MAL31, a sugar transporter involved in pullulan biosynthesis in Aureobasidium pullulans.

To investigate the role of the sugar transporter MAL31 on pullulan biosynthesis, the coding gene mal31 was respectively disrupted and overexpressed in the parental strain A. pullulans CCTCC M 2012259 to construct mutants of A. pullulans Δmal31 and A. pullulans Mal31. Batch pullulan production significantly decreased by 69.1 % in A. pullulans Δmal31 but increased by 15.9 % in A. pullulans Mal31, as compared to the parental strain. We performed kinetics analysis, assays of key enzymes, determination of intracellular UDPG, NADH, and ATP contents, and measurement of transcriptional levels of genes associated with pullulan biosynthesis and excretion. The results confirmed that the mal31 disruption decreased the glucose consumption rate, decreased the formation rate and titer of pullulan, but increased the intracellular UDPG supply for ß-glucan accumulation. In contrast, the mal31 overexpression increased the transcriptional levels of genes associated with pullulan biosynthesis, and accelerated the rates of glucose consumption and pullulan formation, thereby increased pullulan production. Our findings revealed that MAL31 is involved in the transport of precursors for pullulan biosynthesis. This study provides an accurate operating site for genetic modification of A. pullulans for improving pullulan production and also presents a feasible technique route for the overproduction of other polysaccharides.

Improved production of ß-glucan by a T-DNA-based mutant of Aureobasidium pullulans.

To improve ß-1,3-1,6-D-glucan (ß-glucan) production by Aureobasidium pullulans, an Agrobacterium tumefaciens-mediated transformation method was developed to screen a mutant A. pullulans CGMCC 19650. Based on thermal asymmetric-interlaced PCR detection, DNA sequencing, BLAST analysis, and quantitative real-time PCR assay, the T-DNA was identified to be inserted in the coding region of mal31 gene, which encodes a sugar transporter involved in pullulan biosynthesis in the mutant. The maximal biomass and ß-glucan production under batch fermentation were significantly increased by 47.6% and 78.6%, respectively, while pullulan production was decreased by 41.7% in the mutant, as compared to the parental strain A. pullulans CCTCC M 2012259. Analysis of the physiological mechanism of these changes revealed that mal31 gene disruption increased the transcriptional levels of pgm2, ugp, fks1, and kre6 genes; increased the amounts of key enzymes associated with UDPG and ß-glucan biosynthesis; and improved intracellular UDPG contents and energy supply, all of which favored ß-glucan production. However, the T-DNA insertion decreased the transcriptional levels of ags2 genes, and reduced the biosynthetic capability to form pullulan, resulting in the decrease in pullulan production. This study not only provides an effective approach for improved ß-glucan production by A. pullulans, but also presents an accurate and useful gene for metabolic engineering of the producer for efficient polysaccharide production. KEY POINTS: • A mutant A. pullulans CGMCC 19650 was screened by using the ATMT method. • The mal31 gene encoding a sugar transporter was disrupted in the mutant. • ß-Glucan produced by the mutant was significantly improved.

Triton X-100 improves co-production of ß-1,3-D-glucan and pullulan by Aureobasidium pullulans.

The effects of several surfactants on the biosynthesis of ß-1,3-D-glucan (ß-glucan) and pullulan by Aureobasidium pullulans CCTCC M 2012259 were investigated, and Triton X-100 was found to decrease biomass formation but increase ß-glucan and pullulan production. The addition of 5 g/L Triton X-100 to the fermentation medium and bioconversion broth significantly increased ß-glucan production by 76.6% and 69.9%, respectively, when compared to the control without surfactant addition. To reveal the physiological mechanism underlying the effect of Triton X-100 on polysaccharides production, the cell morphology and viability, membrane permeability, key enzyme activities, and intracellular levels of UDPG, NADH, and ATP were determined. The results indicated that Triton X-100 increased the activities of key enzymes involved in ß-glucan and pullulan biosynthesis, improved intracellular UDPG and energy supply, and accelerated the transportation rate of precursors across the cell membrane, all of which contributed to the enhanced production of ß-glucan and pullulan. Moreover, a two-stage culture strategy with combined processes of batch fermentation and bioconversion was applied, and co-production of ß-glucan and pullulan in the presence of 5 g/L Triton X-100 additions was further improved. The present study not only provides insights into the effect of surfactant on ß-glucan and pullulan production but also presents a feasible approach for efficient production of analogue exopolysaccharides. KEY POINTS: • Triton X-100 increased ß-glucan and pullulan production under either batch fermentation or bioconversion. • Triton X-100 increased the permeability of cell membrane and accelerated the transportation rate of precursors across cell membrane. • Activities of key enzymes involved in ß-glucan and pullulan biosynthesis were increased in the presence of Triton X-100. • Intracellular UDPG levels and energy supply were improved by Triton X-100 addition.

Sodium chloride improves pullulan production by Aureobasidium pullulans but reduces the molecular weight of pullulan.

The effect of sodium chloride (NaCl) on pullulan production by batch culture of Aureobasidium pullulans CCTCC M 2012259 was investigated. NaCl at 3 g/L improved the pullulan titer by 26.7% but reduced the molecular weight of pullulan to only 46.8% of that obtained in the control without NaCl. In order to elucidate the physiological mechanism underlying the effect of NaCl on pullulan production, assays of key enzyme activity, gene expression, energy metabolism, and intracellular uridine diphosphate glucose (UDP-glucose) content were performed. Results indicated that NaCl increased the activities of α-phosphoglucose mutase and glucosyltransferase involved in pullulan biosynthesis, increased the activities of α-amylase being responsible for pullulan degradation, upregulated the transcriptional levels of pgm1, fks, and amy2 genes, enhanced the driving force for ATP supply, and helped to maintain intracellular UDP-glucose at a high level in A. pullulans CCTCC M 2012259. All these results illuminate the reason by which NaCl increases pullulan titer but reduces the molecular weight of pullulan.

The mechanism of improved intracellular organic selenium and glutathione contents in selenium-enriched Candida utilis by acid stress.

Batch culture of Candida utilis CCTCC M 209298 for the preparation of selenium (Se)-enriched yeast was carried out under different pH conditions, and maximal intracellular organic Se and glutathione (GSH) contents were obtained in a moderate acid stress environment (pH 3.5). In order to elucidate the physiological mechanism of improved performance of Se-enriched yeast by acid stress, assays of the key enzymes involved in GSH biosynthesis and determinations of energy supply and regeneration were performed. The results indicated that moderate acid stress increased the activity of γ-glutamylcysteine synthetase and the ratios of NADH/NAD+ and ATP/ADP, although no significant changes in intracellular pH were observed. In addition, the molecular mechanism of moderate acid stress favoring the improvement of Se-yeast performance was revealed by comparing whole transcriptomes of yeast cells cultured at pH 3.5 and 5.5. Comparative analysis of RNA-Seq data indicated that 882 genes were significantly up-regulated by moderate acid stress. Functional annotation of the up-regulated genes based on gene ontology and the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway showed that these genes are involved in ATP synthesis and sulfur metabolism, including the biosynthesis of methionine, cysteine, and GSH in yeast cells. Increased intracellular ATP supply and more amounts of sulfur-containing substances in turn contributed to Na2SeO3 assimilation and biotransformation, which ultimately improved the performance of the Se-enriched C. utilis.

Glutathione is involved in physiological response of Candida utilis to acid stress.

Candida utilis often encounters an acid stress environment when hexose and pentose are metabolized to produce acidic bio-based materials. In order to reveal the physiological role of glutathione (GSH) in the response of cells of this industrial yeast to acid stress, an efficient GSH-producing strain of C. utilis CCTCC M 209298 and its mutants deficient in GSH biosynthesis, C. utilis Δgsh1 and Δgsh2, were used in this study. A long-term mild acid challenge (pH 3.5 for 6 h) and a short-term severe acid challenge (pH 1.5 for 2 h) were conducted at 18 h during batch culture of the yeast to generate acid stress conditions. Differences in the physiological performances among the three strains under acid stress were analyzed in terms of GSH biosynthesis and distribution; intracellular pH; activities of γ-glutamylcysteine synthetase, catalase, and superoxide dismutase; intracellular ATP level; and ATP/ADP ratio. The intracellular GSH content of the yeast was found to be correlated with changes in physiological data, and a higher intracellular GSH content led to greater relief of cells to the acid stress, suggesting that GSH may be involved in protecting C. utilis against acid stress. Results presented in this manuscript not only increase our understanding of the impact of GSH on the physiology of C. utilis but also help us to comprehend the mechanism underlying the response to acid stress of eukaryotic microorganisms.

Efficient pullulan production by bioconversion using Aureobasidium pullulans as the whole-cell catalyst.

In this study, pullulan production was achieved by whole-cell bioconversion with Aureobasidium pullulans CCTCC M 2012259. Response surface methodology was applied to optimize the seed medium for incubating cells with high capability of pullulan bioconversion. Three medium components, namely, yeast extract, MgSO4·7H2O, and glucose were identified by Plackett-Berman design as significant factors affecting the cells' pullulan bioconversion capability. A three-level Box-Behnken design was then employed to determine the optimal levels of the three components. A mathematical model was developed to show the influence of each medium component and its effects on the cells' pullulan bioconversion capability. The model predicted a maximum pullulan bioconversion capability of 32.28 mg/g/h at the optimal yeast extract, MgSO4·7H2O, and glucose concentrations of 3.57, 0.18, and 63.97 g/l, respectively. The validation experiments showed that the cells' pullulan bioconversion capability was improved by 23.1% when the optimal medium was used, as compared with that obtained with the basic medium. Subsequently, the gene expression and activities of the key enzymes involved in pullulan biosynthesis were evaluated. When the optimal medium was employed, the transcriptional levels of pgm1 and fks were up-regulated by 2.5- and 1.2-fold, respectively, and the α-phosphoglucose mutase and glucosyltransferase activities were increased by 17 and 19%, respectively, when compared with those achieved using the basic medium. These results indicated that pullulan bioconversion using A. pullulans CCTCC M 2012259 as the whole-cell catalyst is an attractive approach for efficient pullulan production and can be applied for the production of other polysaccharides.

Kinetic study on the pretreatment and enzymatic saccharification of rice hull for the production of fermentable sugars.

The production of fermentable sugars from rice hull was studied by dilute acid pretreatment and enzymatic saccharification. Rice hull (15%, w/v) was pretreated by 1% (v/v) sulfuric acid at high temperature (120 approximately 160 degrees C) for 15, 30, 45, and 60 min, respectively. The maximum sugar concentration from rice hull in the prehydrolysate was obtained at 140 degrees C for 30 min, but the enzymatic saccharification yield from the corresponding pretreated rice hull is not high. To another aspect, the maximum enzymatic saccharification yield was achieved at 160 degrees C for 60 min, while the recovery of fermentable sugars was the poorest. To take account of fermentable sugars from pretreatment and enzymatic saccharification, the maximum yield of sugars was obtained only when rice hull was treated at 140 degrees C for 30 min. Under this condition, 72.5% (w/w) of all sugars generated from the raw material can be recovered. The kinetic study on the enzymatic saccharification of dilute acid pretreated rice hull was also performed in this work by a modified Michaelis-Menten model and a diffusion-limited model. After calculation by a linear and a non-linear regression analysis, both models showed good relation with the experimental results.

[Kinetic models for the effect of temperature on batch glutathione fermentation by Candida utilis].

Glutathione (L-gamma-glutamyl-L-cysteinylglycine), one of the major non-protein thiol compounds, is widely distributed in living cells and plays an important role in maintaining the normal redox environment of cells as an antioxidant. In the production of glutathione by fermentation, temperature is one of the most important environmental factors that affect the yield and the productivity of glutathione. Here the effect of temperature, varied from 24 degrees C to 32 degrees C, on the batch fermentation of glutathione in a 7 L stirred fermenter by Candida utilis WSH 02-08 was investigated. It was found that cell growth was hastened along with the increase of temperature. The maximum dry cell weight was achieved approximately 16 g/L under various temperatures, as soon as the glucose was exhausted. The effect of temperature on glutathione production was different from that on cell growth: the lower the temperature, the higher the glutathione production, i.e. the maximum glutathione concentration at 32 degrees C (235 mg/L) was only 75% and 64% of that at 30 degrees C and 26 degrees C, respectively. The maximum average specific growth rate (0.13 h(-1)) was achieved at 30 degrees C while the maximum glutathione concentration (366 mg x L(-1)) and the maximum intracellular glutathione content (2.3%) were obtained at 26 degrees C. Therefore, the optimum temperatures for cell growth and glutathione production are quite different in the batch fermentation. A modified Logistic equation was successfully applied to estimate the kinetics of cell growth. The maximum specific growth rate and the substrate inhibition constant, calculated from this equation, were both increased along with the temperature. In addition, the glutathione fermentation by C. utilis WSH 02-08 under various temperatures was proven to be a partial growth-associated process by estimating the process with the Luedeking-Piret equation. Based on the estimated parameon the estimated parameters, the effect of temperature on the kinetics of cell growth was further studied. An equation, dX / dt = [0.0224(T + 1.7)]2 X(1-X/Xmax) / 1 + S/ {8.26 x 10(6) x exp [-31477/R/(T+273)]}, was developed and applied to interlink the relationship between biomass concentration and temperature as well as substrate concentration in the batch glutathione fermentation. The experiment results showed that this model could predict the growth pattern very well.

[Effects of dissolved oxygen and pH on Candida utilis batch fermentation of glutathione].

The effects of dissolved oxygen (DO) and pH on glutathione batch fermentation by Candida utilis WSH-02-08 in a 7 liters stirred fermentor were investigated. It was shown that DO concentration is an important factor in glutathione production. With the initial glucose concentration of 30 g/L and a 5 L/min air flow rate, and the agitation rate less than 250 r/min, the DO concentration was not sufficient to satisfy the oxygen requirement during the fermentation. With an agitation rate of more than 300 r/min, the cell growth and glutathione production were enhanced significantly, with the dry cell mass and glutathione production were 20% and 25% higher than that at 200 r/min. When C. utilis WSH 02-08 was cultivated in a batch process without pH control, cell growth and glutathione production were inhibited, likely due to a dramatic decrease in the pH. Intracellular glutathione leakages were observed when the pH was 1.5 or less. To assess the effect of pH on glutathione production, six batch processes controlled at pH 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5 were conducted. The yield was highest at pH 5.5, when the dry cell mass and yield were 27% and 95% respectively higher than fermentation without pH control. The maximal intracellular glutathione content (2.15 %) was also achieved at the pH. To improve our understandings on the effect of pH on the batch glutathione production, a modified Logistic equation and Luedeking-Piret equation were used to simulate cell growth and glutathione production, respectively, under different pH. Based on the parameters obtained by the nonlinear estimation, kinetic analysis was performed to elucidate the effect of pH on the batch glutathione production. The process controlled at pH 5.5 was proven to be the best due to the higher value of K(I) (substrate inhibitory constant in the Logistic equation), lower value of a and higher value of beta (slope and intercept in the Luedeking-Piret equation, respectively).