Activation And Characterization Of Two Cryptic Gene Clusters In Streptomyces Sp. LC6 And LZ35

Natural products are the resource of new drug discovery and microbial natural product is one of the most important source of natural products. The biosynthesis genes of microbial secondary metabolites are usually clustered in bacterial genomes, thus it is convenient for people to predict their product and biosynthesis. With the development of gene sequencing and gene analysis techniques, people find that there are a large number of secondary metabolism synthesis gene clusters in most of the bacterial genomes. However only a fraction of the gene clusters can be related to already known metabolites, with the remaining classified as "cryptic gene clusters" with unknown function, indicating the great potential for Streptomyces as novel natural products arsenal.This research paper mainly focuses on sequence-guided genome mining strategies, trying to awake two cryptic gene clusters in two Streptomyces and get their novel natural products, including:1) "Activation and identification of a pentaketide ansamycin gene cluster in Streptomyces sp. LC6"; 2) "Activation and heterologous expression of a NRPS-FAS hybrid gene cluster in Streptomyces sp. LZ35".Ansamycins are type I PKS polyketide macrolactams derived from 3-amino-5-hydroxy benzoic acid (AHBA), mainly produced by Actinomycetes with broad biological activities. Streptomyces sp. LC6 is an AHBA synthase PCR positive strain isolated from the leaves of mangrove plants Kandelia candel (Linn.) Druce collected from Zhangzhou, Fujian Province. Our previous research has indicated that there is a cryptic pentaketide ansamycin biosynthetic gene cluster, jlm, in its genome. However, we did not isolated any kind of ansamycins from its fermentation products under standard laboratory cultivation conditions, indicating that this gene cluster is silent or low expressed. Several researches have been done to study about this gene cluster:1) The proposed pathway specific regulator genes in this gene cluster were overexpressed respectively by constitutive expression. Miss Juanli Zhang had two novel ansamycin macrodilactams with unprecedented structure, named juanlimycins, isolated and structure elucidated from an SARP overexpression mutant.2) The juanlimycin biosynthetic gene cluster was verified by gene disruption and HPLC-MS analysis.3) In order to study about how the macrodilactams formed, the core gene region of the jlm gene cluster(jlm A-E) was integrated into Streptomyces sp. LZ35, which has sufficient AHBA as precusor, by heterologous expression. HPLC analysis of the mutant strains showed an distinct peak. However, the compound relating to this peak was not isolated mainly because of low production.Streptomyces sp. LZ35 was isolated from the rhizosphere soil of mangrove plants Kandelia candel (Linn.) Druce. Genome sequencing shows that there is a cryptic NRPS-FAS gene cluster (has), proposed to encode a lipopeptide, in its genome. Two kind of strategies, including the constitutive expression of the regulatory genes and heterologous expression, were performed to try to get the novel lipopeptide product encoded by this gene cluster:1) The proposed pathway specific regulatory gene belonging to Ic1R or LuxR family protein was overexpressed in Streptomyces sp. LZ35 by constitutive expression, respectively. The constitutive expression of an Ic1R family regulatory gene resulted in a light green accumulating in the culture medium and HPLC analysis showed a distinct peak with a strong UV absorption. Product isolation led to a chorismic acid derivative and a metal ion chelating agent, while no lipopeptide was identified. The constitutive expression of a LuxR family regulatory gene resulted in the metabolic profiling distinct from the wide type, and the distinct peaks were proved to be an family of lipoamino acid discovered by other strategy.2) A recombinant DNA fragment containg has gene cluster (52.4 kb) was constructed. However, no product was detected after introducing this recombinant DNA into Streptomyces lividans SBT5.To sum up, in this paper we have successfully awaken a cryptic ansamycin biosynthetic gene cluster in Streptomyces sp. LC6 and a cryptic lipoamino acid biosynthetic gene cluster in Streptomyces sp. LZ35 finally. On one hand it is a good example for sequence-guided natural product discovery; On the other hand, it also proves that the numerous cryptic gene clusters in Streptomyces could be activated and be the source of novel natural products, provide reference for people to exploit novel the cryptic gene clusters for novel natural products.