altering the glycosylation pattern of bioactive compounds

Carmen Méndez

José A. Salas*

Dept Biologia Funcional eInstituto Universitario deOncología del Principadode Asturias (IUOPA),Universidad de Oviedo,33006 Oviedo, Spain.*e-mail: [email protected]

Nature produces an amazing number of products

that have useful biological activities, including

compounds with clinical application, such as

antibiotics, antitumor or immunosuppressive

agents, and also compounds with veterinary or

agricultural applications, such as growth promoters,

insecticides, herbicides and antiparasitic agents.

These bioactive compounds are the end products of

complex multistep biosynthetic pathways. Among

microorganisms, approximately three-quarters of

all known bioactive compounds are produced by

members of the actinomycete family, mainly

belonging to the genus Streptomyces1.

Polyketides are probably the most interesting

subgroup of bioactive natural products, because they

have a great diversity of chemical structures and

biological activities2. Commercially important

polyketides include antibiotics (e.g. erythromycin,

rifamycin, tetracyclins and oleandomycin),

antitumor drugs (e.g. doxorubicin, aclacinomycin),

cholesterol-lowering agents (e.g. lovastatin),

immunosuppressive agents (e.g. rapamycin),

insecticides (e.g. spinosyn) and antiparasitic agents

(e.g. avermectins). During the past few years, there

has been a great increase in our knowledge of the

biosynthetic pathways by which microorganisms

produce antibiotics and other bioactive secondary

metabolites. This increase has prompted

researchers in this field to manipulate antibiotic

gene clusters to alter the structure of natural

compounds. This new technology, named

combinatorial biosynthesis3, manipulates genes in

natural-product biosynthesis pathways as a way of

producing natural-product analogs and thus

forming novel derivatives with potential

bioactivity3–8.

An important source of structural biodiversity in

natural products are the sugars that are attached to

specific positions on the aglycon core. These sugar

components usually participate in the molecular

recognition of its cellular target9. Their presence is

Many bioactive natural products are glycosylated compounds in which the

sugars are important or essential for biological activity. The isolation of

several sugar biosynthesis gene clusters and glycosyltransferases from

different antibiotic-producing organisms, and the increasing knowledge

about these biosynthetic pathways opens up the possibility of generating

novel bioactive compounds through combinatorial biosynthesis in the near

future. Recent advances in this area indicate that antibiotic

glycosyltransferases show some substrate flexibility that might allow us to

alter the types of sugar transferred to the different aglycons or, less

frequently, to change the position of its attachment.

Altering the glycosylation pattern of

bioactive compounds

Carmen Méndez and José A. Salas

TRENDS in Biotechnology Vol.19 No.11 November 2001

http://tibtech.trends.com 0167-7799/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(01)01765-6

449Review

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70 Blaser, H.U. and Spindler, F. (1999) The chiral

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biotransformations, Wiley-VCH

Review

therefore important for the biological activity of the

compounds, even, in many cases, essential9. These

sugars can be linked to the aglycon as

monosaccharides, disaccharides or oligosaccharides

of variable sugar length through C-, N- or (most

often) O-glycosylation (Fig. 1). These sugars are

transferred to the corresponding aglycon by

glycosyltransferases, which are generally sugar,

aglycon and site specific. The importance of these

sugar moieties in biological activity has led to the

idea of using combinatorial biosynthesis to

incorporate different sugars into an aglycon or to

glycosylate a different position of the aglycon. This

requires an understanding of how these sugars are

synthesized and knowledge about the

glycosyltransferases responsible for sugar transfer

and their specificity. Great progress has been made

in the identification and characterization of genes

involved in the biosynthesis of these sugar moieties

and in the isolation of genes coding for

glycosyltransferases from many different

antibiotic-producing microorganisms.

Sugar biosynthesis genes

The most abundant and diverse group of secondary

carbohydrate moieties in bioactive compounds are

the 6-deoxyhexoses (6DOHs), which occur in a wide

range of plants, fungi and bacteria; 70 different

6DOHs form part of bioactive compounds10–12

(Table 1). The 6DOHs are formed from nucleoside

diphosphate-activated hexoses (mainly D-glucose)

via a 4-keto-6-deoxy intermediate. The two common

enzymatic steps leading to the biosynthesis of this

intermediate are catalyzed by a dNDP–D-hexose

synthase and a dNDP–D-hexose-4,6-dehydratase,

and structural diversification into either D or L forms

is the result of the action of a 5- or a 3,5-epimerase.

The different 6DOHs will also vary depending on the

substituents and/or the stereochemistry of carbon

atoms at positions 2–5 of the hexose carbon

chain13–15. These typically include further

deoxygenations, transaminations and C-, N- or

O-methylations, thus generating the large family of

6DOHs (Fig. 2).

More and more genes involved in the

biosynthesis of various 6DOHs from antibiotic-

producing actinomycetes are being reported

(Table 1). The involvement of these genes in sugar

biosynthesis has usually been proved by the

insertional inactivation of individual genes and the

isolation of biosynthetic intermediates that either

lack the sugar or possess a modified sugar linked to

the aglycon. For most of them, the assignment of a

role for their gene products in 6DOH biosynthesis

has been made by comparison with similar proteins

in databases. So far, evidence has been provided in

only a few cases to show which set of genes are

necessary to direct the biosynthesis of the sugar.

This is the case for L-daunosamine in the

daunorubicin producer Streptomyces peucetius16,


http://tibtech.trends.com

450 Review

TRENDS in Biotechnology

OH O

OHO

O

O

OH

OCH3

CH3

OCH3

OHNH2

OH OOH

H3CO

O

CH3

OHO

OH

H

CH3

O OOOO

OHOHCH3

CH3CH3CH3

OH

HO

OOO

CH3OH

O

CH3OH

OH

O

O

O

CH3

CH3

O

CH3

OH

CH3

O

OHCH3

OHCH3

CH3

O CH3

N(CH3)2OH

OCH3

OHCH3

OMe

Doxorubicin

Mithramycin

Erythromycin A

Urdamycin AO

OHOH

CH3

O

O

O

CH3

OOHCH3

OOH

O CH3

OH

OH

OO

OCH3

OH

O OH

OH

OMe

OH

Rebeccamycin

NH

N NH

O O

Cl Cl

Fig. 1.The chemicalstructures of someglycosylated bioactivecompounds produced byactinomycetes. Examples areshown that differ in thenumber of sugars(monosaccharides such aserythromycin A, doxorubicin,urdamycin A andrebeccamycin, disaccharidessuch as mithramycin, andtrisaccharides such asmithramycin and urdamycinA [mithramycin contains botha di- and a trisaccharideattached to the aglycon andurdamycin A contains amono- and a trisaccharideattached to the aglycon]) andthe linkage to the aglycon(O-glycosylation as inerythromycin A, mithramycin,doxorubicin and urdamycin A,C-glycosylation as inurdamycin A, andN-glycosylation as inrebeccamycin).

Table 1. 6DOH gene clusters from bioactive compounds that have been totally

or partially identified and characterized

6DOH Bioactive compound Producer organisma Refs

D-Desosamine Erythromycin Sacc. erythraea 29,40,50

Oleandomycin S. antibioticus 17,26,39

Pikromycin S. venezuelae 51

Megalomicin M. megalomicea 52

D-Olivose Mithramycin S. argillaceus 20,31

Urdamycin S. fradiae 32

Landomycin S. cyanogenus 53

D-Oliose Mithramycin S. argillaceus 20,31

D-Mycarose Mithramycin S. argillaceus 20,31

D-Mycaminose Tylosin S. fradiae 19,41

D-Mycinose Tylosin S. fradiae 54

D-Mycosamine Nystatin S. noursei 55

L-Dihydrostreptose Streptomycin S. griseus 14

L-Oleandrose Oleandomycin S. antibioticus 17,39

Avermectin S. avermitilis 56

L-Mycarose Erythromycin Sacc. erythraea 29,49,50

Megalomicin M. megalomicea 52

Tylosin S. fradiae 19,41

L-Noviose Novobiocin S. spheroides 57

L-Rhodinose Urdamycin S. fradiae 32

Landomycin S. cyanogenus 53

Granaticin S. violaceoruber 58

L-Daunosamine Daunorubicin S. peucetius 59,60

L-Nogalose Nogalamycin S. nogalater 61

L-Megosamineb Megalomicin M. megalomicea 52

L-Rhodosamine Aclacinomycins S. galilaeus 62

L-Epivancosamine Chloroeremomycin A. orientalis 63

2-Deoxy-L-fucose Aclacinomycins S. galilaeus 62aAbbreviations: A., Amycolatopsis; M., Micromonospora; Sacc., Saccharopolyspora; S., Streptomyces.bAlso named L-rhodosamine.

L-olivose and L-oleandrose in the oleandomycin

producer Streptomyces antibioticus17, and

L-oleandrose in the avermectin producer

Streptomyces avermitilis18. Plasmid constructs were

generated containing all the genes involved in the

biosynthesis of these 6DOHs and shown to

incorporate these sugars into the corresponding

aglycon in the presence of the respective

glycosyltransferases. Some sugar gene clusters have

been cloned and characterized from more than one

producer organism, including the amino sugar

D-desosamine, whose biosynthesis gene cluster has

been cloned from erythromycin-, oleandomycin-,

pikromycin- and megalomicin-producing organisms.

In a few (but increasing) cases, the activity of the

gene products on different sugar intermediates has

been shown using in vitro enzymatic assays19–23.

Recently, all enzymes involved in the biosynthesis

of L-epivancosamine from the chloroeremomycin

pathway have been expressed in Escherichia coli,

purified and characterized for their enzymatic

activities23. This is the first report of the

reconstitution of TDP–deoxysugar biosynthesis

from the individual enzymes from an antibiotic

biosynthetic pathway.

Glycosyltransferase genes

With the increasing knowledge of the organization of

antibiotic-biosynthesis gene clusters, many genes

encoding glycosyltransferases are being reported.

More than 50 glycosyltransferase genes have been

reported from antibiotic-producing actinomycetes.

All of these belong to glycosyltransferase family 1 in

the Henrissat classification24 (Fig. 3). Most

glycosyltransferases share a conserved motif region

(glycine-rich) close to the C terminus that is

characteristic of UDP–glycosyl and

UDP–glucuronosyl transferases. Four different

desosaminyl glycosyltransferases from various

antibiotic-producing organisms have been identified.

These have amino acid identities ranging from 50.1%

to 53.8% (with the exception of EryCIII and MegCIII,

which share 83.4% identical amino acids but

recognize the same aglycon). It is worth mentioning

that two urdamycin-A glycosyltransferases

(UrdGT1b and UrdGT1c) show a very high degree of

similarity: they share 91% identical amino acids and

only differ in a few amino acids that are mostly

located within a 31-amino acid region25.

Interestingly, these two glycosyltransferases

transfer different 6DOHs: UrdGT1c incorporates

L-rhodinose and UrdTG1b transfers D-olivose.

Three other glycosyltransferases have been

described in antibiotic producers: OleD and OleI

from the oleandomycin producer S. antibioticus26

and MgtA from the spiramycin producer

Streptomyces ambofaciens27. These transferases do

not incorporate any sugar present in the final

antibiotic molecule but rather glycosylate the

antibiotic (or a biosynthetic intermediate), leading

to its inactivation, and therefore behave as self-

resistance mechanisms.

Recently, the glucosyltransferase GtfB has been

crystallized and its structure has been determined by

X-ray analysis at 1.8 Å resolution28. This

glucosyltransferase transfers the glucose residue

from UDP–glucose to the vancomycin aglycon. The

enzyme has been shown to have a two-domain

structure, with a deep interdomain cleft that

probably contains the UDP–glucose-binding site.

Determining the structure of this transferase will

allow us to get further insight into the specificity of

the glycosyltransferases and will facilitate

engineering of antibiotics through the incorporation

of novel sugars.

Using producer organisms as cell factories

Microorganisms producing glycosylated compounds

can be used as cell factories to generate novel

glycosylated compounds. This strategy takes

advantage of the existence of an intracellular pool of

nucleotide-activated sugars and intermediates in

these microorganisms. Evidence of the ability of

some antibiotic biosynthesis glycosyltransferases to

incorporate different sugars (‘sugar flexibility’)

came from insertional inactivation experiments of

genes that encode enzymes involved in sugar

biosynthesis. To assign functions to putative sugar

biosynthesis genes, knockout mutants are

frequently generated either by inserting an

antibiotic resistance marker within the coding

region of a selected gene or by ‘in-frame’ deletion of

part of the gene. This approach usually leads to the



451Review


O

OH

OH

CH3

O

OH

OH

CH3

NH2

L-Rhodinose L-Daunosamine

O

OH

OH

CH3

N(CH3)2

L-Rhodosamine

O

OH

OHCH3OH

CH3

L-Mycarose

O

OH

OH

CH3

NH2 OH

O

OH

NH2

CH3

OH OH

O

OH

OH

CH3

OMe OMe

D-MycosamineD-Perosamine

D-Mycinose

O

OH

OH

CH3

N(CH3)2

OH

D-Mycaminose

O

OH

CH3

N(CH3)2

OH

D-Desosamine

O

OH

OH

CH3

OH OH

CH3

D-Mycarose

O

OH

OH

CH3

OH

D-Olivose

O

OH

OHCH3

OH

D-Oliose

O

OH

N(CH3)2

CH3

D-Forosamine

O

OH

CH3

OH

OMe

D-Chalcose

D-Deoxyhexoses

L-Deoxyhexoses

O

OH

OHCH3

OMe

L-Oleandrose

O

OH

OHCH3

CH3

OMe

L-Cladinose

O

OH

OHCH3

OH OH

CH3

L-Nogalose

O

OH

OH

CH3

OH

L-Oliose

O

OH

OHCH3

OH OH

CH3

L-Noviose

O

OH

OHCH3CH3

NH2

L-Epivancosamine

Fig. 2. The structures ofsome D- and L-6-deoxyhexoses that areused to glycosylatebioactive compounds.

generation of nonproducing mutants, many of which

accumulate biosynthesis intermediates that lack

the sugar owing to the inability of the

glycosyltransferase to recognize and transfer the

synthesized sugar biosynthetic intermediate.

However, in some cases, the glycosyltransferases

are able to transfer (although usually less

efficiently) some sugar biosynthesis intermediates

to the aglycon. This type of mutant has been

generated in erythromycin29,30, mithramycin31,

urdamycin32 and methymycin and/or

pikromycin33–35 producers, and the new compounds

accumulated by these mutants have been purified

and their structures elucidated (Fig. 4).

Mutants affected in specific sugar biosynthesis

genes can also be used as hosts for the expression of

genes involved in sugar biosynthesis in other

pathways. These recombinant strains can synthesize

novel sugar derivatives through the joint action of the

host genes and the newly acquired genes. An

outstanding and ingenious strategy was designed for

the production of the antitumor drugs epirubicin

(4′-epidoxorubicin) and 4′-epidaunorubicin36. These

are important cancer chemotherapy drugs that are

usually produced by low-yielding semisynthetic

processes in which some chemical modification steps

follow the fermentative production of doxorubicin and

daunorubicin by S. peucetius. Combining gene

inactivation and gene expression led to a less time-

consuming and less expensive procedure to produce

these two compounds. Doxorubicin and daunorubicin

differ from epirubicin and 4′-epidaunorubicin only in

the configuration at C4 of the sugar moiety,

L-daunosamine. A mutant in dnmV (which encodes a

4-ketoreductase) produces a 4-keto intermediate. One

of two other genes from different sugar pathways

(eryBIV or avrE from the L-mycarose and L-oleandrose

sugar pathways, respectively), both encoding

4-ketoreductases with different diastereoselectivity

at C4 from that of the daunosamine ketoreductase,

were then expressed independently in this mutant. In

this way, the 4′-epimeric anthracycline derivatives

were formed and recovered from the fermentation

broths with satisfactory yield. One of the implications

of this experiment was the demonstration that the

daunosaminyl glycosyltransferase could recognize

both C4 epimers of this sugar (L-daunosamine and

L-epidaunosamine).

Transfer of glycosyltransferases between species

of antibiotic producers can be another strategy to

promote the incorporation of different sugars into

an aglycon. This is usually achieved by transferring

glycosyltransferases between organisms producing

structurally related bioactive compounds. The host

strain will act as a cell factory providing the source

of nucleotide-activated sugars for the glycosylation

events. The aglycon can be synthesized

endogenously by the host strain, either governing its

biosynthesis by chromosomal genes from the host or

by exogenous genes acquired by the incorporation of

an appropriate replicative or integrative vector

harboring the coding genes. Alternatively, the

aglycon can be fed to the engineered strain. The first

example of this type of strategy was the production

of a hybrid glycopeptide antibiotic by expression of

the glycosyltransferase gene gtfE′ from the

vancomycin producer Amycolatopsis orientalis

C329.4 into the producer of the nonglycosylated

glycopeptide A47934 Streptomyces toyocaensis37.

This resulted in the formation of a new glucosyl

A47934 derivative.



452 Review

SpcFSpcGNysDIPimKBgtfAGtfABgtfBGtfBGtfEBgtfCGtfCGtfDTylNElmGTSnogDLanGT2UrdGT2Gra-orf14CumHNovNMtmGIILanGT4UrdGT1aDauHDnrHAknKEryBVMegBVAknSSnogEDnrSDesVIITylMIIEryCIIIMegCIIIMegDIOleG1OleG2MtmGIIISnogZUrdGT1bUrdGT1cLanGT1LanGT3MtmGIMtmGIVGimAOleDOleIAveBITylCVNgtAMitBArd-orf5


Fig. 3. A dendrogram showing the relationships among differentglycosyltransferases from antibiotic-producing actinomycetes. Theseglycosyltransferases are involved in the biosynthesis of the differentbioactive compounds mentioned (glycosyltransferases, products):SpcF and SpcG, spectinomycin (accession number U70376); NysDI,nystatin55; PimK, pimaricin64; BgtfA, BgtfB and BgtfC, balhimycin65;GtfA, GtfB and GtfC, chloroeremomycin63; GtfD and GtfE, vancomycin37;TylN, TylM2 and TylCV, tylosin41,54,66; ElmGT, elloramycin42; SnogD,SnogE and SnogZ, nogalamycin (accession number AF187532);LanGT1, LanGT2, LanGT3 and LanGT4, landomycin53; UrdGT1a,UrdGT1b, UrdGT1c and UrdGT2, urdamycin25,67; Gra-orf14,granaticin58; CumH, coumermycin A1 (Ref. 68); NovN, novobiocin57;MtmGI, MtmGII, MtmGIII and MtmGIV, mithramycin46,69; DauH,daunomycin70; DnrS, daunorubicin/doxorubicin71; DnrH, baumycin59;AknK and AknS, aclacinomycin62; EryBV and EryCIII, erythromycin50;DesVII, pikromycin51; MegDI, MegBV and MegCIII, megalomycin52;OleG1 and OleG2, oleandomycin39; GimA, spiramycin72; OleD and OleI,oleandomycin26; AveBI, avermectin56; Ngt, rebeccamycin73; Ard-orf5,antibiotic A201A (Ref. 74); MitB, mitomycin75.

Other interesting examples of the formation of

novel compounds by heterologous expression of

glycosyltransferases are the production of novel

macrolide derivatives. In this case, the

glycosyltransferases transferred different sugars

from those they usually did. One example is the

formation of a new erythromycin derivative by

heterologous expression of a glycosyltransferase

from the oleandomycin producer38 (Fig. 5a). The

oleGII gene from S. antibioticus (oleandomycin

producer) codes for an oleandrosyl

glycosyltransferase responsible for the transfer of

this sugar into the 3-hydroxy group of the

8,8a-deoxyoleandolide aglycon17,39. In the

erythromycin producer Saccharopolyspora

erythraea, the EryBV transferase mediates the

transfer of L-mycarose into the same position of the

6-deoxyerythronolide aglycon. Expression of oleGII

in an eryBV deletion mutant produced, with a good

yield, several new active compounds in which the

neutral sugar L-rhamnose was attached to position

C3 of the aglycon. One of these new compounds was

identified as 3-rhamnosyl-6-deoxyerythromycin B,

which showed antibiotic activity38 (Fig. 5a).

The second example involves the formation of a

tylosin derivative in an erythromycin mutant40

(Fig. 5b). The tylM2 glycosyltransferase gene from

Streptomyces fradiae is responsible for the

incorporation of mycaminose during tylosin

biosynthesis41. The ability of this transferase to use

a different amino sugar was examined by

integrating the tylM2 glycosyltransferase gene into

the chromosome of a S. erythraea triple mutant

SGT2, in which the genes coding for the polyketide

synthase and the mycarosyl and desosaminyl

transferases were deleted40. This mutant still

retains the ability to synthesize L-mycarose and

D-desosamine. By feeding this recombinant strain

with the 16-membered aglycone tylactone (the

TylM2 transferase substrate), a new hybrid

glycoside was produced and identified as

5-O-desosaminyl-tylactone (Fig. 5b), indicating

that TylM2 can recognize and transfer a different

amino sugar40. These two examples illustrate the

existence of some sugar flexibility in antibiotic

glycosyltransferases.

The ElmGT glycosyltransferase from S. olivaceus

is an interesting example of a glycosyltransferase

that can transfer different deoxysugars. This

transferase incorporates an L-rhamnose moiety

(which is later permethylated) to the elloramycinone

aglycon during the biosynthesis of the anthracycline-

like antitumor drug elloramycin42. ElmGT has a

broader substrate specificity range than any other

glycosyltransferase from antibiotic biosynthesis

pathways; it can transfer to its natural substrate

aglycon (8-demethyl-tetracenomycin C) L-sugars

(L-rhamnose, L-olivose or L-rhodinose), D-sugars

(D-olivose or D-mycarose) and even a D-olivose

disaccharide42–45 (Fig. 6). The transfer of the

D-diolivosyl disaccharide was achieved by expression

of elmGT in the producer of the antitumor drug

mithramycin (S. argillaceus) fed with the precursor

8-demethyl-tetracenomycin C (Ref. 42). Formation of

this diglycosylated derivative did not occur in a

mutant of the mithramycin producer in which all four

mithramycin glycosyltransferases were

simultaneously deleted. However, interestingly, the

diolivosyl derivative was formed through the

simultaneous expression of the genes encoding two



453Review


O

CH3

OH

CH3

O

O

CH3

CH3

OCH3

O

CH3

OH OH

O CH3

CH3

O

OH N

CH3

CH3CH3

CH3

OH

O

O

OH

OCH3

OH OH

OH

OO

CH3CH3

OOOOH

O

CH3CH3OH

O

Sacc. erythraea eryBIV-minus mutant

OCH3

OH

OOH

O

OHCH3

O CH3

OH

OH

OO

S. fradiae urdR-minus mutantS. argillaceus mtmC-minus mutant

O

O

O

CH3

CH3CH3

CH3CH3

O OOH

CH3OH

OH

S. venezuelae desI-minus mutant

Fig. 4. Someglycosylated derivativesgenerated by inactivationof sugar biosynthesisgenes in the erythromycinproducerSaccharopolysporaerythraea, in themethymycin/pikromycinproducer Streptomycesvenezuelae, in themithramycin producerStreptomyces argillaceusand in the urdamycinproducer Streptomycesfradiae. The proposedfunctions for the deducedproducts of theinactivated genes are:eryBIV, 4-ketoreductase30;desI, 4-dehydrase33;mtmC, 3-C-methyltransferase31;urdR, 4-ketoreductase32.


S. erythraea BV88 (eryBV–)(+oleGII)

Sugars

oleGIIGTF

3-O-rhamnosyl-6-deoxyerythromycin B

S. erythraea SGT2(eryA–, eryBV–, eryCIII–)

(+tylM2)

Sugars

tylM2

GTF

5-O-desosaminyl-tylactone

O

O

CH3

CH3 O

OH

CH3

CH3

CH3

CH3OH

O

O

CH3

CH3 O

OH

CH3

CH3

CH3

CH3

O OOH N(CH3)2

CH3

O

CH3

OH

CH3

O

O

CH3

CH3

CH3

CH3

CH3OH

OH

(a) (b)

O

CH3

OH

CH3

O

O

CH3

CH3

OCH3

CH3

OOH N(CH3)2

CH3CH3

O

O CH3

OHOH

OH

Tylactone

S. argillaceus M3∆MG(+elmGT, mtmGI, mtmGII)

GTFs

D-Diolivosyl-tetracenomycin C

(c)

CH3 OH O O

O

OH

OH

H3COOC

OCH3

OHHO

elmGT

8-Demethyl-tetracenomycin C

D-olivose

GII GI

D-diolivose

OCH3OHOCH3

OHOH O

CH3 OH O O

OOH

H3COOC

OHO

OH

OCH3

Fig. 5. Generation of glycosylated derivatives by interspecific expression of glycosyltransferases.(a) Formation of an erythromycin derivative in a glycosyltransferase-deficient mutant ofSaccharopolyspora erythraea by expression of the oleandomycin oleGII glycosyltransferase gene38.(b) Formation of a tylosin derivative in a polyketide-synthase- and glycosyltransferase-deficientmutant of S. erythraea by expression of the tylosin tylM2 glycosyltransferase gene40. (c) Formation ofa diglycosylated tetracenomycin C derivative in a glycosyltransferase-deficient mutant ofStreptomyces argillaceus by the simultaneous expression of the mithramycin mtmGI and mtmGIIglycosyltransferase genes, and the elloramycin elmGT glycosyltransferase gene42. The aglycon isendogenously synthesized (a) or exogenously supplied by feeding (b,c).

mithramycin transferases (MtmGI and MtmGII) and

the elloramycin transferase (ElmGT) (Fig. 5c). These

two mithramycin glycosyltransferases cooperate in

the formation and transfer of a diolivosyl disaccharide

during the biosynthesis of mithramycin46. Production

of this diglycosylated derivative is the first example of

the use of combinatorial biosynthesis of

glycosyltransferases to create novel glycoside

antibiotics through the cooperative and joint action of

three glycosyltransferases42.

The different sugars transferred by the

glycosyltransferases described in all the examples

shown were incorporated at the position of the

aglycon at which the glycosyltransferase usually

acts. It is an attractive idea to use genetic

engineering to alter the position of sugar

attachment. This has been recently achieved by

making novel hybrid compounds between the

antitumor drugs urdamycin and mithramycin.

UrdGT2 is a glycosyltransferase from the urdamycin

producer S. fradiae Tü2717, which catalyzes the

transfer of D-olivose to the urdamycin aglycon

through a C-glycoside linkage47. The urdGT2 gene

was expressed from a multicopy plasmid in

a mithramycin-non-producing mutant of

S. argillaceus. This mutant, M3∆MG, lacks all the

mithramycin glycosyltransferases48. However, it

contains all the enzymatic machinery necessary for

the biosynthesis of the sugars forming part of the

mithramycin molecule (D-olivose, D-oliose and

D-mycarose) and the ability to synthesize the

mithramycin tetracyclic aglycon. Introduction of the

urdGT2 gene in this mutant caused the formation of

two novel compounds containing the mithramycin

aglycon 4-demethyl-premithramycinone and either

D-olivose or D-mycarose attached through a

C-glycoside linkage to a position other than that at

which mithramycin is usually glycosylated

(A. Trefzer et al., unpublished). The fact that a

different aglycon (4-demethyl-premithramycinone)

and a different 6DOH (D-mycarose) were substrates

for UrdGT2 is another example of the flexibility of

antibiotic glycosyltransferases.

Using nonproducing hosts as cell factories

Appropriate recombinant strains can be constructed

using genetic manipulation to give them the ability

to produce the necessary antibiotic building blocks.

Consequently, they can be used as alternative,

useful cell factories for producing novel compounds.

In these recombinant strains, the aglycon can be

synthesized by the host strain through the

incorporation of a plasmid harboring the genes

necessary for aglycon biosynthesis. Alternatively,

the recombinant strain can be used as a

biotransformation host and the aglycon added to the

culture medium. Selected glycosyltransferases and

the ability to synthesize sugars can be supplied to

the host strains by transformation with one or more

plasmids harboring the corresponding gene sets.

Some plasmids directing the biosynthesis of

different deoxyhexoses have been developed during

the past few years16,17,45 as important tools for the

generation of new glycosylated compounds.

Several outstanding approaches have been

undertaken in this area. The macrolide antibiotic

oleandomycin contains the neutral sugar

L-oleandrose in its molecule and all the genes

involved in its biosynthesis have been identified17,39.

Using different subsets of L-oleandrose biosynthesis

genes, three plasmids were constructed, each

containing the genes encoding the enzymatic

functions required to direct the biosynthesis of a

different sugar, L-rhamnose45, L-olivose or

L-oleandrose17. A biotransformation host was

constructed using Streptomyces albus in which the

oleGII glycosyltransferase gene was stably integrated

into the chromosome. When the plasmids coding for



454 Review


CH3 OH O O

O

OR1

OH

H3COOC

OCH3

OH

OR2

OCH3

OH

OHOH

OCH3

OH

OCH3

OH

OH

OCH3

OH

MeO

OCH3

MeO

MeO

OCH3

OHOH

OCH3

OH

OH

CH3

OCH3

OHOCH3

OHOH

O

R2 R2

L-Rhamnose

L-Rhodinose

L-Olivose

L-Oleandrose

3,4-Dimethoxy-L-olivose

D-Olivose

D-Mycarose

D-Diolivose

Fig. 6. Structures of8-demethyl-tetracenomycin Cderivatives generated withthe participation of theElmGT glycosyltransferasefrom Streptomycesolivaceus. The ElmGTglycosyltransferase hasbeen shown to be able torecognize and transferdifferent monosaccharidesand a disaccharide to theelloramycin aglycon42,43,45.


S. albus(+oleGII, pOLE)

L-Oleandrose

GTF

pOLEoleGII

L-Oleandrosyl-erythronolide B

O

CH3

OH

CH3

O

O

CH3

CH3

CH3

CH3

CH3OH

OH

OH

(a)

Erythronolide B

CH3 OH O O

O

OH

OH

H3COOC

OCH3OH

OH

S. albus(+elmGT, pOLV)

L-Olivose

GTF

pOLVelmGT

L-Olivosyl-tetracenomycin C

(b)

8-Demethyl-tetracenomycin C

O

CH3

OH

CH3

O

O

CH3

CH3

CH3

CH3

CH3O

OH

O CH3OHOMe

OOH

CH3 OH O O

OOH

H3COOC

OHO

OH

OCH3

OH

CH3

Fig. 7. The use of genetically engineered biotransformation hosts for the generation of novelglycosylated derivatives. (a) Formation of a glycosylated derivative from erythronolide B in aStreptomyces albus strain harboring the oleandomycin oleGII glycosyltransferase gene and aplasmid (pOLE) directing the biosynthesis of L-oleandrose17. (b) Formation of a tetracenomycin Cderivative in an S. albus strain harboring the elloramycin elmGT glycosyltransferase gene and aplasmid (pOLV) directing the biosynthesis of L-olivose42. In both cases, the precursor was suppliedby feeding.

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455Review

the biosynthesis of L-olivose and L-oleandrose were

introduced into this recombinant host, the

erythromycin aglycon erythronolide B was converted

into monoglycosylated derivatives after feeding this

compound to the recombinant strains (Fig. 7a).

The three plasmids in combination with the

ElmGT glycosyltransferase described above were

also used to produce novel hybrid elloramycin

analogs45. In this case, coexpression in Streptomyces

lividans of cosmid 16F4 (coding for the biosynthesis

of the elloramycin intermediate 8-demethyl-

tetracenomycin and containing the elmGT gene) and

any of these three sugar plasmids produced three

new compounds: 8-demethyl-8-α-L-olivosyl-

tetracenomycin C, 8-demethyl-8-α-L-oleandrosyl-

tetracenomycin C and 2′-demethoxy-elloramycin45,

in which the sugar moieties were encoded by the

macrolide oleandomycin genes and the aglycon and

sugar transfer were encoded by the elloramycin

cluster. The ElmGT glycosyltransferase described

above is responsible for the incorporation of the

different sugars, as was confirmed by the formation

of L-olivosyl-tetracenomycin C in a S. albus

recombinant strain containing an L-olivose-

synthesizing plasmid and expressing elmGT when

fed with 8-demethyl-tetracenomycin C (Fig. 7b).

Conclusions

In the near future, glycosylation could be an

attractive target for generating novel antibiotics

because the specific attachment of one or more sugars

is required for the activity of many bioactive

compounds. Understanding the functionality of the

different domains of glycosyltransferases might allow

engineering of the domains that contribute to

substrate specificity, thus making it possible to alter

the specificity of the glycosyltransferases. Recent

advances in the understanding of antibiotic

glycosyltransferases show that substrate flexibility

(mainly related to the sugar transferred) is fairly

common in antibiotic glycosyltransferases from

producer organisms. The availability of in vitro

enzymatic systems for the assay of

glycosyltransferases will also contribute to the

possible enzymatic synthesis of a wide variety of

glycosylated derivatives.

Acknowledgements

Work in our laboratorywas supported by grantsof the Spanish Ministry ofEducation and Sciencethrough the Plan Nacionalen Biotecnologia(BIO97-0771), Plan FEDER(1FD97-0040) and theEuropean Union(BIO4-CT96-0080 andQLK3-CT-1999-00095).We thank all the peoplethat have worked in ourlaboratory for helpfulsuggestions anddiscussions, especiallythose contributing toresearch on sugarbiosynthesis andglycosylation.

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456 Review

altering the glycosylation pattern of bioactive compounds

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