This in vitro study (2021) developed a novel method of transcriptional gene repression within E. coli that increased the yield of psilocybin biosynthesis by 302.9% without affecting cell growth.
Introduction
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.
Methods
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.Results/Discussion: 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.
The authors argue that conventional metabolic optimisation in bacteria is time consuming and costly because it typically requires extensive identification and modification of competing branch pathways across the genome. They set out a set of desirable features for a pre‑screening regulatory approach: fast and inexpensive control that avoids genomic modification, the ability to target genes across the genome stably and specifically, the capacity to manipulate essential pathways without harming host growth, and support for combinatorial repression to redirect multiple fluxes simultaneously. To meet these needs they developed a plasmid‑based, quorum‑sensing (QS) controlled small RNA (sRNA) system that dynamically downregulates multiple genomic genes in a cell‑density‑dependent manner, enabling high‑throughput screening and flexible targeting by changing sRNA target binding sites (TBSs) on the plasmid. S‑H. and colleagues report several technical and functional points about the system. Fine‑tuning the cellular level of the Hfq chaperone is critical: when Hfq levels match the total sRNA level, more genes can be repressed simultaneously without disrupting growth. Different LuxR transcription factor variants permit switching sRNA transcription on at different cell‑density thresholds; LuxR I58N was selected as the optimal variant for tuning sRNA transcription in shake‑flask pinene experiments. The QS‑driven sRNA transcription allowed repression of genomic targets of up to 87% (sthA) during stationary phase while avoiding severe metabolic burden. Comparing dynamic, density‑dependent repression with constant repression driven by a strong constitutive promoter, the authors conclude that excessive early repression impairs growth, whereas strong repression initiated during stationary phase does not noticeably interrupt growth; accordingly, promoters with increasing strength over time (or tuned constitutive promoters) are preferable for repression control. The authors applied the strategy to redirect metabolic fluxes for several products. For pinene, combinatorial‑repression screening indicated that simply combining genes that were effective when repressed individually does not necessarily maximise titre; repressing multiple enzymes within the same competing pathway can be inefficient. Monitoring key substrate pools such as NADPH/NADP+ helped select effective targets. After two rounds of screening that increased NADPH, acetyl‑CoA and geranyl diphosphate (GPP) simultaneously, pinene titre improved by 365.3%. The approach was also applied to pentalenene production, increasing titre from 341.5 to 612.9 mg/L by repressing farnesyl diphosphate (FPP) and NADPH‑consuming pathways, and to psilocybin production, increasing titre from 6.9 to 27.7 mg/L by repressing an indole‑efflux enzyme and tryptophan‑ and SAM‑consuming pathways. The authors note that accumulating intermediates by downregulating branch pathways can improve flux into the production pathway when substrates are limiting, as explained by Michaelis–Menten kinetics. Finally, the authors emphasise that their strategy focuses on dynamic, direct regulation of genomic genes via a plasmid sRNA system and can be used for high‑throughput screening. To create a fully autonomous production system, they recommend pairing this regulatory approach with dynamic promoters or tunable constitutive promoters to control expression of the production pathway itself.
Papers cited by this study that are also in Blossom
Adams, A. M., Kaplan, N. A., Wei, Z. et al. · Metabolic Engineering (2019)
Krediet, E., Bostoen, T., Breeksema, J. J. et al. · International Journal of Neuropsychopharmacology (2020)
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.
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