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

CONCLUSION

Before optimizing metabolic networks, necessary preliminary work, such as identifying competing branch pathways from the extremely massive genome, is time consuming and highly costly. Therefore, an approach with the following advantages is desired before performing this groundwork. First, strategies for gene expression control should be fast and inexpensive. In most cases, genomic modification is relatively complicated and not suitable for high-throughput screening. A plasmid system consisting of bio-bricks that can effectively tune the expression levels of genomic genes is usually the prior choice. Second, to obtain access to a complete map of metabolic fluxes, approaches to stably and specifically target genes in the entire genome are required. Third, a crucial part of microbial metabolism consists of essential pathways, of which interruptions may severely impair the growth of the host and lower productivity. Therefore, the manipulation of essential pathways should be carried out without affecting cellular conditions. Finally, combinatorial repression allows redirection of multiple metabolic fluxes, which help regulate complicated pathways and determine the additive effects when repressing multiple genes. To meet all the requirements, we proposed a strategy using an en-gineered plasmid to dynamically downregulate multiple genes in a cell-density-dependent manner. The QS-based sRNA regulation system can be adopted in high-throughput gene screening for metabolic studies. Only a small modification of sRNA TBSs in the plasmid allows the repression of another three genes. In addition, it is expected that as long as the level of the Hfq chaperone is sufficient and Article matches with the total sRNA level, more genes can be repressed simultaneously without interrupting cell growth. It has also been demonstrated that sRNA transcription can be switched on at different cell density thresholds by using different variants of the transcription factor LuxR, enabling effective repressions at varying growth stages. Genes can be repressed up to 87% (sthA) during the stationary phase while preventing the host from serious metabolic burden. By comparing the growth curves of dynamic repression with those of constant repression using the strong constitutive promoter P R , it is concluded that excessive repression at early phases probably results in severely impaired cell growth, while strong repression that occurs during the stationary phase cannot cause obvious interruptions in cell growth. Therefore, dynamic promoters with increasing strengths should be used to control the repression. The problem can also be addressed by using constitutive promoters with tuned strengths. Endogenous bacterial metabolites are constrained at low levels. According to the Michaelis-Menten equation, accumulating intermediates by downregulating branch pathways improves the enzymatic reaction rate of the production pathway when substrates are limiting. Here, we report the application of the dynamic sRNA regulation strategy to redirect metabolic fluxes for the improvement of pinene synthesis. By comparison, LuxR I58N was decided to be the optimal LuxR variant to tune sRNA transcription in shake flask culture. Through the combinatorial-repression screening, it was determined that simply superimposing candidate genes that outperformed when repressed alone may not lead to the highest titer because repressing multiple enzymes that are within the same competing pathway or that produce the same substrate is often inefficient. The monitoring of key substrate (NADPH/NADP + ) levels can help select candidate genes and reach the highest titer. After two rounds of screening, simultaneously increasing levels of NADPH, AC-CoA, and GPP resulted in a 365.3% improvement in pinene titer, indicating that these substrates are largely needed for pinene synthesis. Moreover, to show a broad applicability of this strategy, the production titer of pentalenene was improved from 341.5 to 612.9 mg/L by repressing FPP and NADPH-consuming pathways, and the titer of psilocybin was improved from 6.9 to 27.7 mg/L by repressing an indole-efflux enzyme and tryptophan and SAM-consuming pathways. Furthermore, our strategy focuses on only the dynamic and direct regulation of genomic genes. To establish a fully autonomous regulation system, dynamic promoters or tunable constitutive promoters should be used to control the production pathway.