Unlocking the bacterial domain for industrial biotechnology applications using orthogonal parts and tools

Industrial biotechnology is limited by the availability of standardized genetic parts and tools to control gene expression in a wide range of host organisms. As such, predictably engineering species from all over the bacterial domain is a major bottleneck restricting industrial biotechnology to the use of well-known host organisms such as E. coli and S. cerevisiae. These hosts require extensive engineering as they are often suboptimal for certain processes, which can ultimately result in increased development times and costs. These issues can however largely be resolved by wisely selecting the preferred production host, using the enormous heterogeneity of the bacterial domain. Therefore, orthogonal and standardized parts must be developed, allowing genetic engineering of better suited host organisms such as native producers, hosts with superior precursor turnover and extremophiles. Hence, a Cross-Bacterial Expression System (CBES) consisting of an orthogonal sigma factor and its corresponding orthogonal promoter sequences is developed to allow transcription initiation across the bacterial domain. To ensure reliable and robust gene expression, the system is balanced using host-independent genetic circuitry consisting either of an orthogonal anti-sigma factor, orthogonal antisense-transcription or small RNAs. This system will thus allow orthogonal transcription initiation based on well-characterized and balanced interactions and therefore contribute to the predictable and reliable engineering of up to now unexplored bacterial species.