Pullulan Biosynthesis Research Articles

Overexpression of the pullulan synthetase gene enhanced pullulan production and its molecular weight by a mutant of Aureobasidium melanogenum P16

The pullulan synthetase gene (PUL1), involved in pullulan biosynthesis in Aureobasidium species, remains poorly understood. The open reading frame (ORF) of the PUL1 gene from the high pullulan-producing yeast Aureobasidium melanogenum P16 strain was cloned and characterized. The ORF of the PUL1 gene was determined to be 592 bp in length, encoding 178 amino acid residues. It was observed that an intron of 55 bp disrupted the gene. The promoter of the PUL1 gene contained a CAAT box, a TATA box, and a 5’-HGATAR-3′ sequence. The deduced protein possessed a signal peptide comprising 18 amino acids and harbored five potential N-glycosylation sites. Following the disruption of the PUL1 gene in strain P16, the disruptant DP108 yielded 34.7 ± 0.3 g/L of pullulan from sucrose, significantly lower than the production by its wild-type strain P16. This discrepancy underscores the close association between the PUL1 gene and pullulan biosynthesis. The majority of the fused Gfp-Pul1 proteins were found to be localized in the cell membrane and on the surface of vacuoles within the yeast-like fungal cells, indicating that pullulan biosynthesis occurred at these subcellular sites. Following the overexpression of the PUL1 gene, strain G14 produced >72.0 g/L of pullulan from sucrose, surpassing the production of its wild-type counterpart strain P16, which yielded 65.5 g/L of pullulan under the identical conditions. This outcome demonstrated that the overexpression of the PUL1 gene significantly enhanced pullulan production. The apparent molecular mass of the purified pullulan increased to 4.4 × 105 Da. As an auxiliary protein, Pul1 was predicted to bind to AmAgs2, the key enzyme in pullulan biosynthesis, facilitating enhanced pullulan production.

NsdD, a GATA-type transcription factor is involved in regulation and biosynthesis of macromolecules melanin, pullulan, and polymalate in Aureobasidium melanogenum

In this study, an NSDD gene, which encoded a GATA-type transcription factor involved in the regulation and biosynthesis of melanin, pullulan, and polymalate (PMA) in Aureobasidium melanogenum, was characterized. After the NSDD gene was completely removed, melanin production by the Δnsd mutants was enhanced, while pullulan and polymalate production was significantly reduced. Transcription levels of the genes involved in melanin biosynthesis were up-regulated while expression levels of the genes responsible for pullulan and PMA biosynthesis were down-regulated in the Δnsdd mutants. In contrast, the complementation of the NSDD gene in the Δnsdd mutants made the overexpressing mutants restore melanin production and transcription levels of the genes responsible for melanin biosynthesis. Inversely, the complementation strains, compared to the wild type strains, showed enhanced pullulan and PMA yields. These results demonstrated that the NsdD was not only a negative regulator for melanin biosynthesis, but also a key positive regulator for pullulan and PMA biosynthesis in A. melanogenum. It was proposed how the same transcriptional factor could play a negative role in melanin biosynthesis and a positive role in pullulan and PMA biosynthesis. This study provided novel insights into the regulatory mechanisms of multiple A. melanogenum metabolites and the possibility for improving its yields of some industrial products through genetic approaches.

Light calcium carbonate improves pullulan biosynthesis by Aureobasidium pullulans under high concentration of sugar.

The effects of light calcium carbonate (CaCO3) on pullulan biosynthesis by Aureobasidium pullulans NCPS2016 were investigated. Light CaCO3 enhanced pullulan production by 12.4% when added to the low concentration of fructose broth compared with K2HPO4. Pullulan production was further improved when increasing both the concentrations of light CaCO3 and fructose. Compared to K2HPO4, light CaCO3 improved the activities of UDP-glucose pyrophosphorylase, α-phosphoglucose mutase, UDP-glucosyltransferase, and glucosyltransferase relevant to pullulan biosynthesis, and the gene transcriptional levels of UDP-glucose pyrophosphorylase, α-phosphoglucose mutase, UDP-glucosyltransferase, and glucose kinase were enhanced. During 30-liter fermentation, 144.3g/L of purified pullulan was produced from 200g/L of fructose and 15g/L of light CaCO3 within 168h, with the yield and productivity of 0.72g/g and 0.86g/L/h respectively. This is the first report that light CaCO3 improves pullulan production significantly.

The role of pH transcription factor Appacc in upregulation of pullulan biosynthesis in Aureobasidium pullulans using potato waste as a substrate

pH is one of the important environmental factors affecting the growth, development and secondary metabolites of fungi. To better utilize potato waste for the production of pullulan by fermentation, in this study, the amino acid sequence and structural domain of pH transcription factor Appacc were analyzed using the bioinformatics methods. Appacc showed three typically conserved zinc finger domains, with the closest homology to Zymoseptoria brevis. The function of Appacc was characterized by ΔAppacc and OEXpacc mutants. The mycelium growth of ΔApacc mutants was inhibited, especially, under alkaline conditions. Furthermore, the pullulan production of ΔAppacc mutant was reduced and the expression of pullulan synthetic genes also decreased. Moreover, the OEXpacc mutant further demonstrated that pacc could regulate the expression of pullulan synthesis genes. The yield of pullulan polysaccharide increased from 13.6 g/L to 17.8 g/L by direct fermentation without changing the pH of potato waste. These results suggest that Appacc played a vital role in the growth of Aureobasidium pullulans and that the production of pullulan from potato waste can be increased by overexpression of pacc gene.

Exclusive Biosynthesis of Pullulan Using Taguchi's Approach and Decision Tree Learning Algorithm by a Novel Endophytic Aureobasidium pullulans Strain.

Pullulan is a biodegradable, renewable, and environmentally friendly hydrogel biopolymer, with potential uses in food, medicine, and cosmetics. New endophytic Aureobasidium pullulans (accession number; OP924554) was used for the biosynthesis of pullulan. Innovatively, the fermentation process was optimized using both Taguchi's approach and the decision tree learning algorithm for the determination of important variables for pullulan biosynthesis. The relative importance of the seven tested variables that were obtained by Taguchi and the decision tree model was accurate and followed each other's, confirming the accuracy of the experimental design. The decision tree model was more economical by reducing the quantity of medium sucrose content by 33% without a negative reduction in the biosynthesis of pullulan. The optimum nutritional conditions (g/L) were sucrose (60 or 40), K2HPO4 (6.0), NaCl (1.5), MgSO4 (0.3), and yeast extract (1.0) at pH 5.5, and short incubation time (48 h), yielding 7.23% pullulan. The spectroscopic characterization (FT-IR and 1H-NMR spectroscopy) confirmed the structure of the obtained pullulan. This is the first report on using Taguchi and the decision tree for pullulan production by a new endophyte. Further research is encouraged for additional studies on using artificial intelligence to maximize fermentation conditions.

High-level production of pullulan and its biosynthesis regulation in Aureobasidium pullulans BL06.

Pullulan has many potential applications in the food, pharmaceutical, cosmetic and environmental industries. However, the yield and molecular properties of pullulan produced by various strains still need to be promoted to fit the application needs. A novel yeast-like strain Aureobasidium pullulans BL06 producing high molecular weight (Mw) pullulan (3.3 × 106Da) was isolated and identified in this study. The remarkable Mw of pullulan produced by A. pullulans BL06 was the highest level ever reported thus far. To further regulate the biosynthesis of pullulan in A. pullulans BL06, three gene knockout strains A. pullulans BL06 ΔPMAs, A. pullulans BL06 Δmel, and A. pullulans BL06 ΔPMAsΔmel, were constructed. The results showed that A. pullulans BL06 ΔPMAs could produce 140.2g/L of moderate Mw (1.3 × 105Da) pullulan after 120h of fermentation. The highest yield level of pullulan to date could vastly reduce its production cost and expand its application scope and potential. The application experiments in food preservation showed that the moderate-Mw pullulan obtained in this work could reduce the weight loss of celery cabbages and mangos by 12.5% and 22%, respectively. Thus, the novel strains A. pullulans BL06 and A. pullulans BL06 ΔPMAs possessed unlimited development prospects in pullulan production at various Mw ranges and pullulan applications in multiple fields.

Whole-crop biorefinery of corn biomass for pullulan production by Aureobasidium pullulans

In the present study, corn starch, cob, and straw were biorefined and used as feedstocks for the production of pullulan. The titer and molecular weight (Mw) of pullulan significantly decreased when corn cob and straw hydrolysates were utilized by the parental strain Aureobasidium pullulans CCTCC M 2012259 (PS). Based on adaptive laboratory evolution of PS, an evolved strain A. pullulans EV6 with strong adaptability to the whole corn biomass hydrolysate and high capability of pullulan biosynthesis was screened. Batch pullulan fermentation results indicated that EV6 produced an increased titer of pullulan with a higher Mw than PS. The underlying reasons for these increases were revealed by assaying key enzymes activities and measuring intracellular uridine diphosphate glucose levels. Subsequently, whole-crop biorefinery of corn biomass was conducted, and the results confirmed that whole corn crop has immense potential for efficient pullulan production.

High-level production of pullulan from high concentration of glucose by mutagenesis and adaptive laboratory evolution of Aureobasidium pullulans

The cost of carbon sources and the low efficiency of the fermentation titer limit the industrial application of pullulan. In this study, a hypertonic-tolerant strain with efficient utilization of glucose was obtained using a double strategy. Initially, a strain for efficient synthesis of pullulan from glucose was generated by mutagenesis. Subsequently, the mutant was directionally evolved on the plate containing a high glucose concentration to enhance high osmotic resistance. The enzyme activities and the transcriptional levels involved in pullulan biosynthesis and high osmotic tolerance in mutants were increased. Nitrogen source and inorganic salts also significantly affected the production of pullulan by M233-20 from high concentration of glucose. The pullulan titer of 162.1 g/L was obtained using the response surface methodology in the flask. The strain M233-20 produced 162.3 g/L pullulan in a 30-L bioreactor with a yield of 0.82 g/g glucose. Hence, this work provides a potential industrial pullulan producer M233-20 and a strategy to develop excellent strain.

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 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.

Emerging Trends in Pullulan-Based Antimicrobial Systems for Various Applications.

Global reports on multidrug resistance (MDR) and life-threatening pathogens such as SARS-CoV-2 and Candida cruris have stimulated researchers to explore new antimicrobials that are eco-friendly and economically viable. In this context, biodegradable polymers such as nisin, chitin, and pullulan play an important role in solving the problem. Pullulan is an important edible, biocompatible, water-soluble polymer secreted by Aureobasidium pullulans that occurs ubiquitously. It consists of maltotriose units linked with α-1,6 glycosidic bonds and is classed as Generally Regarded as Safe (GRAS) by the Food and Drug Administration (FDA) in the USA. Pullulan is known for its antibacterial, antifungal, antiviral, and antitumor activities when incorporated with other additives such as antibiotics, drugs, nanoparticles, and so on. Considering the importance of its antimicrobial activities, this polymer can be used as a potential antimicrobial agent against various pathogenic microorganisms including the multidrug-resistant (MDR) pathogens. Moreover, pullulan has ability to synthesize biogenic silver nanoparticles (AgNPs), which are remarkably efficacious against pathogenic microbes. The pullulan-based nanocomposites can be applied for wound healing, food packaging, and also enhancing the shelf-life of fruits and vegetables. In this review, we have discussed biosynthesis of pullulan and its role as antibacterial, antiviral, and antifungal agent. Pullulan-based films impregnated with different antimicrobials such as AgNPs, chitosan, essential oils, and so on, forming nanocomposites have also been discussed as natural alternatives to combat the problems posed by pathogens.

The GATA type transcriptional factors regulate pullulan biosynthesis in Aureobasidium melanogenum P16

Aureobasidium melanogenum P16, the high pullulan producer, had only one GATA type transcriptional activator AreA and one GATA type transcriptional repressor AreB. It was found that 2.4 g/L of (NH4)2SO4 had obvious nitrogen repression on pullulan biosynthesis by A. melanogenum P16. Removal of the AreB gene could make the disruptant DA6 produce 34.8 g/L pullulan while the P16 strain only produced 28.8 g/L pullulan at the efficient nitrogen condition. Further both removal of the native AreA gene and overexpression of the mutated AreAS628-S678 gene with non-phosphorylatable residues could render the transformant DEA12 to produce 39.8 g/L pullulan. The transcriptional levels of most of the genes related to pullulan biosynthesis in the transformant DEA12 were greatly enhanced. The mutated AreAS628-S678 was localized in the nuclei of the transformant DEA12 while the native AreA was distributed in the cytoplasm in A. melanogenum P16. This meant that nitrogen repression on pullulan biosynthesis in the transformant DEA12 was indeed significantly relieved. This was the first time to report that the GATA type transcriptional factors of nitrogen catabolite repression system could regulate pullulan biosynthesis in Aureobasidium spp.

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

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.

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

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 5g/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 5g/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.

Glycerol, trehalose and vacuoles had relations to pullulan synthesis and osmotic tolerance by the whole genome duplicated strain Aureobasidium melanogenum TN3-1 isolated from natural honey

In our previous study, it was found that Aureobasidium melanogenum TN3-1 was a high pullulan producing and osmotic tolerant yeast-like fungal strain. In this study, the HOG1 signaling pathway controlling glycerol synthesis, glycerol, trehalose and vacuoles were found to be closely related to its pullulan biosynthesis and high osmotic tolerance. Therefore, deletion of the key genes for the HOG1 signaling pathway, glycerol and trehalose biosynthesis and vacuole formation made all the mutants reduce pullulan biosynthesis and increase sensitivity of the growth of the mutants to high glucose concentration. Especially, abolishment of both the VSP11 and VSP12 genes which controlled the fission/fusion balance of vacuoles could cause big reduction in pullulan production (less than 7.4 ± 0.4 g/L) by the double mutant ΔDV-5 and increased sensitivity to high concentration glucose, while expression of the VSP11 gene in the double mutant ΔDV-5 made the transformants EV-2 restore pullulan production and tolerance to high concentration glucose. But cell growth of them were the similar. The double mutant ΔDV-5 had much bigger vacuoles and less numbers of vacuoles than the transformant EV-2 and its wild type strain TN3-1 while it grew weakly on the plate with 40% (w/v) glucose while the transformant EV-2 and its wild type strain TN3-1 could grow normally on the plate even with 60% (w/v) glucose. The double mutant ΔDV-5 also had high level of pigment and its cells were swollen. This was the first time to give the evidence that glycerol, trehalose and vacuoles were closely related to pullulan biosynthesis and high osmotic tolerance by A. melanogenum.

The differences between fungal α-glucan synthase determining pullulan synthesis and that controlling cell wall α-1,3 glucan synthesis

The fungal α-glucan synthases (Agss) are multi-domain proteins catalyzing biosynthesis of cell wall α-1,3-glucan which determines cell wall integrity or fungal pathogenicity and pullulan which is a maltotriosyl polymer made of α-1,4 and α-1,6 bound glucose units. The Agss family can be divided into 11 groups, some of which lost the original functions due to accumulation of harmful mutations or gene loss. Schizosaccharomyces pombe kept five kinds of Agss in the genome while Aspergillus spp. and Penicillium spp. lost one or two or three kinds of Agss. All the human, animal and plant pathogens kept only one single kind of Ags or only one active Ags for synthesis of cell wall α-1,3-glucan, a virulence factor. While the genus Aureobasidium spp. contained three kinds of Agss, of which only some of the Ags2 was involved in pullulan biosynthesis. Although many Agss contained Big_5 domain, only the Big_5 domain with conserved amino acids LQS from some strains of A. melanogenum could catalyze pullulan biosynthesis. This whole amino acid sequence and phylogenetic differences may cause non-α-1,3-glucan synthesizing activity of some fungal Agss.

Pullulan biosynthesis in yeast-like fungal cells is regulated by the transcriptional activator Msn2 and cAMP-PKA signaling pathway

Pullulan is an important polysaccharide. Although its synthetic pathway in Aureobasidium melanogenum has been elucidated, the mechanism underlying its biosynthesis as regulated by signaling pathway and transcriptional regulator is still unknown. In this study, it was found that the expression of the UGP1 gene encoding UDPG-pyrophosphorylase (Ugp1) and other genes which were involved in pullulan biosynthesis was controlled by the transcriptional activator Msn2 in the nuclei of yeast-like fungal cells. The Ugp1 was a rate-limiting enzyme for pullulan biosynthesis. In addition, the activity and subcellular localization of the Msn2 were regulated only by the cAMP-PKA signaling pathway. When the cAMP-PKA activity was low, the Msn2 was localized in the nuclei, the UGP1 gene was highly expressed, and pullulan was actively synthesized. By contrast, when the cAMP-PKA activity was high, the Msn2 was localized in the cytoplasm and the UGP1 gene expression was disabled so that pullulan was stopped, but lipid biosynthesis was actively enhanced. This study was the first to report that pullulan and lipid biosynthesis in yeast-like fungal cells were regulated by the Msn2 and cAMP-PKA signaling pathway. Elucidating the regulation mechanisms was important to understand their functions and enhance pullulan and lipid biosynthesis.

Alternative primers are required for pullulan biosynthesis in Aureobasidium melanogenum P16

Although pullulan has many uses in industry, the detailed mechanisms of its biosynthesis still require clarification. In this study, it was found that a short α-1,4-glucosyl chain (pullulan primer) synthesized by the glycogenins Glg1 and Glg2 for initiation of glycogen biosynthesis was also needed for pullulan synthesis. The primers were also synthesized on sterol glycosides and glucosylceramides by catalysis of sterol glucosyltransferase (Sgt1) and ceramide β-glucosyltransferase (Gcs1). All the primers might be elongated to be long α-1,4-glucosyl chain (pullulan precursor) by catalysis of the glycogen synthetase domain of the AmAgs2 as previously reported. Then, the amylase domain of the same AmAgs2 was responsible for pullulan biosynthesis. Removal of all the genes encoding Glg1, Glg2, Gcs1 and Sgt1 made all the mutants produce much less pullulan than the strain P16. However, pullulan synthesis could not be stopped totally in these mutants, suggesting that any other unknown alternative pullulan primers may exist in the yeast cells. Complementation of all the genes in the mutants restored pullulan biosynthesis. This is the first time to report that like starch and glycogen biosynthesis, alternative primers are also required for pullulan biosynthesis in Aureobasidium melanogenum P16.

Enhanced β-glucan and pullulan production by Aureobasidium pullulans with zinc sulfate supplementation.

The effects of mineral salts on the production of exopolysaccharides, including β-glucan and pullulan, by Aureobasidium pullulans CCTCC M 2012259 were investigated. Zinc sulfate at certain concentrations decreased dry biomass but favored to the biosynthesis of both exopolysaccharides. When 100mg/L zinc sulfate was added to the fermentation medium, production of β-glucan and pullulan increased by 141.7 and 10.2%, respectively, when compared with that noted in the control without zinc sulfate addition. To reveal the physiological mechanism underlying improved β-glucan and pullulan production, key enzymes activities, energy metabolism substances, intracellular uridine diphosphate glucose (UDPG) levels, and gene expression were determined. The results indicated that zinc sulfate up-regulated the transcriptional levels of pgm1, ugp, fks, and kre6 genes, increased activities of key enzymes involved in the biosynthesis of UDPG, β-glucan and pullulan, enhanced intracellular UDPG content, and improved energy supply, all of which contributed to the increment in β-glucan and pullulan production. The present study not only provides a feasible approach to improve the production of exopolysaccharides but also contributes to better understanding of the physiological characteristics of A. pullulans.

A multidomain α-glucan synthetase 2 (AmAgs2) is the key enzyme for pullulan biosynthesis in Aureobasidium melanogenum P16

Pullulan, a biological macromolecule, has many applications. However, it is completely unknown how and where it is synthesized. In this study, it was found that the multidomain AmAgs2 (α-glucan synthase 2) encoded by an AmAGS2 gene in Aureobasidium melanogenum P16 contained the amylase domain (Amy_D), the glycogen synthetase domain (Gys_D) and the transmembrane regions in which the exopolysaccharide transporter domain (EPST_D) was embedded. Removal of the AmAGS2 gene in A. melanogenum P16 rendered the disruptants not to synthesize any pullulan and complementation of the AmAGS2 gene in the disruptants restored pullulan synthesis. Overexpression of the gene in Aureobasidium melanogenum CBS105.22, a non-pullulan producer, resulted in the transformants producing pullulan. Therefore, the AmAGS2 gene was the key gene responsible for pullulan biosynthesis in A. melanogenum P16. It was speculated that the short α-1,4-glucosyl chains (pullulan primers) were elongated by the Gys_D of the AmAgs2 to form long α-1,4-glucosyl chains (precursors of pullulan). All the precursors were transported to outside plasma membrane by the EPST_D in the transmembrane regions of the AmAgs2. Then, the Amy_D of the AmAgs2 was responsible for both hydrolysis of the endo-α-1,4-linkages in the precursors to release maltotriose and transfer of the maltotriose to Lph-glucose to form α-1,6 glucosidic bonds between maltotrioses in pullulan molecule. This is the first time to report that the AmAgs2 can play the key role in pullulan biosynthesis.

Improved pullulan production by a mutant of Aureobasidium melanogenum TN3-1 from a natural honey and capsule shell preparation

Aureobasidium melanogenum TN3-1 isolated from a natural honey was a highly genome-duplicated yeast-like fungal strain and a very high pullulan producer. In this study, simultaneous removal of both duplicated AMY1 genes encoding α-amylase and duplicated PKS1 genes responsible for melanin biosynthesis in A. melanogenum TN3-1 rendered a mutant AMY-PKS-11 to transform 140.0 g/L of glucose to produce 103.50 g/L of pigment-free pullulan with molecular weight (Mw) of 3.2 × 105 g/mol. α-Amylase activity produced by the mutant AMY-PKS-11 and expression of the AMY1 genes and PKS genes in it was reduced, but expression of various genes responsible for pullulan biosynthesis in the mutant AMY-PKS-11 was up-regulated. The produced pullulan was used to make the capsule shells successfully and the prepared pullulan capsule shells had various advantages such as high strength, good oxygen barrier properties, raw materials availability, tightness, lightness and high water resistance and may be suitable for all the consumers. Therefore, the prepared capsule shells had highly potential applications in food and pharmaceutical industries.