Search

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 β-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.

In this study, cost-effective substrates such as cassava starch, corn steep liquor (CSL) and soybean meal hydrolysate (SMH) were used for pullulan production by Aureobasidium pullulans CCTCC M 2012259. The medium was optimized using response surface methodology (RSM) and artificial neural network (ANN) approaches, and analysis of variance indicated that the ANN model achieved higher prediction accuracy. The optimal medium predicted by ANN was used to produce high molecular weight pullulan in high yield. SMH substrates increased both biomass and pullulan titer, while CSL substrates maintained higher pullulan molecular weight. Results of kinetic parameters, key enzyme activities and intracellular uridine diphosphate glucose contents revealed the physiological mechanism of changes in pullulan titer and molecular weight using different substrates. Economic analysis of batch pullulan production using different substrates was performed, and the cost of nutrimental materials for CSL and SMH substrates was decreased by 46.1% and 49.9%, respectively, compared to the control using glucose and yeast extract as substrates, which could improve the competitiveness of pullulan against other polysaccharides in industrial applications.

To improve β-1,3-1,6-D-glucan (β-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 β-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 β-glucan biosynthesis; and improved intracellular UDPG contents and energy supply, all of which favored β-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 β-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. • β-Glucan produced by the mutant was significantly improved.

To improve β-1,3-1,6-D-glucan (β-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 β-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 β-glucan biosynthesis; and improved intracellular UDPG contents and energy supply, all of which favored β-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 β-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. • β-Glucan produced by the mutant was significantly improved.

The effects of several surfactants on the biosynthesis of β-1,3-D-glucan (β-glucan) and pullulan by Aureobasidium pullulans CCTCC M 2012259 were investigated, and Triton X-100 was found to decrease biomass formation but increase β-glucan and pullulan production. The addition of 5 g/L Triton X-100 to the fermentation medium and bioconversion broth significantly increased β-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 β-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 β-glucan and pullulan. Moreover, a two-stage culture strategy with combined processes of batch fermentation and bioconversion was applied, and co-production of β-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 β-glucan and pullulan production but also presents a feasible approach for efficient production of analogue exopolysaccharides. KEY POINTS: • Triton X-100 increased β-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 β-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.

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.

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.

ATP is indispensable to the biosynthesis of both S-adenosylmethionine (SAM) and glutathione (GSH) in yeast cells. To improve ATP supply for overproduction of SAM and GSH in Candida utilis CCTCC M 209298, an exogenous ATP6 gene from Arabidopsis thaliana was expressed in the parental strain to construct the mutant C. utilis ATP6 by genomic integration. The maximal production of SAM and GSH in the mutant increased by 46.6 and 28.7%, respectively, when compared with those obtained in the parental strain. The mechanism underlying improved SAM and GSH biosynthesis by exogenous ATP6 gene expression revealed that the mutant had higher activities of key enzymes involved in SAM and GSH biosynthesis as well as energy metabolism. Increased NADH availability and F0 F1 -ATPase activity subsequently resulted in improved ATP regeneration and intracellular ATP supply for SAM and GSH overproduction. The present study not only developed an effective method for improving SAM and GSH biosynthesis by energy metabolism regulation, but also offered a novel approach for efficient production of similar energy-consuming products in eukaryotic cells.

The effects of mineral salts on pullulan production by bioconversion using whole cells of Aureobasidium pullulans CCTCC M 2012259 as the catalyst were investigated. Copper sulfate (CuSO4) improved pullulan production by 36.2% and 42.3% when added at the optimum concentration of 0.2mg/L to the bioconversion broth or seed medium, respectively, as compared with controls without CuSO4 addition. Pullulan production was further enhanced when CuSO4 was added to both seed medium and bioconversion broth simultaneously. In order to probe the mechanism of CuSO4 improvement, cell viability, membrane integrity, intracellular adenosine triphosphate (ATP) levels and the activities of key enzymes involved in pullulan biosynthesis were determined. As a result, CuSO4 increased the activities of key biosynthetic enzymes, maintained intracellular ATP at a higher level, and accelerated the rate of pullulan secretion, all of which contributed to improved pullulan production by bioconversion.

The hydrolyzate of banana peels containing abundant fermentable sugars as glucose, xylose, mannose, and arabinose was successfully used as a novel substrate for the efficient production of glutathione by Candida utilis SZU 07-01. Xylose was first selected as the sole carbon source for glutathione production, medium optimization for better cell growth and higher glutathione using response surface methodology consisting of PB design, the steepest ascent experiment and CCD was carried out, and the optimal combination of nutrients was obtained as follows: xylose 20 g/L, (NH4)2SO4 9.59 g/L, KH2PO4 3 g/L, L-methionine 5.72 g/L and MgSO4 0.20 g/L. The maximum dry cell weight and glutathione achieved using the optimized medium were 7.36 g/L and 154.32 mg/L, respectively. Following with the content in this medium, other sugars like glucose, mannose and arabinose were chosen as the sole carbon source and all tested available for glutathione production. Based on these results, the hydrolyzate of banana peels was selected as a novel substrate, and a high DCW of 7.68 g/L and glutathione yield of 111.33 mg/L were obtained with the initial sugar concentration of 20 g/L in the hydrolyzate of banana peels.

An effective S-adenosylmethionine and glutathione enriching yeast mutant of Candida utilis CCTCC M 209298 was first screened from plates containing 0.5 g/L of DL-ethionine by complex mutagenesis with UV and γ-ray in this study. Medium components optimization for enhanced co-production of S-adenosylmethionine and glutathione by C. utilis CCTCC M 209298 was further carried out using response surface methodology. The significant factors influencing S-adenosylmethionine and glutathione co-production were selected by Plackett-Burman design as sucrose, KH2PO4 and L-methionine, and Box-Behnken design was applied for further optimization studies. Based on these approaches, the optimized concentrations on medium components for higher co-production of S-adenosylmethionine and glutathione were sucrose 35.4 g/L, (NH4)2SO4 10 g/L, KH2PO4 12.3 g/L, MgSO4·7H2O, 0.05 g/L, CaCl2 0.05 g/L and L-methionine 4.6 g/L. The medium optimization by response surface methodology led to a total production of 589.3 mg/L on S-adenosylmethionine and glutathione, which was 2.4-fold increased compared with the medium without optimization.

Several inorganic/organic N-containing substances were tested as nitrogen source for efficient glutathione production by C. utilis WSH 02-08. Although the strain could assimilate all the inorganic/organic nitrogen, urea and ammonium sulfate were found more favorable to cell growth and glutathione biosynthesis in a flask, respectively, and an optimal C/N ratio existed for each as 5.6 mol/mol and 8.3 mol/mol. A mixed nitrogen source of urea and ammonium under diverse C/N ratios could not boost glutathione fermentation despite the many mixed strategies that were introduced. Batch glutathione production in a stirred fermentor, using the sole or mixed nitrogen sources of urea and ammonium sulfate under their optimal C/N ratios, were conducted; urea was further proved to be the best nitrogen source for glutathione production. The reason was then quantitatively described by kinetic model, together with the distribution of flux for metabolites in metabolic network of glutathione biosynthesis by C. utilis WSH 02-08.

Hydrolytic conditions of rice hulls by acid and alkaline treatments before enzymatic saccharification were optimized in this study. Based on the results of single-factor experiments and an orthogonal array experiment, reaction time was found to be the most important factor for the acidic hydrolysis of rice hulls. Maximum yield of sugars from 1 g of rice hulls by acidic treatment under optimized conditions was 213.6 mg. The yield of lignin removal from acidic pretreated rice hulls by alkaline treatment increased with increase in reaction temperature and time. The amount of sugars obtained from 1 g of pretreated rice hulls by enzymatic saccharification was 307.7 mg, and the conversion rate of sugars from crude fibers in pretreated rice hulls was about 72%. Instrumental analyses with FTIR and SEM indicated that lignin in rice hulls was partially removed by alkaline treatment, and the structure of rice hulls became deformed and more fibers were exposed to cellulases after acidic treatment.

The present study investigated the effects of three constituent amino acids on glutathione production in flask culture of Candida utilis. Although l-glutamic acid and glycine had little impact on cell growth and glutathione biosynthesis, l-cysteine positively influenced glutathione production, despite inhibiting cell growth when it was added prior to stationary phase. Adding 8 mmol/L of l-cysteine to the culture broth at 16 h boosted glutathione production by 91%, increasing the intracellular glutathione content by 106% compared to untreated controls. A temperature-shift strategy, in which we shifted batch and fed-batch cultures of C. utilis from 30 to 26°C, also significantly enhanced glutathione production. Applying both strategies (i.e. adding 20 mmol/L l-cysteine and shifting the temperature from 30 to 26°C) at 33 h enhanced the glutathione concentration and the intracellular glutathione content to 1,312 mg/L and 3.75%, respectively, during fed-batch cultivation (glucose feeding at a constant rate of 18.3 g/h). The average specific glutathione production rate under this condition was 129% higher than that of the control without strategy.

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.

The effects of representative surfactants on the growth of Saccharomyces cerevisiae WSH-J701 and glutathione (GSH) production were studied. The anionic surfactant sodium dedecyl sulfate (SDS) and the cationic surfactant cetyltrimethyl ammonium bromide (CTAB) had critical inhibitory concentrations of 0.5 and 0.025 g/l, respectively, to cell growth during cultivation. Above these concentrations, cell growth was greatly inhibited or even ceased. Nonionic surfactants such as polyoxyethylene lauryl ether (Brij30) and polyoxyethylene sorbitan monooleate (Tween80) could also affect cell growth only when their concentrations reached much higher levels. Under the critical concentration, SDS and CTAB increased the extracellular GSH concentration by 10 and 15%, but above this level, they improved extracellular GSH concentration by 35 and 60%, respectively. Otherwise, total GSH concentration and intracellular GSH content decreased slightly. The effect of Brij30 was somewhat like ionic surfactants, extracellular GSH concentration could be increased by 50% but the intracellular GSH content was reduced by 33%. Tween80 seemed to have no effect on extracellular GSH accumulation, but total GSH concentration and intracellular GSH content decreased gradually by increasing its concentration.

In batch culture for glutathione production with Candida utilis, a higher temperature (30 °C) was required to hasten cell growth while a lower temperature (26 °C) was needed to increase the production of glutathione. A two-stage temperature control strategy was used to enhance both the yield and the productivity of glutathione. As a result, glutathione production was increased by 5% and 23% of that at 26 °C and 30 °C, respectively, and the intracellular glutathione content reached 2.5% (w/w).

Selenium-enriched Candida utilis has attracted much attention due to its expanding application in food and feed additives. The objective of this study was to efficiently prepare selenium-enriched C. utilis and to investigate the effects of the prepared yeast on antioxidant capacity in rats. A batch culture of selenium-enriched C. utilis was first carried out, and the addition of sodium selenite (Na2SeO3) after all glucose had been consumed was found to favor higher intracellular glutathione and organic selenium content. Moreover, l -methionine boosted yeast cell growth and glutathione biosynthesis, and prevented glutathione from leaking to the extracellular space that can be caused by Na2SeO3. We therefore developed a two-stage culture strategy involving supplementation with l -methionine and Na2SeO3 at separate culture phases to improve the performance of selenized C. utilis. Using this two-stage culture strategy, intracellular glutathione content reached 18.6 mg/g and 15.5 mg/g, respectively, in batch and fed-batch systems, and organic selenium content reached 905.2 μg/g and 984.7 μg/g, respectively. The effects of selenium-enriched C. utilis on the activities of antioxidant related enzymes in rats were investigated, and the prepared selenium-enriched C. utilis was shown to be an optimal dietary supplement for enhancing antioxidant capacity in rats.

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.

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).

Aims: To study the effect of low pH stress on glutathione (GSH) synthesis and excretion capability of GSH fermentation production in Candida utilis. Methods and Results: When C. utilis WSH 02–08 was cultivated in a glucose–ammonium sulfate medium without pH control, GSH leakage occurred when the pH of the medium decreased to 1·5. However, analysis of the cell viability indicated that the cells were not lysed. To further study the effect of low pH stress on GSH production, pH-controlled batch cultures were conducted, where the pH was switched from 5·5 to 1·2 at 24 h and maintained at 1·2 for 6 h. Nearly all intracellular GSH was leaked into the medium and the cell viability decreased dramatically, conceiving a long-term exposure of strain WSH 02–08 at low pH environment led to a complete cell lysis. A critical point (treated at pH 1·2 for 3 h) was experimentally determined, where most cells were alive but suffering a low pH stress. Low pH-stressed C. utilis cells displayed an increased intracellular GSH synthesis and export capability, which protected the cells against short-term low pH treatment. Conclusions: Using this knowledge, a low pH-stress strategy was developed and applied in fed-batch production of GSH and 197·3 mg l−1 of GSH was secreted into the medium. The GSH-specific production yield could be increased from 2·11 to 2·67% (w/w), and the total GSH concentration could reach 737·1 mg l−1 and increased by 24·9%. Significance and Impact of the Study: This is the first report of GSH secretion of C. utilis at low pH. This study demonstrated the importance of the physiology-based fermentation strategy in the production of useful metabolites.