Functional predictability of universal gene circuits in diverse microbial hosts

Although the principles of synthetic biology were initially established in model bacteria,microbial producers,extremophiles and gut microbes have now emerged as valuable prokaryotic chassis for biological engineering.Extending the host range in which designed circuits can function reliably and predictably presents a major challenge for the concept of synthetic biology to materialize.In this work,we systematically characterized the cross-species universality of two transcriptional regulatory modules—the T7 RNA polymerase activator module and the repressors module—in three non-model microbes.We found striking linear relationships in circuit activities among different organisms for both modules.Parametrized model fitting revealed host non-specific parameters defining the universality of both modules.Lastly,a genetic NOT gate and a band-pass filter circuit were constructed from these modules and tested in non-model organisms.Combined models employing host non-specific parameters were successful in quantitatively predicting circuit behaviors,underscoring the potential of universal biological parts and predictive modeling in synthetic bioengineering.

Novel switchable ECF sigma factor transcription system for improving thaxtomin A production in Streptomyces

The application of the valuable natural product thaxtomin A,a potent bioherbicide from the potato scab pathogenic Streptomyces strains,has been greatly hindered by the low yields from its native producers.Here,we developed an orthogonal transcription system,leveraging extra-cytoplasmic function(ECF)sigma(σ)factor 17(ECF17)and its cognate promoter Pecf17,to express the thaxtomin gene cluster and improve the production of thaxtomin A.The minimal Pecf17 promoter was determined,and a Pecf17 promoter library with a wide range of strengths was constructed.Furthermore,a cumate inducible system was developed for precise temporal control of the ECF17 transcription system in S.venezuelae ISP5230.Theoretically,the switchable ECF17 transcription system could reduce the unwanted influences from host and alleviate the burdens introduced by overexpression of heterologous genes.The yield of thaxtomin A was significantly improved to 202.1±15.3μg/mL using the switchable ECF17 transcription system for heterologous expression of the thaxtomin gene cluster in S.venezuelae ISP5230.Besides,the applicability of this transcription system was also tested in Streptomyces albus J1074,and the titer of thaxtomin A was raised to as high as 239.3±30.6μg/mL.Therefore,the inducible ECF17 transcription system could serve as a complement of the generally used transcription systems based on strong native constitutive promoters and housekeepingσfactors for the heterologous expression of valuable products in diverse Streptomyces hosts.