Hui Hong, Yuhui Sun, Yongjun Zhou, Emily Stephens, Markiyan Samborskyy, and Peter F. Leadlay*
Beilstein Journal of Organic Chemistry 2016, 12:2164-2172
Epub Date: 11 October 2016
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.
Jianzhao Qi, Dan Wan, Hongmin Ma, Yuanzhen Liu,Rong Gong, Xudong Qu, Yuhui Sun, Zixin Deng, and Wenqing Chen*
Cell Chemical Biology 2016, 23(8):935-944
Epub Date: 18 August 2016
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.
Yujie Geng, Zixin Deng, and Yuhui Sun*
RSC Advances 2016, 6:33514-33522
Epub Date: 29 March 2016
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.
Weixin Tao#, Marie E.Yurkovich#, Shishi Wen#, Karen E. Lebe, Markiyan Samborskyy, Yuanzhen Liu, Anna Yang, Yunkun Liu,Yingchen Ju, Zixin Deng, Manuela Tosin, Yuhui Sun*, and Peter F. Leadlay*
Chemical Science 2016, 7(1):376-385
Epub Date: 8 October 2015
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.
Yanling Ma*, FulaiLiu, Min Zhang, Yuhui Sun, and Kui Hong
生物技术通报 2016, 32(4):198-202
Biotechnology Bulletin 2016, 32(4):198-202 (Chinese)
Published online: 22 April 2016
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.