7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Prof. Dr.

Chair

Novel CRISPR/Cas system for highly efficient genome editing in A. niger

Biotechnology & Synthetic Microbiology

The filamentous fungus Aspergillus niger (A. niger) is known for its high capacity for protein secretion. CRISPR/Cas-based systems are currently being used to generate high-yielding A. niger production strains. Given the difficult patent situation for the CRISPR nucleases known and used so far, limiting commercial use, we identified new Cas nucleases for genome editing based on metagenomic DNA analyses from environmental samples. The generated dataset allowed us to identify several novel nucleases, so-called G-dase M, with little or no homology to Cas proteins from public databases. To obtain a proprietary CRISPR tool for strain development of A. niger, a previously characterized G-dase M nuclease was established for genome editing.We have successfully shown that the selected G-dase M nuclease can be programmed to efficiently and precisely introduce DNA double-strand breaks into the A. niger genome. An AMA1-based plasmid was adapted for optimized G-dase M expression and tRNA-gRNA transcription in A. niger. The functionality of the novel CRISPR/G-dase M single plasmid system was evaluated by clone reduction assays for different chromosomal genes in the NHEJ-deficient A. niger host strain. Furthermore, CRISPR/G-dase M activity induced HDR-mediated gene-specific integration of a reporter gene into various chromosomal targets with an efficiency of up to 99 %. In addition, we have developed a system to reduce the metabolic burden of plasmid-based overexpression of the nuclease by uncoupling the expression of G-dase M and tRNA-gRNA. For this purpose, the G‑dase M expression cassette was integrated into the genome of A. niger, while the regulatory elements for tRNA-gRNA transcription were provided on the AMA1-based plasmid. This strategy effectively reduced the metabolic burden caused by overexpression of the episomal expressed nuclease.In summary, we have developed a highly efficient CRISPR system for genome editing in A. niger using the novel class 2 type V G-dase M nuclease. These results have shown the functionality of our proprietary CRISPR nuclease G-dase M in A. niger and will thus expand the molecular toolbox for genetic modification of filamentous fungi.

Oral Presentation

A global search for high-susceptibility targets of programmable RNA antibiotics

The growing emergence of multidrug-resistant bacterial pathogens urgently demands the development of alternative antibiotics. Antisense oligomers (ASOs), such as based on peptide nucleic acid (PNA), can exert potent bactericidal effects when designed to sequester the ribosome binding site of an mRNA of an essential gene. Over the years, various essential genes have been investigated for their vulnerability to PNA targeting in enteric bacteria, with the acpP gene encoding acyl carrier protein being considered the most susceptible PNA target. However, it remains unknown what makes for a potent PNA or PNA target. We present a systematic investigation of the efficacy of antisense PNAs for 293 selected essential genes, tested in both E. coli K12 and uropathogenic E. coli (UPEC). Our screen has produced 23 and 55 potent antibacterial PNAs, corresponding to 21 and 47 essential target genes, in K12 and UPEC, respectively. In addition, we discover both strain-independent and strain-specific antibacterial PNA activities. Bioinformatic analysis will enable us to decipher common traits of the identified most efficient PNA sequences and determine core features of target susceptibility. Further, characterization and testing of UPEC-specific PNAs on a broader panel of Enterobacteriaceae may help to generate species-specific antibacterials. Overall, this study gives new insights into sequence- and target gene-dependent differences in PNA susceptibility and provides information on putative universal predictors of PNA efficacy.

Harnessing synthetic biology tools for chemical production in Lithoautotrophic Biorefineries

Introduction: Cupriavidus necator efficiently sequesters CO2, utilizing H2 to recycle cofactors through its native hydrogenases1. Advancing synthetic biology tools is crucial to redirect carbon to valuable products like N-heterocycles. Since C. necator is an obligate aerobe, autotrophic cultivation usually entails hazardous pressurized knallgas.Goals: We developed synthetic biology tools and safe electro-fermentation methods for C. necator, enabling electricity-driven biotransformations and pioneering the development of novel pathways for N-heterocycle synthesis2,3.Methods: Our engineered C. necator features two genomic integrations: 1) rhamnose-inducible dCas9 for gene repression; 2) arabinose-inducible T7 RNA polymerase-cytidine deaminase fusion (MT7) for gene expression and pathway hypermutation. A newly developed reactor incorporating a biocompatible electrolyzer regenerates hydrogen in-situ4, with integrated sensors for precise monitoring of dissolved- and headspace gasses.Results: Genomic integration of inducible MT7 and dCas9 systems regulated gene expression and enabled pathway evolution. These tools were applied to accelerate the development of new-to-nature pathways for N-heterocycle biosynthesis. Furthermore, an electro-fermentation reactor ensured safe and efficient synthesis of isopropanol, using a previously engineered strain5, as a proof-of-concept.Conclusions: Genomic integration of molecular tools significantly enhanced future C. necator strain development. Additionally, the developed electro-fermentation reactor was successfully implemented for isopropanol production. The utility of this reactor extends to newly developed strains for biosynthesis of N-heterocycles.References:Al-Shameri A., Siebert D. L., Sutiono S., Lauterbach L. &amp; Sieber V. Nat. Commun. 14, (2023).2. Al-Shameri A., Petrich M-C M., Puring K.j., Apfel U.-P., Nestl B.M., Lauterbach L., Chemie - Int. Ed. 59, 10929–10933 (2020).3. Al-Shameri A., Borlinghaus N., Schneller P., Nestl B.M., Lauterbach L., Green Chem. 21, 1396–1400 (2019).4. Rad, R. et al. Cell Reports Phys. Sci. 4, 101526 (2023)Grousseau E. et al. Appl. Microbiol. Biotechnol. 98, 4277–4290 (2014).

Beyond rational : Independent biosensor-guided evolution of 100 strain variants and comparative analysis of their genomes

In contrast to rational metabolic engineering, classical strain development strongly relies on random mutagenesis and screening for the desired production phenotype. Nowadays, with the availability of biosensor-based FACS-screening strategies, these random approaches are coming back into fashion.Here, we employ this technology in combination with comparative genome analyses to identify novel mutations contributing to product formation in the genome of a Corynebacterium glutamicum L‑histidine producer. Since all known genetic targets contributing to L‑histidine production have been already rationally engineered in this strain, identification of novel beneficial mutations can be regarded as challenging, as they might not be intuitively linkable to L‑histidine biosynthesis. For the identification of 100 improved strain variants, we performed &gt; 600 chemical mutagenesis, &gt; 200 biosensor-based FACS-screenings, isolated &gt; 50,000 variants with increased fluorescence, and characterized &gt; 4,500 variants with regard to biomass formation and L-histidine production. Based on comparative genome analyses of these 100 variants accumulating 10-80% more L‑histidine, we discovered several beneficial mutations. Combination of selected genetic modifications allowed for the construction of a strain variant characterized by a doubled L-histidine titer (29 mM) and product yield (0.13 C-mol C-mol-1) in comparison to the starting variant.This study may serve as a blueprint for the identification of novel beneficial mutations in microbial producers in a more systematic manner. This way, also previously unexplored genes or genes with previously unknown contribution to the respective production phenotype can be identified. We believe that this technology has a great potential to push industrial production strains towards maximum performance.Baumann P.T., Dal Molin M., Aring H., Krumbach K., Müller M.-F., Vroling B., van Summeren-Wesenhagen P.V., Noack N., Marienhagen J. (2023). Beyond rational - biosensor-guided isolation of 100 independently evolved bacterial strain variants and comparative analysis of their genomes. BMC Biology 21:183. (https://doi.org/10.1186/s12915-023-01688-x)

Dr.-Ing

Prof. Dr.-Ing

A step-by-step roadmap involving physiological characterization, genomics, fluxomics, and tailored fermentation to empower Paracoccus pantotrophus DSM 2944 as a metabolically versatile SynBio chassis

Introduction:Paracoccus pantotrophus DSM 2944, a Gram-negative bacterium, was selected for its metabolic robustness and potential as a SynBio (synthetic biology) chassis due to its diverse substrate utilization including cheap and renewable feedstocks, CO2, and other C1 and C2 compounds coupled with the production of biopolymer polyhydroxyalkanoates (PHAs).Goals:The study aims to transform P. pantotrophus DSM 2944 into a proficient SynBio chassis through in-depth physiological, metabolic, computational, and phylogenetic studies coupled with industrially related bioreactor scale-up.Results:To investigate the suitability as a new chassis organism, extensive studies exploring substrate utilization and tolerance, optimal growth temperature, pH values, and phylogenetic studies were performed in P. pantotrophus DSM 2944. Moreover, to elucidate the strain&quot;s metabolic flux in vivo, an in-depth analysis of flux distributions was also using 13C labeled glucose. The flux map showed that P. pantotrophus DSM 2944 prefers the pentose phosphate and Entner-Doudoroff pathways over glycolysis offering a surplus co-factor regeneration coupled with energy generation. Furthermore, to facilitate in-silico simulation of an organism&quot;s growth and metabolism, the genome-scale metabolic model was constructed and validated with experimental data. A genetic toolbox was also designed comprising promoters with tunable strengths, gene integration and deletion strategies, and compatible origins of replication. Highlights include improved growth on the non-native carbon-source terephthalic acid along with native carbon-source ethylene glycol, the two monomers from polyethylene terephthalate (PET). Finally, tailor-made DO-based fermentation strategies were established, showcasing the applicability of the strain in bioreactors regarding PHA production.Conclusions:Through the implementation of the chassis roadmap and comprehensive research, P. pantotrophus DSM 2944 emerges as a promising SynBio chassis. Its metabolic versatility and engineered capabilities highlight its potential in advancing sustainable bioeconomy initiatives, paving the way for a greener future.

Utilizing synthetic yeast chromosomes to map and phenotype large scale-deletions in high-throughput.

Question: The Synthetic Yeast Genome Project (Sc2.0) aims to produce the first synthetic eukaryotic genome. All synthetic chromosomes have been synthesised and characterised in individual strains. The sixteen synthetic chromosomes and the tRNA neochromosome are being consolidated in a single cell.1 All chromosomes have been redesigned according to strict principles. One notable feature is the insertion of symmetrical loxP sites downstream of almost every non-essential gene. Upon activation of Cre recombinase, this leads to highly complex structural variations, turning a single genotype into a population. Here we present our strategy and technology for using synthetic yeast to systematically study the effects of large-scale deletions, complementing our knowledge of single gene deletion libraries.Methods: The loxP sites are 34 nucleotides in length. We use this as a landing pad to randomly integrate a modified marker by homologous recombination (HR). The resulting population is transformed with a plasmid overexpressing a system to introduce a double-strand break in the marker. The DNA is degraded in vivo until HR occurs at loxP sites. Counter selection ensures that only candidates that have lost the marker are obtained. We use laboratory automation to select and characterise large numbers of candidates. Based on our high-throughput, low-cost end-point genotyping, we can rapidly identify the deletion size for each strain.2,3 This is followed by phenotyping of the relevant strains.Results: We have developed and established a strategy for targeted introduction of large-scale deletions into synthetic yeast chromosomes. The large deletions will be characterised using molecular and phenotyping techniques to understand their impact on cell viability. References(1) Richardson S, et al. (2017) Design of a synthetic yeast genome. Science, 355(6329), 1040-1044.(2) Mitchell LA, et al. (2015) qPCRTag Analysis--A high throughput, real time PCR assay for Sc2.0 genotyping. J Vis Exp 25:(99):e52941.(3) Lindeboom TA, Sánchez Olmos MdC, et al. (2022) L-SCRaMbLE creates large-scale genome rearrangements in synthetic Sc2.0 chromosomes. bioRxiv.

Oral Session
Biotechnology & Synthetic Microbiology 3

Appointment

Chair

Topic

Programme

Oral SessionBiotechnology & Synthetic Microbiology 3

Appointment

Chair

Topic

Programme

Oral Session
Biotechnology & Synthetic Microbiology 3