Engineered geranyl diphosphate methyltransferase produces 2-methyl-dimethylallyl diphosphate as a noncanonical C6 unit for terpenoid biosynthesis.

Terpenoids constitute the largest class of natural products with complex structures, essential functions, and versatile applications. Creation of new building blocks beyond the conventional five-carbon (C5) units, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate, expands significantly the chemical space of terpenoids. Structure-guided engineering of an S-adenosylmethionine-dependent geranyl diphosphate (GPP) C2-methyltransferase from Streptomyces coelicolor yielded variants converting DMAPP to a new C6 unit, 2-methyl-DMAPP. Mutation of the Gly residue at the position 202 resulted in a smaller substrate-binding pocket to fit DMAPP instead of its native substrate GPP. Replacement of Phe residue at the position 222 with a Tyr residue contributed to DMAPP binding via hydrogen bond. Furthermore, using Escherichia coli as the chassis, we demonstrated that 2-methyl-DMAPP was accepted as a start unit to generate noncanonical trans- and cis-prenyl diphosphates (C5n+1) and terpenoids. This work provides insights into substrate recognition of prenyl diphosphate methyltransferases, and strategies to diversify terpenoids by expanding the building block portfolio.

The Sur7/PalI family transmembrane protein Tos7 (Yol019w) plays a role in secretion in budding yeast.

Tos7 (Yol019w) is a Sur7/PalI family transmembrane protein in the budding yeast Saccharomyces cerevisiae. Since the deletion of TOS7 did not affect growth or cell morphology, the cellular roles of Tos7 have not been established previously. Here, we show that high-copy TOS7 expression suppressed the growth defect of the secretion-defective RGA1-C term-overexpressing mutant and sec15-1 mutant. Moreover, Tos7 physically interacted with Boi2 and the Rho GTPase Rho3, two key regulators of exocyst assembly, suggesting that Tos7 plays a role in secretion. We also show that the deletion of TOS7 rendered the cells more sensitive to the cell wall-disrupting agents Congo red and calcofluor white while high-copy TOS7 expression had an opposite effect, suggesting that Tos7 affects cell wall organization. Finally, we show that Tos7 localized to punctate patches on the plasma membrane that were largely co-localized with the plasma membrane microdomains named MCC (membrane compartment of Can1). Together, these results suggest that Tos7 contributes to cell surface-related functions. Tos7 is likely an auxiliary component of MCC/eisosome that specifically interacts with the secretory pathway.

Roles of the PH, coiled-coil and SAM domains of the yeast polarity protein Boi2 in polarity-site localization and function in polarized growth.

Boi1 and Boi2 are paralogous proteins essential for bud formation in budding yeast. So far, the domains that target Boi1/Boi2 to the polarity sites and function in bud formation are not well understood. Here, we report that a coiled-coil domain of Boi2 cooperates with the adjacent PH domain to confer Boi2's bud-cortex localization and major function in cell growth. The PH domain portion of the PH-CC bi-domain interacts with the Rho GTPases Cdc42 and Rho3 and both interactions are independent of the GTP/GDP-bound state of each GTPase. Interestingly, high-copy RHO3 and BOI2 but not CDC42 suppressed the growth defect of RGA1-C538 overexpression and the sec15-1 mutant and this BOI2 function depends on RHO3, suggesting that Boi2 may function in the Rho3 pathway. The SAM domain of Boi2 plays an essential role in high-copy suppression of the two mutants as well as in the early bud-neck localization of Boi2. The SAM domain and the CC domain also interact homotypically. They are likely involved in the formation of Boi2-containing protein complex. Our results provide new insights in the localization and function of Boi2 and highlight the importance of the PH-CC bi-domain and the SAM domain in Boi2's localization and function.

Genetic variation of Echinococcus spp. in yaks and sheep in the Tibet Autonomous Region of China based on mitochondrial DNA.

BACKGROUND: Cystic echinococcosis (CE) in humans and livestock is caused by Echinococcus granulosus (sensu lato). In China where CE is endemic, a number of studies have shown that Echinococcus granulosus (sensu stricto) is majorly responsible for CE. However, E. canadensis (G6) which is the second leading cause of CE is now being detected in most parts of the country. In this study, the species diversity and genetic variation of Echinococcus granulosus (s.l.) in four counties in Tibet Autonomous Region of China were investigated. METHODS: Infection with Echinococcus granulosus (s.s.) in yaks and sheep was identified using NADH dehydrogenase subunit 1 and 5 (nad1 and nad5) mitochondrial genes while the genotype G6 of E. canadensis initially diagnosed with NADH dehydrogenase subunit 1 (nad1) was further confirmed by analysis of the complete mitochondrial genome and a phylogenetic network constructed based on the nad2 and nad5 genes. RESULTS: Out of 85 hydatid cyst samples collected from slaughtered sheep (n = 54) and yaks (n = 31), 83 were identified as E. granulosus (s.s.) G1 (n = 77), G3 (n = 6) and 2 were identified as E. canadensis G6. Analysis of the nad1/nad5 genes revealed 16/17 mutations with 9/14 parsimony informative sites resulting in 15/14 haplotypes, respectively. Haplotype diversity (Hd) and nucleotide diversity (π) of E. granulosus (s.s.) population were 0.650 and 0.00127 for nad1 and 0.782 and 0.00306 for nad5, respectively, with an overall negative Tajima's D and Fu's Fs. A low FST indicated no genetic difference between isolates from sheep and yaks. CONCLUSION: Pockets of infection with E. canadensis (G6, G7, G8 and G10) have been previously reported in sheep, goats, yaks and/or humans in different parts of China. While the G6 genotype has been previously reported in sheep in the Tibet Autonomous Region, the detection in a yak in the present study represents the first to the best of our knowledge. Therefore, we recommend future surveys and control efforts to comprehensively investigate other potential intermediate hosts for the prevalence and genetic diversity of the E. canadensis group (G6, G7, G8 and G10) across the country and their inclusion into the existing CE control programme.

Prevalence and genetic characterization of Enterocytozoon bieneusi and Giardia duodenalis in Tibetan pigs in Tibet, China.

Enterocytozoon bieneusi and Giardia duodenalis are important opportunistic enteric zoonotic pathogens that cause diarrhoea and intestinal diseases in animals and humans. China is the largest producer of pigs, but whether Tibetan pigs, a unique pig breed in Tibet, are infected with E. bieneusi and G. duodenalis is unknown. Therefore, we conducted a molecular epidemiological survey to determine the prevalence of E. bieneusi and G. duodenalis in Tibetan pigs in Tibet, China, and identified the genotypes of these causative agents. A total of 345 faecal specimens were collected from Tibetan pigs from three Tibet counties (Milin, Cuona and Gongbujiangda), examined by nested PCR and sequenced utilizing genetic markers in the ribosomal internal transcribed spacer (ITS) region of the rRNA and glutamate dehydrogenase (gdh) gene for E. bieneusi and G. duodenalis, respectively. Moreover, using multilocus sequence typing, the subtypes of E. bieneusi were identified based on four loci (MS1, MS3, MS4 and MS7). A total of 41 (11.88%) faecal samples from Tibetan pigs were E. bieneusi-positive, and 2 (0.58%) were G. duodenalis-positive. The multivariate logistic regression analysis showed that age was considered a risk factor for Tibetan pig infection of E. bieneusi. Two novel (GB11, GB31) and four known E. bieneusi genotypes (EbpC, EbpD, PigEBITS5 and CHS12) were identified and were all classified as zoonotic group 1 according to the phylogenetic analysis. Two MLGs (MLGI and MLGII) were further identified in the E. bieneusi EbpC genotype by multilocus sequence typing analysis. In addition, two G. duodenalis assemblages (D and E) were found in the present study. To our knowledge, the current study is the first to detect the prevalence and perform genetic characterization of G. duodenalis in Tibetan pigs in Tibet, China. The results could provide essential data for controlling E. bieneusi and G. duodenalis infections in Tibetan pigs that are in contact with other animals and humans, as Tibetan pigs could be a potential source for human infection by these pathogens.

Identification and characterization of new Muscodor endophytes from gramineous plants in Xishuangbanna, China.

The endophytic fungi Muscodor spp. produce volatile organic compounds (VOCs) which can inhibit and even kill pathogenic fungi, bacteria, and nematodes. Nine endophytic fungal strains, isolated from the shoots of gramineous plants including Arthraxon hispidus, Eleusine indica, Oplismenus undulatifolius, and Oryza granulata, were identified as Muscodor through phylogenetic analysis of the internal transcribed spacer. Through an SPSS K-means cluster analysis, the nine Muscodor strains were divided into four groups based on the antifungal activities of the VOCs produced by these fungi determined by a two-section confrontation test. The first group contains the strains Y-L-54, W-S-41, Y-S-35, W-T-27, and Y-L-56, which showed the strongest activity. The second and third groups contain W-S-35 and Y-L-43, which showed stronger and moderate activity, respectively. The fourth group contains W-S-38 and N-L-7, which were the weakest in inhibiting the tested pathogens. Thirty-five compounds and the relative amounts of VOCs were determined by SPME-GC-MS and comparison with the NIST14 mass spectrometry database and Agilent MassHunter qualitative and quantitative analyses. These 35 compounds were classified into two different categories: (a) the product of fatty acid degradation, and (b) the intermediate and final metabolite of the metabolic pathway with the precursor of mevalonic acid. SPSS clustering analysis showed that the chemical components of VOCs might be correlated with their bioactivity rather than their phylogenetic assignment and some of the identified compounds might be responsible for antifungal activity. In conclusion, new Muscodor endophytes were recorded in tropical gramineous plants and a number of strains showed remarkable bioactive properties. Therefore, they have important potential applications in the fields of plant disease control.

[Helminth Infections in Goats in Nimu County of Tibet].

Postmortem examinations were made in 99 goats in Nimu County of Tibe, and parasites were collected and identified based on morphology. The collected parasites were categorized, and infection status was analyzed. The helminth infection rate was 100% among the goats, and all showed a pattern of mixed infection. The identified parasites belonged to 21 species, 15 genera, and 9 families. The Trichuris genus（36.4%） was the most prevailing among nematodes in the gastrointestinal tract; Paramphistomum cervi（60.6%） and Paramphistomum gotoi（60.6%） were predominant among trematodes detected; Cysticercus tenuicollis（52.5%） was the predominant cestode detected; and Orientobilharzia turkestanicum was the major parasite detected in the portal vein (69.7%).