(665b) Transcriptional Control through Synthetic Genetic Regulation Devices in Clostridium sensu Stricto

Predictable control of transcription within bacterial systems is essential to achieve maximum production yields and titers by reducing carbon flux through undesirable pathways. To achieve such control, genetic parts must be developed that can allow user-defined control. Although much recent progress has been made in transcription factor-based biosensors for workhorse organisms, non-model organisms such as Clostridium still lack tools for rapid and customizable gene expression control¹. Clostridium species are promising microorganisms for the production of advanced biofuels (e.g., butanol) and platform chemicals, yet a lack of efficiency reduces their economic viability. While effort has been dedicated to studying and engineering Clostridium species, progress has been slow compared to the common workhorse organisms, due to the dearth of synthetic biology tools. Additionally, low throughput screening methods such as liquid chromatography are a bottleneck in evaluating more than a handful of strain designs at once, limiting the ‘test’ step in the design-build-test cycle.

In this work, we have developed two systems for transcriptional control in Clostridium sensu stricto. First, we have developed a regulatable CRISPRi gene repression for the fine-tuning of biosynthetic pathways using multiple Cas12a effector proteins, which are better suited for use in Clostridium due to the genus’ AT-rich genomes. We demonstrate tunable repression based on proximity to regulation elements, strand, and number of targeted sites through reporter genes, transcription levels, and metabolic alteration. We propose a set of heuristics for such control. In addition, we demonstrate a set of engineered product-responsive transcription factor regulated promoters (biosensors) for real-time single cell monitoring of butanol accumulation. We demonstrate tunability of gene expression though altering the number, location, and sequence of the transcription factor binding sites. Such TF biosensors can be used for isolation of hyperproducing mutants and rewiring metabolism during fermentation production.

[1] Joseph, R. C., Kim, N. M., and Sandoval, N. R. (2018) Recent Developments of the Synthetic Biology Toolkit for Clostridium, Front. Microbiol. 9.