Herboxidiene biosynthesis, production, and structural ...

Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6860-2

MINI-REVIEW

Herboxidiene biosynthesis, production, and structural modifications: prospect for hybrids with related polyketide Anaya Raj Pokhrel 1 & Dipesh Dhakal 1 & Amit Kumar Jha 1 & Jae Kyung Sohng 1

Received: 6 May 2015 / Revised: 13 July 2015 / Accepted: 16 July 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Herboxidiene is a polyketide with a diverse range of activities, including herbicidal, anti-cholesterol, and premRNA splicing inhibitory effects. Thus, production of the compound on the industrial scale is in high demand, and various rational metabolic engineering approaches have been employed to enhance the yield. Directing the precursors and cofactors pool toward the production of polyketide compounds provides a rationale for developing a good host for polyketide production. Due to multiple promising biological activities, the production of a number of herboxidiene derivatives has been attempted in recent years in a search for the key to improve its potency and to introduce new activities. Structural diversification through combinatorial biosynthesis was attempted, utilizing the heterologous expression of substrate-flexible glucosyltransferase (GT) and cytochrome P450 in Streptomyces chromofuscus to generate structurally and functionally diverse derivatives of herboxidiene. The successful attempt confirmed that the strain was amenable to heterologous expression of foreign polyketide synthase (PKS) or post-PKS modification genes, providing the foundation for generating novel or hybrid polyketides.

Keywords Herboxidiene . Polyketide . Enhancement . Combinatorial biosynthesis . Hybrid polyketide Anaya Raj Pokhrel and Dipesh Dhakal contributed equally to this work. * Jae Kyung Sohng [email protected] 1

Department of BT-Convergent Pharmaceutical Engineering, Institute of Biomolecule Reconstruction (iBR), Sun Moon University, 70, Sunmoon-Ro 221, Tangjeong-myeon, Asan, Chungnam 333-708, Republic of Korea

Introduction Herboxidiene aka GEX1A (Fig. 1) is a polyketide, named so due to its structural properties and biological features. The compound consists of a tetrahydropyran acetic acid moiety and a conjugated diene system (Edmunds et al. 1997). Since the identification of herboxidiene in 1992, the compound has been used successfully as herbicide as well as anti-cholesterol and anti-tumor agent. Herboxidiene was first reported as an exceptionally potent selective herbicide active against a variety of weed species, produced from Streptomyces chromofuscus A7847 in 1991 (Miller-Wideman et al. 1992; Isaac et al. 1992). The herboxidiene producer strain S. chromofuscus A7847 is designated as S. chromofuscus ATCC 49982. Another strain of Streptomyces, Streptomyces sp. GEX1 (FERM BP-5347) has been reported to produce herboxidiene and six other analogs (Fig. 2). All of those analogs showed anti-tumor activity in vitro, with herboxidiene showing the most effective cytotoxicity (Sakai et al. 2002a, b). The compounds that display pharmacological properties similar to herboxidiene, such as anti-cholesterol or anti-cancer activities or pre-mRNA splicing inhibition can be considered its functional analogs. Compounds such as lovastatin, tricholstatin A, and TMC49A exhibit anti-cholesterol activity by reducing the plasma cholesterol level. Herboxidiene demonstrates better lowdensity lipoprotein (LDL) receptor upregulation activity than these compounds (Koguchi et al. 1997; Shao et al. 2012). The most promising activity of herboxidiene is as a pre-mRNA splicing inhibitor. Only a handful of natural compounds that showed such activity by targeting the SF3b sub-complex of spliceosome are pladienolides, FR901464, and thailanstatins (Fig. 2). Pladienolide is a 12-membered macrolide polyketide identified from the culture broth of S. platensis Mer-11107

Appl Microbiol Biotechnol

synthase in combination with NRPS, and is structurally distinct from herboxidiene (Nakajima et al. 1996a, b). Thailanstatins are produced by Burkholderia thailandensis MSMB43 by a biosynthetic gene cluster similar to the FR901464. Thailanstatins differ from FR901464 with the lack of a hydroxyl group and the presence of an extra carboxyl moiety. These differences provide thailanstatins with greater stability than FR901464 (Liu et al. 2013).

Paradigm of herboxidiene from herbicide to pre-mRNA splicing inhibitor Fig. 1 Chemical structure of herboxidiene (GEXA1)

targeting the splicing factor SF3b (Kotake et al. 2007). FR901464, isolated from the culture broth of Pseudomonas sp. no. 2663, was found to be produced by AT-less polyketide Fig. 2 Herboxidiene and its structural and functional analogs

New activities in existing compounds are sometimes encountered during their use. For example, epothilones were initially discovered with a narrow anti-fungal activity, but the drug established itself as a prominent anti-cancer compound with effective activity against paclitaxel-resistant tumors (Gerth et al. 1996; Goodin et al. 2004). Similarly, in 1997,


herboxidiene was found to upregulate the expression of the LDL receptor (known to bind cholesterol from plasma for cellular uptake) and improved binding by 30 % (Brown and Goldstein 1986; Koguchi et al. 1997; Sakai et al. 2002a). In 2002, herboxidiene was discovered to have in vivo anti-cancer activities by inducing cell cycle arrest in the G1 and G2/M phases (Sakai et al. 2002b). One of the exons was found to be skipped during the course of splicing (Sakai et al. 2002a), inciting the study of herboxidiene as a pre-mRNA splicing inhibitor. Alternative splicing leads to different isoforms of vascular endothelial growth factor, which is responsible for either inhibiting or facilitating the formation of blood vessels, and is known to be a key event in tumor growth and metastasis (Bonnal et al. 2012). mRNA splicing is performed by spliceosome, a macromolecular complex composed of five small nuclear ribonucleoproteins (snRNPs) (U1, U2, U4, U5, and U6) (Lagisetti et al. 2013). The U2 snRNPs contain splicing factor 3b (SF3b) complex, which is critical for the accurate selection of 3′ splice sites (Gao et al. 2013). Herboxidiene is one of the splicing inhibitors that bind to SAP155 protein of the SF3b complex and impair the function of SF3b (Hasegawa et al. 2011). Small molecules that act by affecting the functions of spliceosome are limited in number (Roybal and Jurica 2010). Because of its multiple promising biological activities, various approaches have been used for the biosynthesis and production of a number of herboxidiene derivatives (Blakemore and Kocieński 1999; Edmunds et al. 2000; Zhang and Panek 2007; Ghosh and Li 2010; Jha et al. 2015).

Biosynthesis of herboxidiene from S. chromofuscus The biosynthetic pathway of herboxidiene from S. chromofuscus ATCC 49982 was reported by Shao et al. 2012. They sequenced the genome of S. chromofuscus to yield 9.6 Mb sequence data (not published) and reported a gene cluster of type I polyketide (PKS) covering a 53 kb region containing seven open reading frames (ORFs) responsible for the biosynthesis of herboxidiene. Three ORFs code for multimodular type I PKS with nine modules, with the first module serving as the loading module. The organization of these nine modules for producing the polyketide backbone is shown in Fig. 3. During herboxidiene biosynthesis, reductive modifications of extender units take place in all of the extension modules except the loading module. The active site of the KS domain of the loading module was found to be occupied by Glu in place of Cys. This is responsible for the decarboxylation of methylmalonyl-ACP to form propionyl-ACP, which is proposed to serve as the starting unit (Bisang et al. 1999). Based on observations of the resulting molecules, ATs in the loading module and in modules 1, 2, 3, 5, and 6 are considered to be specific for methylmalonyl-CoA, while the other ATs are

believed to select malonyl-CoA for chain extension. ACP domain is present in all nine modules containing Ser as a conserved residue required for cofactor binding (Byers and Gong 2007). The KR domains of modules 3, 4, 5, 6, 7, and 8 belong to type B1, whereas KR domain of module 1 is predicted to be a type A1. Herboxidiene PKS contains a total of six DH domains: one domain each in modules 2, 3, 4, 5, 7, and 8. The ER domain is present only in modules 3 and 7. All of the catalytic domains described above are responsible for the formation of a linear 19-carbon nonaketide by chain initiation, elongations, and modifications, which is finally released from polyketide synthase by the thioesterase (TE) domain present at the end of module 8. The TE domain is known to contain the serine as the active residue (Tsai et al. 2002). Cyclization occurs at C7 and C3 of the linear nonaketide chain to give rise to a tetrahydropyran ring, most likely in a spontaneous process, suggesting that the TE domain may have no role in tetrahydropyran ring formation. The obtained polyketide containing the tetrahydropyran ring is sequentially tailored by epoxidase (herE), cytochrome P450 hydroxylase (herG), and methyltransferase (herF) to form herboxidiene (Yu et al. 2013; Yu et al. 2014). HerE is a dedicated epoxidase showing the sequence similarity with PldD of S. platensis Mer-11107. Sequence alignment of HerG with well-characterized cytochrome P450 hydroxylases revealed the presence of a highly conserved EXXR motif (Yu et al. 2014; Hasemann et al. 1995). Threonine260 (T260) residue present in conserved motif(G/A)GX(D/E)T of HerG is responsible for the formation of the oxygen-binding pocket of CYP 450 hydroxylase (Yu et al. 2014; Poulos et al. 1987; Danielson 2002). HerF belongs to the family of S-adenosyl-Lmethionine (SAM)-dependent methyltransferases. HerF shares 48 % identity with MitM, which is involved in mitomycin biosynthesis. In order to identify the sequence of modifications taking place during the biosynthesis of herboxidiene, in vitro studies were performed. The results indicated that 18-deoxy-25-demethyl-herboxidiene is first hydroxylated at C-18 by HerG, followed by methylation at the 25-hydroxyl group to form herboxidiene (Yu et al. 2014). Yu et al. 2013 characterized the methylation of the biosynthetic intermediate 25-demethyl-herboxidiene by HerF, yielding herboxidiene. The preference of HerF for 25-demethylherboxidiene over 18-deoxy-25-demethyl-herboxidiene suggests that the methylation at C25 is the last tailoring step during herboxidiene biosynthesis (Fig. 3) (Yu et al. 2013).

Illustration of biosynthetic mechanism of herboxidiene in comparison to structurally related compounds Pladienolides and herboxidiene are the closest structural and functional analogs with many striking factors in common.


Fig. 3 Biosynthetic pathway of herboxidiene

Both are polyketides with pre-mRNA splicing inhibitory functions (Hasegawa et al. 2011; Kotake et al. 2007) produced by Streptomyces and share a very similar side chain with 12 members containing consecutive dienes and an epoxide (Kumar and Chandrasekhar 2013). Thus, chemical synthesis of the hybrid of pladienolide and herboxidiene has been attempted to improve the efficacy of the inhibition of premRNA splicing. Looking closely into the gene clusters for biosynthesis of herboxidiene and pladienolides, three ORFs from herboxidiene and four ORFs from pladienolide are involved in polyketide biosynthesis (Shao et al. 2012; Machida et al. 2008a). Herboxidiene and pladienolide biosynthetic gene clusters share notable similarities with over 60 % identity in each module, with the exception of the last modules of both PKS and PldAIII enclosing modules 8 and 9 in pladienolide PKS (Fig. 4a). Modules 8 and 9 in pladienolide PKS are responsible for the C3–C6 backbone of the macrolide ring structure of pladienolide as shown in Fig. 4b. In herboxidiene,

which only contains a single tetrahydropyran ring, the biosynthetic gene cluster does not appear to contain the homologue of PldAIII. The last module of pladienolide PKS would be able to process a longer chain than that of herboxidiene PKS, which could be the reason for the lower amino acid identity between these modules. Besides, there seems to be obvious differences in amino acids in the terminal TE domains of these two polyketides. The TE domain from pladienolide PKS is responsible for catalyzing the lactonization of the polyketide chain (Machida et al. 2008b), while the tetrahydropyran ring of herboxidiene is suspected to be formed by simultaneous cyclization (Shao et al. 2012), suggesting that the TE domain of herboxidiene PKS is only responsible for terminating the growing polyketide chain. Additionally, both polyketides are regulated by transcriptional regulators. Herboxidiene is regulated by a putative LacI family transcriptional regulator (Shao et al. 2012), while pladienolide is regulated by a LuxR family transcriptional


Fig. 4 a Comparison of herboxidiene and pladienolide gene cluster. b Pladienolide biosynthetic pathway

regulator (Machida et al. 2008a). Both polyketides are tailored by three enzymes: HerE, HerF, and HerG for herboxidiene biosynthesis and PldB, PldC, and PldD for pladienolide biosynthesis. HerE, HerF, and HerG respectively code for epoxidase (putative), O-methyltransferase, and cytochrome P450 monooxygenase while PldB, PldC, and PldD encode C6-hydrolylase (cytochrome P450), putative acyltransferase, and putative epoxidase, respectively (Shao et al. 2012; Machida et al. 2008b). Rational combination and reconstitution of the biosynthetic machinery to produce any of these two polyketides together in a herboxidiene or pladienolide producing host is likely to result in hybrid analogs. This can be a potential approach for generating the novel biosynthetic analogs, as herboxidiene and pladienolide hybrid have previously been synthesized chemically (Lagisetti et al. 2013). These two

polyketide synthases can be brought together to modify the polyketide biosynthesis process with different approaches such as alteration of domains within modules, joining of intact modules, combining and engineering multimodular subunits, and mutasynthesis. Furthermore, post-PKS modification by enzymes can introduce additional diversity, which can be extended by reconstitution with diverse post-PKS modification enzymes from similar biosynthetic pathways.

Approaches for enhancement of herboxidiene production from S. chromofuscus Secondary metabolites such as herboxidiene with potential utilities in pharmacology, agriculture, or other fields need to


be produced on the industrial scale either by fermentation, chemical synthesis, or semisynthetic approaches. Among these options, large-scale production of drugs from microbial fermentation has been the basis of industrial production for many years (Olano et al. 2008). The application of different Bomics^ techniques, along with the intervention of synthetic biological approaches, provides new opportunities for the optimal production of desired secondary metabolites (Medema et al. 2011; Chaudhary et al. 2013; Nguyen et al. 2012). Enhancement of herboxidiene by optimization of media components, engineering precursor pathways, and cofactor supplies In general, the selection of media components and their optimum level is critical for secondary metabolite production (Greasham 1983). The presence of favorable or unfavorable carbon and nitrogen sources directly affects production (Aharonowitz and Demain 1978; McDowall et al. 1999; Aharonowitz 1980). For enhanced production of herboxidiene, a media optimization strategy was adopted to develop a suitable formulation called medium no. 6A6, with glycerol as the major carbon source resulting yield of ∼0.76 g/ L (Jha et al. 2015). Similar approaches for glycerol utilization and metabolic engineering have been employed for the enhancement of clavulanic acid (Kun et al. 2013; Jnawali et al. 2010; Li and Townsend 2006), teicoplanin (Park et al. 2007), and ε-poly-L-lysine (Chen et al. 2011). Glycerol and ProFlo can be utilized as effective sources for increasing the titer of major precursors for polyketide biogenesis (Fig. 5). Moreover, glycerol utilization has been reported to be superior to glucose in terms of the production of secondary metabolites (Chen and Mao 2013). Polyketides like herboxidiene are produced by type I PKS consisting of multifunctional enzymes whose domains are organized into modules that each control the incorporation of precursors into a polyketide backbone during chain elongation (Shen 2003; Staunton and Weissman 2001). Malonyl-CoA and methyl-malonyl CoA are the most redundantly utilized extender units and are generated from the starting metabolites through a cascade of events (Chan et al. 2009), as shown in Fig. 5. The enhancement of actinorhodin from S. coelicolor is an example of the enhanced production of a polyketide by modification of the precursor supplies. Overexpression of acetyl-CoA carboxylase (ACC) in S. coelicolor resulted in enhanced carbon flux to malonyl-CoA, leading to a six fold increase in actinorhodin production (Ryu et al. 2006). Similarly, heterologous expression of acetyl-CoA carboxylase complex from S. coelicolor A3 (2) in Nocardia sp. CS682 resulted in a substantial increase in the pool of malonyl-CoA (Maharjan et al. 2012), and subsequent feeding led to

the enhancement of nargenicin A1 (Koju et al. 2012; Dhakal et al. 2015). With herboxidiene, the introduction of an expression cassette containing the ACC complex from S. coelicolor A3 (2) in a pSET152 vector (Bierman et al. 1992) reduced the herboxidiene yield. Similarly, chromosomal integration into the ϕC31 attB1 site resulted in a negative effect on the secondary metabolite yield from Saccharopolyspora spinosa and S. toyocaensis (Baltz 1998). It indicates that site-specific integration may sometimes cause pleiotropic effect such as decline in secondary metabolite production. SAM is a ubiquitous methyl donor and a crucial cofactor in the biosynthesis of different secondary metabolites (Fontecave et al. 2004). The biogenesis of SAM is catalyzed by SAM synthase (Fig. 5), which transfers the adenosyl portion of ATP to methionine (Chiang et al. 1996). Heterologous overexpression of the SAM synthase gene metK leads to the improved production of various secondary metabolites from various Streptomyces spp., such as pristinamycin, granaticin, oleandomycin, and avermectin (Huh et al. 2004; Kim et al. 2003; Okamoto et al. 2003). SAM can also activate secondary metabolite production by the global modulation of multiple regulatory components, independent of its role as a methyl donor (Zhao et al. 2006). The heterologous expression of the metK1-sp gene from S. peucetius enhanced the production of pikromycin from S. venezuelae (Maharjan et al. 2008) and that of Nargenicin A1 from Nocardia sp. CS682 (Koju et al. 2012; Dhakal et al. 2015). In herboxidiene, the polyketide is decorated with a methyl ester at 25-OH (Fig. 1) by HerF (Yu et al. 2013). Thus, the overexpression of the metK1-sp gene from S. peucetius in a multicopy plasmid increased herboxidiene production to ∼0.98 g/L (Jha et al. 2014). Modulation of regulatory and biosynthetic genes for the enhancement of herboxidiene The regulation and signal transduction mechanisms by a variety of regulators in response to the availability or depletion of medium components or specific signaling molecules fine tune the metabolic switching of secondary metabolite production (Hwang et al. 2014). The majority of pathway specific activators in actinomycetes belong to the Streptomyces antibiotics regulatory protein (SARP) family (Wietzorrek and Bibb 1997). One of the pleiotropic regulatory genes belonging to the SARP family is the afsR gene from S. coelicolor. This gene can increase actinorhodin and undecylprodigiosin production upon overexpression in S. lividans (Horinouchi et al. 1983. One afsR homolog, afsR-p, characterized from S. peucetius, plays a positive role in doxorubicin biosynthesis (Parajuli et al. 2005). The overexpression of afsR-p in S. lividans, S. clavuligerus, S. griseus, and S. venezuelae leads to the overproduction of actinorhodin, clavulanic acid, streptomycin, and pikromycin, respectively (Parajuli et al. 2005;


Fig. 5 Different strategies employed for enhancement of herboxidiene illustrating the proposed pathways or mechanisms involved. The red color indicates the route for redirection of precursor supplies initiating from glycerol or ProFlo; blue color indicates the modulation in postmodification steps (for e.g., methylation); and green color indicates the

global effect of AfsR and SAM on complete biosynthesis. ACC acetylCoA carboxylase, MCM methylmalonyl-CoA mutase, PCC propionylCoA carboxylase, SAM S-adenosylmethionine, ATP adenosine triphosphate

Maharjan et al. 2008). With S. chromofuscus, the overexpression of afsR-p by genomic integration increased the titer of herboxidiene to ∼1.26 g/L, whereas overexpression based on multicopy plasmid surmounted the herboxidiene production to ∼2.89 g/L, which is the highest titer of this particular secondary metabolite to date (Jha et al. 2014). Besides regulatory genes, structural genes are key players for the biogenesis of all secondary metabolites. Thus, the

overexpression or inactivation of targeted genes to increase the gene dose has been utilized to boost the production of secondary metabolites (Olano et al. 2008). More than that, the replication of multiple copies of complete gene clusters has been an instrumental approach for enhancement (Chaudhary et al. 2013; Li et al. 2015). Simultaneous measurements of all the components involved in metabolic networks facilitate the identification of bottlenecks resulting from


metabolic flux distribution or regulation systems (Breitling et al. 2013). Since the information about the biosynthetic mechanism of herboxidiene is prevailed, such modulation of biosynthetic genes, regulatory genes, or complete biosynthetic gene cluster can be utilized for maximizing the production from S. chromofuscus.

Engineering of post-PKS modifications and novel analogs through post-PKS engineering BCombinatorial biosynthesis^ utilizes the in vivo pathway engineering or genetic manipulation of enzymes for biosynthesis in order to produce new or altered natural products (Menzella and Reeves 2007; Blanchard and Thorson 2006; Floss 2006; Wong and Khosla 2012). Generally, most enzymes, including those involved in biosynthetic pathways, are classically renowned for their precision and ability to act as highly stereoand regio-specific catalysts. However, the substrate promiscuity of some enzymes within biosynthetic pathways is a valuable asset for the diversification of natural products (Patrikainen et al. 2012). The basic carbon framework of polyketides is modified by oxidation to introduce hydroxy or carbonyl groups, methylation at oxygen, nitrogen or carbon centers, or decoration with sugar molecules by discrete enzymes (Staunton and Weissman 2001). These modifications are responsible for the physiochemical properties of natural products such as hydrophobicity, solubility, or the introduction of important functionalities, which are precise for bioactivity (Rix et al. 2002). Thus, the rational engineering of postPKS processing can be exploited for creating structural and functional diversity in polyketides. For example, various glycosyltransferases were assessed for their biocatalytic potential, creating structurally and functionally diverse compounds (Gantt et al. 2011). YjiC, a substrate-flexible glycosyltransferase characterized for its promiscuous activity toward polyphenols and polyketide compounds (Dhakal et al. 2015; Parajuli et al. 2014; Le et al. 2014), was utilized as a suitable enzyme for the generation of 18-O-β-D-glucopyranoside herboxidiene and 18-O-β-D-glucopyranoside-25-demethyl-herboxidiene (Fig. 6) in S. chromofuscus based in vivo cell factory (Jha et al. 2015). Cytochrome P450 can accomplish versatile biocatalytic actions such as aliphatic and aromatic bond hydroxylation, double-bond epoxidation, heterocyclization, aryl and phenolic ring coupling, oxidative rearrangement of the carbon skeleton, and C–C bond cleavage in diverse natural products, all with precise chemo-, regio-, and stereoselectivity (Podust and Sherman 2012). Specifically, oxidations such as hydroxylation and epoxidation catalyzed by cytochrome P450 have been reported to be important for specific bioactivity (Rix et al. 2002). Meanwhile, some of these monooxygenases are promiscuous to non-native substrates for the generation of

novel products with higher or altered biological activities (Yoon et al. 2002; Lee et al. 2005). PikC, a substrate-flexible cytochrome P450 monooxygenase belonging to the pikromycin gene cluster of S. venezuelae, can accept different ring-membered aglycons, YC-17, narbomycin, methymycin, and pikromycin as natural substrates (Zhang and Sherman 2001; Lee et al. 2006). It can also accept diverse unnatural macrolactones (Yoon et al. 2002; Lee et al. 2005; Basnet et al. 2008). The P450 monooxygenase, EryF, from the erythromycin gene cluster of Saccharopolyspora erythraea, is responsible for the hydroxylation of 6-deoxyerythronolide B (Shafiee and Hutchinson 1988) but shows unequivocal flexibility for several substrates related to 6-deoxyerythronolide B (Andersen et al. 1993). Hence, utilizing the fascinating feature of substrate promiscuity and amenability of the heterologous expression of monooxygenase PikC and EryF on S. chromofuscus, herboxidiene was assessed for modification by hydroxylation. Interestingly, two novel isomers of herboxidiene, i.e., herboxidiene A1 and herboxidiene B1, were also isolated and characterized as the products of intramolecular rearrangement. Herboxidiene A1 contained an extra tetrahydrofuran ring, whereas herboxidiene B1 contained an extra tetrahydropyran ring in addition to the single tetrahydropyran present in the parental structure (Fig. 6). Similarly, other novel analogs such as 5-hydroxyherboxidiene A1, 5-hydroxy-25-demethyl-herboxidiene A1, 5,6-dihydroxy-herboxidiene A1, 5,6-dihydroxy-25demethyl-herboxidiene A1, 5-hydroxy-herboxidiene B1, 5hydroxy-25-demethyl-herboxidiene B1, 5,6-dihydroxyherboxidiene B1, and 5,6-dihydroxy-25-demethylherboxidiene B1, were characterized (Fig. 6). The achievement of versatile compounds characterized with putative hydroxylation validates the substrate promiscuity of these monooxygenases for herboxidiene and its biosynthetic intermediates. Similarly, some epoxidized derivatives of herboxidiene were achieved (Fig. 6) using the in vivo catalytic machinery of S. chromofuscus furbished with a heterologous expression system for EpoF, a dedicated epoxidase from the epothilone gene cluster from Sorangium cellolosum, confirming its applicability for combinatorial biosynthesis. The epoxidated compounds produced were herboxidiene C and 25-demethyl-herboxidiene C (Jha et al. 2015). Similarly for pladienolide, which is highly related to herboxidiene, the cytochrome P450-mediated structural modification was manifested by the overexpression of PsmA, a 16-OH hydroxylase specific for pladienolide B from S. bungoensis A-1544. (Machida et al. 2008a) in the native host S. platensis Mer11107, resulting in the formation of pladienolide D (Machida et al. 2009). Thus, a combinatorial biosynthetic approach using substrate-flexible enzymes provides a strong rationale for diversifying the targeted secondary metabolites with beneficial structural and functional properties. Generally, the key


Fig. 6 Different novel analogs of herboxidiene generated by the pathway engineering approaches using substrate-flexible glycosyltransferase and cytochrome P450 monooxygenases. Glycosylated products are obtained with S. chromofuscus containing heterologous expression of GT, Yjic.

Novel hydroxylated derivatives were attained from S. chromofuscus by heterologous expression of PikC or EryF, whereas heterologous expression of EpoF generated epoxidized derivatives

challenge in combinatorial biosynthesis involving modular PKS is the significant reduction in the productivity of the hybrid system, complicating the process of purification and structural characterization. Production with S. chromofuscus in optimized media overcomes such hurdles to some extent (Jha et al. 2014). Hence, it opens up new avenues for a rational modification of herboxidiene by flexible enzymes utilizing the physiological and biochemical machinery of this super host.

editing (Cobb et al. 2014; Fernández-Martínez and Bibb 2014) have revolutionized the scope of metabolic engineering approaches for producing versatile secondary metabolites through host engineering (Pickens et al. 2011; Siegl and Luzhetskyy 2012; Weber et al. 2015). Regarding the feasibility of producing herboxidiene as a major metabolite with a yield surpassing ∼3 g/L (Jha et al. 2014) or its successful modification using different substrate-flexible enzymes (Jha et al. 2015), S. chromofuscus presents itself as an amenable strain for established genetic engineering tools. Genome sequences and their annotation data are important ingredients for genome mining that reveal novel gene clusters associated with the biosynthesis of various secondary metabolites (Nett et al. 2009; Helfrich et al. 2014) in various Streptomyces spp. Some of these metabolites may be cryptic and require the use of suitable heterologous hosts for production in significant quantities (Chaudhary et al. 2013). As a prolific producer, the wildtype strain of S. chromofuscus produces 0.74 g/L of polyketide herboxidiene, indicating that the host bears a naturally higher pool of starting and extension units for polyketide biosynthesis. These foundations establish this strain as a strong candidate that can be developed for efficient production or as a

Future perspectives A stable, well-behaved chassis (host cell) is essential for the production of a desired molecule at a significant level (Keasling 2012). Moreover, the system biological tools can help with the study of the connections between the primary and secondary metabolites of particular strains along with their regulation and signal transduction cascades (Hwang et al. 2014). The current tools for advanced DNA synthesis and assembly of large fragments along with precise techniques for modulation at the gene level and high-throughput genome


heterologous expression host for generating novel or hybrid polyketide analogs of herboxidiene by the engineering of native PKS. Equally, there is enormous potential for better expression of foreign PKS utilizing the higher precursor pool. Utilizing cutting-edge synthetic biological or system biological impetus, S. chromofuscus can be developed into an attractive platform for heterologous production or structural diversification of different secondary metabolites.

Compliance with ethical standards This article does not contain any studies with human participants or animals performed by any of the authors. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (NRF-2014R1A2A2A01002875). Conflict of interest The authors declare that they have no conflict of interest.

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