Enantioselective resolution of (R,S)-DMPM to prepare (R)-DMPM by an adsorbed-covalent crosslinked esterase PAE07 from Pseudochrobactrum asaccharolyticum WZZ003.

(R)-N-(2,6-dimethylphenyl) aminopropionic acid methyl ester ((R)-DMPM) is an important chiral intermediate of the fungicide N-(2,6-Dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester ((R)-Metalaxyl). In this study, (1) D3520 (macroporous acrylic anion resin), selected from the ten resins, was used to immobilize the esterase from Pseudochrobactrum asaccharolyticum WZZ003 (PAE07) for resoluting the (R,S)-DMPM to obtain (R)-DMPM. (2) Up to 20 g/L PAE07 could be immobilized onto D3520 with a high enzymatic activity of 32.4 U/g. Moreover, the Km and Vmax values of 19.1 mM and 2.8 mM/min for D3520-immobilized PAE07 indicated its high activity and stereoselectivity. (3) The optimal temperature and pH for the immobilized PAE07 were 40 ℃ and 8.0, and substrate concentration was up to 0.35 M. After 15 h reaction, the conversion rate from (R,S)-DMPM to (R)-DMPM was 48.0% and the e.e.p and E values were 99.5% and 1393.0, respectively. In scale-up resolution, 200 g/L substrate and 12.5 g immobilized esterase PAE07 condition, a conversion rate from substrate to product of 48.1% and a product e.e.p of 98% were obtained within 12 h, with the activity of immobilized PAE07 retained 80.2% after 5 cycles of reactions. These results indicated that the D3520-immobilized esterase PAE07 had great potential for enzymatic resolution of (R,S)-DMPM to prepare (R)-Metalaxyl.

Production, Biosynthesis, and Commercial Applications of Fatty Acids From Oleaginous Fungi.

Oleaginous fungi (including fungus-like protists) are attractive in lipid production due to their short growth cycle, large biomass and high yield of lipids. Some typical oleaginous fungi including Galactomyces geotrichum, Thraustochytrids, Mortierella isabellina, and Mucor circinelloides, have been well studied for the ability to accumulate fatty acids with commercial application. Here, we review recent progress toward fermentation, extraction, of fungal fatty acids. To reduce cost of the fatty acids, fatty acid productions from raw materials were also summarized. Then, the synthesis mechanism of fatty acids was introduced. We also review recent studies of the metabolic engineering strategies have been developed as efficient tools in oleaginous fungi to overcome the biochemical limit and to improve production efficiency of the special fatty acids. It also can be predictable that metabolic engineering can further enhance biosynthesis of fatty acids and change the storage mode of fatty acids.

One-step utilization of inulin for docosahexaenoic acid (DHA) production by recombinant Aurantiochytrium sp. carrying Kluyveromyces marxianus inulinase.

This study aimed to express an inulinase gene from the yeast Kluyveromyces marxianus (KmINU) in Aurantiochytrium sp. and realized one-step utilization of inulin resource for DHA production without any chemical pretreatment. An expression cassette with a length of 6052 bp for expressing the inulinase gene was constructed by a fast two-step PCR method and then was transferred into the Aurantiochytrium sp. cells. The Aurantiochytrium sp. recombinant T39 was selected with an inulinase activity up to 50.1 U/mL in 72 h. In a 5-l fed-batch fermentation, as high as 148.9 g/L of inulin was directly used within 120 h, and only 1.2 g/L of total sugar was left in the medium at the end of fermentation. The biomass of 51.4 g/L with a lipid content of 69.2% DCW and a DHA yield of 14.9 g/L was obtained.

Protein Engineering of a Pyridoxal-5'-Phosphate-Dependent l-Aspartate-α-Decarboxylase from Tribolium castaneum for ß-Alanine Production.

In the present study, a pyridoxal-5'-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from Tribolium castaneum (TcPanD) was selected for protein engineering to efficiently produce ß-alanine. A mutant TcPanD-R98H/K305S with a 2.45-fold higher activity than the wide type was selected through error-prone PCR, site-saturation mutagenesis, and 96-well plate screening technologies. The characterization of purified enzyme TcPanD-R98H/K305S showed that the optimal cofactor PLP concentration, temperature, and pH were 0.04% (m/v), 50 °C, and 7.0, respectively. The 1mM of Na+, Ni2+, Co2+, K+, and Ca2+ stimulated the activity of TcPanD-R98H/K305S, while only 5 mM of Ni2+ and Na+ could increase its activity. The kinetic analysis indicated that TcPanD-R98H/K305S had a higher substrate affinity and enzymatic reaction rate than the wild enzyme. A total of 267 g/L substrate l-aspartic acid was consumed and 170.5 g/L of ß-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.

Transcriptome Mechanism of Utilizing Corn Steep Liquor as the Sole Nitrogen Resource for Lipid and DHA Biosynthesis in Marine Oleaginous Protist Aurantiochytrium sp.

In the current study, corn steep liquor (CSL) is evaluated as an ideal raw agro-material for efficient lipid and docosahexaenoic acid DHA production by Aurantiochytrium sp. Low CSL level in medium (nitrogen deficiency) stimulated the biosynthesis of lipids and DHA while inhibiting cellular growth. The transcriptomic profiles of the Aurantiochytrium sp. cells are analyzed and compared when cultured under high (H group), normal (N group), and low (L group) levels of CSL in the medium. The discriminated transcriptomic profiles from the three groups indicates that changes in CSL level in medium result in a global change in transcriptome of Aurantiochytrium sp. The overall de novo assembly of cDNA sequence data generated 61,163 unigenes, and 18,129 of them were annotated in at least one database. A total of 5105 differently expressed (DE) genes were found in the N group versus the H group, with 2218 downregulated and 2887 upregulated. A total of 3625 DE genes were found in the N group versus the L group, with 1904 downregulated and 1721 upregulated. The analysis and categorization of the DE genes indicates that the regulation mechanism of CSL involved in the perception and transduction of the limited nitrogen signal, the interactions between the transcription factors (TFs) and multiple downstream genes, and the variations in downstream genes and metabolites, in sequence, are illuminated for the first time in the current study.

Integrated Approaches to Reveal Genes Crucial for Tannin Degradation in Aureobasidium melanogenum T9.

Tannins biodegradation by a microorganism is one of the most efficient ways to produce bioproducts of high value. However, the mechanism of tannins biodegradation by yeast has been little explored. In this study, Aureobasidium melanogenum T9 isolated from red wine starter showed the ability for tannins degradation and had its highest biomass when the initial tannic acid concentration was 20 g/L. Furthermore, the genes involved in the tannin degradation process were analyzed. Genes tan A, tan B and tan C encoding three different tannases respectively were identified in the A. melanogenum T9. Among these genes, tan A and tan B can be induced by tannin acid simultaneously at both gene transcription and protein expression levels. Our assay result showed that the deletion of tanA and tanB resulted in tannase activity decline with 51.3 ± 4.1 and 64.1 ± 1.9 U/mL, respectively, which is much lower than that of A. melanogenum T9 with 91.3 ± 5.8 U/mL. In addition, another gene coding gallic acid decarboxylase (gad) was knocked out to better clarify its function. Mutant Δgad completely lost gallic acid decarboxylase activity and no pyrogallic acid was seen during the entire cultivation process, confirming that there was a sole gene encoding decarboxylase in the A. melanogenum T9. These results demonstrated that tanA, tanB and gad were crucial for tannin degradation and provided new insights for the mechanism of tannins biodegradation by yeast. This finding showed that A. melanogenum has potential in the production of tannase and metabolites, such as gall acid and pyrogallol.

Whole Conversion of Soybean Molasses into Isomaltulose and Ethanol by Combining Enzymatic Hydrolysis and Successive Selective Fermentations.

Isomaltulose is mainly produced from sucrose by microbial fermentation, when the utilization of sucrose contributes a high production cost. To achieve a low-cost isomaltulose production, soy molasses was introduced as an alternative substrate. Firstly, α-galactosidase gene from Rhizomucor miehei was expressed in Yarrowia lipolytica, which then showed a galactosidase activity of 121.6 U/mL. Under the effects of the recombinant α-galactosidase, most of the raffinose-family oligosaccharides in soy molasses were hydrolyzed into sucrose. Then the soy molasses hydrolysate with high sucrose content (22.04%, w/w) was supplemented into the medium, with an isomaltulose production of 209.4 g/L, and the yield of 0.95 g/g. Finally, by virtue of the bioremoval process using Pichia stipitis, sugar byproducts in broth were transformed into ethanol at the end of fermentation, thus resulting in high isomaltulose purity (97.8%). The bioprocess employed in this study provides a novel strategy for low-cost and efficient isomaltulose production from soybean molasses.

High-Throughput Biochemical Fingerprinting of Oleaginous Aurantiochytrium sp. Strains by Fourier Transform Infrared Spectroscopy (FT-IR) for Lipid and Carbohydrate Productions.

The traditional biochemical methods for analyzing cellular composition of oleaginous microorganisms are time-consuming, polluting, and expensive. In the present study, an FT-IR method was used to analyze the cellular composition of the marine oleaginous protist Aurantiochytrium sp. during various research processes, such as strains screening, medium optimization, and fermentation, and was evaluated as a green, low-cost, high throughput, and accurate method compared with the traditional methods. A total of 109 Aurantiochytrium sp. strains were screened for lipid and carbohydrate production and the best results were found for the strains No. 6 and No. 32. The yields and productivities could reach up to 47.2 g/L and 0.72 g/L/h for lipid, 21.6 g/L and 0.33 g/L/h for docosahexaenoic acid (DHA) in the strain No. 6, and 15.4 g/L and 0.18 g/L/h for carbohydrate in the strain No. 32, under the optimal conditions, respectively. These results confirmed potentials of the two Aurantiochytrium sp. strains for lipid, DHA, and carbohydrate productions at industrial scales. The FT-IR method in this study will facilitate research on the oleaginous Aurantiochytrium sp., and the obtained two strains for lipid and carbohydrate productions will provide the foundations for their applications in medical, food, and feed industries.

Transcriptomic Mechanism of the Phytohormone 6-Benzylaminopurine (6-BAP) Stimulating Lipid and DHA Synthesis in Aurantiochytrium sp.

The phytohormone 6-benzylaminopurine (6-BAP) significantly improves lipid synthesis of oleaginous microorganisms with the great potential applied in lipid production. In the current study, the lipid and DHA productions in oleaginous Aurantiochytrium sp. were found to be improved by 48.7% and 55.3%, respectively, induced by 6-BAP treatments. Then, using high-throughput RNA-seq technology, the overall de novo assembly of the cDNA sequence data generated 53871 unigenes, and 15902 of these were annotated in at least one database. The comparative transcriptomic profiles of cells with and without 6-BAP treatments revealed that a total of 717 were differently expressed genes (DE), with 472 upregulated and 245 downregulated. Further annotation and categorization indicated that some DE genes were involved in pathways crucial to lipid and DHA productions, such as fatty acid synthesis, central carbon metabolism, transcriptional factor, signal transduction, and mevalonate pathway. A regulation mode of 6-BAP, in turn, perception and transduction of 6-BAP signal, transcription factor, expression regulations of the downstream genes, and metabolic changes, respectively, was put forward for the first time in the present study. This research illuminates the transcriptomic mechanism of phytohormone stimulation of lipid and DHA production in an oleaginous microorganism and provides the potential targets modified using genetic engineering for improving lipid and DHA productivity.

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.

Metabolomics analysis reveals 6-benzylaminopurine as a stimulator for improving lipid and DHA accumulation of Aurantiochytriumsp.

BACKGROUND: Phytohormones are chemical messengers that have a positive effect on biodiesel production of microalgae at low concentrations. However, the effect of phytohormone 6-benzylaminopurine on lipid and docosahexaenoic acid (DHA) production in marine DHA-producer Aurantiochytrium has never been reported. In this study, a GC-MS-based metabolomics method combined with a multivariate analysis is applied to reveal the metabolic mechanism of 6-benzylaminopurine enhancing production of lipid and DHA in Aurantiochytrium sp.YLH70. RESULTS: In total, 71 metabolites were identified by GC-MS. The PCA model revealed that 76.9% of metabolite variation was related to 6-benzylaminopurine treatment, and overall metabolomics profiles between the 6-benzylaminopurine and control groups were clearly discriminated. Forty-six metabolites identified by the PLS-DA model were responsible for responding to 6-benzylaminopurine. Metabolic analysis showed that 6-benzylaminopurine could accelerate the rate of utilization of glucose in Aurantiochytrium sp. YLH70, and the metabolic flux from glycolysis, TCA cycle and mevalonate pathway to fatty acids biosynthesis was promoted. Moreover, the anti-stress mechanism in Aurantiochytrium sp.YLH70 might be induced by 6-benzylaminopurine. CONCLUSION: Metabolomics is a suitable tool to discover the metabolic mechanism for improving lipid and DHA accumulation in a microorganism. 6-benzylaminopurine has the potential to stimulate lipid and DHA production of Aurantiochytrium sp.YLH70 for industrial purposes. © 2015 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Utilization of High-Fructose Corn Syrup for Biomass Production Containing High Levels of Docosahexaenoic Acid by a Newly Isolated Aurantiochytrium sp. YLH70.

High-fructose corn syrup (HFCS) is an agro-source product and has been the most commonly used substitute for sugar as sweetener in food industry due to its low price and high solution property. In this study, the F55 HFCS, rich in fructose and glucose, was first tested for biomass and docosahexaenoic acid productions as a mixed carbon source by a newly isolated Aurantiochytrium sp.YLH70. After the compositions of the HFCS media were optimized, the results showed that the HFCS with additions of metal ion and vitamin at low concentrations was suitable for biomass and docosahexaenoic acid productions and the metal ion and sea salt had the most significant effects on biomass production. During the 5-l fed-batch fermentation, total HFCS containing 180 g l(-1) reducing sugar was consumed and yields of biomass, lipid, and DHA could reach 78.5, 51, and 20.1 g l(-1), respectively, at 114 h. Meanwhile, the daily productivity and the reducing sugar conversion yield for docosahexaenoic acid were up to 4.23 g l(-1)day(-1) and 0.11 g g(-1). The fatty acid profile of Aurantiochytrium sp.YLH70 showed that 46.4% of total fatty acid was docosahexaenoic acid, suggesting that Aurantiochytrium sp.YLH70 was a promising DHA producer.

Reduced genome size of Helicobacter pylori originating from East Asia.

Helicobacter pylori (H. pylori), a major pathogen colonizing the human stomach, shows great genetic variation. Comparative analysis of strains from different H. pylori populations revealed that the genome size of strains from East Asia decreased to 1.60 Mbp, which is significantly smaller than that from Europe or Africa. In parallel with the genome reduction, the number of protein coding genes was decreased, and the guanine-cytosine content was lowered to 38.9%. Elimination of non-essential genes by mutations is likely to be a major cause of the genome reduction. Bacteria with a small genome cost less energy. Thus, H. pylori strains from East Asia may have proliferation and growth advantages over those from Western countries. This could result in enhanced capacity of bacterial spreading. Therefore, the reduced genome size potentially contributes to the high prevalence of H. pylori in East Asia.

Expression of the acid protease gene from Saccharomycopsis fibuligera in the marine-derived Yarrowia lipolytica for both milk clotting and single cell protein production.

In this study, the native acid protease gene in Yarrowia lipolytica 22a-2 with high content of protein was disrupted, and the disruptant 3-13-10 obtained had very low acid protease activity. Then, the acid protease gene (AP1 gene) from Saccharomycopsis fibuligera A11 was actively expressed in the disruptant 3-13-10, and the transformant 43 carrying the AP1 gene had high specific acid protease activity (46.7 U/mg). The recombinant acid protease produced by the transformant 43 was found to be able to actively clot milk, and the transformant 43 still kept high content of protein. The hydrolysis products of κ-casein under catalysis of the recombinant acid protease and the commercial calf rennet had the same molecular mass, suggesting that the recombinant acid protease and its producer can be used both in cheese manufacturing and as protein source in food industry.

Cloning of exo-ß-1,3-glucanase gene from a marine yeast Williopsis saturnus and its overexpression in Yarrowia lipolytica.

The exo-ß-1,3-glucanase structural gene (WsEXG1 gene, accession number: FJ875997.2) was isolated from both the genomic DNA and cDNA of the marine yeast Williopsis saturnus WC91-2 by inverse PCR and RT-PCR. An open reading frame of 1,254 bp encoding a 417 amino acid protein (isoelectric point: 4.5) with calculated molecular weight of 46.2 kDa was characterized. The promoter of the gene (intronless) was located from -28 to -77 and had one TATA box while its terminator contained the sequence AAGAACAATAAACAA from +1,386 to +1,401. The protein had the Family 5 glycoside hydrolase signature IGLELLNEPL and a fragment with the sequence of NLCGEWSAA, where the Glu-310 (E) was considered to be the catalytic nucleophile. The WsEXG1 gene was overexpressed in Yarrowia lipolytica Po1h and the recombinant WsEXG1 was purified and characterized. The molecular weight of the purified rWsEXG1 was 46.0 kDa. The optimal pH and temperature of the purified rWsEXG1 were 5.0°C and 40°C, respectively. The purified rWsEXG1 had high exo-ß-1,3-glucanase activity. Therefore, the recombinant ß-1,3-glucanase may have highly potential applications in food and pharmaceutical industries.

The clinical manifestations and pathophysiology of cerebral small vessel disease.

Small vessel disease (SVD) is responsible for brain chronic circular disorder, and accounts for 20%-30% cases of ischemic stroke as well as cerebral hemorrhage, and to a great extent, encephalopathy. Binswanger's disease and multiple small strokes, which are common in older people, are also closely associated with SVD. These disorders often cause decline in cognition, vascular dementia, impairment in gait and balance, mood depression, and urinary incontinence, and often brings great social and economic burdens. SVD-related encephalopathy increases the incidences of fall, disability and death in elderly people. With the aging of the society, more attention should be paid to the importance of early diagnosis and prophylactic treatment of SVD. Here the clinical manifestations and pathophysiology of SVD are reviewed.

Surface display of acid protease on the cells of Yarrowia lipolytica for milk clotting.

The acid protease structural gene was amplified from the genomic DNA of Saccharomycopsis fibuligera A11. When the gene was cloned into the multiple cloning site of the surface display vector pINA1317-YlCWP110 and expressed in the cells of Yarrowia lipolytica, the cells displaying the acid protease could form clear zone on the plate-containing milk indicating that they had extracellular acid protease activity. The cells displaying the acid protease can be used to effectively clot skimmed milk. The highest clotting milk activity (1,142.9 U/ml) was observed under the conditions of pH 3.0, 40 degrees C, 20 mM of CaCl(2), and 10% skimmed milk powder. We found that the acid protease displayed on the cells of Y. lipolytica which has generally regarded as safe status could be easily isolated and concentrated compared to the free acid protease. Therefore, the displayed acid protease may have many potential applications in food and cheese industries. This is the first report that the yeast cells displaying the acid protease were used to clot milk.