Castano-Cerezo, S. | bioRxiv Science

bioRxiv2022

Optogenetic control of beta-carotene bioproduction in yeast across multiple lab-scales

Optogenetics arises as a valuable tool to precisely control genetic circuits in microbial cell factories. Light control holds the promise of optimizing bioproduction methods and maximize yields, but its implementation at different steps of the strain development process and at different culture scales remains challenging. In this study, we aim to control beta-carotene bioproduction using optogenetics in Saccharomyces cerevisiae and investigate how its performance translates across culture scales. We built four lab-scale illumination devices, each handling different culture volumes, and each having specific illumination characteristics and cultivating conditions. We evaluated optogenetic activation and beta-carotene production across devices and optimized them both independently. Then, we combined optogenetic induction and beta-carotene production to make a light-inducible beta-carotene producer strain. This was achieved by placing the transcription of the bifunctional lycopene cyclase / phytoene synthase CrtYB under the control of the pC120 optogenetic promoter regulated by the EL222-VP16 light-activated transcription factor, while other carotenogenic enzymes (CrtI, CrtE, tHMG) were expressed constitutively. We show that illumination, culture volume and shaking impact differently optogenetic activation and beta-carotene production across devices. This enabled us to determine the best culture conditions to maximize light-induced beta-carotene production in each of the devices, reaching a content of up to 880 g/gCDW. Our study exemplifies the stakes of scaling up optogenetics in devices of different lab scales and sheds light on the interplays and potential conflicts between optogenetic control and metabolic pathway efficiency. As a general principle, we propose that it is important to first optimize both components of the system independently, before combining them into optogenetic producing strains to avoid extensive troubleshooting. We anticipate that our results can help designing both strains and devices that could eventually lead to larger scale systems in an effort to bring optogenetics to the industrial scale.

synthetic biology↗

bioRxiv2022

Inferring assembly-curving trends of bacterial micro-compartment shell hexamers from crystal structure arrangements

Bacterial microcompartments (BMC) are complex macromolecular assemblies that participate to varied chemical processes in about one fourth of bacterial species. BMC-encapsulated enzymatic activities are segregated from other cell contents by means of semipermeable shells, justifying why BMC are viewed as prototype nano-reactors for biotechnological applications. Herein, we undertook a comparative study of trends of self-assembly of BMC hexamers (BMC-H), the most abundant shell constituents. Published and new microscopy data show that some BMC-H, like {beta}-carboxysomal CcmK, tend to assemble flat whereas other BMC-H often build curved-implying objects. Inspection of available crystal structures presenting BMC-H in tiled arrangements permitted to identify two major assembly modes with a striking connection with experimental trends. All-atom molecular dynamics (MD) supported that BMC-H bending is triggered robustly only from the disposition adopted by BMC-H that form curved objects experimentally, conducting to almost identical arrangements to those found in structures of recomposed BMC shells. Simulations on ensembles of planar-behaving hexamers, which were previously reconfigured to comply with such disposition, confirmed that bending is defined by assembly details, rather than by BMC-H identity. Finally, although no common atomic determinants could be identified as responsible of BMC-H spontaneous curvature, an inter-hexamer ionic pair was pinpointed as contributor to hold a subset of BMC-H in low bending dispositions. These results are expected to improve our understanding of the variable mechanisms of biogenesis characterized for BMC, and of possible strategies to regulate BMC size and shape.

biochemistry↗