Probing efficient microbial CO2 utilisation through metabolic and process modelling.

Acetogenic gas fermentation is increasingly studied as a promising technology to upcycle carbon-rich waste gasses. Currently the product range is limited, and production yields, rates and titres for a number of interesting products do not allow for economically viable processes. By pairing process modelling and host-agnostic metabolic modelling, we compare fermentation conditions and various products to optimise the processes. The models were then used in a simulation of an industrial-scale bubble column reactor. We find that increased temperatures favour gas transfer rates, particularly for the valuable and limiting H2 , while furthermore predicting an optimal feed composition of 9:1 mol H2 to mol CO2 . Metabolically, the increased non-growth associated maintenance requirements of thermophiles favours the formation of catabolic products. To assess the expansion of the product portfolio beyond acetate, both a product volatility analysis and a metabolic pathway model were implemented. In-situ recovery of volatile products is shown to be within range for acetone but challenging due to the extensive evaporation of water, while the direct production of more valuable compounds by acetogens is metabolically unfavourable compared to acetate and ethanol. We discuss alternative approaches to overcome these challenges to utilise acetogenic CO2 fixation to produce a wider range of carbon negative chemicals.

High-throughput colorimetric assays optimized for detection of ketones and aldehydes produced by microbial cell factories.

Randomized strain and pathway engineering are critical to improving microbial cell factory performance, calling for the development of high-throughput screening and selection systems. To facilitate this effort, we have developed two 96-well plate format colorimetric assays for reliable quantification of various ketones and aldehydes from culture supernatants, based on either a vanillin-acetone reaction or the 2,4-dinitrophenylhydrazine (2,4-DNPH) reagent. The vanillin-acetone assay enabled accurate and selective measurement of acetone titers up to 2 g l-1 in a minimal culture medium. The 2,4-DNPH-based assay can be used for a wide range of aldehydes and ketones, shown here through the optimization of conditions for 15 different compounds. Both assays were implemented to improve acetone production from different substrates by an engineered Escherichia coli strain. The fast and user-friendly colorimetric assays proposed here open the potential for iterative rounds of (automated) strain and pathway engineering and screening, facilitating the efforts towards further boosting production titers of industrially relevant ketones and aldehydes.

The ProUSER2.0 Toolbox: Genetic Parts and Highly Customizable Plasmids for Synthetic Biology in Bacillus subtilis.

Versatile DNA assembly standards and compatible, well-characterized part libraries are essential tools for creating effective designs in synthetic biology. However, to date, vector standards for Gram-positive hosts have limited flexibility. As a result, users often revert to PCR-based methods for building the desired genetic constructs. These methods are inherently prone to introducing mutations, which is problematic considering vector backbone parts are often left unsequenced in cloning workflows. To circumvent this, we present the ProUSER2.0 toolbox: a standardized vector platform for building both integrative and replicative shuttle vectors forBacillus subtilis. The ProUSER2.0 vectors consist of a ProUSER cassette for easy and efficient insertion of cargo sequences and six exchangeable modules. Furthermore, the standard is semicompatible with several previously developed standards, allowing the user to utilize the parts developed for these. To provide parts for the toolbox, seven novel integration sites and six promoters were thoroughly characterized in B. subtilis. Finally, the capacity of the ProUSER2.0 system was demonstrated through the construction of signal peptide libraries for two industrially relevant proteins. Altogether, the ProUSER2.0 toolbox is a powerful and flexible framework for use in B. subtilis.

Genome-scale metabolic modeling of P. thermoglucosidasius NCIMB 11955 reveals metabolic bottlenecks in anaerobic metabolism.

Parageobacillus thermoglucosidasius represents a thermophilic, facultative anaerobic bacterial chassis, with several desirable traits for metabolic engineering and industrial production. To further optimize strain productivity, a systems level understanding of its metabolism is needed, which can be facilitated by a genome-scale metabolic model. Here, we present p-thermo, the most complete, curated and validated genome-scale model (to date) of Parageobacillus thermoglucosidasius NCIMB 11955. It spans a total of 890 metabolites, 1175 reactions and 917 metabolic genes, forming an extensive knowledge base for P. thermoglucosidasius NCIMB 11955 metabolism. The model accurately predicts aerobic utilization of 22 carbon sources, and the predictive quality of internal fluxes was validated with previously published 13C-fluxomics data. In an application case, p-thermo was used to facilitate more in-depth analysis of reported metabolic engineering efforts, giving additional insight into fermentative metabolism. Finally, p-thermo was used to resolve a previously uncharacterised bottleneck in anaerobic metabolism, by identifying the minimal required supplemented nutrients (thiamin, biotin and iron(III)) needed to sustain anaerobic growth. This highlights the usefulness of p-thermo for guiding the generation of experimental hypotheses and for facilitating data-driven metabolic engineering, expanding the use of P. thermoglucosidasius as a high yield production platform.

Physical decoupling of XylS/Pm regulatory elements and conditional proteolysis enable precise control of gene expression in Pseudomonas putida.

Most of the gene expression systems available for Gram-negative bacteria are afflicted by relatively high levels of basal (i.e. leaky) expression of the target gene(s). This occurrence affects the system dynamics, ultimately reducing the output and productivity of engineered pathways and synthetic circuits. In order to circumvent this problem, we have designed a novel expression system based on the well-known XylS/Pm transcriptional regulator/promoter pair from the soil bacterium Pseudomonas putida mt-2, in which the key functional elements are physically decoupled. By integrating the xylS gene into the chromosome of the platform strain KT2440, while placing the Pm promoter into a set of standard plasmid vectors, the inducibility of the system (i.e. the output difference between the induced and uninduced state) improved up to 170-fold. We further combined this modular system with an extra layer of post-translational control by means of conditional proteolysis. In this setup, the target gene is tagged with a synthetic motif dictating protein degradation. When the system features were characterized using the monomeric superfolder GFP as a model protein, the basal levels of fluorescence were brought down to zero (i.e. below the limit of detection). In all, these novel expression systems constitute an alternative tool to altogether suppress leaky gene expression, and they can be easily adapted to other vector formats and plugged-in into different Gram-negative bacterial species at the user's will.