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Metabolic engineering by interspecies
protoplast fusion of angucycline
producing streptomycetes
A.Kobylyanskyy 1, A.Luzhetskyy 2 , A.Bechthold
2
and V.Fedorenko
1
Ivan Franko National University, Department of Genetics and Biotechnology,
Hrushevskyy str. 4, 79005 Lviv, Ukraine1,
Albert-Ludwigs-Universität, Institut für Pharmazeutische Wissenschaften,
Stefan-Meier-Straße 19, 79104 Freiburg, Germany2
Protoplast fusion proved itself as an important molecular genetic method widely used for the improvement of secondary metabolite production. It allows
recombination events to occur throughout the genome and at a long range thereby modifying bunches of genes in order to improve it. This signifies reorientation of
attention from directed evolution of single enzymes to optimization of pathways. The approach used in this work involves protoplast fusion of two or more
actinomycete strains, belonging to different species. The particularity is however that the parental strains carry resistance marker insertions in angucycline
biosynthetic genes clusters. The presence of resistance cassettes within the clusters stipulates for the recombination between them in course of protoplast fusion.
The recombination is directed in the way that double resistant fused mutants carry the hymeric cluster, consistent of the fragments of both parental ones. By
applying this technology we aimed to provide the production of novel biologically active secondary metabolites and extend our knowledge about recombination
events and enzyme substrate specificity.
Angucyclic antibiotics are quinone natural polyketide
products bearing a characteristic four-ring frame of the
aglycon moiety, which is assembled in an angular
manner. There are few hundreds of them characterised
so far, all of which to various measure possess
antitumor activity.
For three decades protoplast fusion has been
successfully used to modify the phenotypic traits of
streptomycetes (Fig.1). In contrast to other established
techniques, a high frequency of gene transfer and
recombination can be achieved by fusing complete
protoplast genomes. Recently this process was
optimized towards a recursive multiparental fusion and
combined with high-throughput screening formats and
that greatly accelerated the overall process of strain
development.
Such fused mutants should be able to restore the
missing glycosylation or oxygenation steps owing to
complementation of metabolic pathways by the
enzymes of both parental metabolic pools. Moreover,
the flexibility of post-PKS modifying enzymes allows the
introduction of new functional groups or attachment of
additional sugars in unusual for both parents positions.
The morphology of the majority of obtained fusants
resembled one of the parental strains. Nevertheless,
several of them displayed completely different shape
and color of colonies. The most remarkable were the
recombinants of S. fradiae ∆urdQ/R x S. globisporus
M12 called Rec5 (Fig.2).
In order to ascertain that Rec5 recombinant includes
genes from both parental biosynthetic clusters we
managed to amplify urdGT2 and lndGT1 native
glycosyltransferase genes (Fig.2) from its genomic DNA
1
2
3
4
5
6
7
Lanes:
1.1kb DNA ladder
2. Rec5 with lndGT2 primers
3. Rec5 with urdGT2 primers
Positive controls
4. S. fradiae ΔurdQ/R with urdGT2
5. S. globisporus M12 with lndGT2
Negative controls
6. S. fradiae ΔurdQ/R withlndGT2
7. S. globisporus M12 with urdGT2
1,1
kb
S. globisporus M12
Fig.4. PCR of Rec5 genomic DNA with primers
complementing urdGT2 and lndGT1
S. fradiae ∆urdQ/R
Rec5
To our astonishment, generated Rec5 fused mutant
turned out to produce about 40! different compounds of
angucyclic nature. We succeeded to ascertain the
structure only for some of them (tab.2). Namely we
detected
putative
aquayamycin,
tetrangomycin,
tetrangulol and urdamycinone derivatives.
Tab.2. Some angucyclic products of the
recombinant Rec5 strain
Ret.t [min] Mass [M-H]-
Fig.1. Fused protoplasts of Streptomyces cells
under an electron microscope
The mutant strains recently generated in our
laboratory (table1) have been used in our experiments
of protoplast fusion. All of them carry risistance marker
insertions within different post-polyketidesynthetic
(PKS) tailoring genes. Since PKS genes themselves
are homologous in genomes of mentioned strains, we
provide recombination directly within the biosynthetic
clusters of corresponding antibiotics.
Fig.2. The morfology of recombinant strain Rec5
The parental strains of Rec5 are the insertional
oxygenase gene mutant of S. globisporus 1912
(producer of landomycin E) and olivose-biosynthesis
deficient mutant of S. fradiae Tü2717 (Fig.3). Both
parents secrete unglycosylated and not fully
oxygenated angucyclic intermediates.
S.globisporus М12
lnd-cluster
prx
I
EFA BCD
M2
O P G
H Q
R S T
U V W GT2 X GT1
J Z1
Tab.1. Strains used in the experiments of
protoplast fusion.
Strain name
Gene
Gene function
inactivated
simA8
monooxygenase
S.cyanogenus ∆lanGT1
lanGT1
glycosyltransferase
S.cyanogenus ∆lanGT2
+urdGT2
lanGT1
glycosyltransferase
S.antibioticus ∆simA8
S. fradiae ΔurdQ/R
sugar dehydraurdQ, urdR
tase, reductase
S. globisporus Е7
lndE
S. globisporus М12
lndM2
Micromonospora
sp.Tü6368 ΔsaqGT3
saqGT3
Major II
metabolites
Resistance
marker
12-dehydrorabelomycin
spectinomycin
tetrangulol
9-C-D-olivosyl- spectinomycin,
tetrangulol
apramycin
prejadomycin
hygromycin
oxygenasetetrangulol,
dehydrogenase tetrangomycin
hygromycin
SaqAE3
spectinomycin
4-hydroxytetrangomycin
tetrangulol
UV/Vis spectrum
3,7
565
100-1
tetrangomycin
3,8
4
4,8
6,1
8,5
8,8
9,2
11,2
11,9
14,2
17,3
22
22,4
22,7
485
599
581
581
469
337
695
469
827
321
697
433
563
693
Aquayamycin
12b-DUG, 100-2
Diolyvosyltetrangomycin
Urdamycin A
Urdamycin M
Hydroxytetrangomycin
Urdamycin B
Urdamycin K
Urdamycin O
Tetrangomycin
Urdamycin R
Olivosyltetrangulol
Diolivosyltetrangulol
Triolivosyltetrangulol
aquayamycin
aquayamycin
tetrangomycin
aquayamycin
aquayamycin
tetrangomycin
aquayamycin
aquayamycin
aquayamycin
tetrangomycin
aquayamycin
tetrangulol
tetrangulol
tetrangulol
Among the others there are glycosylated urdamycin
derivatives and even fully oxygenated and glycosylated
end product – urdamycin A, wich is an evidence of
mutual complementarity of missing enzymes in both
clusters. So far we isolated four novel compounds,
secreted by Rec5 recombinant shown on figure 5.
Diolivosyltetrangulol: R=
S. fradiae ΔurdQ/R
urd-cluster
GT1a GT1b Int GT1c K J
apramycin
glycosyltransferase
11-hydroxytetrangomycin
spectinomycin
urdamycin X
monooxygenase
tetrangomycin
Compound
O E F A
B CD L
M
J2 Z1 GT2 G H Z3 Q
R
S T
urdamycin X
Fig 3. The mutations and major secondary metabolites of the strains applied for protplast fusion
Triolivosyltetrangulol: R=
Olivosyltetrangulol:
R=H
Diolyvosyltetrangomycin
Fig 5. Novel metabolites produced by Rec5
CONCLUSION
Metabolic engineering by means of interspecies protoplast fusion with the consequent screening of antibiotic resistant recombinants turned out to be enormously
fruitful in the case of angucycline producing mutants with damadged post-PKS steps. The recombination within antibiotic biosynthetic gene clusters is thus being
provoked by the selective antibiotic pressure. As a result of one fusion of properly selected parental strains we obtained a recombinant producing a whole collection
of compounds. Generating it by means of sequential unit step targeted mutagenesis took perennial effort. Protoplast fusion is a promising method to proceed in strain
modification when inductive mutagenesis reaches its deadlock. It allows breeding and improvement of subgenomic DNA fragments thereby modifying metabolic
pathways. Beside pure combinatorial profit simultaneously a great deal of data on gene functions and nature of enzymes interaction can be won.