*3.3. Proposed Biosynthetic Pathway of Albucidin*

Structurally, albucidin is closely related to oxetanocin A (Figure 2b), which has been isolated from the culture of *Bacillus megaterium* NK84-0218 [22]. Both compounds are the only known naturally occurring nucleosides featuring four membered oxetane rings in their structure. From a structural view, albucidin is 2 -dehydroxymethyl oxetanocin A. Two genes, *oxsA* and *oxsB,* encoding a putative HD domain phosphohydrolase and a cobalamin-dependent S-adenosylmethionine radical enzyme

have been reported to be responsible for oxetanocin biosynthesis [23]. dAMP, dADP and dATP were identified as direct oxetanocin precursors [24]. The product of *oxsB* catalyses the contraction of the deoxyribose ring, while the product of *oxsA* is responsible for the removal of one or multiple phosphates from a phosphorylated 2 -deoxyadenosine derivative [24,25]. Through the simultaneous actions of OxsA and OxsB, the phosphorylated 2 -deoxyadenosine is converted to the oxetanocin A precursor, its aldehyde form, which must be reduced to complete biosynthesis [24]. This reaction is not encoded by the genes within the oxetanocin A cluster and is likely to be carried by an unidentified enzyme of *B. megaterium* NK84-0218.

Gene inactivation studies have given evidence that two genes, *albA* and *albB*, are required for the production of albucidin. Both genes encode putative SAM radical proteins. At the protein level, the *albA* gene shows homology to biotin synthases and the *albB* gene shows homology to the product of the oxetanocin biosynthetic gene *oxsB*. Despite the high structural similarity of albucidin and oxetanocin A, the homologue of the second oxetanocin biosynthetic gene *oxsA* cannot be found within the albucidin cluster or in the genome of albucidin producer *S. albus* subsp. *chlorinus* NRRL B-24108. The homology of the *albB* gene to *oxsB* implies that the product of *albB* might also be responsible for the ring contraction reaction in albucidin biosynthesis. However, the structural differences between albucidin and oxetanocin and the absence of an *oxsA* homologue imply substantial differences in biosynthetic routes leading to the biosynthesis of the nucleosides. Due to the lack of an *oxsA* homologue that is responsible for the dephosphorylation of adenine deoxyribonucleotides during oxetanocin biosynthesis, we propose that deoxyadenosine is used instead of dAMP, dADP or dATP as a precursor for albucidin production. The product of *albB* is likely responsible for the contraction of the deoxyribose ring of deoxyadenosine (Figure 3) in a similar manner as its homologue OxsB catalyses oxetane ring formation in oxetanocin A biosynthesis [24]. As a result of this reaction, the aldehyde form of oxetanocine A is formed. The conversion of the latter into albucidin is likely to be catalysed by the product of *albA,* which removes the aldehyde group from the 2'-position (Figure 3).

**Figure 3.** The proposed scheme of albucidin biosynthesis. 2 -Deoxyadenosine (**1**) is converted into the aldehyde form of oxetanocin A (**2**) by the product of *albB*. The latter is then converted into albucidin (**3**) by the product of *albA*.

In this paper, we report the identification, cloning, and heterologous expression of the albucidin biosynthetic gene cluster from *Streptomyces albus* subsp. *chlorinus* NRRL B-24108. Albucidin is a nucleoside phytotoxin featuring a rare oxetane ring in its structure. This metabolite shows herbicidal activity against a broad spectrum of grass and broadleaf weeds. In treated plants, albucidin induces metabolic perturbation, chlorosis, and bleaching [10]. The exact MOA of the compound remains unknown. The identification of the albucidin cluster presented in this paper enables biosynthetic studies of albucidin, optimization of its production as well as albucidin supply for the determination of its MOA.
