First report of anthracnose on Ophiopogon jaburan caused by Colletotrichum liriopes in China.

Ophiopogon jaburan (Liliaceae), named white lilyturf, is widely cultivated as an ornamental plant in south China. During 2017-2019, leaf spots on O. jaburan were observed all year in Zhanjiang, Guangdong, China (N21°9'3"; E110°17'47"). Almost all plants were infected and the disease incidence on affected leaves was about 80% in the field. Initially, spots were brown, round or oval, and gradually enlarged to irregular shapes. The color of the spots changed from rusty-brown to grayish-white with rusty-brown borders. Subsequently, the spots expanded until the leaves withered and died. Infected tissues were surface-sterilized with 75% ethanol for 30s followed by 1% NaClO solution for 1 min, then rinsed thrice with sterile water, before placed on potato dextrose agar (PDA) containing 50mg/L ampicillin, and incubated in darkness at 25℃ with 90% relative humidity. Colonies growing on PDA were cushion-like, pale greenish grey to grayish black on the front side and clearly dark gray on the reverse. Colony diameter was av. 86.0 mm (n = 15) grown in the dark at 25 ℃ for 10 days. Conidia with oil droplets were colorless, hyaline, smooth-walled, aseptate, slightly curved, and tapered gradually to each end, 12.3-28.9 × 2.2-6.6 µm (av. 20.9×4.2µm, n=200). Setae were brown to dark brown, 2-4 septate, with the base slightly inflated, and measured 40.0-130.3 × 2.2-5µm (av. 84.3 × 3.3µm, n=23). On PDA, scattered or loosely clustered appressoria were elliptical or irregular, smooth-walled, aseptate, and dark brown. To confirm the identification, partial regions of the internal transcribed spacer (White et al. 1990), beta-tubulin (Aveskamp et al. 2009) and actin (Carbone et al 1999) were amplified and sequenced (MW989743, MZ014461 and MZ014462). The blast results showed these sequences had >99.59% homology with sequences of Colletotrichum liriopes holotype strain CBS 119444 (NR_111449, GU228098 and GU227902). Maximum likelihood analysis and Bayesian inference were performed from concatenated sequences using RAxML v.1.0.0 and MrBayes v.3.2.1 software respectively. Several C. liriopes strains clustered in the same clade. Based on morphological-molecular characteristics, the fungus was identified as C. liriopes (Damm et al 2009; Chen et al. 2019). To confirm pathogenicity, healthy leaves were surface disinfected with 75% ethanol and rinsed thrice with sterile water. On ten leaves, three sites were wounded by pricking with needles, and inoculated 20 µL of 106 conidia/ml suspension or mycelium in contact with blade surface using 6-mm mycelial plugs. Similarly, the inoculation was done for three unwounded sites each leaf. Sterile water and medium plugs (without fungus) served as controls. All leaves were incubated on sterile wet filter paper at 25-28℃ with 90% relative humidity. After 7 days, all the inoculated leaves showed symptoms similar to those of field diseases, whereas control leaves remained healthy. The fungus with morphological-molecular features identical to the original isolate was reisolated from the disease lesions. C. liriopes causes anthracnose on Bletilla ochracea, Eria coronaria, Hemerocallis fulva, Pleione bulbocodioides (Jayawardena et al 2016) and Liriope sp. (Yang et al 2020; Chen et al 2019) in China. This is the first report of C. liriopes causing anthracnose on O. jaburan in China. Anthracnose could greatly affect ornamental value of O. jaburan, and this work can alert gardeners to prevent and control of the disease.

Molecularly imprinted polymer as efficient sorbent of solid-phase extraction for determination of gonyautoxin 1,4 in seawater followed by high-performance liquid chromatography-fluorescence detection.

A kind of new molecularly imprinted polymer (MIP) was synthesized by bulk polymerization using guanosine as dummy template molecule, α-methacrylic acid as functional monomer and ethylene glycol dimethyl acrylic ester as crosslinker. Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) showed that the MIP had homogenous and uniform-sized cavities. It was confirmed that the MIP had higher binding affinity and selectivity towards gonyautoxins 1,4 (GTX 1,4) than the non-imprinted polymer (NIP) according to the static equilibrium adsorption. An off-line molecularly imprinted solid-phase extraction (MISPE) method followed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) was established for the analysis of GTX 1,4. 0.1 mol/L acetic acid and 95:5 (v:v) methanol/water were optimized as the washing and elution solutions, respectively. The recoveries of spiked cultured seawater samples were satisfactory, as high as 88 %. Using this method, the concentrations of GTX 1,4 from cultured seawater samples of Alexandrium minutum and Alexandrium tamarense were detected to be 1.10 µg/L and 0.99 µg/L, respectively. Graphical Abstract The synthesis of molecularly imprinted polymer and molecularly imprinted solid-phase extraction analysis for gonyautoxin 1,4.

Improvement of robustness and ethanol production of ethanologenic Saccharomyces cerevisiae under co-stress of heat and inhibitors.

Bioethanol is an attractive alternative to fossil fuels. Saccharomyces cerevisiae is the most important ethanol producer. However, yeast cells are challenged by various environmental stresses during the industrial process of ethanol production. The robustness under heat, acetic acid, and furfural stresses was improved for ethanologenic S. cerevisiae in this work using genome shuffling. Recombinant yeast strain R32 could grow at 45°C, and resist 0.55% (v/v) acetic acid and 0.3% (v/v) furfural at 40°C. When ethanol fermentation was conducted at temperatures ranging from 30 to 42°C, recombinant strain R32 always gave high ethanol production. After 42 h of fermentation at 42°C, 187.6 ± 1.4 g/l glucose was utilized by recombinant strain R32 to produce 81.4 ± 2.7 g/l ethanol, which were respectively 3.4 and 4.1 times those of CE25. After 36 h of fermentation at 40°C with 0.5% (v/v) acetic acid, 194.4 ± 1.2 g/l glucose in the medium was utilized by recombinant strain R32 to produce 84.2 ± 4.6 g/l of ethanol. The extent of glucose utilization and ethanol concentration of recombinant strain R32 were 6.3 and 7.9 times those of strain CE25. The ethanol concentration produced by recombinant strain R32 was 8.9 times that of strain CE25 after fermentation for 48 h under 0.2% (v/v) furfural stress at 40°C. The strong physiological robustness and fitness of yeast strain R32 support its potential application for industrial production of bioethanol from renewable resources such as lignocelluloses.

Integrated expression of the α-amylase, dextranase and glutathione gene in an industrial brewer's yeast strain.

Genetic engineering is widely used to meliorate biological characteristics of industrial brewing yeast. But how to solve multiple problems at one time has become the bottle neck in the genetic modifications of industrial yeast strains. In a newly constructed strain TYRL21, dextranase gene was expressed in addition of α-amylase to make up α-amylase's shortcoming which can only hydrolyze α-1,4-glycosidic bond. Meanwhile, 18s rDNA repeated sequence was used as the homologous sequence for an effective and stable expression of LSD1 gene. As a result, TYRL21 consumed about twice much starch than the host strain. Moreover TYRL21 speeded up the fermentation which achieved the maximum cell number only within 3 days during EBC tube fermentation. Besides, flavor evaluation comparing TYRL21 and wild type brewing strain Y31 also confirmed TYRL21's better performances regarding its better saccharides utilization (83% less in residual saccharides), less off-flavor compounds (57% less in diacetyl, 39% less in acetaldehyde, 67% less in pentanedione), and improved stability index (increased by 49%) which correlated with sensory evaluation of final beer product.

Enhancement of Intracellular Accumulation of Copper by Biogenesis of Lipid Droplets in Saccharomyces cerevisiae Revealed by Transcriptomic Analysis.

Copper is an essential micronutrient for life, whose homeostasis is rigorously regulated to meet the demands of normal biological processes and to minimize the potential toxicity. Copper enriched by yeast is regarded as a safe and bioavailable form of copper supplements. Here, a Saccharomyces cerevisiae mutant strain H247 with expanded storage capability of copper was obtained through atmospheric and room-temperature plasma treatment. Transcriptomic analyses found that transcriptional upregulation of DGA1 might be the major contributor to the enhancement of intracellular copper accumulation in strain H247. The positive correlation between biogenesis of lipid droplets and intracellular accumulation of copper was confirmed by overexpression of the diacylglycerol acyltransferase encoding genes DGA1 and LRO1 or knockout of DGA1. Lipid droplets are not only the storage pool of copper but might prompt the copper trafficking to mitochondria, vacuoles, and Golgi apparatus. These results provide new insights into the sophisticated copper homeostatic mechanisms and the biological functions of lipid droplets.

Enhancement of Copper Uptake of Yeast Through Systematic Optimization of Medium and the Cultivation Process of Saccharomyces cerevisiae.

Copper is an essential trace element for living organisms. Copper enriched by yeast of Saccharomyces cerevisiae is regarded as the biologically available organic copper supplement with great potentiality for application. However, the lower uptake ratio of copper ions makes the production of copper enriched by yeast uneconomically and environmentally unfriendly. In this study, S. cerevisiae Cu-5 with higher copper tolerance and intracellular copper accumulation was obtained by screening of our yeast strains collection. To increase the uptake ratio of copper ions, the medium composition and cultivation conditions for strain Cu-5 were optimized systematically. A medium comprised of glucose, yeast extract, (NH4)2SO4, and inorganic salts was determined, then a novel cultivation strategy including pH control at 5.5 and increasing amounts of yeast extract for a higher concentration of copper ion in the medium was developed. The uptake ratios of copper ions were more than 90% after combining 50 to 100 mg/L copper ions with 3.5 to 5.0 g/L yeast extract, which is the highest until now and is conducive to the cost-effective and environmentally friendly production of bioactive copper in yeast-enriched form.

Improvement of acetic acid tolerance and fermentation performance of Saccharomyces cerevisiae by disruption of the FPS1 aquaglyceroporin gene.

The FPS1 gene coding for the Fps1p aquaglyceroporin protein of an industrial strain of Saccharomyces cerevisiae was disrupted by inserting CUP1 gene. Wild-type strain, CE25, could only grow on YPD medium containing less than 0.45% (v/v) acetic acid, while recombinant strain T12 with FPS1 disruption could grow on YPD medium with 0.6% (v/v) acetic acid. Under 0.4% (v/v) acetic acid stress (pH 4.26), ethanol production and cell growth rates of T12 were 1.7 ± 0.1 and 0.061 ± 0.003 g/l h, while those of CE25 were 1.2 ± 0.1 and 0.048 ± 0.003 g/l h, respectively. FPS1 gene disruption in an industrial ethanologenic yeast thus increases cell growth and ethanol yield under acetic acid stress, which suggests the potential utility of FPS1 gene disruption for bioethanol production from renewable resources such as lignocelluloses.

Engineering of cis-Element in Saccharomyces cerevisiae for Efficient Accumulation of Value-Added Compound Squalene via Downregulation of the Downstream Metabolic Flux.

Transcriptional downregulation is widely used for metabolic flux control. Here, marO, a cis-element of Escherichia coli mar operator, was explored to engineer promoters of Saccharomyces cerevisiae for downregulation. First, the ADH1 promoter (PADH1) and its enhanced variant PUADH1 were engineered by insertion of marO into different sites, which resulted in decrease in both gfp5 transcription and GFP fluorescence intensity to various degrees. Then, marO was applied to engineer the native ERG1 and ERG11 promoters due to their importance for accumulation of value-added intermediates squalene and lanosterol. Elevated squalene content (4.9-fold) or lanosterol content (4.8-fold) and 91 or 28% decrease in ergosterol content resulted from the marO-engineered promoter PERG1(M5) or PERG11(M3), respectively, indicating the validity of the marO-engineered promoters in metabolic flux control. Furthermore, squalene production of 3.53 g/L from cane molasses, a cheap and bulk substrate, suggested the cost-effective and promising potential for squalene production.

Construction of amylolytic industrial brewing yeast strain with high glutathione content for manufacturing beer with improved anti-staling capability and flavor.

Glutathione in beer works as the main antioxidant compounds which correlates with beer flavor stability. High residual sugars in beer contribute to major non-volatile components which correlate to high caloric content. In this work, Saccharomyces cerevisiae GSH1 gene encoding glutamylcysteine synthetase and Scharomycopsis fibuligera ALP1 gene encoding alpha-amylase were co-expressed in industrial brewing yeast strain Y31 targeting at alpha-acetolactate synthase (AHAS) gene (ILV2) and alcohol dehydrogenase gene (ADH2), and new recombinant strain TY3 was constructed. The glutathione content from the fermentation broth of TY3 increased to 43.83 mg/l compared to 33.34 mg/l from Y31. The recombinant strain showed high alpha-amylase activity and utilized more than 46% of starch after 5 days growing on starch as sole carbon source. European Brewery Convention tube fermentation tests comparing the fermentation broth of TY3 and Y31 showed that the flavor stability index increased to 1.3 fold and residual sugar concentration were reduced by 76.8%, respectively. Due to the interruption of ILV2 gene and ADH2 gene, the amounts of off-flavor compounds diacetyl and acetaldehyde were reduced by 56.93% and 31.25%, comparing with the amounts of these from Y31 fermentation broth. In addition, as no drug-resistance genes were introduced to new recombinant strain, consequently, it should be more suitable for use in beer industry because of its better flavor stability and other beneficial characteristics.

Construction of an industrial brewing yeast strain to manufacture beer with low caloric content and improved flavor.

In this study, the problems of high caloric content, increased maturation time and off-flavors in commercial beer manufacture arising from residual sugar, diacetyl, and acetaldehyde levels were addressed. A recombinant industrial brewing yeast strain (TQ1) was generated from T1 [Lipomyces starkeyi dextranase gene (LSD1) introduced, alpha-acetohydroxyacid synthase gene (ILV2) disrupted] by introducing Saccharomyces cerevisiae glucoamylase (SGA1) and a strong promoter PGK1 while disrupting the genes coding alcohol dehydrogenase (ADH2). The highest glucoamylase activity for TQ1 was 93.26 U/ml compared with host strain T1 (12.36 U/ml) and wild-type industrial yeast strain YSF5 (10.39 U/ml), respectively. European Brewery Convention (EBC) tube fermentation tests comparing the fermentation broths of TQ1 with T1 and YSF5 showed that the real extract were reduced by 15.79% and 22.47%; the main residual maltotriose concentration were reduced by 13.75% and 18.82%; the caloric content were reduced by 27.18 and 35.39 calories per 12 oz. Due to the disruption of ADH2 gene in TQ1, the off-flavor acetaldehyde concentration in the fermentation broth were 9.43% and 13.28% respectively lower than that of T1 and YSF5. No heterologous DNA sequences or drug-resistance genes were introduced into TQ1. So, the gene manipulations in this work properly solved the addressed problems in commercial beer manufacture.

Recombinant industrial brewing yeast strains with ADH2 interruption using self-cloning GSH1+CUP1 cassette.

A self-cloning module for gene knock-out and knock-in in industrial brewing yeast strain was constructed that contains copper resistance and gamma-glutamylcysteine synthetase gene cassette, flanked by alcohol dehydrogenase II gene (ADH2) of Saccharomyces cerevisiae. The module was used to obtain recombined strains RY1 and RY2 by targeting the ADH2 locus of host Y1. RY1 and RY2 were genetically stable. PCR and enzyme activity analysis of RY1 and RY2 cells showed that one copy of ADH2 was deleted by GSH1+CUP1 insertion, and an additional copy of wild type was still present. The fermentation ability of the recombinants was not changed after genetic modification, and a high level of glutathione (GSH) was secreted, resulting from GSH1 overexpression, which codes for gamma-glutamylcysteine synthetase. A pilot-scale brewing test for RY1 and RY2 indicated that acetaldehyde content in fermenting liquor decreased by 21-22%, GSH content increased by 20-22% compared with the host, the antioxidizability of the recombinants was improved, and the sensorial evaluation was also better than that of the host. No heterologous DNA was harbored in the recombinants; therefore, they could be applied in the beer industry in terms of their biosafety.

New industrial brewing yeast strains with ILV2 disruption and LSD1 expression.

New industrial brewing yeast strains, free of vector sequences and drug-resistance genes, were constructed by disrupting alpha-acetohydroxyacid synthase (AHAS) gene (ILV2) and introducing Lipomyces starkeyi dextranase (DEX) gene (LSD1) as a selective marker. The resulting recombinant strains can survive on YNB minimal medium plate with dextran T-70 as sole carbon source and showed lower AHAS activity. Fermentation test with recombinant strains in 500 ml conical flask confirmed DEX activity and lower AHAS activity compared with their host strain. Moreover, the fermentative performance of recombinant strains T1 and Q9 was better than their host, and the residual sugar content was reduced by 20-25% in fermented wort with recombinant strains compared to their host, too.

Over-expression of GSH1 gene and disruption of PEP4 gene in self-cloning industrial brewer's yeast.

Foam stability is often influenced by proteinase A, and flavor stability is often affected by oxidation during beer storage. In this study, PEP4, the gene coding for proteinase A, was disrupted in industrial brewing yeast. In the meantime, one copy of GSH1 gene increased in the same strain. GSH1 is responsible for gamma-glutamylcysteine synthetase, a rate-limiting enzyme for synthesis of glutathione which is one kind of important antioxidant and beneficial to beer flavor stability. In order to improve the brewer's yeast, plasmid pYPEP, pPC and pPCG1 were firstly constructed, which were recombined plasmids with PEP4 gene, PEP4's disruption and PEP4's disruption+GSH1 gene respectively. These plasmids were verified to be correct by restriction enzymes' assay. By digesting pPCG1 with AatII and PstI, the DNA fragment for homologous recombination was obtained carrying PEP4 sequence in the flank and GSH1 gene internal to the fragment. Since self-cloning technique was applied in the study and the modified genes were from industrial brewing yeast itself, the improved strains, self-cloning strains, were safe to public. The genetic stability of the improved strains was 100%. The results of PCR analysis of genome DNA showed that coding sequence of PEP4 gene had been deleted and GSH1 gene had been inserted into the locus of PEP4 gene in self-cloning strains. The fermentation ability of self-cloning strain, SZ-1, was similar to that of the host. Proteinase A could not be detected in beer brewed with SZ-1, and GSH content in the beer increased 35% compared to that of the host, Z-1.

[Effect of over-expression of sterol C-22 desaturase on ergosterol production in yeast strains].

Ergosterol, the main sterol in yeast, is responsible for structural membrane features such as fluidity and permeability. Additionally, ergosterol is economically important as a precursor of vitamin D2. The biosynthesis of sterols in yeast is complex. As an enzyme of the later ergosterol biosynthesis, the sterol C-22 desaturase encoded by ERG5 gene is required to form the C-22 (23) double bond in the sterol side chain. In order to know the regulation of C-22 sterol desaturase in the ergosterol biosynthesis, ERG5 gene was cloned and over-expressed in the Saccharomyces cerevisiae. Primer 1 (5'-GTCGGTACCTCCAATGACAATAAATACC-3', Kpn I) and primer 2 (5'-AAGGATCCTAGCAGATCATTAGCTGTAG-3', BamH I) were designed according to the ERG5 sequence in GenBank. A 1.8 kb DNA fragment containing the open reading frame and terminator of ERG5 gene was amplified from Saccharomyces cerevisiae YSF-20 by PCR and inserted into YEp352 to generate recombinant plasmid pYE5. To express ERG5 gene properly in S. cerevisiae, the recombinant expression plasmid pYPE5 containing ERG5 from pYE5 under the control of PGK1 promoter, the URA3 gene as the selection marker and the plasmid YEp352 as the vector was constructed, and then they were introduced into Saccharomyces cerevisiae YS58. To make sure the plasmid pYPE5 in the YS58 acted properly, the disruptant (YSE5) was created by deleting a 0.4 kb fragment of ERG5 gene and inserting the CUP1 gene into the ERG5 and transforming the YS58. And then the disruptant (YSE5) was transformed with the plasmid pYPE5 carrying the corresponding complementing ERG5 gene to control the activity of the over-expressed ERG5 gene and restauration of the wild-type sterol pattern. The sterol profile of the disruptant (YSE5) demonstrated that ergosta-5, 7-dien-3beta-ol was accumulated which was very similar to ergosterol but with a saturated side chain. In contrast, the YSE5 (pYPE5) strain contains predominantly ergosterol. The sterol content of the transformant was analyzed using gas chromatography (GC) analysis. The result shows that ergosterol production in recombinant strains was reduced. And the experiment of the effect of culturing time shows that ergosterol productions in recombinant strains were always lower than YS58 (pYPE5) from 24-48 h culturing time. Under the optimal culture condition, ergosterol content in recombinant strain YS58 (pYPE5) was about 0.70-fold of that in the referring strain.

[Distribution pattern and influencing factors of vegetation carbon storage of Tamarix chinense in the coastal wetland of Laizhou Bay, China]. / èŽ±å·žæ¹¾æ»¨æµ·æŸ½æŸ³æž—æ¹¿åœ°æ¤è¢«ç¢³å‚¨é‡çš„åˆ†å¸ƒç‰¹å¾åŠå ¶å½±å“å› ç´ .

The tamarisk shrub wetland located at the south of Laizhou Bay is the largest tamarisk area existing in the northern China, which is also the important part of the wetland ecological rehabilitation project 'Southern Mangrove Northern Tamarisk' in China. Based on the field data from Changyi National Marine Ecological Special Reserve surveyed in August 2014, we investigated the spatial patterns of vegetation, biomass, carbon content, and the associated environmental parameters in this area. The results showed that the average vegetation biomass and carbon storage were 949.0 g·m-2 and 393.1 g·m-2, respectively. They were higher in the central area than in the eastern and the western parts, and were generally the highest for the shrub part aboveground, followed by that underground, and the lowest for the litter. There were two single-species communities (dominated by Tamarix chinensis and Suaeda salsa, respectively) and four mixed communities. The carbon storage was the highest for the T. chinensis community, followed by the mixed communities and the lowest for S. salsa community. The water content and conductivity of surface soil in this area were generally low, probably due to the reduced intertidal waves blocked by the dam in the north. The vegetation carbon storage was most influenced by soil nutrients (total nitrogen and total phosphorus) and silt particle content rather than salinity. Furthermore, the alteration of the soil hydrologic condition caused the succession of vegetation communities in this area. When the salt tolerance community (e.g., S. salsa) shifted to the light salt tolerance community (e.g., Setaria viridis, Artemisia capillaries), the vegetation carbon storage increased significantly.

[Construction of high sulphite-producing industrial strain of Saccharomyces cerevisiae].

In the process of beer storage and transportation, off-flavor can be produced for oxidation of beer. Sulphite is important for stabilizing the beer flavor because of its antioxidant activity. However, the low level of sulphite synthesized by the brewing yeast is not enough to stabilize beer flavor. Three enzymes involve sulphite biosynthesis in yeast. One of them, APS kinase (encoded by MET14) plays important role in the process of sulphite formation. In order to construct high sulphite-producing brewing yeast strain for beer production, MET14 gene was cloned and overexpressed in industrial strain of Saccharomyces cerevisiae. Primer 1 (5'-TGTGAATTCCTGTACACCAATGGCTACT-3', EcoR I) and primer 2 (5'-TATAAGCTTGATGA GGTGGATGAAGACG-3', HindIII) were designed according to the MET14 sequence in GenBank. A 1.1kb DNA fragment containing the open reading frame and terminator of MET14 gene was amplified from Saccharomyces cerevisiae YSF-5 by PCR, and inserted into YEp352 to generate recombinant plasmid pMET14. To express MET14 gene properly in S. cerevisiae, the recombinant expression plasmids pPM with URA3 gene as the selection marker and pCPM with URA3 gene and copper resistance gene as the selection marker for yeast transformation were constructed. In plasmid pPM, the PGK1 promoter from plasmid pVC727 was fused with the MET14 gene from pMET14, and the expression cassette was inserted into the plasmid YEp352. The dominant selection marker, copper-resistance gene expression cassette CUP1-MTI was inserted in plasmid pPM to result in pCPM. Restriction enzyme analysis showed that plasmids pPM and pCPM were constructed correctly. The laboratory strain of S. cerevisiae YS58 with ura3, trp1, leu2, his4 auxotroph was transformed with plasmid pPM. Yeast transformants were screened on synthetic minimal medium (SD) containing leucine, histidine and tryptophan. The sulphite production of the transformants carrying pPM was 2 fold of that in the control strain YS58, which showed that the MET14 gene on plasmid pPM was expressed functionally in YS58. The industrial brewing yeast strain YSF-38 was transformed with the plasmid pCPM and yeast transformants were selected on YEPD medium containing 4mmol/L copper sulphate. The recombinant strain carrying pCPM showed a 3.2-fold increase in sulphite production when compared to the host strain YSF-38 under laboratory culture conditions. Flask fermentation under brewing-like conditions was performed in Tsingtao Beer Brewery. The sulphite production of the recombinant strain began to be higher than that of the host strain YSF-38 at the fourth day and reached the maximum at the eighth day. At the end of fermentation, the sulphite produced by recombinant strain is 1.4 fold of that in the host strain. The overexpression of MET14 gene in both laboratory and industrial strains of S. cerevisiae increases the sulphite formation. It is the first time to construct high sulphite-producing industrial strain by functional expression of MET14 in S. cerevisiae. Such study provides the foundation for construction of an excellent brewing yeast strain that can produce proper sulphite and can be used in commercial beer production.

Preparation and characterization of magnetic molecularly imprinted polymers for selective trace extraction of dienestrol in seawater.

Highly selective and efficient magnetic molecularly imprinted polymers (MMIPs) were prepared using Fe3O4@SiO2 as a magnetic supporter, 3-methacryloxypropyltrimethoxy-silane (MPS) as a silane coupling agent, DIS as a template, methacrylic acid (MAA) as a functional monomer and ethyleneglycol dimethacrylate (EGDMA) as a cross-linker for the extraction of trace residuals of the synthetic estrogen dienestrol (DIS) in seawater, which is a concern worldwide for its endocrine disruption and carcinogenic danger to human health. The obtained MMIPs were demonstrated to have spherical morphologies, core-shell structures, large binding capacities, high efficiency and selectivity. These were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and adsorption experiments. Owing to the specific binding sites, the MMIPs exhibited an almost three times higher adsorption capacity towards DIS (Qmax=4.68mgg-1) than magnetic molecularly non-imprinted polymers (MNIPs) (Qmax=1.72mgg-1). DIS in spiked seawater samples from the Weihai Bay of China was extracted and enriched by MMIPs, and satisfactory recoveries (87.3%-96.4%) with low relative standard deviation (RSD) values (2.03%-5.18%, n=5) were obtained. The limit of detection (LOD) of the method obtained was 0.16µgL-1, and the limit of quantitation was 0.52µgL-1 after MMIPs. No significant deterioration of the adsorption capacity of the MMIPs was observed after six rounds of regeneration. The results further demonstrated the applicability of the MMIPs method, a simple and straightforward method for the extraction and enrichment of DIS in seawater without any time-consuming procedures.

Deletion of tandem repeats causes flocculation phenotype conversion from Flo1 to NewFlo in Saccharomyces cerevisiae.

A gene, FLONS, conferring NewFlo-type flocculation ability in yeast was cloned. The 3,396-bp ORF encoded a peptide of 1,132 amino acids with high identity to Flo1 protein. Aligned with the FLO1 gene, two repeated regions (675 and 540 bp) were lost in the middle of FLONS, revealing that this gene was a derived form of the FLO1 gene. The missing repeated sequence contained three highly homologous repeat units. Although the flocculation phenotype of the transformant YTS-S with the FLONS gene was inhibited by both mannose and glucose, it exhibited some distinguished physiological characteristics from the reported typical NewFlo-type flocculation during detailed investigation. The deletion of repeats was suspected to cause conversion of the flocculation phenotype from Flo1 to NewFlo, suggesting that intragenic tandem repeats generated functional variability in Flo1 protein.

Genetic basis of flocculation phenotype conversion in Saccharomyces cerevisiae.

Two NewFlo-type flocculent transformants Saccharomyces cerevisiae YTS-S and YTS-L were obtained from a partial yeast genomic library. Even though both of the transformants displayed the same flocculation phenotype, they represented different physiological characteristics during detailed investigation. Analysis of the two transformants YTS-L and YTS-S confirmed the presence of FLONL and FLONS genes, respectively. The 3396-bp ORF of FLONS encoded a protein of 1132 amino acids. Meanwhile, the presence of a 1686-bp ORF encoding a 562-amino acid protein was revealed in FLONL. Both FLONL and FLONS showed high identity to FLO1 gene. Aligned with the intact FLO1 gene, FLONS lost two internal repeated regions, whereas one repeated sequence was inserted into the middle of the FLONL gene. All of the altered regions could be found in the middle repetitive sequence of the FLO1 gene. The results indicate that FLONL and FLONS are both derived forms of the FLO1 gene. Genetic variability triggered by tandem repeats in FLO1 gene is believed to be responsible for the differential phenotypic properties of the yeast strains YTS-S and YTS-L.

[Genetically modified industrial brewing yeast with high-glutathione and low-diacetyl production].

Recombinant plasmid pICG was constructed by replacing the internal fragment of a-acetohydroxyacid synthase (AHAS) gene (ILV2) with a copy of gamma-glutamylcysteine synthetase gene (GSH1) and copper chelatin gene (CUP1) from the industrial brewing yeast strain YSF31. YSF31 was transformed with plasmid pICG linearized by Kpn I and Pst I. A recombinant strain with high-glutathione and low-diacetyl production was selected. The results of fermentation in 100-L bioreactor showed that the lagering time of beer produced for recombinant strain T2 was shortened by 3 days and the shelf life of the beer was prolonged about 50%. It may be more acceptable for the commercial application, as it does not contain foreign DNA.