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bioRxiv · 10.1101/2025.10.31.685836

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

Kostanjsek, M.·Raynal, A.·Dimopoulos, G.·Behrendt, G.·Martins dos Santos, V. A. P.·Beekwilder, J.·Batianis, C.·Weusthuis, R. A.·Asin-Garcia, E.·Bisschops, M. M. M.
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Abstract

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

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Kostanjsek, M., Raynal, A., Dimopoulos, G., Behrendt, G., Martins dos Santos, V. A. P., Beekwilder, J., Batianis, C., Weusthuis, R. A., Asin-Garcia, E., Bisschops, M. M. M.. 2025-11-01. Rhodo-Box: a Synthetic Biology Toolbox to Facilitate Metabolic Engineering of Rhodobacter sphaeroides. https://doi.org/10.1101/2025.10.31.685836

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