Rhodo-Box: A Synthetic Biology Toolbox to Facilitate Metabolic Engineering of <i>Rhodobacter sphaeroides</i>

<i>Rhodobacter sphaeroides</i> is a purple nonsulfur 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 <i>R. sphaeroides</i> 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-P_{<i>salTTC</i>}, LacI-P_{<i>lacT7A1_O3O4</i>}, VanR-P_<i>vanCC</i>, and XylS-P_m), (d) 11 RBSs, and (e) four transcriptional terminators. Furthermore, we present a semiautomated, user-friendly cloning approach which enables rapid construction of <i>R. sphaeroides</i> strains. The Rhodo-Box toolkit equips <i>R. sphaeroides</i> with a standardized, automation-compatible collection of parts and workflows essential for efficient design-build-test-learn cycles and advanced metabolic engineering.