Insights about the biosynthesis of the avermectin deoxysugar L-oleandrose through heterologous expression of Streptomyces avermitilis deoxysugar genes in Streptomyces lividans

Wohlert, Sven-Eric; Lomovskaya, Natalia; Kulowski, Kerry; Fonstein, Leonid; Occi, James; Gewain, Keith M.; MacNeil, Douglas J.; Hutchinson, C. Richard

doi:10.1016/s1074-5521(01)00043-6

Cited by 54 publications

(43 citation statements)

References 45 publications

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“…Epirubicin (compound 3b) production in the S. venezuelae system required the TDP-4-ketohexose reduction step which confers an equatorial C-4 hydroxyl group to TDP-4-keto-L-daunosamine, the glycosylation step attaching TDP-4-epi-L-daunosamine to ε-RHO, producing 4Ј-epi-rhodomycin D (compound 1b), and the postglycosylation steps leading to epirubicin (compound 3b). Glycosyltransferase/auxiliary protein pairs capable of converting ε-RHO to 4Ј-epi-rhodomycin D (compound 1b) efficiently were first screened using a TDP-4-epi-L-daunosamine biosynthetic gene cassette containing avrE, a gene encoding TDP-4-ketohexose reductase in the L-oleandrose biosynthetic pathway from S. avermitilis (46), in place of dmnV since it has already been reported that avrE can support 4Ј-epi-daunorubicin (compound 2b) production in S. peucetius (24). Four plasmids were constructed: pEDNS1, pEDNS2, pEDNS3, and pEDNS4 carrying aknS/aknT, dnmS/dnmQ, stfG/stfPII, and snogE/snogN, respectively (Table 2).…”

Section: Resultsmentioning

confidence: 99%

Development of a Streptomyces venezuelae-Based Combinatorial Biosynthetic System for the Production of Glycosylated Derivatives of Doxorubicin and Its Biosynthetic Intermediates

Han

Park

Lee

et al. 2011

Appl Environ Microbiol

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Doxorubicin, one of the most widely used anticancer drugs, is composed of a tetracyclic polyketide aglycone and L-daunosamine as a deoxysugar moiety, which acts as an important determinant of its biological activity. This is exemplified by the fewer side effects of semisynthetic epirubicin (4-epi-doxorubicin). An efficient combinatorial biosynthetic system that can convert the exogenous aglycone -rhodomycinone into diverse glycosylated derivatives of doxorubicin or its biosynthetic intermediates, rhodomycin D and daunorubicin, was developed through the use of Streptomyces venezuelae mutants carrying plasmids that direct the biosynthesis of different nucleotide deoxysugars and their transfer onto aglycone, as well as the postglycosylation modifications. This system improved epirubicin production from -rhodomycinone by selecting a substrate flexible glycosyltransferase, AknS, which was able to transfer the unnatural sugar donors and a TDP-4-ketohexose reductase, AvrE, which efficiently supported the biosynthesis of TDP-4-epi-L-daunosamine. Furthermore, a range of doxorubicin analogs containing diverse deoxysugar moieties, seven of which are novel rhodomycin D derivatives, were generated. This provides new insights into the functions of deoxysugar biosynthetic enzymes and demonstrates the potential of the S. venezuelae-based combinatorial biosynthetic system as a simple biological tool for modifying structurally complex sugar moieties attached to anthracyclines as an alternative to chemical syntheses for improving anticancer agents.

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Section: Resultsmentioning

confidence: 99%

Development of a Streptomyces venezuelae-Based Combinatorial Biosynthetic System for the Production of Glycosylated Derivatives of Doxorubicin and Its Biosynthetic Intermediates

Han

Park

Lee

et al. 2011

Appl Environ Microbiol

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“…Interestingly, the producer organisms of these two compounds seem to have evolved two different pathways for synthesis and incorporation of the same sugar into the aglycon. In S. avermitilis, L-oleandrose is synthesized as a nucleotide-activated sugar that is transferred to the aglycon by a glycosyltransferase which incorporates two successive L-oleandrose moieties (12,27). In contrast, the oleandomycin producer S. antibioticus synthesizes the unmethylated derivative, L-olivose, which is transferred to the aglycon by the OleG2 glycosyltransferase and then converted into L-oleandrose by the OleY methyltransferase (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…pQE60 (Qiagen) and pUWL201 (7) were used as expression vectors in E. coli and S. lividans, respectively. pWHM2109 is a plasmid containing all the L-oleandrose genes from the avermectin biosynthetic gene cluster in Streptomyces avermitilis (27). When antibiotic selection of transformants was needed, 100 g of ampicillin/ml, 25 g of thiostrepton/ml, or 25 g of apramycin/ml was used.…”

Section: Methodsmentioning

confidence: 99%

“…In the course of the present work, an L-oleandrose-synthesizing plasmid (pWHM2109) was made available to us. This plasmid contains seven genes (avrCDEFGHI) of the L-oleandrose gene cluster from the avermectin producer S. avermitilis; it directs the biosynthesis of L-oleandrose as shown by the incorporation of this neutral sugar into the avermectin aglycon (27). Protoplasts of S. albus NAG2 were transformed with pOLV, pOLE, and pWHM2109, and selected transformants from each transformation were fed with either EB or OLV-EB for bioconversion.…”

Section: Expression Of Thementioning

confidence: 99%

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Functional Analysis of OleY l -Oleandrosyl 3- O -Methyltransferase of the Oleandomycin Biosynthetic Pathway in Streptomyces antibioticus

Rodrı́guez

Olano

et al. 2001

J Bacteriol

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Oleandomycin, a macrolide antibiotic produced by Streptomyces antibioticus, contains two sugars attached to the aglycon: L-oleandrose and D-desosamine. oleY codes for a methyltransferase involved in the biosynthesis of L-oleandrose. This gene was overexpressed in Escherichia coli to form inclusion bodies and in Streptomyces lividans, producing a soluble protein. S. lividans overexpressing oleY was used as a biotransformation host, and it converted the precursor L-olivosyl-erythronolide B into its 3-O-methylated derivative, L-oleandrosyl-erythronolide B. Two other monoglycosylated derivatives were also substrates for the OleY methyltransferase: L-rhamnosyl-and L-mycarosyl-erythronolide B. OleY methyltransferase was purified yielding a 43-kDa single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme showed a molecular mass of 87 kDa by gel filtration chromatography, indicating that the enzyme acts as a dimer. It showed a narrow pH range for optimal activity, and its activity was clearly stimulated by the presence of several divalent cations, being maximal with Co 2؉ . The S. antibioticus OleG2 glycosyltransferase is proposed to transfer L-olivose to the oleandolide aglycon, which is then converted into L-oleandrose by the OleY methyltransferase. This represents an alternative route for L-oleandrose biosynthesis from that in the avermectin producer Streptomyces avermitilis, in which L-oleandrose is transferred to the aglycon by a glycosyltransferase.Oleandomycin (Fig. 1A) is a clinically important 14-membered macrolide antibiotic produced by Streptomyces antibioticus. Structurally it contains a macrolactone ring (oleandolide) which is synthesized by the assembly of one starter acetylcoenzyme A (CoA) and six extender methylmalonyl CoA units. These reactions are catalyzed by a modular type I polyketide synthase comprising three large polypeptides, named OleA1, OleA2, and OleA3 (24,25). The oleandomycin biosynthetic gene cluster has been fully sequenced and characterized (1,15,16,18,19,24,25) (Fig. 1B). Two DNA regions flanking the genes encoding the three multifunctional polypeptides of the polyketide synthase contain a number of genes that code for other enzymatic functions required for the biosynthesis of the two sugars and their transfer to the aglycon (1, 16, 18), genes encoding an ABC transporter system responsible for secretion (15), genes encoding an inactivating-reactivating enzymatic system that confers self-resistance to the producer organism (18, 26), and a gene encoding a cytochrome P-450 oxygenase involved in epoxidation of the macrolactone ring at carbon 8 (19, 24). The oleandomycin macrolactone ring is glycosylated by two 6-deoxysugars (L-oleandrose and D-desosamine). Two glycosyltransferases (OleG1 and OleG2) are responsible for sugar transfer. Insertional inactivation of oleG1 produced a mutant that accumulates the macrolactone 8,8a-deoxyoleandolide, because both OleG1 and OleG2 glycosyltransferases were affected in this mutant by a polar effect (16). Complementatio...

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“…The PCR product was cloned into ZERO-Blunt (Invitrogen), then a 0?7 kb NsiI-NsiI (one NsiI site is from the vector) fragment containing the ery-ORF5 gene was transferred into NsiI-cut pKOS146-83A (Table 1) to give plasmid pKOS146-101A. pKOS146-83A was made from pUC119 (Vieira & Messing, 1987), in which the HindIII-EcoRI polylinker was replaced by a HindIII-EcoRI fragment containing the actII-ORF4 gene and the divergent actI and actIII promoters from pWHM467 (Wohlert et al, 2001). Three fragments, the EcoRI-PacI fragment of pKOS146-101A, the HindIII-PacI fragment of pKOS146-88A (Table 1) and the EcoRI-HindIII fragment of pKOS146-87B (Table 1) were ligated together and packaged using a GigapackIII-plus (Stratagene) in vitro packaging kit.…”

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confidence: 99%

A specific role of the Saccharopolyspora erythraea thioesterase II gene in the function of modular polyketide synthases

Hu¹,

Pfeifer

Chao

et al. 2003

Microbiology

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Bacterial modular polyketide synthase (PKS) genes are commonly associated with another gene that encodes a thioesterase II (TEII) believed to remove aberrantly loaded substrates from the PKS. Co-expression of the Saccharopolyspora erythraea ery-ORF5 TEII and eryA genes encoding 6-deoxyerythronolide B synthase (DEBS) in Streptomyces hosts eliminated or significantly lowered production of 8,89-deoxyoleandolide [15-nor-6-deoxyerythronolide B (15-nor-6dEB)], which arises from an acetate instead of a propionate starter unit. Disruption of the TEII gene in an industrial Sac. erythraea strain caused a notable amount of 15-norerythromycins to be produced by utilization of an acetate instead of a propionate starter unit and also resulted in moderately lowered production of erythromycin compared with the amount produced by the parental strain. A similar behaviour of the TEII gene was observed in Escherichia coli strains that produce 6dEB and 15-methyl-6dEB. Direct biochemical analysis showed that the ery-ORF5 TEII enzyme favours hydrolysis of acetyl groups bound to the loading acyl carrier protein domain (ACP L ) of DEBS. These results point to a clear role of the TEII enzyme, i.e. removal of a specific type of acyl group from the ACP L domain of the DEBS1 loading module.

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Insights about the biosynthesis of the avermectin deoxysugar L-oleandrose through heterologous expression of Streptomyces avermitilis deoxysugar genes in Streptomyces lividans

Cited by 54 publications

References 45 publications

Development of a Streptomyces venezuelae-Based Combinatorial Biosynthetic System for the Production of Glycosylated Derivatives of Doxorubicin and Its Biosynthetic Intermediates

Development of a Streptomyces venezuelae-Based Combinatorial Biosynthetic System for the Production of Glycosylated Derivatives of Doxorubicin and Its Biosynthetic Intermediates

Functional Analysis of OleY l -Oleandrosyl 3- O -Methyltransferase of the Oleandomycin Biosynthetic Pathway in Streptomyces antibioticus

A specific role of the Saccharopolyspora erythraea thioesterase II gene in the function of modular polyketide synthases

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