Regulation of methanol utilisation pathway genes in yeasts

Methylotrophic yeasts such as Candida boidinii, Hansenula polymorpha, Pichia methanolica and Pichia pastoris are an emerging group of eukaryotic hosts for recombinant protein production with an ever increasing number of applications during the last 30 years. Their applications are linked to the use of strong methanol-inducible promoters derived from genes of the methanol utilisation pathway. These promoters are tightly regulated, highly repressed in presence of non-limiting concentrations of glucose in the medium and strongly induced if methanol is used as carbon source. Several factors involved in this tight control and their regulatory effects have been described so far. This review summarises available data about the regulation of promoters from methanol utilisation pathway genes. Furthermore, the role of cis and trans acting factors (e.g. transcription factors, glucose processing enzymes) in the expression of methanol utilisation pathway genes is reviewed both in the context of the native cell environment as well as in heterologous hosts

Deletion of the Pichia pastoris KU70 Homologue Facilitates Platform Strain Generation for Gene Expression and Synthetic Biology

Targeted gene replacement to generate knock-outs and knock-ins is a commonly used method to study the function of unknown genes. In the methylotrophic yeast Pichia pastoris, the importance of specific gene targeting has increased since the genome sequencing projects of the most commonly used strains have been accomplished, but rapid progress in the field has been impeded by inefficient mechanisms for accurate integration. To improve gene targeting efficiency in P. pastoris, we identified and deleted the P. pastoris KU70 homologue. We observed a substantial increase in the targeting efficiency using the two commonly known and used integration loci HIS4 and ADE1, reaching over 90% targeting efficiencies with only 250-bp flanking homologous DNA. Although the ku70 deletion strain was noted to be more sensitive to UV rays than the corresponding wild-type strain, no lethality, severe growth retardation or loss of gene copy numbers could be detected during repetitive rounds of cultivation and induction of heterologous protein production. Furthermore, we demonstrated the use of the ku70 deletion strain for fast and simple screening of genes in the search of new auxotrophic markers by targeting dihydroxyacetone synthase and glycerol kinase genes. Precise knock-out strains for the well-known P. pastoris AOX1, ARG4 and HIS4 genes and a whole series of expression vectors were generated based on the wild-type platform strain, providing a broad spectrum of precise tools for both intracellular and secreted production of heterologous proteins utilizing various selection markers and integration strategies for targeted or random integration of single and multiple genes. The simplicity of targeted integration in the ku70 deletion strain will further support protein production strain generation and synthetic biology using P. pastoris strains as platform hosts

Measurement of (anti)deuteron and (anti)proton production in DIS at HERA

The first observation of (anti)deuterons in deep inelastic scattering at HERA has been made with the ZEUS detector at a centre-of-mass energy of 300--318 GeV using an integrated luminosity of 120 pb-1. The measurement was performed in the central rapidity region for transverse momentum per unit of mass in the range 0.3<p_T/M<0.7. The particle rates have been extracted and interpreted in terms of the coalescence model. The (anti)deuteron production yield is smaller than the (anti)proton yield by approximately three orders of magnitude, consistent with the world measurements.Comment: 26 pages, 9 figures, 5 tables, submitted to Nucl. Phys.

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Synthetic Pichia pastoris promoters based on AOX1 regulatory elements

During the last decade, the methylotrophic yeast, Pichia pastoris, has become a major eukaryotic host for recombinant protein production in both academic and industrial research. Up to now, the expression of more than 500 proteins has been reported. One major reason for the success of this yeast as an expression system is the inducible promoter of its alcohol oxidase I (AOX1) gene. Its key features include an exceptional expression strength as well as a very strong glucose repression. By computational sequence analysis several putative cis-acting elements could be identified within the AOX1 promoter sequence. Based on this sequence analyses, we performed deletion studies and identified both, positively and negatively acting promoter elements. Consequently, these elements were tested by adding them to basal promoter elements and finally they were rearranged to generate synthetic and hybrid promoter libraries with different expression levels and regulation patterns

Synthetic Pichia pastoris promoters based on AOX1 regulatory elements

During the last decade, the methylotrophic yeast, Pichia pastoris, has become a major eukaryotic host for recombinant protein production in both academic and industrial research. Up to now, the expression of more than 500 proteins has been reported. One major reason for the success of this yeast as an expression system is the inducible promoter of its alcohol oxidase I (AOX1) gene. Its key features include an exceptional expression strength as well as a very strong glucose repression. By computational sequence analysis several putative cis-acting elements could be identified within the AOX1 promoter sequence. Based on this sequence analyses, we performed deletion studies and identified both, positively and negatively acting promoter elements. Consequently, these elements were tested by adding them to basal promoter elements and finally they were rearranged to generate synthetic and hybrid promoter libraries with different expression levels and regulation patterns

Engineering Pichia pastoris for improved NADH regeneration: A novel chassis strain for whole-cell catalysis

Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy

New shuttle vectors constructed during this study.

a<p>Promoter to regulate the expression of the gene of interest.</p>b<p>Localization of the recombinant protein. Vectors aimed for intracellular production can be used for the secretory production by adding a signal sequence.</p

Strains of <i>P. pastoris</i> used and constructed during this work.

<p>The growth rates reported correspond to the maximal growth rates (^h-1) reached in minimal media during the exponential growth phase. The standard deviation reported is calculated according to the growth rates of three biological replicates. c. = complemented. BM = buffered minimal media with glucose (D), glycerol (G) or methanol (M).</p>a<p>NRRL Y-11430, ATCC 76273.</p>b<p>Centraalbureau voor Schimmelcultures.</p

Integration cassette composition and function.

<p>a) <i>KU70</i> disruption cassette based on the <i>S. cerevisiae</i> FLP recombinase system. On both sides the flipper cassette with <i>AOX1</i> promoter (<i>P_AOX1</i>), FLP recombinase (<i>FLP</i>), CYC1 terminator (<i>CYC1_TT</i>) and Zeocin™ resistance cassette are surrounded by recombinase target sequences (<i>FRT</i>) and locus specific integration sequences (5′int and 3′int). Cassette components are not drawn to scale. b) After methanol induced (<i>P_AOX1</i>) FLP production and subsequent <i>FRT</i> recognition leading to cassette excision only one <i>FRT</i> (34 bp) is left in the locus in between the 3′ and 5′ integration sequences. c) The lengths of the homologous sequences at 5′ and 3′ ends of the disruption cassettes used to compare the homologous recombination frequencies in wt and ku70 deletion strains varied from 100 bp to 1350 bp in the <i>HIS4</i> locus. Zeocin™ resistance cassette was placed in between the homologous sequences. Cassette components are not drawn to scale.</p