Cordycepin and pentostatin biosynthesis gene identified through transcriptome and proteomics analysis of Cordyceps kyushuensis Kob.

Cordyceps kyushuensis is the only species of cordyceps growing on the larvae of Clanis bilineata Walker, and has been demonstrated that there are lots of pharmacological components including cordycepin. Cordycepin shows lots of pharmacological action but it could be converted to 3'-deoxyinosine by adenosine deaminase in vivo, which weakens the efficiency of cordycepin. That pentostatin, which has been reported to inhibit adenosine deaminase, combining cordycepin could enhance the efficiency of cordycepin in vivo. During transcriptome and proteomics analysis of Cordyceps kyushuensis, a single gene cluster including four genes we named ck1-ck4 which can synthesis both cordycepin and pentostatin has been identified using BLAST. Meanwhile, KEGG, KOG, GO analysis and differentially expressed genes were analyzed in transcriptome and proteomics. This study first sequenced transcriptome and proteomics of C. kyushuensis, and demonstrated that there is a single gene cluster related to biosynthesis of cordycepin and pentostatin, which can be employed to improve the yield of cordycepin and find more functional proteins.

Fungal Cordycepin Biosynthesis Is Coupled with the Production of the Safeguard Molecule Pentostatin.

Cordycepin (COR) and pentostatin (PTN) are adenosine analogs with related bioactivity profiles as both mimic adenosine and can inhibit some of the processes that are adenosine dependent. Both COR and PTN are also natural products and were originally isolated from the fungus Cordyceps militaris and the bacterium Streptomyces antibioticus, respectively. Here, we report that not only is PTN produced by C. militaris but that biosynthesis of COR is coupled with PTN production by a single gene cluster. We also demonstrate that this coupling is an important point of metabolic regulation where PTN safeguards COR from deamination by inhibiting adenosine deaminase (ADA) activity. ADA is not inhibited until COR reaches self-toxic levels, at which point ADA derepression occurs allowing for detoxification of COR to 3'-deoxyinosine. Finally, we show that using our biosynthetic insights, we can engineer C. militaris to produce higher levels of COR and PTN.

Biosynthesis of 2'-Chloropentostatin and 2'-Amino-2'-Deoxyadenosine Highlights a Single Gene Cluster Responsible for Two Independent Pathways in Actinomadura sp. Strain ATCC 39365.

2'-Chloropentostatin (2'-Cl PTN, 2'-chloro-2'-deoxycoformycin) and 2'-amino-2'-deoxyadenosine (2'-amino dA) are two adenosine-derived nucleoside antibiotics coproduced by Actinomadura sp. strain ATCC 39365. 2'-Cl PTN is a potent adenosine deaminase (ADA) inhibitor featuring an intriguing 1,3-diazepine ring, as well as a chlorination at C-2' of ribose, and 2'-amino dA is an adenosine analog showing bioactivity against RNA-type virus infection. However, the biosynthetic logic of them has remained poorly understood. Here, we report the identification of a single gene cluster (ada) essential for the biosynthesis of 2'-Cl PTN and 2'-amino dA. Further systematic genetic investigations suggest that 2'-Cl PTN and 2'-amino dA are biosynthesized by independent pathways. Moreover, we provide evidence that a predicted cation/H+ antiporter, AdaE, is involved in the chlorination step during 2'-Cl PTN biosynthesis. Notably, we demonstrate that 2'-amino dA biosynthesis is initiated by a Nudix hydrolase, AdaJ, catalyzing the hydrolysis of ATP. Finally, we reveal that the host ADA (designated ADA1), capable of converting adenosine/2'-amino dA to inosine/2'-amino dI, is not very sensitive to the powerful ADA inhibitor pentostatin. These findings provide a basis for the further rational pathway engineering of 2'-Cl PTN and 2'-amino dA production.IMPORTANCE 2'-Cl PTN/PTN and 2'-amino dA have captivated the great interests of scientists, owing to their unusual chemical structures and remarkable bioactivities. However, the precise logic for their biosynthesis has been elusive for decades. Actually, the identification and elucidation of their biosynthetic pathways not only enrich the biochemical repertoire of novel enzymatic reactions but may also lay solid foundations for the pathway engineering and combinatorial biosynthesis of this family of purine nucleoside antibiotics to generate novel hybrid analogs with improved features.

An Unusual Protector-Protégé Strategy for the Biosynthesis of Purine Nucleoside Antibiotics.

Pentostatin (PTN, deoxycoformycin) and arabinofuranosyladenine (Ara-A, vidarabine) are purine nucleoside antibiotics used clinically to treat hematological cancers and human DNA virus infections, respectively. PTN has a 1,3-diazepine ring, and Ara-A is an adenosine analog with an intriguing epimerization at the C-2' hydroxyl group. However, the logic underlying the biosynthesis of these interesting molecules has long remained elusive. Here, we report that the biosynthesis of PTN and Ara-A employs an unusual protector-protégé strategy. To our surprise, we determined that a single gene cluster governs PTN and Ara-A biosynthesis via two independent pathways. Moreover, we verified that PenB functions as a reversible oxidoreductase for the final step of PTN. Remarkably, we provided the first direct biochemical evidence that PTN can protect Ara-A from deamination by selective inhibition of the host adenosine deaminase. These findings expand our knowledge of natural product biosynthesis and open the way for target-directed genome mining of Ara-A/PTN-related antibiotics.

[Determination of pentostatin in culture broth using high performance liquid chromatography-mass spectrometry].

A method for the determination of pentostatin in culture broth was established using high performance liquid chromatography-mass spectrometry (HPLC-MS). The chromatographic conditions used were as follows: the analytical column was Hypersil ODS2 (250 mm x 4.6 mm, 5 microm); the mobile phase was methanol/acetonitrile/10 mmol/L ammonium acetate (pH 7.6) (2.5/2. 5/95, v/v/v) with a flow rate of 1.0 mL/min; the detection wavelength was 280 nm; the injection volume was 10 microL; the temperature was 40 degrees C. The linear range of the method for pentostatin was 1.0 - 100 mg/L (r = 0.999 9). The method is characterized by simplicity, higher sensitivity and accuracy.