Research Progress on Cordycepin Synthesis and Methods for Enhancement of Cordycepin Production in Cordyceps militaris

Abstract

C. militaris is an insect-born fungus that belongs to Ascomycota and Cordyceps. It has a variety of biological activities that can be applied in medicine, health-care products, cosmeceuticals and other fields. Cordycepin (COR) is one of the major bioactive components identified from C. militaris. Thus, C. militaris and COR have attracted extensive attention. In this study, chemical synthetic methods and the biosynthesis pathway of COR were reviewed. As commercially COR was mainly isolated from C. militaris fermentation, the optimizations for liquid and solid fermentation and genetic modifications of C. militaris to increase COR content were also summarized. Moreover, the research progress of genetic modifications of C. militaris and methods for separation and purification COR were introduced. Finally, the existing problems and future research direction of C. militaris were discussed. This study provides a reference for the production of COR in the future.

Keywords: Cordyceps militaris; biosynthesis; cordycepin; genetic modification.

Figure 1

The adenosine metabolism pathway and possible COR biosynthesis pathway [60,61,62]. Abbreviations: IMP: inosine monophosphate; AMP: adenosine monophosphate; ADA, adenosine deaminase; ADE, adenine deaminase; ADK: adenosine kinase; ADEK, adenylate kinase; ADN, adenosine nucleosidase; ADK, adenosine kinase; DADK, deoxyadenylate kinase; NDK, nucleoside-diphosphate kinase; AMPD, AMP deaminase; dAMP: deoxyadenosine monophosphate; ADP: adenosine diphosphate; dADP: deoxyadenosine diphosphate; RNR: ribonucleotide reductases; APRT, adenine phosphoribosyltransferase; NT5E, 5′-nucleotidase; PNP, purine nucleoside phosphorylase; RNR, ribonucleotide triphosphate reductase.

Figure 2

Delineation of COR biosynthetic gene cluster and the COR biosynthesis pathway [63]. (A) The gene cluster for synthesize cordycepin C. militaris. (B) Schematic structure of the proteins. (C) Delineation of the biosynthetic pathway of cordycepin. Abbreviations: PRPP, phosphoribosyl pyrophosphate; 2′-C-3′-dA: 2′-carbonyl-3′-deoxyadenosine; 2′,3′-cAMP: 2′,3′-cyclic monophosphate; NK: an N-terminal nucleoside kinase; HisG: a C-terminal HisG family of ATP phosphoribosyltransferases; PTN: pentostatin; 3′-dI: 3′-deoxyinosine.

Figure 3

Genetic modification methods of C. militaris. Constructed plasmid with target gene was extracted from E. coli and transformed by ATMT, PMT and PB methods. For the ATMT method, the plasmid was transferred into Agrobacterium, followed by co-cultivation with C. militaris spores. For the PMT method, the plasmid (including split marker DNA) was transferred into protoplast mediated by PEG (polyethylene glycol). For the PE method, the plasmid was coated with gold powder, and the broken mycelium suspension of C. militaris plated in medium was used as the receptor material for transformation. Finally, the transformant was screened in the corresponding medium for all methods. The transformants were further confirmed by polymerase chain reaction (PCR) or the expression of a reporter gene.

Figure 4

Schematic diagram of COR separation, purification and detection. SFE and MIPs techniques were used to separate the fruiting bodies of solid fermentation or the fermentation broth of liquid fermentation. HSCCC/RPC techniques were used to separate and purify the crystallization of the crude samples, and then the COR samples were detected and analyzed by high-performance liquid chromatography (HPLC).