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