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

Verification of Dscam expression in rat microglia

Mikrogliazellen sind spezialisierte Makrophagen, die im Gehirn und Rückenmark vorkommen. Basierend auf der Expression eines Antikörper (5D4)- reaktiven Keratansufat- Proteoglykans auf der Oberfläche der Zellen konnten zwei funktionell unterschiedliche Populationen charakterisiert werden. Zellen mit geringerer Expression (5D4low) sind immunologisch aktiver, als jene mit hoher Expression (5D4high). Ein Vergleich von 5D4high- und 5D4low- Microarray Daten führte zur Entdeckung vieler Gene, welche im Bezug auf Entwicklung und Funktion von Mikrogliazellen von großem Interesse sein könnten. Fünf Gene, welche eine erhöhte Expressionsrate in 5D4high Mikrogliazellen zeigten, wurden untersucht: Cadm1, Dlx2, Dscam, Tef und Twist-2. Die Expression aller Gene außer Cadm1 konnte auf mRNA Ebene nachgewiesen werden. Für unsere Fragestellung war Dscam aufgrund seiner Funktion im Immunsystem von Drosophila und wegen seiner homo- und heterophiler Interaktionen sehr interessant. Deshalb entschieden wir uns, die Expression von Dscam in Mikrogliazellen auf mRNA- und Protein- Ebene genauer zu untersuchen. Zusätzlich führten wir ein funktionelles Knock-Down Experiment durch. Obwohl wir Dscam mehrmals nachweisen konnten, war es uns nicht möglich seine Expression in Ratten- Mikrogliazellen definitiv zu bestätigen, da mögliche falsch positive Zellen nicht mit Sicherheit ausgeschlossen werden konnten. Zukünftige Studien sollten die Rolle von Dscam im Säugetier- Immunsystem verifizieren. Weitere Erkenntnisse über Mikroglia- Diversität und die Entwicklung eines neuen, geeigneten Antikörpers könnten dabei zielführend sein.Microglia cells are specialized macrophages, found throughout the brain and spinal cord. Two functional distinct populations have been characterized on the basis of the expression of an antibody (5D4) reactive keratan sulfate proteoglycan on their cell surface. Cells with lower expression (5D4low) are more immunologically active than their highly expressing (5D4high) counterparts. 5D4high/5D4low- comparative microarray data revealed many differentially expressed genes which were of particular interest with respect to microglia development and function. Five genes, which showed an increased expression rate in 5D4high microglia cells, were analyzed: Cadm1, Dlx2, Dscam, Tef and Twist-2. All of them, besides Cadm1, were found to be active on the mRNA level in rat microglia cells. The role of Dscam in the immune system of Drosophila and its ability to make homo- and heterophilic interactions was very interesting in this aspect. Hence, it was the most promising aspirant for further investigations. We focused on Dscam and examined its expression on mRNA and protein level in rat microglia cells. Additionally, a functional knock-down experiment was performed. Even though Dscam expression was shown several times, we could not clearly confirm its expression in rat microglia cells, since the presence of false positive cells, possible due to Dscam positive contaminants could not be completely excluded. Future studies should be done to further address the possible role of Dscam in mammalian immune responses. Helpful in this regard would be more and novel literature about microglia diversity as well as the development of an appropriate antibody

Promoter and process engineering for recombinant protein production in Pichia pastoris towards simple, fast and methanol-free cultivation regimes and high product titers

Protein production in Pichia pastoris often applies methanol-induced gene promoters such as PAOX1 to drive the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties independent of methanol such as the PGTH1 promoter which we reported recently [1]. In order to further increase its potential, we investigated its regulation in more detail by screening of promoter variants harbouring deletions and mutations. Thereby we could identify the main regulatory region and important transcription factor binding sites of PGTH1. We also created a PGTH1 variant, called PG1-3, with greatly enhanced induction properties compared to the wild type promoter. Model based process engineering could successfully be implemented for PG1-3 to outperform the PAOX1-driven production in a simple feed regime, and to establish a speed fermentation with high titers after only two days total fermentation time. [1] Prielhofer, R.; Maurer, M.; Klein, J.; Wenger, J.; Kiziak, C.; Gasser, B.; Mattanovich, D., Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris. Microb Cell Fact 2013, 12 (1), 5

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

Hijacked then lost in translation:the plight of the recombinant host cell in membrane protein structural biology projects

Membrane protein structural biology is critically dependent upon the supply of high-quality protein. Over the last few years, the value of crystallising biochemically characterised, recombinant targets that incorporate stabilising mutations has been established. Nonetheless, obtaining sufficient yields of many recombinant membrane proteins is still a major challenge. Solutions are now emerging based on an improved understanding of recombinant host cells; as a 'cell factory' each cell is tasked with managing limited resources to simultaneously balance its own growth demands with those imposed by an expression plasmid. This review examines emerging insights into the role of translation and protein folding in defining high-yielding recombinant membrane protein production in a range of host cells

Detection and elimination of cellular bottlenecks in protein-producing yeasts

Yeasts are efficient cell factories and are commonly used for the production of recombinant proteins for biopharmaceutical and industrial purposes. For such products high levels of correctly folded proteins are needed, which sometimes requires improvement and engineering of the expression system. The article summarizes major breakthroughs that led to the efficient use of yeasts as production platforms and reviews bottlenecks occurring during protein production. Special focus is given to the metabolic impact of protein production. Furthermore, strategies that were shown to enhance secretion of recombinant proteins in different yeast species are presented

Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level

Background: The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. Results: We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. Conclusions: P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength

Expression and purification of recombinant G protein-coupled receptors: A review

Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques