Functional production of transporters from biomass-degrading anaerobic fungi for metabolic engineering

Membrane-embedded transporters and receptors are increasingly becoming targets for the metabolic engineering community that aims to enhance the performance and stability of microbial production strains. Anaerobic gut fungi inhabit the digestive tract of herbivores such as cows and sheep, and excel at degrading raw plant biomass into fermentable sugars. Recently, a transcriptomic analysis of three strains of gut fungi suggested that they display a plethora of carbohydrate binding proteins on their surface, including G-protein coupled receptors with a novel architecture; and possess a multitude of small-solute transporters that are of chief biotechnological interest: transporters for sugars, amino acids, lipids, drugs, and metals. Here, we introduced genes encoding gut fungal fluoride transporters into Saccharomyces cerevisiae, and show that with codon optimization, the yeast produce large quantities of functional and correctly membrane-localized transporters capable of bolstering solvent tolerance. We are currently expanding our approach to putative drug- and sugar-transporters and receptors sourced from the anaerobic fungi. These results in part explain the physiology of these understudied fungi, and highlight the critical role that their membrane proteins play towards their existence in competitive, extreme environments. Notably, the work expands on the toolbox of receptor and transporter proteins that can be used to enhance the performance and stability of model microbial cell factory strains. Please click Additional Files below to see the full abstract

A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity

Background: Bacterial surface display is an attractive technique for the production of cell-anchored, functional proteins and engineering of whole-cell catalysts. Although various outer membrane proteins have been used for surface display, an easy and versatile high-throughput-compatible assay for evaluating and developing surface display systems is missing.Results: Using a single domain antibody (also called nanobody) with high affinity for green fluorescent protein (GFP), we constructed a system that allows for fast, fluorescence-based detection of displayed proteins. The outer membrane hybrid protein LppOmpA and the autotransporter C-IgAP exposed the nanobody on the surface of Escherichia coli with very different efficiency. Both anchors were capable of functionally displaying the enzyme Chitinase A as a fusion with the nanobody, and this considerably increased expression levels compared to displaying the nanobody alone. We used flow cytometry to analyse display capability on single-cell versus population level and found that the signal peptide of the anchor has great effect on display efficiency.Conclusions: We have developed an inexpensive and easy read-out assay for surface display using nanobody:GFP interactions. The assay is compatible with the most common fluorescence detection methods, including multi-well plate whole-cell fluorescence detection, SDS-PAGE in-gel fluorescence, microscopy and flow cytometry. We anticipate that the platform will facilitate future in-depth studies on the mechanism of protein transport to the surface of living cells, as well as the optimisation of applications in industrial biotech

Accurate DNA assembly and genome engineering with optimized uracil excision cloning

Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of <i>gfp</i> and a four-gene pathway that produces β-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly facilitating the advanced engineering of robust cell factories

An expression tag toolbox for microbial production of membrane bound plant cytochromes P450

Membrane-associated Cytochromes P450 (P450s) are one of the most important enzyme families for biosynthesis of plant-derived medicinal compounds. However, the hydrophobic nature of P450s makes their use in robust cell factories a challenge. Here we explore a small library of N-terminal expression tag chimeras of the model plant P450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs. Analysis of long-term cultures by flow cytometry showed homogeneous populations for some of the conditions. Three chimeric designs were chosen for a more complex combinatorial assembly of a multigene pathway consisting of two P450s and a redox partner. Cells expressing these recombinant enzymes catalysed the conversion of the substrate to highly different ratios of the intermediate and the final product of the pathway. Finally, the effect of a robustly performing expression tag was explored with a library of 49 different P450s from medicinal plants and nearly half of these were improved in expression by more than 2-fold. The developed toolbox serves as platform to tune P450 performance in microbial cells, thereby facilitating recombinant production of complex plant P450-derived biochemicals

Effectiveness of a workplace intervention to reduce workplace bullying and violence at work : study protocol for a two-wave quasi-experimental intervention study

Introduction Bullying and violence at work are relatively common in Finnish public sector workplaces. Previous research has demonstrated their association with increased risk of poor health and well-being, but only few intervention studies exist. The aim of this protocol paper is to describe the development and assessment of the effectiveness of a workplace intervention aimed at reducing these harmful phenomena. Methods and analysis This protocol describes a two-wave quasi-experimental intervention. Each of the three participating Finnish public sector organisations (cities) will select four work units (a total of 450-500 employees) to participate in an intervention including 2-3 workshops for the work unit, 2-3 consultative meetings with the supervisor of the work unit, a follow-up meeting for the entire work unit (a maximum of 6-month time lag) and online meetings with the supervisor to monitor achievements and discuss about difficult cases, if any. Three age-matched, sex-matched and occupation-matched controls for each participants of the intervention group will be randomly selected, a total 1350-1500 individuals in the control group. For intervention and control groups, premeasurement is based on responses to a survey that was conducted in 2020. Postintervention measurement will be based on survey responses in 2022. Data will be analysed using latent change score modelling or difference-in-difference analysis. Ethics and dissemination Ethics approvals are from the Ethics committees of the Helsinki and Uusimaa hospital district and the Finnish institute of Occupational Health. Results will be made available to participating organisations and their employees, the funder and other researchers via open access article in a peer-reviewed journal and subsequent reporting of the results via social media channels and press release to the public.Peer reviewe

Genome-Wide Meta-Analysis of Sciatica in Finnish Population

Sciatica or the sciatic syndrome is a common and often disabling low back disorder in the working-age population. It has a relatively high heritability but poorly understood molecular mechanisms. The Finnish population is a genetic isolate where small founder population and bottleneck events have led to enrichment of certain rare and low frequency variants. We performed here the first genome-wide association (GWAS) and meta-analysis of sciatica. The meta-analysis was conducted across two GWAS covering 291 Finnish sciatica cases and 3671 controls genotyped and imputed at 7.7 million autosomal variants. The most promising loci (pPeer reviewe

Co‑cultivation of the anaerobic fungus Caecomyces churrovis with Methanobacterium bryantii enhances transcription of carbohydrate binding modules, dockerins, and pyruvate formate lyases on specific substrates

International audienceAbstract Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungus Caecomyces churrovis and the methanogen Methanobacterium bryantii (not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated in C. churrovis across a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome of C. churrovis was obtained and annotated, which is the first sequenced genome of a non-rhizoid-forming anaerobic fungus. C. churrovis possess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative to C. churrovis monoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2-fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of the C. churrovis strain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal–methanogen physical associations and fungal cell wall development and remodeling