Abstract:The substrate flexibilities of several cytochrome P450 monooxygenases involved in macrolide biosynthesis were investigated to test their potential for the generation of novel macrolides. PikC hydroxylase in the pikromycin producer Streptomyces venezuelae accepted oleandomycin as an alternative substrate and introduced a hydroxy group at the C-4 position, which is different from the intrinsic C-12 hydroxylation position in the natural substrate. This is the first report of C-4 hydroxylation activity of cytochro… Show more
“…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).…”
Section: Engineering Of Post-pks Modifications and Novel Analogs Thromentioning
confidence: 99%
“…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).…”
Section: Engineering Of Post-pks Modifications and Novel Analogs Thromentioning
Herboxidiene is a polyketide with a diverse range of activities, including herbicidal, anti-cholesterol, and pre-mRNA 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.
“…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).…”
Section: Engineering Of Post-pks Modifications and Novel Analogs Thromentioning
confidence: 99%
“…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).…”
Section: Engineering Of Post-pks Modifications and Novel Analogs Thromentioning
Herboxidiene is a polyketide with a diverse range of activities, including herbicidal, anti-cholesterol, and pre-mRNA 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.
“…Hence, by utilizing the substrate flexible cytochromes for combinatorial biosynthesis novel analogs were generated. An engineered S. venezuelae HK954 mutant strain (with deletion of the last module of pikromycin PKS ( pikAIV ) but intact substrate flexible cytochrome P450, pikC) was used for generating novel hydroxylated analogs of oleandomycin [67]. Similarly, S. venezuelae mutant strain YJ003 blocked in desosamine biosynthesis pathway was used for the expression of substrate flexible cytochrome P450s, EryF from erythromycin gene cluster and OleP from oleandomycin gene cluster to generate novel analogs of 12 and 14 membered macrolactones inherently produced by the host strain (Fig.…”
Section: Combinatorial Biosynthesis For Diversification Of Antibioticsmentioning
confidence: 99%
“…Similarly, S. venezuelae mutant strain YJ003 blocked in desosamine biosynthesis pathway was used for the expression of substrate flexible cytochrome P450s, EryF from erythromycin gene cluster and OleP from oleandomycin gene cluster to generate novel analogs of 12 and 14 membered macrolactones inherently produced by the host strain (Fig. 6) [67]. Fig.…”
Section: Combinatorial Biosynthesis For Diversification Of Antibioticsmentioning
Actinobacteria are characterized as the most prominent producer of natural products (NPs) with pharmaceutical importance. The production of NPs from these actinobacteria is associated with particular biosynthetic gene clusters (BGCs) in these microorganisms. The majority of these BGCs include polyketide synthase (PKS) or non-ribosomal peptide synthase (NRPS) or a combination of both PKS and NRPS. Macrolides compounds contain a core macro-lactone ring (aglycone) decorated with diverse functional groups in their chemical structures. The aglycon is generated by megaenzyme polyketide synthases (PKSs) from diverse acyl-CoA as precursor substrates. Further, post-PKS enzymes are responsible for allocating the structural diversity and functional characteristics for their biological activities. Macrolides are biologically important for their uses in therapeutics as antibiotics, anti-tumor agents, immunosuppressants, anti-parasites and many more. Thus, precise genetic/metabolic engineering of actinobacteria along with the application of various chemical/biological approaches have made it plausible for production of macrolides in industrial scale or generation of their novel derivatives with more effective biological properties. In this review, we have discussed versatile approaches for generating a wide range of macrolide structures by engineering the PKS and post-PKS cascades at either enzyme or cellular level in actinobacteria species, either the native or heterologous producer strains.
“…Among several well-known actinomycete heterologous hosts, a pikromycin (Pik)/methymycin-producing strain, Streptomyces venezuelae ATCC 15439, has recently been developed as a promising host in that it is amenable to genetic manipulation and requires a short culture period (3 to 4 days) for metabolite production compared to other Streptomyces species (7,8,19,23,24). In addition, the presence of two post-polyketide synthase (PKS) tailoring enzymes that have unusual substrate flexibility, DesVII glycosyltransferase (2,3,6,25) and PikC P450 monooxygenase (11,13,18), offers it another advantage as a heterologous host for combinatorial biosynthesis of novel compounds. However, one of the key challenges in developing S. venezuelae as a competent heterologous host is the relatively low production of heterologous polyketides (8).…”
To elevate the production level of heterologous polyketide in Streptomyces venezuelae, an additional copy of the positive regulatory gene pikD was introduced into the pikromycin (Pik) polyketide synthase (PKS) deletion mutant of S. venezuelae ATCC 15439 expressing tylosin PKS genes. The resulting mutant strain showed enhanced production of both tylactone (TL) and desosaminyl tylactone (DesTL) of 2.7-and 17.1-fold, respectively. The notable increase in DesTL production strongly suggested that PikD upregulates the expression of the desosamine (des) biosynthetic gene cluster. In addition, two hydroxylated forms of DesTL were newly detected from the extract of this mutant. These hydroxylated forms presumably resulted from a PikDdependent increase in expression of the pikC gene that encodes P450 hydroxylase. Gene expression analysis by reverse transcriptase PCR and bioconversion experiments of 10-deoxymethynolide, narbonolide, and TL into the corresponding desosaminyl macrolides indicated that PikD is a positive regulator of the des and pikC genes, as well as the Pik PKS genes. These results demonstrate the role of PikD as a pathway-specific positive regulator of the entire Pik biosynthetic pathway and its usefulness in the development of a host-vector system for efficient heterologous production of desosaminyl macrolides and novel hydroxylated compounds.
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