Functional Analysis of Ribonucleotide Reductase from Cordyceps militaris Expressed in Escherichia coli

Abstract

Cordyceps militaris produces cordycepin (3'-deoxyadenosine), which has various activities, including anti-oxidant, anti-tumoral, anti-viral, and anti-inflammatory. Ribonucleotide reductase (RNR) seems to be a candidate to produce cordycepin in C. militaris because RNR catalyzes the reduction of nucleotides to 2'-deoxynucleotides, whose structures are similar to that of cordycepin. However, the role of RNR has not been confirmed yet. In this study, complementary DNAs (cDNAs) of C. militaris RNR (CmRNR) large and small subunits (CmR1 and CmR2) were cloned from C. militaris NBRC9787 to investigate the function of CmRNR for its cordycepin production. C. militaris NBRC9787 began to produce cordycepin when grown in a liquid surface culture in medium composed of glucose and yeast extract for 15 days. CmR1 cDNA and CmR2 cDNA were obtained from its genomic DNA and from total RNA extracted from its mycelia after cultivation for 21 days, respectively. Recombinant CmR1 and CmR2 were expressed individually in Escherichia coli and purified. Purified recombinant CmR1 and CmR2 showed RNR activity toward adenosine diphosphate (ADP) only when two subunits were mixed but only show the reduction of ADP to 2'-deoxyADP. These results indicate that the pathway from ADP to 3'deoxyADP via CmRNR does not exist in C. militaris and cordycepin production in C. militaris may be mediated by other enzymes.

Genome resequencing and transcriptome analysis reveal the molecular mechanism of albinism in Cordyceps militaris

“…In earlier studies, cordycepin was polymerized via 3′-deoxyribose and adenine using a glycosidic bond ( Kredich and Guarino, 1961 ). However, adenosine can also be used as the precursor of cordycepin synthesis and produces cordycepin through multiple enzymes such as adenylate kinase (ADEK), ferritin/ribonucleotide reductase-like protein (RNR), 5’-nucleotidase (NT5E), adenosine nucleosidase, oxidoreductase domain-containing protein (Cns1), and phosphoribosyl-aminoimidazole-succinocarboxamide synthase (Cns2) ( Suhadolnik and Cory, 1964 ; Lennon and Suhadolnik, 1976 ; Kato et al, 2017 ; Liu et al, 2018 ). In addition, a mutation of CmWC-1 repressed cordycepin biosynthesis and fruiting body development ( Yang et al, 2016 ).…”

Immunostimulatory Activity of Cordyceps militaris Fermented with Pediococcus pentosaceus SC11 Isolated from a Salted Small Octopus in Cyclophosphamide-Induced Immunocompromised Mice and Its Inhibitory Activity against SARS-CoV 3CL Protease

“…The relationship between the mechanisms of cordycepin synthesis and LAB fermentation has not yet been elucidated. However, the production of cordycepin through lactic acid fermentation may be similar to the synthesis of 2-deoxrtivonucleotides from adenosine, adenosine monophosphate, or adenosine diphosphate by ribonucleotide reductase [51][52][53]. Recent studies have reported that cordycepin showed anti-viral activity by targeting the spike protein, main protease, and RNAdependent RNA polymerase of viruses [54,55].…”

New Insights Into the Biosynthesis of Typical Bioactive Components in the Traditional Chinese Medicinal Fungus Cordyceps militaris

“…Thus, according to the similarity to 2′deoxyadenosine biosynthesis, Xiang et al predicted that the biosynthesis of cordycepin may proceed through a reductive mechanism after transcriptome analysis of the O. sinensis fruiting body (Xiang et al, 2014), but they didn't verify the cordycepin biosynthesis pathway. Untill 2017, the recombinant E. coli which expressed CmRNR showed RNR activity on ADP but did not produce 3' -deoxy ADP (Kato et al, 2017), which seemed to deny the participation of RNR in cordycepin biosynthesis in C. militaris. But in 2020, Zhang et al indicated that the RNR consisted of two subunits: a large one (RNRL) and a small one (RNRM), RNRM could regulate the biosynthesis of cordycepin directly via the reduction of adenosine, as demonstrated by overexpressing RNRM in C. militaris (Zhang et al, 2020b).…”