Activation and Characterization of Cryptic Gene Cluster: Two Series of Aromatic Polyketides Biosynthesized by Divergent Pathways

Abstract

One biosynthetic gene cluster (BGC) usually governs the biosynthesis of a series of compounds exhibiting either the same or similar molecular scaffolds. Reported here is a multiplex activation strategy to awaken a cryptic BGC associated with tetracycline polyketides, resulting in the discovery of compounds having different core structures. By constitutively expressing a positive regulator gene in tandem mode, a single BGC directed the biosynthesis of eight aromatic polyketides with two types of frameworks, two pentacyclic isomers and six glycosylated tetracyclines. The proposed biosynthetic pathway, based on systematic gene inactivation and identification of intermediates, employs two sets of tailoring enzymes with a branching point from the same intermediate. These findings not only provide new insights into the role of tailoring enzymes in the diversification of polyketides, but also highlight a reliable strategy for genome mining of natural products.

Characterization of Highly Reductive Modification of Tetracycline D-Ring Reveals Enzymatic Conversion of Enone to Alkane

“…In addition to tjhO5, gene knockout experiments verified three genes, including tjhC5, tjhD2, and tjhD4, to be closely related to the post-modifications of tetracyclines. 23 On the basis of a bioinformatic analysis, TjhC5 contains a cyclase domain, TjhD2 is an aldo-keto reductase, sharing moderate homology (33% amino acid identity) with SsfF which can reduce the C-4 carbonyl of 2,4-keto-ATC, and TjhD4 belongs to the shortchain dehydrogenase (SDR) superfamily and NAD + -dependent epimerase/dehydratase subfamily, which is most likely to participate in subsequent reduction. To verify this hypothesis, we performed various biochemical assays on TjhD4 from E. coli BL21(DE3) (Figure S4).…”

“…19−22 In a recent study, we activated and characterized a silent gene cluster tjh in Streptomyces aureus suzhoueusis governing the biosynthesis of two types of type II polyketides (pentacyclic and tetracyclic structures). 23 In comparison with typical tetracyclines containing α,β-unsaturated ketones, the Drings of these noncanonical tetracyclines are highly reduced to either a CC (5−7) or C−C bond (9−11) (Figure 1a). On consideration that three new compounds (12−14) with a core structure similar to that of 4-hydroxy-ATC have been previously identified in ΔtjhO5 mutants, the atypical tetracyclines are likely to be converted from intermediates with an α,β-unsaturated ketone (Figure 1a and Figure S1a).…”

Discovery of a Novel Spiro-Naphthoquinone Compound in Streptomyces aureus SP-371

“…以 pKC1139 为载体构建含左右臂的同框敲除质粒 pNQY-1, 左臂引物为 tjhD5-L-f: TATAAGCTTCACAC-AGGTGACGTACTTC 和 tjhD5-L-r: TATTCTAGATCGT-TGCGGTACGACACC, 右臂引物为 tjhD5-R-f: TATTCT-AGAGATGCTCAAGGGTCTGC 和 tjhD5-R-r: TATGA-ATTCCATGACGTTCTCCATCAGC. 同框敲除质粒经过 接合转移导入 ΔtjhC5 突变株中, 经过筛选得到最终双 敲除突变株, 具体操作详见文献 [3]. 通过 PCR 验证突变 株的基因型, 验证引物为 tjhD5-yz-f: CATCGCAAC-GATCAAGGAATTC 和 tjhD5-yz-r: GCCTGCGAGCAC-AGGAACAG, 目标条带大小为 329 bp.…”

“…II 型聚酮化合物的生物合成 已经得到了广泛的研究, 结构多样性主要来源于它们不 同的后修饰途径 [2a-2c] . 我们 [3] 在金色链霉菌 SP-371 (S. aureus suzhoueusis SP-371)的基因组中发现了一个新型 II 型聚酮化合物的生物合成基因簇 tjh, 通过过表达正调 控基因的方法激活了 tjh, 成功得到了 8 个新化合物, 它 们包含有两种不同的骨架结构(线性四环结构和五元稠 环结构). 根据单基因敲除实验的结果, 推测这两类不 同结构的化合物可能是通过同一中间体经过分叉的生 物合成途径得到的.…”

Activation of Cryptic Antibiotic Biosynthetic Gene Clusters Guided by RNA-seq Data from Both Streptomyces ansochromogenes and ΔwblA

“…There are many successful examples for mining of hidden natural products. For instance, (i) Three natural products, streptothricin, geosmin and strevertene A (polyene), were successfully activated by using the reporter-guided strategy [16]; (ii) Multiplex activation strategies were implemented to awaken a cryptic biosynthetic gene cluster, resulting in the discovery of eight aromatic polyketides with two types of frameworks, two pentacyclic isomers and six glycosylated tetracyclines [17]; (iii) In Streptomyces roseosporus, mureidomycin biosynthesis was activated by the introduction of an exogenous regulatory gene ssaA [18].…”