Biology subjects

Köpke, M.

Publications and source records attributed to Köpke, M..

7 recordsLinked to original sources

bioRxiv2023

Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum

Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C1) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO2) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in the C1-fixing gene cluster and its closely-linked genes is incomplete. Here, we investigate the role of two genes with unclear functions - hypothetical protein (hp; LABRINI_07945) and CooT nickel binding protein (nbp; LABRINI_07950) - directly adjacent and expressed at similar levels to the C1-fixing gene cluster in the gas-fermenting model-acetogen Clostridium autoethanogenum. Targeted deletion of either the hp or nbp gene using CRISPR/nCas9, and phenotypic characterisation in heterotrophic and autotrophic batch and autotrophic bioreactor continuous cultures revealed significant growth defects and altered by-product profiles for both {Delta}hp and {Delta}nbp strains. Variable effects of gene deletion on autotrophic batch growth on rich or minimal media suggest that both genes affect the utilisation of complex nutrients. Autotrophic chemostat cultures showed lower acetate and ethanol production rates and higher carbon flux to CO2 and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of the C1-fixing gene cluster and ethanol synthesis pathways. Our work contributes to a better understanding of genotype-phenotype relationships in acetogens and offers engineering targets to improve carbon fixation efficiency in gas fermentation.

synthetic biology↗

bioRxiv2023

Autotrophic adaptive laboratory evolution of the acetogen Clostridium autoethanogenum delivers the gas-fermenting strain LAbrini with superior growth, products, and robustness

Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has already been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for routine bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on a minimal medium with one strain, named "LAbrini" (LT1), exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of sporulation-related mutations in genes CLAU_3129 (spo0A) and CLAU_1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism, which we named "LAbrini".

bioengineering↗

bioRxiv2022

RNA-seq sample preparation kits strongly affect transcriptome profiles of a gas-fermenting bacterium

Transcriptome analysis via RNA sequencing (RNA-seq) has become a standard technique employed across various biological fields of study. This rapid adoption of the RNA-seq approach has been mediated, in part, by the development of different commercial RNA-seq library preparation kits compatible with standard next-generation sequencing (NGS) platforms. Generally, the essential steps of library preparation such as ribosomal RNA (rRNA) depletion and first-strand cDNA synthesis are tailored to a specific group of organisms (e.g. eukaryotes vs. prokaryotes) or genomic GC content. Therefore, the selection of appropriate commercial products is of crucial importance to capture the transcriptome of interest as closely to the native state as possible without introduction of technical bias. However, researchers rarely have the resources and time to test various commercial RNA-seq kits for their samples. This work reports a side-by-side comparison of RNA-seq data from Clostridium autoethanogenum obtained using three commercial rRNA removal and strand-specific library construction products by NuGEN Technologies, Qiagen, and Zymo Research and assesses their performance relative to published data. While all three vendors advertise their products as suitable for prokaryotes, we found significant differences in their performance regarding rRNA removal, strand-specificity, and, most importantly, transcript abundance distribution profiles. Notably, RNA-seq data obtained with Qiagen products were most similar to published data and delivered the best results in terms of library strandedness and transcript abundance distribution range. Our results highlight the importance of finding appropriate organism-specific workflows and library preparation products for RNA-seq studies. ImportanceRNA-seq is a powerful technique for transcriptome profiling while involving elaborate sample processing before library sequencing. Our work is important as we show that RNA-seq library preparation kits can strongly affect the outcome of the RNA-seq experiment. Although library preparation benefits from the availability of various commercial kits, choosing appropriate products for the specific samples can be challenging for new users or for users working with unconventional organisms. Evaluating the performance of different commercial products requires significant financial and time investment infeasible to most researchers. Therefore, users are often guided in their choice of kits by published data involving similar input samples. We conclude that important consideration should be given to selecting of sample processing workflows for any given organism.

systems biology↗

bioRxiv2021

Spacer2PAM: A computational framework for identification of functional PAM sequences for endogenous CRISPR systems

RNA-guided nucleases from clustered regularly interspaced short palindromic repeats (CRISPR) systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR systems in many bacteria and archaea are particularly attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR effectors. However, each CRISPR system recognizes a unique set of PAM sequences, which requires extensive screening of randomized DNA libraries. This challenge makes it difficult to develop endogenous CRISPR systems, especially in organisms that are slow-growing or have transformation idiosyncrasies. To address this limitation, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to identify potential PAM sequences for any CRISPR system given its corresponding CRISPR array as input. Spacer2PAM can be used in "Quick" mode to generate a single PAM prediction that is likely to be functional or in "Comprehensive" mode to inform targeted, unpooled PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-predicted PAM library for the type I-B CRISPR system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR systems for industrial biotechnology and synthetic biology.

synthetic biology↗

bioRxiv2021

Application of transposon-insertion sequencing to determine gene essentiality in the acetogen Clostridium autoethanogenum

The majority of the genes present in bacterial genomes remain poorly characterised with up to one third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to easily transformable species leaving those with low DNA-transfer rates out of reach. Here we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum using a mariner-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal codA marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into C. autoethanogenum were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as a sole carbon and energy source. IMPORTANCEAlthough microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterised, leaving the function of many as either hypothetical or entirely unknown. High-throughput, transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA-transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here we developed approaches to facilitate transposon-insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases, CO and CO2, into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth providing insights into the utilisation of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon-insertion sequencing to other microbes where DNA-transfer has until now represented a barrier to progress.

molecular biology↗

bioRxiv2020

Modular cell-free expression plasmids to accelerate biological design in cells

Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms--organisms that are easy to use like Escherichia coli--often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have the desired metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has emerged as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institutes DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for C. autoethanogenum expression with efficiencies ranging from 68-90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

synthetic biology↗

bioRxiv2019

In vitro prototyping and rapid optimization of biosynthetic enzymes for cellular design

Microbial cell factories offer an attractive approach for production of biobased products. Unfortunately, designing, building, and optimizing biosynthetic pathways remains a complex challenge, especially for industrially-relevant, non-model organisms. To address this challenge, we describe a platform for in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (iPROBE). In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE with two examples. First, we tested and ranked 54 different pathways for 3-hydroxybutyrate production, improving in vivo production in Clostridium by 20-fold to 14.63 {+/-} 0.48 g/L and identifying a new biosynthetic route to (S)-(+)-1,3-butanediol. Second, we used iPROBE and data-driven design to optimize a 6-step n-butanol pathway, increasing titers 4-fold across 205 pathways, and showed strong correlation between cell-free and cellular performance. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

synthetic biology↗