Impact of PpSpi1, a glycosylphosphatidylinositol-anchored cell wall glycoprotein, on cell wall defects of N-glycosylation-engineered Pichia pastoris.

IMPORTANCE: Engineering of biological pathways in various microorganisms is a promising direction for biotechnology. Since the existing microbial cells have evolved over a long period of time, any artificial engineering may cause some unexpected and harmful effects on them. Systematically studying and evaluating these engineered strains are very important and necessary. In order to produce therapeutic proteins with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. The properties of a P. pastoris strain with humanized N-glycosylation machinery were carefully evaluated in this study. Our work has identified a key glycoprotein (PpSpi1) responsible for the poor growth and morphological defects of this glycoengineered strain. Overexpression of PpSpi1 could significantly rescue the growth defect of the glycoengineered P. pastoris and facilitate its future industrial applications.

Influence of perceived discrimination on problematic smartphone use among Chinese deaf and hard-of-hearing students: Serial mediating effects of sense of security and social avoidance.

BACKGROUND: Reducing the rate and impact of problematic smartphone use (PSU) among deaf and hard-of-hearing (DHH) students has become a social issue. Research has suggested that perceived discrimination is associated with PSU, although the underlying mechanism of this association for DHH students is not clear. AIMS: This study explored the mediating roles of sense of security and social avoidance in the association between perceived discrimination and PSU among Chinese DHH students. METHOD: Five hundred and seventy DHH students, selected through simple random sampling, participated in an online survey, which included measurements of perceived discrimination, sense of security, social avoidance, and PSU. The results were analyzed with SPSS 25.0 and Mplus 7.0. RESULTS: (1) Perceived discrimination, sense of security, and social avoidance were significantly associated with PSU among DHH students; (2) sense of security and social avoidance played mediating roles separately in the association between perceived discrimination and PSU; (3) sense of security and social avoidance played serial mediating roles in the relationship between perceived discrimination and PSU. CONCLUSIONS: The findings suggest that discriminatory experiences of DHH students are related to a relatively low sense of security and high social avoidance, which may lead to PSU. The results have important implications for the prevention of and intervention for PSU among DHH students.

Salinomycin biosynthesis reversely regulates the ß-oxidation pathway in Streptomyces albus by carrying a 3-hydroxyacyl-CoA dehydrogenase gene in its biosynthetic gene cluster.

Streptomyces is well known for synthesis of many biologically active secondary metabolites, such as polyketides and non-ribosomal peptides. Understanding the coupling mechanisms of primary and secondary metabolism can help develop strategies to improve secondary metabolite production in Streptomyces. In this work, Streptomyces albus ZD11, an oil-preferring industrial Streptomyces strain, was proved to have a remarkable capability to generate abundant acyl-CoA precursors for salinomycin biosynthesis with the aid of its enhanced ß-oxidation pathway. It was found that the salinomycin biosynthetic gene cluster contains a predicted 3-hydroxyacyl-CoA dehydrogenase (FadB3), which is the third enzyme of ß-oxidation cycle. Deletion of fadB3 significantly reduced the production of salinomycin. A variety of experimental evidences showed that FadB3 was mainly involved in the ß-oxidation pathway rather than ethylmalonyl-CoA biosynthesis and played a very important role in regulating the rate of ß-oxidation in S. albus ZD11. Our findings elucidate an interesting coupling mechanism by which a PKS biosynthetic gene cluster could regulate the ß-oxidation pathway by carrying ß-oxidation genes, enabling Streptomyces to efficiently synthesize target polyketides and economically utilize environmental nutrients.

An Efficient Markerless Deletion System Suitable for the Industrial Strains of Streptomyces.

The genus Streptomyces is intensively studied due to its excellent ability to produce secondary metabolites with diverse bioactivities. In particular, adequate precursors of secondary metabolites as well as sophisticated post modification systems make some high-yield industrial strains of Streptomyces the promising chassis for the heterologous production of natural products. However, lack of efficient genetic tools for the manipulation of industrial strains, especially the episomal vector independent tools suitable for large DNA fragment deletion, makes it difficult to remold the metabolic pathways and streamline the genomes in these strains. In this respect, we developed an efficient deletion system independent of the episomal vector for large DNA fragment deletion. Based on this system, four large segments of DNA, ranging in length from 10 kb to 200 kb, were knocked out successfully from three industrial Streptomyces strains without any marker left. Notably, compared to the classical deletion system used in Streptomyces, this deletion system takes about 25% less time in our cases. This work provides a very effective tool for further genetic engineering of the industrial Streptomyces.

Screening and evaluation of the strong endogenous promoters in Pichia pastoris.

BACKGROUND: Due to its ability to perform fast and high-density fermentation, Pichia pastoris is not only used as an excellent host for heterologous protein expression but also exhibits good potential for efficient biosynthesis of small-molecule compounds. However, basic research on P. pastoris lags far behind Saccharomyces cerevisiae, resulting in a lack of available biological elements. Especially, fewer strong endogenous promoter elements available for foreign protein expression or construction of biosynthetic pathways were carefully evaluated in P. pastoris. Thus, it will be necessary to identify more available endogenous promoters from P. pastoris. RESULTS: Based on RNA-seq and LacZ reporter system, eight strong endogenous promoters contributing to higher transcriptional expression levels and ß-galactosidase activities in three frequently-used media were screened out. Among them, the transcriptional expression level contributed by P0019, P0107, P0230, P0392, or P0785 was basically unchanged during the logarithmic phase and stationary phase of growth. And the transcriptional level contributed by P0208 or P0627 exhibited a growth-dependent characteristic (a lower expression level during the logarithmic phase and a higher expression level during the stationary phase). After 60 h growth, the ß-galactosidase activity contributed by P0208, P0627, P0019, P0407, P0392, P0230, P0785, or P0107 was relatively lower than PGAP but higher than PACT1. To evaluate the availability of these promoters, several of them were randomly applied to a heterogenous ß-carotene biosynthetic pathway in P. pastoris, and the highest yield of ß-carotene from these mutants was up to 1.07 mg/g. In addition, simultaneously using the same promoter multiple times could result in a notable competitive effect, which might significantly lower the transcriptional expression level of the target gene. CONCLUSIONS: The novel strong endogenous promoter identified in this study adds to the number of promoter elements available in P. pastoris. And the competitive effect observed here suggests that a careful pre-evaluation is needed when simultaneously and multiply using the same promoter in one yeast strain. This work also provides an effective strategy to identify more novel biological elements for engineering applications in P. pastoris.

Enhanced Triacylglycerol Metabolism Contributes to Efficient Oil Utilization and High-Level Production of Salinomycin in Streptomyces albus ZD11.

Streptomyces is well known for biosynthesis of secondary metabolites with diverse bioactivities. Although oils have been employed as carbon sources to produce polyketide antibiotics for several industrial Streptomyces strains, the intrinsic correlation between oil utilization and high production of antibiotics still remains unclear. In this study, we investigated the correlation between oil metabolism and salinomycin biosynthesis in Streptomyces albus ZD11, which employs soybean oil as the main carbon source. Comparative genomic analysis revealed the enrichment of genes related to triacylglycerol (TAG) metabolism in S. albus ZD11. Transcriptomic profiling further confirmed the enhancement of TAG metabolism and acyl coenzyme A biosynthesis in S. albus ZD11. Multiple secreted lipases, which catalyze TAG hydrolysis, were seen to be working in a synergistic and complementary manner in aiding the efficient and stable hydrolyzation of TAGs. Together, our results suggest that enhanced TAG hydrolysis and fatty acid degradation contribute to the high efficiency of oil utilization in S. albus ZD11 in order to provide abundant carbon precursors for cell growth and salinomycin biosynthesis.IMPORTANCE In order to obtain high-level production of antibiotics, oils have been used as the main carbon source for some Streptomyces strains. Based on multiomics analysis, this study provides insight into the relationship between triacylglycerol (TAG) metabolism and antibiotic biosynthesis in S. albus ZD11, an oil-preferring industrial Streptomyces strain. Our investigation into TAG hydrolysis yielded further evidence that this strain utilizes complicated strategies enabling an efficient TAG metabolism. In addition, a novel secreted lipase was identified that exhibited highly hydrolytic activity for medium- and long-chain TAGs. Our findings represent a good start toward clarifying the complicated relationship between TAG catabolism and high-level antibiotic production in the industrial strains.

Response of metal-coordination-based polyelectrolyte complex micelles to added ligands and metals.

Polyelectrolyte complex based micelles have attracted significant attention due to their potential regarding bio-applications. Although the morphology and functions have been studied extensively, dynamic properties, particularly component exchange with other surrounding molecules, have remained elusive to date. Here, we show how micelles based on metal-ligand coordination complex coacervate-core micelles (M-C3Ms) respond to addition of extra ligand and metal ions. The micelles are prepared from a polycationic-neutral diblock copolymer and an anionic coordination polyelectrolyte, which is obtained by coordination between metal ions (lanthanides Ln3+ and Zn2+) and a bis-ligand (LEO) containing two dipicolinic acid (DPA) groups connected by a tetra-ethylene oxide spacer (4EO). Our findings show that the bis-ligand LEO is essential for the growth of coordination polymers and consequently the formation of micelles, leading to equilibrium structures with the same micellar composition and structure independent of the order of mixing. In other words, adding single DPA has no effect on the formed M-C3Ms. As for metal exchange, we find that added Zn2+ can replace some of the Ln3+ from Ln-C3Ms, leading to a hybrid coordination structure with both Ln3+ and Zn2+. We find that component exchange occurs in these coordination polyelectrolyte micelles, but it is more favorable in the direction of replacing the weak binding components with strong ones. Hence, the designed M-C3Ms based on the strong binding components, such as Ln-C3Ms, shall be relatively stable in biological surroundings, paving the way for the application of such particles as bio-imaging probes.

Display of Microbial Glucose Dehydrogenase and Cholesterol Oxidase on the Yeast Cell Surface for the Detection of Blood Biochemical Parameters.

High levels of blood glucose are always associated with numerous complications including cholesterol abnormalities. Therefore, it is important to simultaneously monitor blood glucose and cholesterol levels in patients with diabetes during the management of chronic diseases. In this study, a glucose dehydrogenase from Aspergillus oryzae TI and a cholesterol oxidase from Chromobacterium sp. DS-1 were displayed on the surface of Saccharomyces cerevisiae, respectively, using the yeast surface display system at a high copy number. In addition, two whole-cell biosensors were constructed through the immobilization of the above yeast cells on electrodes, for electrochemical detection of glucose and cholesterol. The assay time was 8.5 s for the glucose biosensors and 30 s for the cholesterol biosensors. Under optimal conditions, the cholesterol biosensor exhibited a linear range from 2 to 6 mmol·L-1. The glucose biosensor responded efficiently to the presence of glucose at a concentration range of 20-600 mg·dL-1 (1.4-33.3 mmol·L-1) and showed excellent anti-xylose interference properties. Both biosensors exhibited good performance at room temperature and remained stable over a three-week storage period.

Genome mining and homologous comparison strategy for digging exporters contributing self-resistance in natamycin-producing Streptomyces strains.

As antibiotics are always toxic to the antibiotic-producing strains themselves, most Streptomyces strains have evolved several self-resistance mechanisms, among which the antibiotic efflux system is understood best and is commonly found. Among the efflux systems, the ATP-binding cassette (ABC) transporter superfamily and the major facilitator superfamily (MFS) are two important transporter families. In this work, the ABC transporters and the MFS transporters from the four reported natamycin-producing Streptomyces strains have been investigated in order to clarify whether these Streptomyces strains share similar efflux strategies for natamycin metabolism. Fifty-one groups of homologous exporter genes were identified as shared by four strains. Differential transcriptional analysis between the natamycin-producing strain Streptomyces chattanoogensis L10 and its ΔscnS0 mutant, which produces no natamycin, reveals that the expression levels of 25 of the above groups of genes were observably changed. The production of natamycin declined over 30% after solely knocking out several of these 25 groups of genes in S. chattanoogensis L10. This indicates that these transporters participate in the efflux of molecules related to natamycin biosynthesis. Our study is the first to demonstrate that the exporters participating in a particular antibiotic metabolism can be excavated and identified quickly by the strategy of genome mining and homologous comparison in the antibiotic-producing strains, leading to deeper understanding of the complex self-resistance mechanisms in Streptomycetes.

Cangrelor or Clopidogrel in Patients with Type 2 Diabetes Mellitus Undergoing Percutaneous Coronary Intervention: A Meta-Analysis of Randomized Controlled Trials.

INTRODUCTION: With recent advances in interventional cardiology where percutaneous coronary intervention (PCI) has become the most preferred invasive strategy and with advances in adjunctive pharmacotherapy, several newer oral P2Y12 inhibitors have reached the market. In this analysis, we aimed to compare the cardiovascular outcomes and bleeding events which were associated with the use of cangrelor versus clopidogrel in patients with type 2 diabetes mellitus (T2DM) 48 h after PCI. METHODS: The electronic databases MEDLINE (PubMed), www.ClinicalTrials.gov , EMBASE, and Cochrane central were searched for relevant publications comparing canagrelor with clopidogrel during PCI. Patients with T2DM were extracted. Adverse cardiovascular outcomes and bleeding events at 48 h follow-up were considered as the end points. This meta-analysis was carried out with the latest RevMan software (5.30). Odds ratios (OR) and 95% confidence intervals (CI) were used to represent the data. RESULTS: This analysis consisted of a total number of 5031 participants with T2DM (enrolled between the years 2006 and 2012). Compared to clopidgrel, use of cangrelor in these patients with T2DM was not associated with significantly different primary end point (OR 0.94, 95% CI 0.75-1.16; P = 0.55), myocardial infarction (OR 0.96, 95% CI 0.76-1.20; P = 0.71), all-cause death (OR 0.70, 95% CI 0.25-1.96; P = 0.49), ischemia-driven revascularization (OR 0.66, 95% CI 0.32-1.36; P = 0.26), and stent thrombosis (OR 0.45, 95% CI 0.17-1.17; P = 0.10). Thrombolysis in myocardial infarction (TIMI)-defined major and minor bleedings were similarly manifested: (OR 1.02, 95% CI 0.38-2.74; P = 0.96) and (OR 1.39, 95% CI 0.70-2.79; P = 0.35), respectively. Global use of strategies to open occluded arteries (GUSTO)-defined moderate and severe bleeding were also not significantly different: (OR 1.36, 95% CI 0.70-2.67; P = 0.37) and (OR 1.21, 95% CI 0.41-3.59; P = 0.74), respectively. However, GUSTO-defined mild bleeding and acute catheterization and urgent intervention triage strategy (ACUITY)-defined major and minor bleedings were significantly in favor of clopidogrel in comparison to cangrelor in these patients with T2DM: (OR 1.28, 95% CI 1.09-1.50; P = 0.003), (OR 1.43, 95% CI 1.05-1.94; P = 0.02), and (OR 1.23, 95% CI 1.04-1.46; P = 0.02), respectively. Other bleeding outcomes were not significantly different. CONCLUSIONS: In these patients with T2DM, cangrelor was comparable to clopidogrel in terms of efficacy at 48 h following PCI. However, it was associated with significantly higher mild GUSTO bleeding and major and minor ACUITY bleeding, therefore requiring further workups on its safety side. This hypothesis should be explored further and confirmed in other forthcoming trials based strictly on patients with T2DM.

Early versus late clinical outcomes following same day discharge after elective percutaneous coronary intervention: A systematic review and meta-analysis.

BACKGROUND: Nowadays 57% of the cardiologists based in the United Kingdom and 32% of the cardiologists based in Canada utilize same day discharge (SDD) following elective percutaneous coronary intervention (PCI) as a routine practice. In this analysis, we aimed to systematically assess early versus late clinical outcomes following SDD after elective PCI. METHODS: The Medical Literature Analysis and Retrieval System Online, the Cochrane Central, the Resources from the United States National Library of Medicine (www.ClinicalTrials.gov: http://www.clinicaltrials.gov) and EMBASE were carefully searched for relevant English publications which reported early versus late clinical outcomes in patients who were discharged on the same day following revascularization by PCI. Relevant clinical outcomes which were reported in the original studies were considered as the endpoints in this analysis. Odd ratios (OR) and 95% confidence intervals (CI) were used to represent the data, and RevMan 5.3 was used as the statistical software. RESULTS: A total number of 21, 687 participants (enrollment time period from the year 1998 to the year 2015) were assigned to this analysis. When early versus late clinical outcomes were compared in patients who were discharged on the same day following elective PCI, major adverse cardiac events (OR: 0.75, 95% CI: 0.31-1.79; P = .51), mortality (OR: 0.26, 95% CI: 0.06-1.06; P = .06), stroke (OR: 1.46, 95% CI: 0.72-2.94; P = .29), arrhythmia (OR: 1.30, 95% CI: 0.64-2.63; P = .47), hematoma (OR: 1.00, 95% CI: 0.60-1.66; P = 1.00) and major bleeding from access site (OR: 1.68, 95% CI: 0.22-12.85; P = .62) were not significantly different. Post-procedural myocardial infarction (OR: 2.01, 95% CI: 0.71-5.70; P = .19) and minor bleeding from access site (OR: 6.61, 95% CI: 0.86-50.66; P = .07) were also similarly manifested. However, re-hospitalization was significantly higher in those patients with late clinical outcomes (OR: 0.18, 95% CI: 0.07-0.44; P = .0002). CONCLUSIONS: In those patients who were discharged from the hospital on the same day following elective PCI, no significant difference was observed in the assessed early versus late clinical outcomes. However, late clinical outcomes resulted in a significantly higher rate of re-hospitalization. Larger studies should confirm this hypothesis.

Choosing between ticagrelor and clopidogrel following percutaneous coronary intervention: A systematic review and Meta-Analysis (2007-2017).

BACKGROUND: Limitations have been observed with the use of clopidogrel following percutaneous coronary intervention (PCI) indicating the urgent need of a more potent anti-platelet agent. We aimed to compare the efficacy and safety of ticagrelor versus clopidogrel following PCI. METHODS: Online databases were searched for relevant studies (published between the years 2007 and 2017) comparing ticagrelor versus clopidogrel following coronary stenting. Primary outcomes assessed efficacy whereas secondary outcomes assessed safety. Odds ratios (OR) with 95% confidence intervals (CIs) based on a random effect model were calculated and the analysis was carried out by the RevMan 5.3 software. RESULTS: A total number of 25,632 patients with acute coronary syndrome (ACS) [12,992 patients with ST segment elevation myocardial infarction (STEMI) and 14,215 patients with non-ST segment elevation myocardial infarction (NSTEMI)] were included in this analysis, of whom 23,714 patients were revascularized by PCI. Results of this analysis did not show any significant difference in all-cause mortality, major adverse cardiac events (MACEs), myocardial infarction, stroke and stent thrombosis observed between ticagrelor and clopidogrel with (OR: 0.83, 95% CI: 0.67-1.03; P = .09), (OR: 0.64, 95% CI: 0.41-1.01; P = .06), (OR: 0.77, 95% CI: 0.57-1.03; P = .08), (OR: 0.85, 95% CI: 0.57-1.26; P = .42) and (OR: 0.70, 95% CI: 0.47-1.05; P =.09).However, ticagrelor was associated with a significantly higher minor and major bleeding with (OR: 1.57, 95% CI: 1.30-1.89; P = .00001) and (OR: 1.52, 95% CI: 1.01-2.29; P = 0.04) respectively. Dyspnea was also significantly higher in the ticagrelor group (OR: 2.64, 95% CI: 1.87-3.72; P = .00001). CONCLUSION: Ticagrelor and clopidogrel were comparable in terms of efficacy in these patients with ACS. However, the safety outcomes of ticagrelor should further be investigated.

Hispidin induces autophagic and necrotic death in SGC-7901 gastric cancer cells through lysosomal membrane permeabilization by inhibiting tubulin polymerization.

Hispidin and its derivatives are widely distributed in edible mushrooms. Hispidin is more cytotoxic to A549, SCL-1, Bel7402 and Capan-1 cancer cells than to MRC5 normal cells; by contrast, hispidin protects H9c2 cardiomyoblast cells from hydrogen peroxide-induced or doxorubicin-induced apoptosis. Consequently, further research on how hispidin affects normal and cancer cells may help treat cancer and reduce chemotherapy-induced side effects. This study showed that hispidin caused caspase-independent death in SGC-7901 cancer cells but not in GES-1 normal cells. Hispidin-induced increases in LC3-II occurred in SGC-7901 cells in a time independent manner. Cell death can be partially inhibited by treatment with ATG5 siRNA but not by autophagy or necroptosis inhibitors. Ultrastructural evidence indicated that hispidin-induced necrotic cell death involved autophagy. Hispidin-induced lysosomal membrane permeabilization (LMP) related to complex cell death occurred more drastically in SGC-7901 cells than in GES-1 cells. Ca2+ rather than cathepsins from LMP contributed more to cell death. Hispidin induced microtubule depolymerization, which can cause LMP, more drastically in SGC-7901 cells than in GES-1 cells. At 4.1 µM, hispidin promoted cell-free tubulin polymerization but at concentrations higher than 41 µM, hispidin inhibited polymerization. Hispidin did not bind to tubulin. Alterations in microtubule regulatory proteins, such as stathmin phosphorylation at Ser16, contributed to hispidin-induced SGC-7901 cell death. In conclusion, hispidin at concentrations higher than 41 µM may inhibit tubulin polymerization by modulating microtubule regulatory proteins, such as stathmin, causing LMP and complex SGC-7901 cell death. This mechanism suggests a promising novel treatment for human cancer.

SlnR is a positive pathway-specific regulator for salinomycin biosynthesis in Streptomyces albus.

Salinomycin, a polyether antibiotic produced by Streptomyces albus, is widely used in animal husbandry as an anticoccidial drug and growth promoter. Situated within the salinomycin biosynthetic gene cluster, slnR encodes a LAL-family transcriptional regulator. The role of slnR in salinomycin production in S. albus was investigated by gene deletion, complementation, and overexpression. Gene replacement of slnR from S. albus chromosome results in almost loss of salinomycin production. Complementation of slnR restored salinomycin production, suggesting that SlnR is a positive regulator of salinomycin biosynthesis. Overexpression of slnR in S. albus led to about 25 % increase in salinomycin production compared to wild type. Quantitative RT-PCR analysis revealed that the expression of most sal structural genes was downregulated in the ΔslnR mutant but upregulated in the slnR overexpression strain. Electrophoretic mobility gel shift assays (EMSAs) also revealed that SlnRDBD binds directly to the three intergenic regions of slnQ-slnA1, slnF-slnT1, and slnC-slnB3. The SlnR binding sites within the three intergenic regions were determined by footprinting analysis and identified a consensus-directed repeat sequence 5'-ACCCCT-3'. These results indicated that SlnR modulated salinomycin biosynthesis as an enhancer via interaction with the promoters of slnA1, slnQ, slnF, slnT1, slnC, and slnB3 and activates the transcription of most of the genes belonging to the salinomycin gene cluster but not its own transcription.

Multiple transporters are involved in natamycin efflux in Streptomyces chattanoogensis L10.

Antibiotic-producing microorganisms have evolved several self-resistance mechanisms to prevent auto-toxicity. Overexpression of specific transporters to improve the efflux of toxic antibiotics has been found one of the most important and intrinsic resistance strategies used by many Streptomyces strains. In this work, two ATP-binding cassette (ABC) transporter-encoding genes located in the natamycin biosynthetic gene cluster, scnA and scnB, were identified as the primary exporter genes for natamycin efflux in Streptomyces chattanoogensis L10. Two other transporters located outside the cluster, a major facilitator superfamily transporter Mfs1 and an ABC transporter NepI/II were found to play a complementary role in natamycin efflux. ScnA/ScnB and Mfs1 also participate in exporting the immediate precursor of natamycin, 4,5-de-epoxynatamycin, which is more toxic to S. chattanoogensis L10 than natamycin. As the major complementary exporter for natamycin efflux, Mfs1 is up-regulated in response to intracellular accumulation of natamycin and 4,5-de-epoxynatamycin, suggesting a key role in the stress response for self-resistance. This article discusses a novel antibiotic-related efflux and response system in Streptomyces, as well as a self-resistance mechanism in antibiotic-producing strains.

Mutations of charged amino acids at the cytoplasmic end of transmembrane helix 2 affect transport activity of the budding yeast multidrug resistance protein Pdr5p.

Pdr5p is a major ATP-binding cassette (ABC) transporter in Saccharomyces cerevisiae. It displays a sequence and functional homology to the pathogenic Candida albicans multidrug resistance protein Cdr1p. The transmembrane helices of Pdr5p act in substrate recognition, binding, translocation and eventual removal of toxic substances out of the plasma membrane via the formation of a binding pocket. In this study, we identify two novel Pdr5 mutants (E574K and E580K), which exhibit impaired substrate efflux functions. Both mutants remained hypersensitive to all tested Pdr5p substrates without affecting their protein expression levels, localization or ATPase activities. As E574 and E580 are both located adjacent to the predicted cytoplasmic end of transmembrane helix 2, this implies that such charged residues are functionally essential for Pdr5p. Molecular docking studies suggest the possibility that oppositely charged substitution at residue E574 may disturb the interaction between the substrates and Pdr5p, resulting in impaired transport activity. Our results present new evidence, suggesting that transmembrane helix 2 plays an important role for the efflux function of Pdr5p.

Genomic evolution of Saccharomyces cerevisiae under Chinese rice wine fermentation.

Rice wine fermentation represents a unique environment for the evolution of the budding yeast, Saccharomyces cerevisiae. To understand how the selection pressure shaped the yeast genome and gene regulation, we determined the genome sequence and transcriptome of a S. cerevisiae strain YHJ7 isolated from Chinese rice wine (Huangjiu), a popular traditional alcoholic beverage in China. By comparing the genome of YHJ7 to the lab strain S288c, a Japanese sake strain K7, and a Chinese industrial bioethanol strain YJSH1, we identified many genomic sequence and structural variations in YHJ7, which are mainly located in subtelomeric regions, suggesting that these regions play an important role in genomic evolution between strains. In addition, our comparative transcriptome analysis between YHJ7 and S288c revealed a set of differentially expressed genes, including those involved in glucose transport (e.g., HXT2, HXT7) and oxidoredutase activity (e.g., AAD10, ADH7). Interestingly, many of these genomic and transcriptional variations are directly or indirectly associated with the adaptation of YHJ7 strain to its specific niches. Our molecular evolution analysis suggested that Japanese sake strains (K7/UC5) were derived from Chinese rice wine strains (YHJ7) at least approximately 2,300 years ago, providing the first molecular evidence elucidating the origin of Japanese sake strains. Our results depict interesting insights regarding the evolution of yeast during rice wine fermentation, and provided a valuable resource for genetic engineering to improve industrial wine-making strains.

Mutations adjacent to the end of transmembrane helices 6 and 7 independently affect drug efflux capacity of yeast ABC transporter Pdr5p.

As a mammalian p-glycoprotein homolog, Pdr5p is a major ATP-binding cassette transporter for cellular detoxification in the yeast Saccharomyces cerevisiae. In this study, two novel loss-of-function mutations located adjacent to the ends of the predicted transmembrane helices of Pdr5p were identified. C793F and S1230L mutations considerably impaired the transport activity of Pdr5p without affecting the ATPase activity and the expression level of the protein. Our results demonstrate that the size of residue 793 and the hydrophobicity of residue 1230 are important for Pdr5p efflux function. It reveals that amino acid residues located near the end of transmembrane helix play an important role in drug efflux of Pdr5p. Molecular docking results further suggest that these two single mutations might have disturbed interactions between the drugs and Pdr5p, preventing the drugs from approaching the intracellular or extracellular portal and subsequently from being exported by Pdr5p.

Improvement of natamycin production by engineering of phosphopantetheinyl transferases in Streptomyces chattanoogensis L10.

Phosphopantetheinyl transferases (PPTases) are essential to the activities of type I/II polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) through converting acyl carrier proteins (ACPs) in PKSs and peptidyl carrier proteins (PCPs) in NRPSs from inactive apo-forms into active holo-forms, leading to biosynthesis of polyketides and nonribosomal peptides. The industrial natamycin (NTM) producer, Streptomyces chattanoogensis L10, contains two PPTases (SchPPT and SchACPS) and five PKSs. Biochemical characterization of these two PPTases shows that SchPPT catalyzes the phosphopantetheinylation of ACPs in both type I PKSs and type II PKSs, SchACPS catalyzes the phosphopantetheinylation of ACPs in type II PKSs and fatty acid synthases (FASs), and the specificity of SchPPT is possibly controlled by its C terminus. Inactivation of SchPPT in S. chattanoogensis L10 abolished production of NTM but not the spore pigment, while overexpression of the SchPPT gene not only increased NTM production by about 40% but also accelerated productions of both NTM and the spore pigment. Thus, we elucidated a comprehensive phosphopantetheinylation network of PKSs and improved polyketide production by engineering the cognate PPTase in bacteria.

A mutation in intracellular loop 4 affects the drug-efflux activity of the yeast multidrug resistance ABC transporter Pdr5p.

Multidrug resistance protein Pdr5p is a yeast ATP-binding cassette (ABC) transporter in the plasma membrane. It confers multidrug resistance by active efflux of intracellular drugs. However, the highly polymorphic Pdr5p from clinical strain YJM789 loses its ability to expel azole and cyclohexmide. To investigate the role of amino acid changes in this functional change, PDR5 chimeras were constructed by segmental replacement of homologous BY4741 PDR5 fragments. Functions of PDR5 chimeras were evaluated by fluconazole and cycloheximide resistance assays. Their expression, ATPase activity, and efflux efficiency for other substrates were also analyzed. Using multiple lines of evidence, we show that an alanine-to-methionine mutation at position 1352 located in the predicted short intracellular loop 4 significantly contributes to the observed transport deficiency. The degree of impairment is likely correlated to the size of the mutant residue.