Efficient pullulan production by Aureobasidium pullulans using cost-effective substrates.

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.

Identification of Fungal Limonene-3-Hydroxylase for Biotechnological Menthol Production.

More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as a medical component. So far, only extraction from plant material and chemical synthesis are possible. An alternative approach for menthol production could be a biotechnological-chemical process with ideally only two conversion steps, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies have already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs, or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low compared to that of the highly selective L3H enzymes from the biosynthetic pathway for menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa. We have discovered additional fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production, and its additional ability to convert α- and ß-pinene to verbenol and pinocarveol, respectively.IMPORTANCE (-)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (-)-menthol production from renewable (+)-limonene.

Production of cellulases by Aureobasidium pullulans LB83: optimization, characterization, and hydrolytic potential for the production of cellulosic sugars.

Aureobasidium pullulans LB83 was evaluated for cellulase production under submerged fermentation conditions. Different process variables such as carbon sources (corn cob, sugarcane bagasse, and sugarcane straw), synthetic (urea, ammonium sulfate, and peptone), and non-synthetic (soybean meal, rice, and corn meal) nitrogen sources and inoculum size were evaluated by one parameter at-a-time strategy. Aureobasidium pullulans LB83 showed maximum cellulase activity (FPase, 2.27 U/mL; CMCase, 7.42 U/mL) on sugarcane bagasse. Among the nitrogen sources, soybean meal as a non-synthetic nitrogen sources showed a maximum cellulase activity (FPase 2.45 U/mL; CMCase, 6.86 U/mL) after 60 hr. The inoculum size of 1.6 × 106 CFU/mL had the maximum FPase and CMCase activities of 3.14 and 8.74 U/mL, respectively. For the enzymatic hydrolysis, both the commercial cellulase (10 FPU/g of Cellic CTec 2 (#A) and 10 FPU/g of crude enzyme extract (CEE) (#B), and varying ratio of CTec 2 and CEE in combination #C (5 FPU/g of CTec 2 + 5 FPU/g CEE), combination #D (2.5 FPU/g of CTec 2 + 7.5 FPU/g CEE), and combination #E (7.5 FPU/g of CTec 2 + 2.5 FPU/g CEE) were assessed for enzymatic hydrolysis of delignified sugarcane bagasse. Enzyme combination #C showed maximum hydrolysis yield of 92.40%. The study shows the hydrolytic potential of cellulolytic enzymes from A. pullulans LB83 for lignocellulosic sugars production from delignified sugarcane bagasse.

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.