Theassembly-line synthases that produce bacterial polyketide natural products follow a modular paradigm in which each round of chain extension is catalysed by a different set or module of enzymes. Examples of deviation from this paradigm, in which a module catalyses either multiple extensions or none are of interest from both a mechanistic and an evolutionary viewpoint. We present evidence that in the biosynthesis of the 36-membered macrocyclic aminopolyol lactones azalomycin and kanchanamycin, the first extension module catalyses both the first and second cycles of polyketide chain extension. To confirm the integrity of the azl gene cluster, it was cloned intact on a bacterial artificial chromosome and transplanted into the heterologous host strain Streptomyces lividans, which does not possess the genes for marginolactone production. When furnished with 4-guanidinobutyramide, a specific precursor of the azalomycin starter unit, the recombinant S. lividans produced azalomycin, showing that the polyketide synthase genes in the sequenced cluster are sufficient to accomplish formation of the full-length polyketide chain. This provides strong support for module iteration in the azalomycin and kanchanamycin biosynthetic pathways. In contrast, re-sequencing of the gene cluster for biosynthesis of the polyketide β-lactone ebelactone in Streptomyces aburaviensis has shown that, contrary to a recently-published proposal, the ebelactone polyketide synthase faith fully follows the colinear modular paradigm.

Polyoxin, produced by Streptomcyes cacaoi var. asoensis and Streptomyces aureochromogenes, contains two non-proteinogenic amino acids, carbamoylpolyoxamic acid (CPOAA) and polyoximic acid. Although the CPOAA moiety is highly unusual, its biosynthetic logic has remained enigmatic for decades. Here, we address CPOAA biosynthesis by reconstitution of its pathway. We demonstrated that its biosynthesis is initiated by a versatile N-acetyltransferase, PolN, catalyzing L-glutamate (1) to N-acetylglutamate (2). Remarkably, we verified that PolM, a previously annotated dehydrogenase, catalyzes an unprecedented tandem reduction of acyl-phosphate toaldehyde, and subsequently to alcohol. We also unveiled a distinctive acetylation cycle catalyzed by PolN to synthesize α-amino-δ-hydroxyvaleric acid (6). Finally, we report that PolL is capable of converting a rare sequential hydroxylation of α-amino-δ-carbamoylhydroxyvaleric acid (7) to CPOAA. PolL represents an intriguing family of Fe(II)-dependent α-ketoglutarate dioxygenase with a cupin fold. These data illustrate several novel enzymatic reactions, and also set a foundation for rational pathway engineering for polyoxin production.

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein, Cas9, is a RNA-guided endonuclease that uses RNA–DNA base pairing torecognize and cleave double-stranded DNA (dsDNA) with a protospacer adjacent motif (PAM). It is widely accepted that the most commonly used Streptococcus pyogenes Cas9 (SpyCas9) protein recognizes a canonical 5′-NGG-3′ sequence in the PAM. In this study, we discovered another critical characteristic required for SpyCas9 cleavage i.e. the interspace between the protospacer and NGG. The results generated from DNA cleavage assays showed that both interspace length and the presence of a GG dinucleotide (particularly the upstream guanosine) are critical components in permitting SpyCas9-mediated cleavage. Interestingly, the interspace length significantly affects the selection of SpyCas9 cleavage sites on thenon-complementary strand. Additionally, the complementary strand cleavage siteis determined by the location of the single-molecular guide RNA (sgRNA). This indicates that PAM and sgRNA play different roles indetermining the SpyCas9 specific cleavage site. Importantly, we also revealed for the first time that in vitro annealing of dsDNA with exogenous PAM-presenting oligonucleotides (PAMmers) stimulated SpyCas9 cleavage of target dsDNA without PAM. This study pertaining to PAM and SpyCas9 is expected to improve our understanding of SpyCas9 with an associated impact on related bioengineering capabilities.

Thiolactomycin (TLM) is a thiotetronate antibiotic that selectively targets bacterial fatty acid biosynthesis through inhibition of the β-ketoacyl-acyl carrier protein synthases (KASI/II) that catalyse chain elongation on the type II fatty acid synthase. It has proved effective in in vivo infection models of Mycobacterium tuberculosis and continues to attract interest as a template for drug discovery. We have used a comparative genomics approach to uncover the biosynthetic pathway to TLM and related thiotetronates. Analysis of the whole-genome sequence of Streptomyces olivaceus Tü 3010 producing the more ramified thiotetronate Tü 3010 provided initial evidence that such thiotetronates are assembled by a novel iterative polyketide synthase-nonribosomal peptide synthetase, and revealed the identity of other pathway enzymes. Subsequent genome sequencing of three other thiotetronate-producing strains confirmed that near-identical clusters were also present in these genomes. In-frame gene deletion within the cluster for Tü 3010 from Streptomyces thiolactonus NRRL 15439, or within the TLM cluster, led to loss of production of the respective thiotetronate, confirming their identity. A separate genetic locus encodes a cysteine desulfurase and a sulfur transferase to supply the sulfur atom for thiotetronate ring formation. These insights have allowed us to propose a mechanism for sulfur insertion, and have opened the way to engineering of the biosynthesis of TLM and other thiotetronates to produce novel analogues.

旨在建立阿扎霉素F产生菌链霉菌211726的基因转移系统，以便基因敲除和外源基因表达等遗传操作。以整合型质粒pSET152和pIB139为出发质粒，通过接合转移构建了阿扎霉素F产生菌链霉菌211726的基因转移系统。结果显示25 μg/mL阿泊拉霉素可有效筛选接合子。经PCR验证，质粒成功整合到菌株链霉菌211726基因组中，接合子经多次传代后，导入的质粒pSET152和pIB139仍稳定整合于接合子基因组上。

The objective of this study is to establish the gene transfer system of strain Streptomyces sp. 211726 producing azalomycin F，which can be used for genetic manipulations such as gene knock-out and expression of foreign genes. Intergeneric genetic transfer system of Streptomyces sp. 211726 producing azalomycin F was constructed by conjugating integrative plasmid pSET152 with pIB139. Results showed that 25 μg/mL apramycin may be used to efficiently screen conjugants. PCR verification revealed that exogenous plasmid was successfully integrated in the chromosomal DNA of Streptomyces sp. 211726. The continuous passage culture experiment demonstrated that transformed pSET152 and pIB139 of conjugants were stably inherited.