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.

Keywords: adjuvant; affinity purification; cyclic dinucleotides; dinucleotide cyclases; microbial preparation.

FIGURE 1

HPLC analysis (UV 254 nm) of the initial CDNs before purification, which were produced by the different engineered Escherichia coli cells expressing the proper DNCs and other related proteins under various conditions. (A) Production of c-di-GMP in E. coli BL21 (DE3) cells expressing only tDGCm or co-expressing tDGCm and STING^CTD under the optimized conditions. (B) Production of 3′3′-cGAMP in E. coli BL21 (DE3) cells: top, cells were grown in LB medium and expressed only the DncVt; second from top, cells were grown in LB medium and co-expressed DncVt, GMK, and NDK; third from top, cells were grown in LB medium and expressed only the DncVtm; bottom, cells were grown in TB medium and co-expressed DncVtm, GMK, and NDK. (C) The secreted production of 2′3′-cGAMP in E. coli BL21-CodonPlus (DE3)-RIL cells expressing mcGAS as a SUMO fusion protein in LB or modified M9 minimal medium with the other optimized conditions.

FIGURE 2

Schematic representation of the microbial production of c-di-GMP (A), 3′3′-cGAMP (B), and 2′3′-cGAMP (C) via engineering the expression of the proper DNCs and other related proteins in E. coli. The expression vectors and inserted genes are simply indicated. Gene symbols and the enzymes they encode: tDGCm, DGC Arg158Ala mutant from Thermotoga maritime; STING^CTD, carboxy-terminal domain of human STING^H232; DncVtm, mutant Thr179Arg of V. cholera DncV truncation; NDK, nucleoside-diphosphate kinase from E. coli; GMK, guanylate kinase from Staphylococcus aureus; SUMO-mcGAS, mouse cGAS as a SUMO fusion protein. Three CDNs structural drawings depict the free acid form of the phosphate moieties.

FIGURE 3

Schematic representation of CDNs purification from the bacteria culture.

FIGURE 4

HPLC-MS/MS chromatogram (UV 254 nm) of the final pure c-di-GMP (A), 3′3′-cGAMP (B), and 2′3′-cGAMP (C). The left insert panels show the MS spectra of the final pure CDNs, the ions at m/z 691.1022 (calculated mass 691.1027), 675.1074 (calculated mass 675.1078), and 675.1075 (calculated mass 675.1078) represent the [M + H]⁺ ion of c-di-GMP, 3′3′-cGAMP and 2′3′-cGAMP, respectively. The right insert panels show the tandem mass spectra of the final pure CDNs resulting from higher-energy collision dissociation (HCD) of the precursor ion ([M + H]⁺ = 691.10 or 675.11).