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.

RedEx: a method for seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters.

Biosynthesis reprograming is an important way to diversify chemical structures. The large repetitive DNA sequences existing in polyketide synthase genes make seamless DNA manipulation of the polyketide biosynthetic gene clusters extremely challenging. In this study, to replace the ethyl group attached to the C-21 of the macrolide insecticide spinosad with a butenyl group by refactoring the 79-kb gene cluster, we developed a RedEx method by combining Redαß mediated linear-circular homologous recombination, ccdB counterselection and exonuclease mediated in vitro annealing to insert an exogenous extension module in the polyketide synthase gene without any extra sequence. RedEx was also applied for seamless deletion of the rhamnose 3'-O-methyltransferase gene in the spinosad gene cluster to produce rhamnosyl-3'-desmethyl derivatives. The advantages of RedEx in seamless mutagenesis will facilitate rational design of complex DNA sequences for diverse purposes.

Hydrocarbons, the advanced biofuels produced by different organisms, the evidence that alkanes in petroleum can be renewable.

It is generally regarded that the petroleum cannot be renewable. However, in recent years, it has been found that many marine cyanobacteria, some eubacteria, engineered Escherichia coli, some endophytic fungi, engineered yeasts, some marine yeasts, plants, and insects can synthesize hydrocarbons with different carbon lengths. If the organisms, especially some native microorganisms and engineered bacteria and yeasts, can synthesize and secret a large amount of hydrocarbons within a short period, alkanes in the petroleum can be renewable. It has been documented that there are eight pathways for hydrocarbon biosynthesis in different organisms. Unfortunately, most of native microorganisms, engineered E. coli and engineered yeasts, only synthesize a small amount of intracellular and extracellular hydrocarbons. Recently, Aureobasidium pullulans var. melanogenum isolated from a mangrove ecosystem has been found to be able to synthesize and secret over 21.5 g/l long-chain hydrocarbons with a yield of 0.275 g/g glucose and a productivity of 0.193 g/l/h within 5 days. The yeast may have highly potential applications in alkane production.

Genetic Modification of the Marine-Isolated Yeast Aureobasidium melanogenum P16 for Efficient Pullulan Production from Inulin.

In this study, in order to directly and efficiently convert inulin into pullulan, the INU1 gene from Kluyveromyces maximum KM was integrated into the genomic DNA and actively expressed in the high pullulan producer Aureobasidium melanogenum P16 isolated from the mangrove ecosystem. After the ability to produce pullulan from inulin by different transformants was examined, it was found that the recombinant strain EI36, one of the transformants, produced 40.92 U/ml of inulinase activity while its wild-type strain P16 only yielded 7.57 U/ml of inulinase activity. Most (99.27 %) of the inulinase produced by the recombinant strain EI36 was secreted into the culture. During the 10-l fermentation, 70.57 ± 1.3 g/l of pullulan in the fermented medium was attained from inulin (138.0 g/l) within 108 h, high inulinase activity (42.03 U/ml) was produced within 60 h, the added inulin was actively hydrolyzed by the secreted inulinase, and most of the reducing sugars were used by the recombinant strain EI36. This confirmed that the genetically engineered yeast of A. melanogenum strain P16 was suitable for direct pullulan production from inulin.

EEMD independent extraction for mixing features of rotating machinery reconstructed in phase space.

Empirical Mode Decomposition (EMD), due to its adaptive decomposition property for the non-linear and non-stationary signals, has been widely used in vibration analyses for rotating machinery. However, EMD suffers from mode mixing, which is difficult to extract features independently. Although the improved EMD, well known as the ensemble EMD (EEMD), has been proposed, mode mixing is alleviated only to a certain degree. Moreover, EEMD needs to determine the amplitude of added noise. In this paper, we propose Phase Space Ensemble Empirical Mode Decomposition (PSEEMD) integrating Phase Space Reconstruction (PSR) and Manifold Learning (ML) for modifying EEMD. We also provide the principle and detailed procedure of PSEEMD, and the analyses on a simulation signal and an actual vibration signal derived from a rubbing rotor are performed. The results show that PSEEMD is more efficient and convenient than EEMD in extracting the mixing features from the investigated signal and in optimizing the amplitude of the necessary added noise. Additionally PSEEMD can extract the weak features interfered with a certain amount of noise.

High-level pullulan production by Aureobasidium pullulans var. melanogenium P16 isolated from mangrove system.

After over 100 strains of Aureobasidium spp. isolated from mangrove system were screened for their ability to produce exopolysaccharide (EPS), it was found that Aureobasidium pullulans var. melanogenium P16 strain among them could produce high level of EPS. Under the optimal conditions, 65.3 g/L EPS was produced by the P16 strain within 120 h at flask level. During 10-L batch fermentation, when the medium contained 120.0 g/L sucrose, 67.4 g/L of EPS and 23.1 g/L of cell dry weight in the culture were obtained within 120 h, leaving 0.78 g/L of reducing sugar and 11.4 g/L of total sugar in the fermented medium. It should be stressed that during the fermentation, no melanin was observed. After purification, the purified EPS was confirmed to be pullulan. This is the first time to report that A. pullulans var. melanogenium P16 strain isolated from the mangrove system can produce high level of pullulan.

The unique role of siderophore in marine-derived Aureobasidium pullulans HN6.2.

The L -ornithine-N (5)-monooxygenase structural gene (SidA gene, accession number: FJ769160) was isolated from both the genomic DNA and cDNA of the marine yeast Aureobasidium pullulans HN6.2 by inverse PCR and RT-PCR. An open reading frame of 1,461 bp encoding a 486 amino acid protein (isoelectric point: 7.79) with calculated molecular weight of 55.4 kDa was characterized. The promoter of the gene (intronless) was located from -1 to -824 and had three HGATAR boxes which were putative binding motifs for the respective DNA-binding motifs and one CATA box. The SidA gene in A. pullulans HN6.2 was disrupted by integrating the hygromycin B phosphotransferase (HPT) gene into Open Reading Frame of the SidA gene using homologous recombination. Of all the disruptants obtained, one strain S6 (∆sidA) did not synthesize both intracellular and extracellular fusigen so that it could not inhibit growth of the pathogenic bacteria Vibrio anguillarum and Vibrio parahaemolyticus. The disruptant S6 did not grow in the iron-deplete medium and seawater medium because cell budding was stopped, but could grow in the iron-replete medium with 10 µM Fe(3+) and Fe(2+). H(2)O(2) in the medium was more toxic to the disruptant S6 than to its wild type HN6.2. Thus, we infer that the fusigen produced by the marine-derived A. pullulans HN6.2 can play a unique role in chelating, uptake and concentration of iron to maintain certain proper physiological functions within the cells and secretion of siderophore may represent an efficient tool to eliminate competitors to compete for limiting nutritional resources in marine environments.