Hyper-Production of Pullulan by a Novel Fungus of Aureobasidium melanogenum ZH27 through Batch Fermentation.

Pullulan, which is a microbial exopolysaccharide, has found widespread applications in foods, biomedicines, and cosmetics. Despite its versatility, most wild-type strains tend to yield low levels of pullulan production, and their mutants present genetic instability, achieving a limited increase in pullulan production. Therefore, mining new wild strains with robust pullulan-producing abilities remains an urgent concern. In this study, we found a novel strain, namely, Aureobasidium melanogenum ZH27, that had a remarkable pullulan-producing capacity and optimized its cultivation conditions using the one-factor-at-a-time method. To elucidate the reasons that drove the hyper-production of pullulan, we scrutinized changes in cell morphology and gene expressions. The results reveal that strain ZH27 achieved 115.4 ± 1.82 g/L pullulan with a productivity of 0.87 g/L/h during batch fermentation within 132 h under the optimized condition (OC). This pullulan titer increased by 105% compared with the initial condition (IC). Intriguingly, under the OC, swollen cells featuring 1-2 large vacuoles predominated during a rapid pullulan accumulation, while these swollen cells with one large vacuole and several smaller ones were prevalent under the IC. Moreover, the expressions of genes associated with pullulan accumulation and by-product synthesis were almost all upregulated. These findings suggest that swollen cells and large vacuoles may play pivotal roles in the high level of pullulan production, and the accumulation of by-products also potentially contributes to pullulan synthesis. This study provides a novel and promising candidate for industrial pullulan production.

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.

Direct Isomaltulose Synthesis From Beet Molasses by Immobilized Sucrose Isomerase.

Isomaltulose is becoming a focus as a functional sweetener for sucrose substitutes; however, isomaltulose production using sucrose as the substrate is not economical. Low-cost feedstocks are needed for their production. In this study, beet molasses (BM) was introduced as the substrate to produce isomaltulose for the first time. Immobilized sucrose isomerase (SIase) was proved as the most efficient biocatalyst for isomaltulose synthesis from sulfuric acid (H2SO4) pretreated BM followed by centrifugation for the removal of insoluble matters and reducing viscosity. The effect of different factors on isomaltulose production is investigated. The isomaltulose still achieved a high concentration of 446.4 ± 5.5 g/L (purity of 85.8%) with a yield of 0.94 ± 0.02 g/g under the best conditions (800 g/L pretreated BM, 15 U immobilized SIase/g dosage, 40°C, pH of 5.5, and 10 h) in the eighth batch. Immobilized SIase used in repeated batch reaction showed good reusability to convert pretreated BM into isomaltulose since the sucrose conversion rate remained 97.5% in the same batch and even above 94% after 11 batches. Significant cost reduction of feedstock costs was also confirmed by economic analysis. The findings indicated that this two-step process to produce isomaltulose using low-cost BM and immobilized SIase is feasible. This process has the potential to be effective and promising for industrial production and application of isomaltulose as a functional sweetener for sucrose substitute.

A novel thermoanalytical method for quantifying microplastics in marine sediments.

Microplastic pollution in marine environments is of particular concern on its risk to the ecosystem. To assess and manage microplastic contaminants, their quantitative detection in environmental samples is a high priority. However, uncertainties of current methods still exist when estimating their abundances, particularly with fine-grained (<1 mm) microplastics. This work reports a novel thermoanalytical method for quantifying microplastics by measuring the contents of microplastic-derived carbon (MPC) in samples under the premise of nearly eliminating the limit of their particle appearances. After validating the method via samples with the spiked microplastics, we have conducted a case study on sediment core H43 that spanned 1925-2009 CE from the Yellow Sea for further illustrating the high reliability and practicability of this method for quantifying microplastics in natural samples. Our results have demonstrated that the proposed method may be a promising technique to determine the mass-related concentrations of the total microplastics in marine sediments for evaluating their pollution status and quantitative contribution to marine carbon storage.

Evaluation of ergosterol composition and esterification rate in fungi isolated from mangrove soil, long-term storage of broken spores, and two soils.

Ergosterol is an important fungal-specific biomarker, but its use for fungal biomass estimation is still varied. It is important to distinguish between free and esterified ergosterols, which are mainly located on the plasma membrane and the cytosolic lipid particles, respectively. The present study analyzes free and esterified ergosterol contents in: (1) the fifty-nine strains of culturable fungi isolated from mangrove soil, (2) the broken spores of the fungus Ganoderma lucidum stored in capsule for more than 12 years, and (3) the mangrove soil and nearby campus wood soil samples by high performance liquid chromatography (HPLC). The results show that the contents of free and esterified ergosterols varied greatly in fifty-nine strains of fungi after 5 days of growth, indicating the diversity of ergosterol composition in fungi. The average contents of free and total ergosterols from the fifty-nine strains of fungi are 4.4 ± 1.5 mg/g and 6.1 ± 1.9 mg/g dry mycelia, respectively, with an average ergosterol esterification rate of 27.4%. The present study suggests that the fungi might be divided into two classes, one is fungi with high esterification rates (e.g., more than 27%) such as Nectria spp. and Fusarium spp., and the other is fungi with low esterification rates (e.g., less than 27%) such as Penicillium spp. and Trichoderma spp. Moreover, the ergosterol esterification rate in the spores of G. lucidum is 91.4% with a very small amount of free ergosterol (0.015 mg/g), compared with 41.9% with a higher level of free ergosterol (0.499 mg/g) reported in our previous study in 2007, indicating that free ergosterol degrades more rapidly than esterified ergosterol. In addition, the ergosterol esterification rates in mangrove soil and nearby campus wood soil samples range from 0 to 39.0%, compared with 80% in an old soil organic matter reported in a previous study, indicating the potential relationship between aging degree of fungi or soil and esterification rate. The present study proposes that both free and esterified ergosterols should be analyzed for fungal biomass estimation. When the ergosterol esterification rates in soils are higher, free ergosterol might be a better marker for fungal biomass. It is speculated that the ergosterol esterification rate in soils might contain some important information, such as the age of old-growth forests over time scales of centuries to millennia, besides the senescence degree of fungal mycelia in soils. KEY POINTS: • Fungi might be divided into two classes depending on ergosterol esterification rates. • Ergosterol esterification rate of broken spores stored for long time raised evidently. • Both free and esterified ergosterols should be analyzed for fungal biomass estimate. • Free ergosterol is a better marker for fungal biomass with a high esterification rate.

Characterization of Matrix Metalloprotease-9 Gene from Nile tilapia (Oreochromis niloticus) and Its High-Level Expression Induced by the Streptococcus agalactiae Challenge.

The bacterial diseases of tilapia caused by Streptococcus agalactiae have resulted in the high mortality and huge economic loss in the tilapia industry. Matrix metalloproteinase-9 (MMP-9) may play an important role in fighting infection. However, the role of MMP-9 in Nile tilapia against S. agalactiae is still unclear. In this work, MMP-9 cDNA of Nile tilapia (NtMMP-9) has been cloned and characterized. NtMMP-9 has 2043 bp and encodes a putative protein of 680 amino acids. NtMMP-9 contains the conserved domains interacting with decorin and inhibitors via binding forces compared to those in other teleosts. Quantitative real-time-polymerase chain reaction (qPCR) analysis reveals that NtMMP-9 distinctly upregulated following S. agalactiae infection in a tissue- and time-dependent response pattern, and the tissues, including liver, spleen, and intestines, are the major organs against a S. agalactiae infection. Besides, the proteolytic activity of NtMMP-9 is also confirmed by heterologous expression and zymography, which proves the active function of NtMMP-9 interacting with other factors. The findings indicate that NtMMP-9 was involved in immune responses against the bacterial challenge at the transcriptional level. Further work will focus on the molecular mechanisms of NtMMP-9 to respond and modulate the signaling pathways in Nile tilapia against S. agalactiae invasion and the development of NtMMP-9-related predictive biomarkers or vaccines for preventing bacterial infection in the tilapia industry.

Efficient Conversion of Cane Molasses Towards High-Purity Isomaltulose and Cellular Lipid Using an Engineered Yarrowia lipolytica Strain in Fed-Batch Fermentation.

: Cane molasses is one of the main by-products of sugar refineries, which is rich in sucrose. In this work, low-cost cane molasses was introduced as an alternative substrate for isomaltulose production. Using the engineered Yarrowia lipolytica, the isomaltulose production reached the highest (102.6 g L-¹) at flask level with pretreated cane molasses of 350 g L-¹ and corn steep liquor of 1.0 g L-¹. During fed-batch fermentation, the maximal isomaltulose concentration (161.2 g L-¹) was achieved with 0.96 g g-¹ yield within 80 h. Simultaneously, monosaccharides were completely depleted, harvesting the high isomaltulose purity (97.4%) and high lipid level (12.2 g L-¹). Additionally, the lipids comprised of 94.29% C16 and C18 fatty acids, were proved suitable for biodiesel production. Therefore, the bioprocess employed using cane molasses in this study was low-cost and eco-friendly for high-purity isomaltulose production, coupling with valuable lipids.

Relationship between ß-d-fructofuranosidase activity, fructooligosaccharides and pullulan biosynthesis in Aureobasidium melanogenum P16.

It has been thought that when different strains of Aureobasidium spp. were grown in sucrose, the produced fructooligosaccharides (FOSs) by ß-d-fructofuranosidase were beneficial for their cell growth and pullulan biosynthesis. However, it is still unknown about how ß-d-fructofuranosidases activity and synthesized FOSs influence on pullulan biosynthesis. It was found that the genomic DNA of Aureobasidium melanogenum P16, a high pullulan producing yeast, contained three genes encoding ß-d-fructofuranosidase1, ß-d-fructofuranosidase2 and ß-d-fructofuranosidase3. The FTR1 factor, a transcriptional activator, activated expression of the three ß-d-fructofuranosidase genes and invertase gene. Disruption of the FTR1 gene rendered a disruptant DF3 to produce less FOSs (12.1 ±â€¯0.4 g/L), less ß-d-fructofuranosidase activity (1.1 ±â€¯0.2 U/mL), lower Mw (3.8 × 105) of the pullulan and more pullulan titer (77.0 ±â€¯2.6 g/L) than the yeast strain P16. Similarly, removal of both the two genes encoding ß-d-fructofuranosidase1 and ß-d-fructofuranosidase3 resulted in a double mutant DF4-7 producing 77.5 ±â€¯3.1 g/L pullulan with Mw of 3.4 × 105, 0.2 ±â€¯0.0 U/mL of ß-d-fructofuranosidase activity and the trace amount of FOSs while its wild type strain P16 yielded 65.7 ±â€¯3.5 g/L pullulan with Mw of 4.4 × 105, 6.8 ±â€¯0.0 U/mL of ß-d-fructofuranosidase activity and 6.2 ±â€¯0.5 g/L of FOSs. These confirmed that high ß-d-fructofuranosidase activity, the presence of high level of FOSs negatively influenced pullulan biosynthesis, but positively increased Mw of the produced pullulan. However, the ß-d-fructofuranosidase2 had no such function. Furthermore, complementation of the FTR1 gene, ß-d-fructofuranosidase1 gene and ß-d-fructofuranosidase3 gene enabled the corresponding transformants to restore ß-d-fructofuranosidase activity, FOSs and pullulan biosynthesis and Mw of the pullulan.

Simultaneous production of both high molecular weight pullulan and oligosaccharides by Aureobasdium melanogenum P16 isolated from a mangrove ecosystem.

After the compositional change of a pullulan production medium, a molecular weight (Mw) of the pullulan produced by Aureobasidium melanogenum P16 was 2.32×106 and a pullulan titer was 44.4g/L while a Mw of the pullulan produced by A. melanogenum P16 grown in the initial medium was only 3.47×105 and a pullulan titer was 65.3g/L. The increased Mw of the pullulan was due to the decreased activities of α-amylase, glucoamylase and pullulanase while the decreased pullulan titer was related to the decreased transcriptional levels of the genes encoding 6-P-glucose kinase, glucosyltransferase, α-phosphoglucose mutase, UDPG-pyrophosphorylase and pullulan synthetase. During the 10-L fermentation, when the yeast strain P16 was grown in the initial medium, the pullulan and oligosaccharide titers were 65.5g/L and 7.8g/L, respectively and the Mw of the produced pullulan was 4.42×105 while when the yeast strain P16 was grown in the compositionally changed medium, the pullulan and oligosaccharide titers were 46.4g/L and 27.8g/L, respectively and the Mw of the produced pullulan was 2.6×106. Most of the oligosaccharides produced by the yeast strain P16 cultivated in the compositionally changed medium had degree of polymerization of 4 and 5. Therefore, both of the high Mw pullulan and oligosaccharides with high levels were produced by the yeast strain P16.

CreA is directly involved in pullulan biosynthesis and regulation of Aureobasidium melanogenum P16.

Aureobasidium melanogenum P16 is a high pullulan-producing yeast. However, glucose repression on its pullulan biosynthesis must be relieved. After the gene encoding a glucose repressor was cloned, characterized and analyzed, it was found that the repressor belonged to one member of the CreA in filamentous fungi, not to one member of the Mig1 in yeasts. After the CREA gene was fully removed from the yeast strain P16, the glucose repression in the disruptant DG41 was relieved. At the same time, the pullulan production by the disruptant DG41 was enhanced compared to that by its wild-type strain P16, and the transcriptional levels of the gene encoding a glucosyltransferase, three genes encoding glucose transporters, the gene encoding a 6-P-glucose kinase and the genes encoding α-amylase, glucoamylase and pullulanase in the disruptant DG41 were also promoted. However, the transcriptional levels of the genes encoding the CreA and another two glucose transporters were greatly reduced. During the 10-liter fermentation, the disruptant DG41 produced 64.93 ± 1.33 g/l pullulan from 120 g/l of glucose, while its wild-type strain P16 produced only 52.0 ± 1.95 g/l pullulan within 132 h. After the CREA gene was complemented in the disruptant D373, the pullulan production by the transformant BC4 was greatly reduced compared to that by its wild-type strain P16, and the transcriptional levels of the many genes in the transformant BC4 were also decreased. All the results confirmed that the CreA played an important role in the regulation of pullulan biosynthesis in A. melanogenum P16, and that glucose derepression on pullulan biosynthesis could improve pullulan production from glucose. This study opened the possibility for improving the industrial production of this exopolysaccharide by genetic engineering.

A glycosyltransferase gene responsible for pullulan biosynthesis in Aureobasidium melanogenum P16.

In this study, one of the glucosyltransferase genes for pullulan production was cloned from Aureobasidum melanogenum P16 and charaterized. It was found that the UGT1 gene had 4774bp with four introns (47, 52, 54 and 46bp). The N-terminal part of the protein displayed a conserved sequence controlling both sugar donor and accepter for substrate specificity whereas its C-terminal part carried a DXD motif that coordinated donor sugar binding. After complete removal of the gene UGT1, the mutant 1152-3 still produced 27.7±3.1g/L of pullulan and 4.6U/g of the specific glucosyltransferase activity while its wild type strain P16 yielded 63.38±2.0g/L of pullulan and 5.7U/g of the specific glucosyltransferase activity. However, after overexpression of the gene UGT1, the transformant G63 could produce 78.0±3.01g/L of pullulan and 19.0U/g of the specific glucosyltransferase activity. It is interesting to note that the molecular weight of the produced pullulan by the wild type strain was 4.6×105 while that of the produced pullulan by the transformant G63 was 6.2×105. During the 10-Litter fermentation, the pullulan titer produced by the transformant G63 reached 80.2±2.0 g/L within 132h.