Enlarging the synthetic biology toolbox for Pichia pastoris: Golden Gate cloning and CRISPR/Cas9

State-of-the-art strain engineering techniques for the protein producing yeast host Pichia pastoris include overexpression of homologous and heterologous genes, and deletion of host genes. For this purpose overexpression vectors and gene deletion methods such as the split marker technique have been established. For metabolic and cell engineering purposes, the simultaneous overexpression of more than one gene is often needed. Previous approaches employing subsequent steps of overexpression and marker recycling were time- and labor-consuming. Therefore, efficient systems allowing multiple gene overexpression are required, that can be stably integrated into the P. pastoris genome. To this end, we developed a synthetic biology toolbox based on Golden Gate cloning to enable efficient construction of complex and versatile over-expression vectors. Up to five different expression cassettes, employing a library of promoters and terminators can be combined into one vector, and successfully integrated into the genomic DNA of P. pastoris at targeted loci in one step. Recent trends in synthetic biology, however, go into the direction of building up large and complex reaction networks. To allow for clean and unscarred genetic engineering, a CRISPR/Cas9 based method for gene insertions, deletions and replacements was developed, which paves the way for precise genomic rearrangements in P. pastoris. By using this technique precise genomic integrations were performed efficiently without integrative selection markers. The repertoire of genetic techniques developed so far, will provide a wide variety of possibilities to engineer P. pastoris. Applications for these synthetic biology tools in cell engineering of recombinant P. pastoris will be presented

Recent Progress in Direct DME Synthesis and Potential of Bifunctional Catalysts

PtX technologies are one major building block of the future energy system based on renewables sources. Dimethyl ether (DME) is an important PtX product that can be used as intermediate the production of CO$_{2}$-neutral base chemicals. New applications lead to an increase of the global production and the optimization of the process efficiency, especially when considering decentralized synthesis. This review article puts some spotlights on recent developments in methanol and the direct DME synthesis with a special focus on the modeling and bifunctional catalyst. This study is expected to provide a foundation for future works in the field of catalysis research based on catalysts design and kinetic modeling

Photoassociation and coherent transient dynamics in the interaction of ultracold rubidium atoms with shaped femtosecond pulses - I. Experiment

We experimentally investigate various processes present in the photoassociative interaction of an ultracold atomic sample with shaped femtosecond laser pulses. We demonstrate the photoassociation of pairs of rubidium atoms into electronically excited, bound molecular states using spectrally cut femtosecond laser pulses tuned below the rubidium D1 or D2 asymptote. Time-resolved pump-probe spectra reveal coherent oscillations of the molecular formation rate, which are due to coherent transient dynamics in the electronic excitation. The oscillation frequency corresponds to the detun-ing of the spectral cut position to the asymptotic transition frequency of the rubidium D1 or D2 lines, respectively. Measurements of the molecular photoassociation signal as a function of the pulse energy reveal a non-linear dependence and indicate a non-perturbative excitation process. Chirping the association laser pulse allowed us to change the phase of the coherent transients. Furthermore, a signature for molecules in the electronic ground state is found, which is attributed to molecule formation by femtosecond photoassociation followed by spontaneous decay. In a subsequent article [A. Merli et al., submitted] quantum mechanical calculations are presented, which compare well with the experimental data and reveal further details about the observed coherent transient dynamics

Coherent control with shaped femtosecond laser pulses applied to ultracold molecules

We report on coherent control of excitation processes of translationally ultracold rubidium dimers in a magneto-optical trap by using shaped femtosecond laser pulses. Evolution strategies are applied in a feedback loop in order to optimize the photoexcitation of the Rb2 molecules, which subsequently undergo ionization or fragmentation. A superior performance of the resulting pulses compared to unshaped pulses of the same pulse energy is obtained by distributing the energy among specific spectral components. The demonstration of coherent control to ultracold ensembles opens a path to actively influence fundamental photo-induced processes in molecular quantum gases

Lower hypoxic ventilatory response in smokers compared to non-smokers during abstinence from cigarettes

Background Carotid body O2-chemosensitivity determines the hypoxic ventilatory response (HVR) as part of crucial regulatory reflex within oxygen homeostasis. Nicotine has been suggested to attenuate HVR in neonates of smoking mothers. However, whether smoking affects HVR in adulthood has remained unclear and probably blurred by acute ventilatory stimulation through cigarette smoke. We hypothesized that HVR is substantially reduced in smokers when studied after an overnight abstinence from cigarettes i.e. after nicotine elimination. Methods We therefore determined the isocapnic HVR of 23 healthy male smokers (age 33.9 ± 2.0 years, BMI 24.2 ± 0.5 kg m−2, mean ± SEM) with a smoking history of >8 years after 12 h of abstinence and compared it to that of 23 healthy male non-smokers matched for age and BMI. Results Smokers and non-smokers were comparable with regard to factors known to affect isocapnic HVR such as plasma levels of glucose and thiols as well as intracellular levels of glutathione in blood mononuclear cells. As a new finding, abstinent smokers had a significantly lower isocapnic HVR (0.024 ± 0.002 vs. 0.037 ± 0.003 l min−1 %−1BMI−1, P = 0.002) compared to non-smokers. However, upon re-exposure to cigarettes the smokers’ HVR increased immediately to the non-smokers’ level. Conclusions This is the first report of a substantial HVR reduction in abstinent adult smokers which appears to be masked by daily smoking routine and may therefore have been previously overlooked. A low HVR may be suggested as a novel link between smoking and aggravated hypoxemia during sleep especially in relevant clinical conditions such as COPD

Differences in cortical contractile properties between healthy epithelial and cancerous mesenchymal breast cells

Cell contractility is mainly imagined as a force dipole-like interaction based on actin stress fibers that pull on cellular adhesion sites. Here, we present a different type of contractility based on isotropic contractions within the actomyosin cortex. Measuring mechanosensitive cortical contractility of suspended cells among various cell lines allowed us to exclude effects caused by stress fibers. We found that epithelial cells display a higher cortical tension than mesenchymal cells, directly contrasting to stress fiber-mediated contractility. These two types of contractility can even be used to distinguish epithelial from mesenchymal cells. These findings from a single cell level correlate to the rearrangement effects of actomyosin cortices within cells assembled in multicellular aggregates. Epithelial cells form a collective contractile actin cortex surrounding multicellular aggregates and further generate a high surface tension reminiscent of tissue boundaries. Hence, we suggest this intercellular structure as to be crucial for epithelial tissue integrity. In contrast, mesenchymal cells do not form collective actomyosin cortices reducing multicellular cohesion and enabling cell escape from the aggregates

Correlation of age and haematoma volume in patients with spontaneous lobar intracerebral haemorrhage

Background Lobar intracerebral haemorrhage (LH) is gaining importance in the ageing population, but there are only limited data regarding specific clinical characteristics and risk factors of older patients with LH. Methods This retrospective analysis of patients with spontaneous supratentorial haemorrhage included 174 consecutive patients (78 LH and 96 deep ICH (DH)). Clinical data including the preadmission status, neuroradiological findings, initial presentation, treatment and outcome were evaluated using institutional databases, patients' medical charts and mailed questionnaires. Logistic regression analyses were calculated for initial parameters predisposing LH and for treatment and outcome parameters associated with LH. Results Age-stratified volume analysis revealed increasing haematoma volumes for LH (≤70 years: 26.2 ml; 70–80 years: 37 ml; >80 years: 61.3 ml), whereas DH showed no relation between volume and age (≤70 years: 10.1 ml; 70–80 years: 23.2 ml; >80 years: 12.1 ml). DH patients had significantly higher HbA1c levels. Post-ICH seizures were more frequent after LH. Logistic regression analyses identified the parameters: age, haematoma volume and post-ICH seizures to be associated with LH, whereas intraventricular haemorrhage, extraventricular drainages and elevated HbA1c were related to DH. Conclusion Haematoma volumes are substantially increasing in LH patients who are older than 70 years. Pathological HbA1c levels are significantly associated and predisposing for DH. These findings further support the ongoing debate of different disease entities for supratentorial ICH (ie, association of cerebral amyloid angiopathy and lobar ICH versus diabetes induced atherosclerosis in deep ICH). Future studies should focus on identifying specific pathological characteristics and risk factors for both bleeding sites to implement specific preventive measures, that is amyloid angiopathy modulating therapies for LH, and to avoid risk factors that are specific for each haemorrhage location

Golden Pi CS : a golden gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris

This work has been supported by the Federal Ministry of Science, Research and Economy (BMWFW), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, the Government of Lower Austria and ZIT - Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG.State-of-the-art strain engineering techniques for the host Pichia pastoris (syn. Komagataella spp.) include overexpression of homologous and heterologous genes, and deletion of host genes. For metabolic and cell engineering purposes the simultaneous overexpression of more than one gene would often be required. Very recently, Golden Gate based libraries were adapted to optimize single expression cassettes for recombinant proteins in P. pastoris. However, an efficient toolbox allowing the overexpression of multiple genes at once was not available for P. pastoris. With the Golden Pi CS system, we provide a flexible modular system for advanced strain engineering in P. pastoris based on Golden Gate cloning. For this purpose, we established a wide variety of standardized genetic parts (20 promoters of different strength, 10 transcription terminators, 4 genome integration loci, 4 resistance marker cassettes). All genetic parts were characterized based on their expression strength measured by eGFP as reporter in up to four production-relevant conditions. The promoters, which are either constitutive or regulatable, cover a broad range of expression strengths in their active conditions (2-192% of the glyceraldehyde-3-phosphate dehydrogenase promoter P ), while all transcription terminators and genome integration loci led to equally high expression strength. These modular genetic parts can be readily combined in versatile order, as exemplified for the simultaneous expression of Cas9 and one or more guide-RNA expression units. Importantly, for constructing multigene constructs (vectors with more than two expression units) it is not only essential to balance the expression of the individual genes, but also to avoid repetitive homologous sequences which were otherwise shown to trigger "loop-out" of vector DNA from the P. pastoris genome. Golden Pi CS, a modular Golden Gate-derived P. pastoris cloning system, is very flexible and efficient and can be used for strain engineering of P. pastoris to accomplish pathway expression, protein production or other applications where the integration of various DNA products is required. It allows for the assembly of up to eight expression units on one plasmid with the ability to use different characterized promoters and terminators for each expression unit. Golden Pi CS vectors are available at Addgene. The online version of this article (10.1186/s12918-017-0492-3) contains supplementary material, which is available to authorized users