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

Abstract

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 A i , A m , and R A (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 LuxR I58N 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.

“Biosynthesis of psilocybin and its nonnatural derivatives by a promiscuous psilocybin synthesis pathway in Escherichia coli”

“…Previously, the full de novo recombinant psilocybin pathway has been used in Aspergillus nidulans (Hoefgen et al, 2018), Saccharomyces cerevisiae (Milne et al, 2020), and E. coli (Bao et al, 2021) to enable de novo production of psilocybin at titers of approx. 100 mg/L, 627 ± 140 mg/L, and 27.7 mg/L, respectively.…”

Quorum Sensing Inhibition Based Drugs to Conquer Antimicrobial Resistance

“…HapR-GFP translational fusion was constructed in E. coli and repressed by Orr sRNAs expression [52]. Early growth phase of bacteria and cell count was impaired based on CRISPRi mediated gene silencing at transcription level may leads Quorum Sensing Inhibition Based Drugs to Conquer Antimicrobial Resistance DOI: http://dx.doi.org/10.5772/intechopen.104125 to higher restriction of endogenous metabolic pathways [53]. Eventually repressive sRNA regulation systems of genetically engineered plasmid, not like CRISPRi mediated gene silencing.…”

“…Eventually repressive sRNA regulation systems of genetically engineered plasmid, not like CRISPRi mediated gene silencing. Its only target QS multiple genes and not disturbing endogenous metabolic pathways responsive genes and cell growth [53]. Combinatorial dynamic repression strategy elucidates P lux promoter control of small RNA transcription complex with overexpression Hfq chaperone and LuxR I58N transcription factor in plasmid involved in quorum-sensing system with increased 3-oxohexanoyl-homoserine lactone (AHL) concentrations [53].…”

“…Its only target QS multiple genes and not disturbing endogenous metabolic pathways responsive genes and cell growth [53]. Combinatorial dynamic repression strategy elucidates P lux promoter control of small RNA transcription complex with overexpression Hfq chaperone and LuxR I58N transcription factor in plasmid involved in quorum-sensing system with increased 3-oxohexanoyl-homoserine lactone (AHL) concentrations [53]. The optimising metabolic networks as well simultaneously control of multiple genes using sRNA from the massive genome in a cell-density-dependent manner without affecting cellular conditions [53].…”

Preventing production escape during scale-up using an engineered glucose-inducible genetic circuit

“…Additionally, recent efforts have been directed at engineering quorum-sensing responsive promoters [13][14][15][16] . However, only a few inducer-responsive promoters are available for B. subtilis responding to cumate, bacitracin, IPTG, pyruvate, xylose, and maltose [17][18][19][20][21] .…”