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

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.

Abstract

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.”

Authors: Shao-Heng Bao, Hui Jiang, Ling-Yun Zhu, Ge Yao, Peng-Gang Han, Xiu-Kun Wan, Kang Wang, Tian-Yu Song, Chang-Jun Liu, Shan Wang, Zhe-Yang Zhang, Dong-Yi Zhang & Er Meng

Notes

The demand for psilocybin is on the rise, yet the cost of pharmaceutical-grade psilocybin is estimated at around $2,000 USD per gram (Fricke et al., 2019) while researchers might pay up to $7,000-$10,000 per gram. This raises the incentive to develop a more cost-effective means of producing the substance. And while the traditional route of chemical synthesis is being pursued most vehemently by COMPASS pathways and the Usona Institute, a number of new companies, such as Psybio Therapeutics, Psygen, Octarine Bio, and CB Therapeutics, are exploring the alternative route of biosynthesis. This involves genetically altering the metabolic networks of yeast or bacteria to convert sugar molecules into the desired compound.

A previous paper that described the first successful attempt of producing psilocybin on the gram scale with the bacteria E. coli, yielded a 32-fold improvement over earlier techniques. But the costs per serving (before any other costs) would then be $40-$50 at 20-30mg per 70kg. This was still higher than the costs of commercially available magic mushrooms or truffles (at $/€10-15). However, the current paper applied a novel gene repression method, which enabled them to selectively inhibit metabolic enzymes that interfere with psilocybin biosynthesis. This effectively improved the yield by an additional 302.9%.

What is novel about this technique?

  • The previous attempt to synthesize psilocybin still required expensive precursor materials, given the difficulty of facilitating trypthophan synthesis within E. coli. To solve this problem, they used a more efficient mutation of the tryptophan synthase enzyme and inserted it into E. coli
  • The efficiency of psilocybin synthesis was previously limited by the efficiency of methyltranferase enzyme (psiM), so the investigators inserted additional RNA strains into E. coli to overexpress this enzyme
  • The investigators identified native pathways in E. coli that interfere with the biosynthesis of psilocybin, for instance transporter enzymes that diminish the cellular concentration of its unfinished precursors. The selective repression of this drug transport enzyme alone increased the production of psilocybin by 107.2%
  • The investigators developed a novel RNA-based method that selectively repressess certain genes in a cell-density-dependent manner. Interfering with the genetic make up of the organism itself may otherwise severely impair its natural growth cycle of the host organism and lower its productivity. However, this technique repressess genes on a transcriptional (RNA) level and they were able to repress interference with psilocybin synthesis without impeding on the cellular growth of E. coli

The combined result of these innovations resulted in a 302.9% improvement in their biosynthesis of psilocybin. In reference to previous cost efficiency estimates, this method puts the cost of psilocybin at around $13 – $16.50 at 20-30mg per 70kg. Hence the current study significantly closes the cost gap between psilocybin biosynthesis and growing psilocybin mushrooms/truffles. And although it raises the competitive potential of the biosynthesis method, it is still questionable whether it can match the synthetic method developed by the non-profit Usona Institute that can produce psilocybin even on a large (1kg) scale.

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