Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker

Background: A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele. Results: A K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth. Conclusions: In this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii

Draft Genome Sequence of the Marine Streptomyces sp. Strain PP-C42, Isolated from the Baltic Sea

Streptomyces, a branch of aerobic Gram-positive bacteria represents the largest genus of actinobacteria. The streptomycetes are characterized by a complex secondary metabolism and produce over two-thirds of the clinically used natural antibiotics today. Here we report the draft genome sequence of a Streptomyces strain PP-C42 isolated from the marine environment. A subset of unique genes and gene clusters for diverse secondary metabolites as well as antimicrobial peptides (AMPs) could be identified from the genome, showing great promise as a source for novel bioactive compound

Draft Genome Sequence of the Marine Streptomyces sp. Strain PP-C42, Isolated from the Baltic Sea

Streptomyces, a branch of aerobic Gram-positive bacteria represents the largest genus of actinobacteria. The streptomycetes are characterized by a complex secondary metabolism and produce over two-thirds of the clinically used natural antibiotics today. Here we report the draft genome sequence of a Streptomyces strain PP-C42 isolated from the marine environment. A subset of unique genes and gene clusters for diverse secondary metabolites as well as antimicrobial peptides (AMPs) could be identified from the genome, showing great promise as a source for novel bioactive compound

Identification and Characterization of Antifungal Compounds Using a Saccharomyces cerevisiae Reporter Bioassay

New antifungal drugs are urgently needed due to the currently limited selection, the emergence of drug resistance, and the toxicity of several commonly used drugs. To identify drug leads, we screened small molecules using a Saccharomyces cerevisiae reporter bioassay in which S. cerevisiae heterologously expresses Hik1, a group III hybrid histidine kinase (HHK) from Magnaporthe grisea. Group III HHKs are integral in fungal cell physiology, and highly conserved throughout this kingdom; they are absent in mammals, making them an attractive drug target. Our screen identified compounds 13 and 33, which showed robust activity against numerous fungal genera including Candida spp., Cryptococcus spp. and molds such as Aspergillus fumigatus and Rhizopus oryzae. Drug-resistant Candida albicans from patients were also highly susceptible to compounds 13 and 33. While the compounds do not act directly on HHKs, microarray analysis showed that compound 13 induced transcripts associated with oxidative stress, and compound 33, transcripts linked with heavy metal stress. Both compounds were highly active against C. albicans biofilm, in vitro and in vivo, and exerted synergy with fluconazole, which was inactive alone. Thus, we identified potent, broad-spectrum antifungal drug leads from a small molecule screen using a high-throughput, S. cerevisiae reporter bioassay

A search for resonant production of ttˉt\bar{t} pairs in $4.8\ \rm{fb}^{-1}ofintegratedluminosityof of integrated luminosity of p\bar{p}collisionsat collisions at \sqrt{s}=1.96\ \rm{TeV}$

We search for resonant production of tt pairs in 4.8 fb^{-1} integrated luminosity of ppbar collision data at sqrt{s}=1.96 TeV in the lepton+jets decay channel, where one top quark decays leptonically and the other hadronically. A matrix element reconstruction technique is used; for each event a probability density function (pdf) of the ttbar candidate invariant mass is sampled. These pdfs are used to construct a likelihood function, whereby the cross section for resonant ttbar production is estimated, given a hypothetical resonance mass and width. The data indicate no evidence of resonant production of ttbar pairs. A benchmark model of leptophobic Z \rightarrow ttbar is excluded with m_{Z'} < 900 GeV at 95% confidence level.Comment: accepted for publication in Physical Review D Sep 21, 201

Precision Top-Quark Mass Measurements at CDF

We present a precision measurement of the top-quark mass using the full sample of Tevatron $\sqrt{s}=1.96$ TeV proton-antiproton collisions collected by the CDF II detector, corresponding to an integrated luminosity of 8.7 $fb^{-1}$. Using a sample of $t\bar{t}$ candidate events decaying into the lepton+jets channel, we obtain distributions of the top-quark masses and the invariant mass of two jets from the $W$ boson decays from data. We then compare these distributions to templates derived from signal and background samples to extract the top-quark mass and the energy scale of the calorimeter jets with {\it in situ} calibration. The likelihood fit of the templates from signal and background events to the data yields the single most-precise measurement of the top-quark mass, \mtop = 172.85 \pm$0.71 (stat)$\pm$0.85 (syst) GeV/c^{2}.$Comment: submitted to Phys. Rev. Let

Evidence for t\bar{t}\gamma Production and Measurement of \sigma_t\bar{t}\gamma / \sigma_t\bar{t}

Using data corresponding to 6.0/fb of ppbar collisions at sqrt(s) = 1.96 TeV collected by the CDF II detector, we present a cross section measurement of top-quark pair production with an additional radiated photon. The events are selected by looking for a lepton, a photon, significant transverse momentum imbalance, large total transverse energy, and three or more jets, with at least one identified as containing a b quark. The ttbar+photon sample requires the photon to have 10 GeV or more of transverse energy, and to be in the central region. Using an event selection optimized for the ttbar+photon candidate sample we measure the production cross section of, and the ratio of cross sections of the two samples. Control samples in the dilepton+photon and lepton+photon+\met, channels are constructed to aid in decay product identification and background measurements. We observe 30 ttbar+photon candidate events compared to the standard model expectation of 26.9 +/- 3.4 events. We measure the ttbar+photon cross section to be 0.18+0.08 pb, and the ratio of the cross section of ttbar+photon to ttbar to be 0.024 +/- 0.009. Assuming no ttbar+photon production, we observe a probability of 0.0015 of the background events alone producing 30 events or more, corresponding to 3.0 standard deviations.Comment: 9 pages, 3 figure

Combined search for the standard model Higgs boson decaying to a bb pair using the full CDF data set

We combine the results of searches for the standard model Higgs boson based on the full CDF Run II data set obtained from sqrt(s) = 1.96 TeV p-pbar collisions at the Fermilab Tevatron corresponding to an integrated luminosity of 9.45/fb. The searches are conducted for Higgs bosons that are produced in association with a W or Z boson, have masses in the range 90-150 GeV/c^2, and decay into bb pairs. An excess of data is present that is inconsistent with the background prediction at the level of 2.5 standard deviations (the most significant local excess is 2.7 standard deviations).Comment: To be published in Phys. Rev. Lett (v2 contains minor updates based on comments from PRL