Biology subjects

Brinkmann, C. K.

Publications and source records attributed to Brinkmann, C. K..

3 recordsLinked to original sources

bioRxiv2024

Advancing chloroplast synthetic biology through high-throughput plastome engineering of Chlamydomonas reinhardtii

Chloroplast synthetic biology holds promise for developing improved crops through improving the function of plastids. However, chloroplast engineering efforts face limitations due to the scarcity of genetic tools and the low throughput of plant-based systems. To address these challenges, we here established Chlamydomonas reinhardtii as a prototyping chassis for chloroplast synthetic biology. We developed an automation workflow that enables the generation, handling, and analysis of thousands of transplastomic strains in parallel, expanded the repertoire of selection markers for chloroplast transformation, established new reporter genes, and characterized over 140 regulatory parts, including native and synthetic promoters, UTRs, and intercistronic expression elements. We integrated the system within the Phytobrick cloning standard and demonstrate several applications, including a library-based approach to develop synthetic promoter designs in plastids. Finally, we provide a proof-of-concept for prototyping novel traits in plastids by introducing a chloroplast-based synthetic photorespiration pathway and demonstrating a twofold increase in biomass production. Overall, our study advances chloroplast engineering, and provides a promising platform to rapidly prototype chloroplast manipulations before their transfer into higher plants and crops.

synthetic biology↗

bioRxiv2024

Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform

Climate change poses a significant threat to global agriculture, necessitating innovative solutions. Plant synthetic biology, particularly chloroplast engineering, holds promise as a viable approach to this challenge. Chloroplasts present a variety of advantageous traits for genetic engineering, but the development of genetic tools and genetic part characterization in these organelles is hindered by the lengthy timescales required to generate transplastomic organisms. To address these challenges, we have established a versatile protocol for generating chloroplast-based cell-free gene expression (CFE) systems derived from a diverse range of plant species, including wheat (monocot), spinach, and poplar trees (dicots). We show that these systems work with conventionally used T7 RNA polymerase, as well as the endogenous chloroplast polymerases, allowing for detailed characterization and prototyping of regulatory sequences at both transcription and translation levels. To demonstrate the platform for characterization of promoters and 5 and 3 untranslated regions (UTRs) in higher plant chloroplast gene expression, we analyze a collection of 23 5UTRs, 10 3UTRs, and 6 chloroplast promoters, assessed their expression in spinach and wheat extracts, and found consistency in expression patterns, suggesting cross-species compatibility. Looking forward, our chloroplast CFE systems open new avenues for plant synthetic biology, offering prototyping tools for both understanding gene expression and developing engineered plants, which could help meet the demands of a changing global climate. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/580994v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@a6b24org.highwire.dtl.DTLVardef@2b62org.highwire.dtl.DTLVardef@1203308org.highwire.dtl.DTLVardef@f674a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

L-SCRaMbLE creates large-scale genome rearrangements in synthetic Sc2.0 chromosomes

Optimization of the metabolic flux through heterologous pathways to improve bioproduction or utilization of alternative substrates requires both fine-tuning of non-native gene expression levels and improvement of the host genome. The SCRaMbLE system incorporated into synthetic Sc2.0 yeast strains enables a rapid approach to rearrange the genome of Saccharomyces cerevisiae in order to create optimized chassis. Here, we show that the light-inducible Cre recombinase L-SCRaMbLE can efficiently generate diverse recombination events when applied to Sc2.0 strains containing a linear or circular synthetic chromosome III. We present an efficient and straightforward workflow for the identification of complex rearranged synthetic chromosomes from SCRaMbLEd isolates without selection pressure. The screening method is based on novel genotyping primers, the loxPsym tags, which indicate not only deletions but also inversions and translocations. Long-read Nanopore sequencing is used to decode the selected genotypes and shows in conjunction with flow cytometry that large-scale karyotype alterations can be a consequence of SCRaMbLE.

synthetic biology↗