Geschmack ist vorhersagbar: Mit FlavorMiner. FlavorMiner heißt das Tool, das IPB-Chemiker und Partner aus Kolumbien jüngst entwickelt haben. Das Programm kann, basierend auf maschinellem Lernen (KI), anhand der…
Seit Februar 2021 bietet Wolfgang Brandt, ehemaliger Leiter der Arbeitsgruppe Computerchemie am IPB, sein Citizen Science-Projekt zur Pilzbestimmung an. Dafür hat er in regelmäßigen Abständen öffentliche Vorträge zur Vielfalt…
Püllmann, P.; Knorrscheidt, A.; Münch, J.; Palme, P. R.; Hoehenwarter, W.; Marillonnet, S.; Alcalde, M.; Westermann, B.; Weissenborn, M. J.;A modular two yeast species secretion system for the production and preparative application of fungal peroxygenasesbioRxiv(2020)DOI: 10.1101/2020.07.22.216432
Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be broadly applicable and consists of the three module types: i) a signal peptide panel guiding secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. We show that optimal signal peptides could be selected for successful UPO secretion by combinatorial testing of 17 signal peptides for each UPO gene. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid workflow from putative UPO gene to preparative scale enantioselective biotransformations.
Preprints
Grützner, R.; Martin, P.; Horn, C.; Mortensen, S.; Cram, E. J.; Lee-Parsons, C. W. T.; Stuttmann, J.; Marillonnet, S.;Addition of Multiple Introns to a Cas9 Gene Results in Dramatic Improvement in Efficiency for Generation of Gene Knockouts in PlantsbioRxiv(2020)DOI: 10.1101/2020.04.03.023036
The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by stably transforming a Cas9 expression construct into the plant genome. The efficiency with which mutations are obtained in genes of interest can vary considerably depending on specific features of the constructs, including the source and nature of the promoters and terminators used for expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency with which mutations could be obtained in target genes in Arabidopsis thaliana with the Cas9 nuclease, we have investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLS) and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on Cas9 activity and that two NLSs work better than one. However, the most important impact on the efficiency of the constructs was obtained by addition of 13 introns into the Cas9 coding sequence, which dramatically improved editing efficiencies of the constructs; none of the primary transformants obtained with a Cas9 lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of primary transformants generated with intronized Cas9 displayed mutant phenotypes. The intronized Cas9 was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.
Preprints
Bassal, M.; Majovsky, P.; Thieme, D.; Herr, T.; Abukhalaf, M.; Ayash, M.; Al Shweiki, M. R.; Proksch, C.; Hmedat, A.; Ziegler, J.; Neumann, S.; Hoehenwarter, W.;Reshaping of the Arabidopsis thaliana proteome landscape and co-regulation of proteins in development and immunitybioRxiv(2020)DOI: 10.1101/2020.03.09.978627
Proteome remodeling is a fundamental adaptive response and proteins in complex and functionally related proteins are often co-expressed. Using a deep sampling strategy we define Arabidopsis thaliana tissue core proteomes at around 10,000 proteins per tissue and absolutely quantify (copy numbers per cell) nearly 16,000 proteins throughout the plant lifecycle. A proteome wide survey of global post translational modification revealed amino acid exchanges pointing to potential conservation of translational infidelity in eukaryotes. Correlation analysis of protein abundance uncovered potentially new tissue and age specific roles of entire signaling modules regulating transcription in photosynthesis, seed development and senescence and abscission. Among others, the data suggest a potential function of RD26 and other NAC transcription factors in seed development related to desiccation tolerance as well as a possible function of Cysteine-rich Receptor-like Kinases (CRKs) as ROS sensors in senescence. All of the components of ribosome biogenesis factor (RBF) complexes were co-expressed tissue and age specifically indicating functional promiscuity in the assembly of these little described protein complexes in Arabidopsis. Treatment of seedlings with flg22 for 16 hours allowed us to characterize proteome architecture in basal immunity in detail. The results were complemented with parallel reaction monitoring (PRM) targeted proteomics, phytohormone, amino acid and transcript measurements. We obtained strong evidence of suppression of jasmonate (JA) and JA-Ile levels by deconjugation and hydroxylation via IAA-ALA RESISTANT3 (IAR3) and JASMONATE-INDUCED OXYGENASE 2 (JOX2) under the control of JASMONATE INSENSITIVE 1 (MYC2). This previously unknown regulatory switch is another part of the puzzle of the as yet understudied role of JA in pattern triggered immunity. The extensive coverage of the Arabidopsis proteome in various biological scenarios presents a rich resource to plant biologists that we make available to the community.
Preprints
Barthel, K.; Martin, P.; Ordon, J.; Lee Erickson, J.; Gantner, J.; Lee Erickson, J.; Herr, R.; Kretschmer, C.; Ferik, F.; Berner, T.; Keilwagen, J.; Marillonnet, S.; Stuttmann, J.; Bonas, U.;One-shot generation of duodecuple (12x) mutant Arabidopsis: Highly efficient routine editing in model speciesbioRxiv(2020)DOI: 10.1101/2020.03.31.018671
Genome editing by RNA-guided nucleases in model species is still hampered by low efficiencies, and isolation of transgene-free individuals often requires tedious PCR screening. Here, we present a toolkit that mitigates these drawbacks for Nicotiana benthamiana and Arabidopsis thaliana. The toolkit is based on an intron-optimized SpCas9-coding gene (zCas9i), which conveys dramatically enhanced editing efficiencies. The zCas9i gene is combined with remaining components of the genome editing system in recipient vectors, which lack only the user-defined guide RNA transcriptional units. Up to 32 guide RNA transcriptional units can be introduced to these recipients by a simple and PCR-free cloning strategy, with the choice of three different RNA polymerase III promoters for guide RNA expression. We developed new markers to aid transgene counter-selection in N. benthamiana, and demonstrate their efficacy for isolation of several genome-edited N. benthamiana lines. In Arabidopsis, we explore the limits of multiplexing by simultaneously targeting 12 genes by 24 sgRNAs. Perhaps surprisingly, the limiting factor in such higher order multiplexing applications is Cas9 availability, rather than recombination or silencing of repetitive sgRNA TU arrays. Through a combination of phenotypic screening and pooled amplicon sequencing, we identify transgene-free duodecuple mutant Arabidopsis plants directly in the T2 generation. This demonstrates high efficiency of the zCas9i gene, and reveals new perspectives for multiplexing to target gene families and to generate higher order mutants.