Yihua Chen

Improvement of secondary metabolite production in Streptomyces by manipulating pathway regulation

Titer improvement is a constant requirement in the fermentation industry. The traditional method of “random mutation and screening” has been very effective despite the considerable amount of time and resources it demands. Rational metabolic engineering, with the use of recombinant DNA technology, provides a novel, alternative strategy for titer improvement that complements the empirical method used in industry. Manipulation of the specific regulatory systems that govern secondary metabolite production is an important aspect of metabolic engineering that can efficiently improve fermentation titers. In this review, we use examples from Streptomyces secondary metabolism, the most prolific source of clinically used drugs, to demonstrate the power and utility of exploiting natural regulatory networks, in particular pathway-specific regulators, for titer improvement. Efforts to improve the titers of fredericamycin, C-1027, platensimycin, and platencin in our lab are highlighted.

Genome Sequence of the Milbemycin-Producing Bacterium Streptomyces bingchenggensis

Streptomyces bingchenggensis is a soil-dwelling bacterium producing the commercially important anthelmintic macrolide milbemycins. Besides milbemycins, the insecticidal polyether antibiotic nanchangmycin and some other antibiotics have also been isolated from this strain. Here we report the complete genome sequence of S. bingchenggensis. The availability of the genome sequence of S. bingchenggensis should enable us to understand the biosynthesis of these structurally intricate antibiotics better and facilitate rational improvement of this strain to increase their titers.

Identification and Utility of FdmR1 as a Streptomyces Antibiotic Regulatory Protein Activator for Fredericamycin Production in Streptomyces griseus ATCC 49344 and Heterologous Hosts

The fredericamycin (FDM) A biosynthetic gene cluster, cloned previously from Streptomyces griseus ATCC 49344, contains three putative regulatory genes, fdmR, fdmR1, and fdmR2. Their deduced gene products show high similarity to members of the Streptomyces antibiotic regulatory protein (SARP) family (FdmR1) or to MarR-like regulators (FdmR and FdmR2). Here we provide experimental data supporting FdmR1 as a SARP-type activator. Inactivation of fdmR1 abolished FDM biosynthesis, and FDM production could be restored to the fdmR1::aac(3)IV mutant by expressing fdmR1 in trans. Reverse transcription-PCR transcriptional analyses revealed that up to 26 of the 28 genes within the fdm gene cluster, with the exception of fdmR and fdmT2, were under the positive control of FdmR1, directly or indirectly. Overexpression of fdmR1 in S. griseus improved the FDM titer 5.6-fold (to about 1.36 g/liter) relative to that of wild-type S. griseus. Cloning of the complete fdm cluster into an integrative plasmid and subsequent expression in heterologous hosts revealed that considerable amounts of FDMs could be produced in Streptomyces albus but not in Streptomyces lividans. However, the S. lividans host could be engineered to produce FDMs via constitutive expression of fdmR1; FDM production in S. lividans could be enhanced further by overexpressing fdmC, encoding a putative ketoreductase, concomitantly with fdmR1. Taken together, these studies demonstrate the viability of engineering FDM biosynthesis and improving FDM titers in both the native producer S. griseus and heterologous hosts, such as S. albus and S. lividans. The approach taken capitalizes on FdmR1, a key activator of the FDM biosynthetic machinery.

Dedicated ent-kaurene and ent-atiserene synthases for platensimycin and platencin biosynthesis

Platensimycin (PTM) and platencin (PTN) are potent and selective inhibitors of bacterial and mammalian fatty acid synthases and have emerged as promising drug leads for both antibacterial and antidiabetic therapies. Comparative analysis of the PTM and PTN biosynthetic machineries in Streptomyces platensis MA7327 and MA7339 revealed that the divergence of PTM and PTN biosynthesis is controlled by dedicated ent-kaurene and ent-atiserene synthases, the latter of which represents a new pathway for diterpenoid biosynthesis. The PTM and PTN biosynthetic machineries provide a rare glimpse at how secondary metabolic pathway evolution increases natural product structural diversity and support the wisdom of applying combinatorial biosynthesis methods for the generation of novel PTM and/or PTN analogues, thereby facilitating drug development efforts based on these privileged natural product scaffolds.

The oxidative ring cleavage in jadomycin biosynthesis: a multistep oxygenation cascade in a biosynthetic black box.

The antibiotic jadomycin B is derived from an angucycline intermediate that undergoes oxidative ring cleavage and the unique incorporation of l-isoleucine into its polyketide backbone. To elucidate the enzymes and substrates involved in this key oxygenation event, we have investigated a region of the jad gene cluster that is located immediately downstream of the previously identified oxygenase genes jadF and jadG and contains a third putative oxygenase gene, jadH, as well as a potential hydrolase gene, jadK. Inactivation of jadG and jadH, respectively, led to the accumulation of several shunt products and a novel potential pathway intermediate, named prejadomycin. Production of these angucyclines and the failure to generate a ring-cleavage product in various mutant strains illustrates the complex protein–protein interaction network within the oxygenase subcluster. Furthermore, these results demonstrate that both JadF and JadH display secondary dehydratase activities that contrary to their oxygenase activities, appear to be independent of the respective protein-complex binding partners. The polyketide glycoside antibiotic jadomycin B (2) and its aglycon jadomycin A (1) (Scheme 1) are produced by the soil bacterium Streptomyces venezuelae ISP5230 under stress conditions such as heat shock, phage infection, and particularly ethanol treatment. The jadomycin family possesses a unique nitrogen-containing pentacyclic benz[b]oxazolophenanthridine backbone that has been shown by precursor-directed biosynthesis with various amino acids as well as feeding experiments with C-labeled acetate to derive from the fusion of an l-amino acid, for exam-

Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes

Enediynes are potent antitumor antibiotics that are classified as 9- or 10-membered according to the size of the enediyne core structure. However, almost nothing is known about enediyne core biosynthesis, and the determinants of 9- versus 10-membered enediyne core biosynthetic divergence remain elusive. Previous work identified enediyne-specific polyketide synthases (PKSEs) that can be phylogenetically distinguished as being involved in 9- versus 10-membered enediyne biosynthesis, suggesting that biosynthetic divergence might originate from differing PKSE chemistries. Recent in vitro studies have identified several compounds produced by the PKSE and associated thioesterase (TE), but condition-dependent product profiles make it difficult to ascertain a true catalytic difference between 9- and 10-membered PKSE-TE systems. Here we report that PKSE chemistry does not direct 9- versus 10-membered enediyne core biosynthetic divergence as revealed by comparing the products from three 9-membered and two 10-membered PKSE-TE systems under identical conditions using robust in vivo assays. Three independent experiments support a common catalytic function for 9- and 10-membered PKSEs by the production of a heptaene metabolite from: (i) all five cognate PKSE-TE pairs in Escherichia coli; (ii) the C-1027 and calicheamicin cognate PKSE-TEs in Streptomyces lividans K4-114; and (iii) selected native producers of both 9- and 10-membered enediynes. Furthermore, PKSEs and TEs from different 9- and 10-membered enediyne biosynthetic machineries are freely interchangeable, revealing that 9- versus 10-membered enediyne core biosynthetic divergence occurs beyond the PKSE-TE level. These findings establish a starting point for determining the origins of this biosynthetic divergence.

iso-Migrastatin, migrastatin, and dorrigocin production in Streptomyces platensis NRRL 18993 is governed by a single biosynthetic machinery featuring an acyltransferase-less type I polyketide synthase.

iso-Migrastatin and related glutarimide-containing polyketides are potent inhibitors of tumor cell migration and their implied potential as antimetastatic agents for human cancers has garnered significant attention. Genome scanning of Streptomyces platensis NRRL 18993 unveiled two candidate gene clusters (088D and mgs); each encodes acyltransferase-less type I polyketide synthases commensurate with iso-migrastatin biosynthesis. Both clusters were inactivated by λ-RED-mediated PCR-targeting mutagenesis in S. platensis; iso-migrastatin production was completely abolished in the ΔmgsF mutant SB11012 strain, whereas inactivation of 088D-orf7 yielded the SB11006 strain that exhibited no discernible change in iso-migrastatin biosynthesis. These data indicate that iso-migrastatin production is governed by the mgs cluster. Systematic gene inactivation allowed determination of the precise boundaries of the mgs cluster and the essentiality of the genes within the mgs cluster in iso-migrastatin production. The mgs cluster consists of 11 open reading frames that encode three acyltransferase-less type I polyketide synthases (MgsEFG), one discrete acyltransferase (MgsH), a type II thioesterase (MgsB), three post-PKS tailoring enzymes (MgsIJK), two glutarimide biosynthesis enzymes (MgsCD), and one regulatory protein (MgsA). A model for iso-migrastatin biosynthesis is proposed based on functional assignments derived from bioinformatics and is further supported by the results of in vivo gene inactivation experiments.

Characterization of JadH as an FAD- and NAD(P)H-Dependent Bifunctional Hydroxylase/Dehydrase in Jadomycin Biosynthesis

National Natural Science Foundation of China [30670017]; Ministry of Science and Technology [2007CB707800]

Cloning and sequencing of the kedarcidin biosynthetic gene cluster from Streptoalloteichus sp. ATCC 53650 revealing new insights into biosynthesis of the enediyne family of antitumor antibiotics

Enediyne natural product biosynthesis is characterized by a convergence of multiple pathways, generating unique peripheral moieties that are appended onto the distinctive enediyne core. Kedarcidin (KED) possesses two unique peripheral moieties, a (R)-2-aza-3-chloro-β-tyrosine and an iso-propoxy-bearing 2-naphthonate moiety, as well as two deoxysugars. The appendage pattern of these peripheral moieties to the enediyne core in KED differs from the other enediynes studied to date with respect to stereochemical configuration. To investigate the biosynthesis of these moieties and expand our understanding of enediyne core formation, the biosynthetic gene cluster for KED was cloned from Streptoalloteichus sp. ATCC 53650 and sequenced. Bioinformatics analysis of the ked cluster revealed the presence of the conserved genes encoding for enediyne core biosynthesis, type I and type II polyketide synthase loci likely responsible for 2-aza-l-tyrosine and 3,6,8-trihydroxy-2-naphthonate formation, and enzymes known for deoxysugar biosynthesis. Genes homologous to those responsible for the biosynthesis, activation, and coupling of the l-tyrosine-derived moieties from C-1027 and maduropeptin and of the naphthonate moiety from neocarzinostatin are present in the ked cluster, supporting 2-aza-l-tyrosine and 3,6,8-trihydroxy-2-naphthoic acid as precursors, respectively, for the (R)-2-aza-3-chloro-β-tyrosine and the 2-naphthonate moieties in KED biosynthesis.

Improvement of the enediyne antitumor antibiotic C-1027 production by manipulating its biosynthetic pathway regulation in Streptomyces globisporus.

The production of C-1027 in Streptomyces globisporus was previously increased 2- to 3-fold by manipulating three pathway-specific activators, SgcR1, SgcR2, and SgcR3. In this study, we have further characterized two putative C-1027 regulatory genes, sgcE1 and sgcR, by in vivo inactivation. The HxlR family DNA-binding protein SgcE1 was not essential for C-1027 biosynthesis, since inactivation of sgcE1 showed no effect on C-1027 production. In contrast, the proposed repressive role of the sgcR gene was confirmed by a 3-fold increase in C-1027 production in the ΔsgcR mutant S. globisporus SB1022 strain relative to the wild-type strain. Considering SgcR shows no significant similarity to any protein of known function, it may be representative of a new family of regulatory proteins. Finally, overexpression of the previously characterized activator sgcR1 in S. globisporus SB1022 increased the C-1027 yield to 37.5 ± 7.7 mg/L, which is about 7-fold higher than the wild-type strain.