A dynamic and multilocus metabolic regulation strategy using quorum-sensing-controlled bacterial small RNA.

Metabolic regulation strategies have been developed to redirect metabolic fluxes to production pathways. However, it is difficult to screen out target genes that, when repressed, improve yield without affecting cell growth. Here, we report a strategy using a quorum-sensing system to control small RNA transcription, allowing cell-density-dependent repression of target genes. This strategy is shown with convenient operation, dynamic repression, and availability for simultaneous regulation of multiple genes. The parameters Ai, Am, and RA (3-oxohexanoyl-homoserine lactone [AHL] concentrations at which half of the maximum repression and the maximum repression were reached and value of the maximum repression when AHL was added manually, respectively) are defined and introduced to characterize repression curves, and the variant LuxRI58N is identified as the most suitable tuning factor for shake flask culture. Moreover, it is shown that dynamic overexpression of the Hfq chaperone is the key to combinatorial repression without disruptions on cell growth. To show a broad applicability, the production titers of pinene, pentalenene, and psilocybin are improved by 365.3%, 79.5%, and 302.9%, respectively, by applying combinatorial dynamic repression.

Quorum-sensing based small RNA regulation for dynamic and tuneable gene expression.

OBJECTIVES: Developing a dynamic regulation strategy is an essential step in establishing an automatic control system for manipulating metabolic fluxes and cellular behaviors. To broaden the extent of the application, a system that can generally control any gene of interest is demanded. RESULTS: Through characterization and optimization, the strategy repressed the immediate expression incrementally from 0 to 90% during culturing. Moreover, by changing single base pair in the lux box of the Plux promoter, the degree of repression of the target genomic gene was tuned to a difference of 70%. This strategy is expected to control metabolic flux without disrupting cell growth. CONCLUSIONS: We engineered bacterial small RNA to develop a pathway-independent strategy that can dynamically repress the expression of any gene at the posttranscription level.

Improving biosynthetic production of pinene through plasmid recombination elimination and pathway optimization.

Pinene is a monoterpene with wide industrial applications, especially as a promising high energy-density jet fuel. Traditional production of pinene on an industrial scale is material consumptive and has a low yield. As an alternative, microbial organisms have been engineered though advanced synthetic biological techniques to produce a variety of heterologous products, including pinene. Here, we investigated the stability of genetic circuits encoding the pinene producing pathway during fermentation and its relationship to the pinene titer. By replacing scar sequences in the genetic elements and modifying the genome of E. coli strain MG1655, plasmid loss caused by serious metabolic burden was eliminated, generating a remarkable increase in the pinene titer. Furthermore, the heterologous mevalonate pathway was analyzed by overexpression of enzymes and intermediates monitoring. Optimized pathway plasmids and strains were combined to increase the pinene titer to 104.6 mg/L.