yeasts, 51% were identified as members of the genus Pichia, and the remaining 49% included species belonging to the genera Clavispora, Debaryomyces, Kluyveromyces, Hanseniaspora, Issatchenkia, Lodderomyces, Kodamaea, Metschnikowia, and Torulaspora. The predominant genera in the basidiomycetous yeasts were Cryptococcus (48.6%), Rhodotorula (29%), and Rhodosporidium (12.1%). The yeast-like fungi were represented by Aureobasidium pullulans (6.7%) and Lecythophora hoffmanii (0.8%). Of the 239 yeast strains tested for Xylanase, only five strains of Aureobasidium sp. produced xylanase on xylan-agar medium. Matrix-assisted laser desorption ionization-time of flight analysis and N-terminal amino-acid sequence of the xylanase of isolate YS67 showed high similarity with endo-1-4-beta-xylanase (EC 3.2.1.8) of Aureobasidium pullulans var. melanigenum.

Acidophilic xylanase from Aureobasidium pullulans: efficient expression and secretion in Pichia pastoris and mutational analysis

A yeast-like fungus Aureobasidium pullulans var. melanigenum strain ATCC 20524 produces an extracellular acidophilic endo-1,4-β-xylanase with an optimum pH of 2.0 [Ohta et al., J. Biosci. Bioeng., 92, 262–270 (2001)]. The xynI cDNA encoding the precursor protein (XynI) was expressed in the methylotrophic yeast Pichia pastoris under the control of the alcohol oxidase I gene promoter. The 34 amino acid prepro-signal peptide of the A. pullulans XynI directed the efficient secretion of 178 mg of active xylanase per liter of the culture medium. The secretion level of the xylanase with its own signal peptide was comparable to that of the mature protein fused to the prepro leader from Saccharomyces cerevisiae α-mating factor and twofold higher than that of the mature protein fused to the pre-type signal peptide from P. pastoris acid phosphatase. The N-terminal amino acid sequence and the apparent M r of 24 kDa of the secreted recombinant protein indicated the native-like processing of the A. pullulans XynI signal sequence in P. pastoris. The three-dimensional model and mutational analysis of the xynI gene product showed that Asp-73 and Glu-157 residues located at the upper and lower edges of the active site cleft, respectively, play a significant role in its low pH optimum.