Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an H. polymorpha replicon-like sequence present on the S. cerevisiae LEU2 gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multi-copy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22% of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in H. polymorpha. In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68% of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.
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This article describes a relatively straightforward procedure to knock out the gene that encodes the invertase enzyme in baker’s yeast. The SUC2 gene, which encodes for the invertase enzyme, is knocked out by a single-step PCR knock out method. The knockout is subsequently confirmed at the genetic level by PCR and agarose gel electrophoresis. The knockout is confirmed at the biochemical level by measuring the activity of the invertase enzyme using a colorimetric assay. This tips and tools article describes an easily scalable, inexpensive, yet challenging research project helping undergraduate students at the Bachelor level to conceptualize the effect of the deletion of a gene encoding an enzyme.
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Carnitine/choline acyltransferases play diverse roles in energy metabolism and neuronal signalling. Our knowledge of their evolutionary relationships, important for functional understanding, is incomplete. Therefore, we aimed to determine the evolutionary relationships of these eukaryotic transferases. We performed extensivephylogenetic and intron position analyses. We found that mammalian intramitochondrial CPT2 is most closely related to cytosolic yeast carnitine transferases (Sc-YAT1 and 2), whereas the other members of the family are related to intraorganellar yeast Sc-CAT2. Therefore, the cytosolically active CPT1 more closely resembles intramitochondrial ancestors than CPT2. The choline acetyltransferase is closely related to carnitine acetyltransferase and shows lower evolutionary rates than long chain acyltransferases. In the CPT1 family several duplications occurred during animal radiation, leading to the isoforms CPT1A, CPT1B and CPT1C. In addition, we found five CPT1-like genes in Caenorhabditis elegans that strongly group to the CPT1 family. The long branch leading to mammalian brain isoform CPT1C suggests that either strong positive or relaxed evolution has taken place on this node. The presented evolutionary delineation of carnitine/choline acyltransferases adds to current knowledge on their functions and provides tangible leads for further experimental research.
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The global market for the industrial manufacturing of recombinant proteins (RPS) is steadily increasing and demand will keep rising in years to come. Currently, RPs are already an integral part of disease therapeutics, agriculture and the chemical industry and RP manufacturing methods rely heavily on host systems such as prokaryotes and, to a lesser extent, mammalian, yeast and plant cells. When comparing these host systems, all have their specific strengths and weaknesses and numerous challenges remain to improve protein manufacturing on an industrial scale. In this project, GLO Biotics proposes an innovative plant-based RP expression platform with the potential of significantly reducing costs and process requirements compared to the current state-of-the-art systems. Specifically, this novel concept is based on the use of coconut water as a natural, cell-free ‘protein production factory’. Coconut water in nuts aged 4-6 months is composed of free-floating cell nuclei devoid of cell walls, and it has been demonstrated these nuclei can express foreign proteins. Compared to existing platforms, the relative ease of delivering foreign protein-coding genes into this system, as well as the ease of recovery of the produced protein, potentially offers an innovative platform with great commercial attractiveness. In summary, the aim of this project is to provide a proof-of-concept for coconut water as a novel and competitive RP production platform by demonstrating the production and recovery of several commercially available RPs. To this end, GLO Biotics intends to collaborate with Zuyd University of Applied Sciences (Zuyd) and the Aachen Maastricht Institute for Biobased Materials (AMIBM) in demonstrating the potential of the ‘GLO-Conuts’ expression system. As a consortium, Zuyd and GLO Biotics will utilize their shared experience in molecular engineering and DNA vector technology and AMIBM will bring their expertise in plant-based RP production and recovery.
The missing link in diagnostic testing for rheumatoid arthritis (RA) is an agglutination assay, easy to perform and dedicated to decentralized testing. Approximately 75% of RA patients produce autoantibodies to citrullinated proteins (ACPA), which can be detected using an agglutination-based diagnostic test. Such a diagnostic test will be cheaper, less laborious and faster than current tests and does not require sophisticated equipment. Novio Catalpa is developing this alternative test for ACPA in collaboration with Radboud University. To develop this test, specifically tagged and citrullinated nanobodies are needed, but the production is still challenging. Current methods for the production of ACPA diagnostics involve chemical synthesis, in which a variety of toxic chemicals are used in each step. The alternative assay involves nanobodies fused with RA-biomarker target entities, which can be completely produced by ‘green synthesis’ in the yeast Pichia pastoris using the expertise of HAN BioCentre. The yeast P. pastoris has proven to be able to produce nanobodies and is a fast and cost-effective platform that often results in high protein yields. Goal of the project is therefore to determine the feasibility and best green route to produce purified nanobodies tagged with citrullinated ACPA targets that can be used for developing an agglutination assay for RA. P. pastoris does not produce endogenous PAD enzymes which are needed for citrullination of the nanobodies in order to be able to use it in a RA agglutination test. Therefore, PAD enzymes from other sources need to be tested and applied. The project results will be directly used by Novio Catalpa to further develop the innovative test for RA. This project will contribute to and finally result in a single-step agglutination assay suitable for both point-of-care testing and automation in clinical laboratories.
Green methanol is emerging as a key player in sustainable biotech, offering a renewable alternative to fossil fuels or sugar based feedstocks. Although methanol has long been considered a promising material for bioproduction, using it on industrial scale has been challenging due to its high oxygen demands, making the process expensive and inefficient. This project focuses on developing a sustainable, but more economical feasible way to produce biochemicals, like Single Cell Protein (SCP). The innovative solution proposed by FeedstocksUnited (FSU) is to use paraformaldehyde, a compound derived from renewable methanol, as feedstock, which requires much less oxygen during fermentation. This new method has already shown promising results in the lab, where it was tested with microorganisms that can use formaldehyde (released from paraformaldehyde) as a source of carbon and energy. FSU’s approach has the potential to significantly reduce the costs and environmental impacts associated with large-scale bioproduction. The process can be managed more efficiently than methods using methanol, since the production of paraformaldehyde from formaldehyde is tunable. This process control will lead to better yields and reduced energy and feedstock consumption. The HAN BioCentre, with its advanced research facilities and experienced team, will conduct further research to optimize this method for industrial applications. This includes studying how organisms metabolize formaldehyde and improving the process through continuous fermentation. The research also supports educational goals by involving students in cutting-edge biotechnological work. Ultimately, the project aims to provide a solid proof-of-concept that can be scaled up to industrial levels, contributing to a more sustainable bioeconomy.
