Biology subjects

Weusthuis, R. A.

Publications and source records attributed to Weusthuis, R. A..

3 recordsLinked to original sources

bioRxiv2026

A rapid HPLC-based method to determine NAD(P)(H) and NMN redox cofactor concentrations and ratios in microbes

Redox cofactors are a key part of cellular physiology as they are involved in most metabolic pathways, and their ratios are linked to cellular robustness. However, measuring their levels in cells remains a challenge. Here, we describe a novel method to rapidly measure NAD(H), NADP(H) and nicotinamide mononucleotide (NMN) levels and their oxidized/reduced ratios using an HPLC connected to a fluorescence detector. By extensively characterizing this method and benchmarking it against the classical iodonitrotetrazolium (INT) assay, we show that this method results in accurate and reproducible measurements of NAD+, NADP+ and NMN levels in bacteria. We further demonstrate that this method can be used to determine intracellular NADH and NADPH concentrations and ratios of nicotinamide nucleotide cofactors in engineered Escherichia coli strains, as well as other bacterial species such as Pseudomonas putida.

biochemistry↗

bioRxiv2025

Rhodo-Box: a Synthetic Biology Toolbox to Facilitate Metabolic Engineering of Rhodobacter sphaeroides

Rhodobacter sphaeroides is a purple non-sulphur alphaproteobacterium with a highly versatile metabolism. This microorganism holds promise as a chassis for sustainable biomanufacturing of numerous chemicals. Yet, its potential is constrained by a lack of standardized, well-characterized genetic elements to tune gene expression such as transcriptional promoters and ribosome binding sites (RBSs). In this study, we present Rhodo-Box, a comprehensive toolkit for R. sphaeroides created by adapting and extending the Zymo-Parts modular cloning framework. Using Rhodo-Box we built and characterized: (a) three broad-host origins of replication (pBBR1, RK2 and RSF1010), (b) a set of 13 promoters, (c) four inducible expression systems (NahR-PsalTTC, LacI-PlacT7A1_O3O4, VanR-PvanCC, and XylS-Pm), (d) 11 RBSs, and (e) four transcriptional terminators. Furthermore, we present a semi-automated, user-friendly cloning approach which enables rapid construction of R. sphaeroides strains. The Rhodo-Box toolkit equips R. sphaeroides with a standardized, automation-compatible collection of parts and workflows essential for efficient design-build-test-learn cycles and advanced metabolic engineering. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/685836v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@fcb172org.highwire.dtl.DTLVardef@1e5eb44org.highwire.dtl.DTLVardef@1b8df28org.highwire.dtl.DTLVardef@42eb2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a

Clostridium species are re-emerging as biotechnological workhorses for industrial acetone-butanol-ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of several CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the strict xylose-inducible promoter, we achieved efficient (25-100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (Spo0A, Upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the Spo0A and Upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome.

microbiology↗