Highly Efficient Preparation of Cyclic Dinucleotides via Engineering of Dinucleotide Cyclases in Escherichia coli

Abstract

Cyclic dinucleotides (CDNs) are widely used secondary signaling molecules in bacterial and mammalian cells. The family of CDNs includes c-di-GMP, c-di-AMP and two distinct versions of hybrid cGAMPs. Studies related to these CDNs require large doses that are relatively expensive to generate by current methods. Here we report what to our knowledge is the first feasible microbial-based method to prepare these CDNs including c-di-GMP, 3′3′-cGAMP and 2′3′-cGAMP. The method mainly includes two parts: producing high yield of CDNs by engineering the overexpression of the proper dinucleotide cyclases (DNCs) and other related proteins in Escherichia coli, and purifying the bacteria-produced CDNs by a unified and simple process involving a STING affinity column, macroporous adsorption resin and C18 reverse-phase liquid chromatography. After purification, we obtained the diammonium salts of c-di-GMP, 3′3′-cGAMP and 2′3′-cGAMP with weight purity of >99, >96, >99% and in yields of >68, >26, and >82 milligrams per liter of culture, respectively. This technological platform enables the production of CDNs from cheaper material, provides a sustainable source of CDNs for scientific investigation, and can easily be further developed to prepare CDNs on a large scale for industry.

Biotechnological production of cyclic dinucleotides—Challenges and opportunities

“…coli (Lv et al, 2019). This is also the first published attempt for the biocatalytic production of 2'3'-cGAMP at a liter scale.…”

“…An overall yield of 44% has been achieved with an 82% (w/w) purity confirmed by NMR analysis and a low endotoxin contamination <0.00005% (w/w) confirmed by the Limulus test. Noteworthy is a sequential chromatography that was applied for the purification of the whole-cell catalysis products (Lv et al, 2019).…”

“…In addition to isolated enzymes, whole cell biocatalysts were demonstrated as promising alternative for the production of CDNs. Due to the comparably large scale of 1 L, 134 mg of crude c‐di‐GMP and 64 mg of crude 3′3′‐cGAMP were produced with recombinant E. coli (Lv et al, 2019). This is also the first published attempt for the biocatalytic production of 2'3'‐cGAMP at a liter scale.…”

2′,3′‐Cyclic GMP‐AMP Dinucleotides for STING‐Mediated Immune Modulation: Principles, Immunotherapeutic Potential, and Synthesis

“…This feature has recently been employed by many researchers to enzymatically synthesize 2',3'-cGAMP analogues for therapeutic applications. [61][62][63][64][65]…”

“…Recently, Zhu and coworkers developed a feasible microbial-based method to produce 2',3'-cGAMP (Figure 16). [63] They transformed a plasmid encoding mcGAS into an E. coli BL21-CodonPlus (DE3)-RIL strain and then added isopropyl β-D-1thiogalactopyranoside (IPTG) to induce mcGAS expression. They found that expressed mcGAS can sense endogenous DNA and synthesize 2',3'-cGAMP.…”