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Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.05781-11 AEM Accepts, published online ahead of print on 5 August 2011
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Initiation of Polyene Macrolide Biosynthesis: Interplay Between PKS Domains and 1
Modules Revealed via Domain Swapping, Mutagenesis and Heterologous 2
Complementation 3
4
Sondre Heia1, Sven E.F. Borgos1, Håvard Sletta2, Leticia Escudero3, Elena M. Seco3, Francisco 5
Malpartida3, Trond E. Ellingsen2, Sergey B. Zotchev1* 6
7
1Department of Biotechnology, NTNU, N-7491 Trondheim, Norway; 8
2Department of Biotechnology, SINTEF Materials and Chemistry, N-7465 Trondheim, 9
Norway. 10
3Centro Nacional de Biotecnologia del CSIC, Darwin 3, 28049 Cantoblanco, Madrid, Spain. 11
12
Running title: cross-complementation, domain swapping and mutagenesis in the polyene PKS 13
loading modules 14
Key words: polyene antibiotics, polyketide synthase, loading modules, acyltransferase 15
domains 16
17
Author for correspondence: Sergey B. Zotchev, Department of Biotechnology, 18
Norwegian University of Science and Technology, N-7491 Trondheim, Norway; 19
Tel. +47 73 59 86 79 20
Fax: +47 73 59 12 83 21
email: sergey.zotchev@nt.ntnu.no 22
23
Abbreviations: PKS, polyketide synthase; KS, ketosynthase; AT, acyltransferase; ACP, acyl 24
carrier protein; DH, dehydratase; CoA ligase, coenzyme A ligase. 25
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ABSTRACT 26
27
Polyene macrolides are important antibiotics used to treat fungal infections in humans. In this 28
work, acyl transferase (AT) domains swaps, mutagenesis, and cross-complementation by with 29
heterologous PKS loading modules were performed in order to facilitate production of new 30
analogues of the polyene macrolide nystatin. Replacement of the AT0 in the nystatin PKS 31
loading module NysA with the propionate-specific AT1 from the nystatin PKS NysB, 32
construction of hybrids between NysA and the loading module of rimocidin PKS RimA, and 33
stepwise exchange of specific amino acids in the AT0 domain by site-directed mutagenesis 34
have beenwere accomplished. However, none of the NysA mutants constructed was able to 35
initiate production of new nystatin analogues. Nevertheless, many NysA mutants and hybrids 36
were functional, providing for different levels of nystatin biosynthesis. An interplay between 37
certain residues in AT0 and an active site residue in the ketosynthase (KS)-like domain of 38
NysA in initiation of nystatin biosynthesis has beenwas revealed. Some hybrids between the 39
NysA and RimA loading modules carrying the NysA AT0 domain were able to prime rimocidin 40
PKS with both acetate and butyrate units upon complementation of rimA-deficient mutant of 41
the rimocidin/CE-108 producer Streptomyces diastaticus. Expression of the PimS0 loading 42
module from the pimaricin producer in the same host, however, resulted in production of the 43
CE-108 only. Taken together, these data strongly suggestindicate relaxed substrate specificity 44
of NysA AT0 domain, which is being counteracted by a strict specificity of the first extender 45
module KS domain in the nystatin PKS of S. noursei. 46
47
INTRODUCTION 48
49
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Biosynthesis of polyketides by by bacteria received much attention during the last two decades 50
due to the high commercial potential of the latter compounds as biologically active molecules 51
for treatment of diverse human diseases. Macrolides represent a large group of polyketides 52
currently used as antibacterial, antuifungal, antitumor and immunosuppressive agents, 53
structurally characterized by the macrolactone ring, which represents a cyclized polyketide 54
chain. The latter is biosynthetically assembled by large enzymes, polyketide synthases (PKS) 55
type I, in a manner similar to the fatty acid biosynthesis (334). PKS responsible for the 56
macrolactone ring assembly are organised in modules, each performing decarboxylative 57
condensation of the carboxylic acid units such as acetate, propionate or butyrate onto the 58
growing polyketide chain. Each ketide unit incorporated into the chain can undergo in-59
synthesis modification by the reductive domains present in the next extension module, thus 60
providing chemical modification that will be reflected in the final product. The PKS type I are 61
thus considered nature’s assembly lines for biosynthesis of macrolides, and a number of 62
alterations have been introduced into these systems, resulting in biosynthesis of “unnatural” 63
polyketides with properties distinctly different from the original molecules (2019). The 64
success of the PKS type I manipulation in terms of obtaining desired product depends on many 65
factors, and in certain cases the failure could not be easily explained. Such factors as 66
availability of starter and extender units, structural integrity of the PKS proteins (including 67
both enzymatic domains and linkers between them), and the ability of the next-in-line module 68
to accept and process altered substrate all seem to have a profound impact on the functionality 69
of these proteins (343). 70
Polyketide chain assembly by the type I PKS begins with the loading of a starter unit, in most 71
cases acetate or propionate, onto the first PKS extender module (13). This process is 72
accomplished by a loading module, which can be either fused to the first extender module, or 73
represented by a separate polypeptide, such as in the case of polyene macrolide PKSs (2). The 74
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mechanism of the polyketide biosynthesis initiation by the loading module depends on the 75
composition of this module. The loading modules containing ketosynthase (KS)-like domains 76
presumably recruit malonyl- or methylmalonyl-CoAs, which are decarboxylated to provide 77
foryield acetate or propionate starter units, respectively. The KS-like domains in such loading 78
modules usually contain Glu instead of Cys in their active sites, and this residue has been 79
assumed to be important for decarboxylase activity of the KS-like domains (4). In certain 80
cases, the KS-like domain is absent from the loading modules, suggesting that they utilize 81
acetyl- or propyonyl-CoA to initiate polyketide synthesis. In any case, the choice of the starter 82
unit is ensured by the acyltransferase (AT) domain in the loading module, which can have 83
either strict or relaxed specificity (19, 310, 343). Since changing the specificity of the AT 84
domain can provide for new engineered polyketides, substantial efforts in dissecting the 85
specificity issues have been made recently. Earlier, it has been noticed that specificity of the 86
AT domains towards different substrates is reflected in the amino acid sequences of these 87
domains (10). Since then, the specificities of several PKS modules towards starter or extender 88
unit were successfully changed by either replacing the whole AT domains, or by introducing 89
specific mutations (9, 18, 19, 221). 90
Nystatin A1 (Figure 1) is a medically important antifungal antibiotic, and it was recently shown 91
that nystatin analogues with high antifungal activity and lower toxicity activity can be obtained 92
via manipulation of the nystatin biosynthetic genes (7). Among the envisaged analogues that 93
would be interesting to produce and analyze are those that would contain extended or modified 94
C-37 side chain resulting from the initiation of biosynthesis with a starting unit other than 95
acetate. Earlier studies on the loading module NysA of the nystatin PKS, which contains KS-96
like domain with Ser instead of Cys residue in its active site suggested revealed that both 97
malonyl-CoA and acetyl-CoA can be used for initiation of nystatin biosynthesis (6, Figure 2). 98
In the current work we attempted to change the specificity of the nystatin PKS loading module 99
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in order to prime nystatin biosynthesis with either propionate or butyrate rather than acetate. 100
We used the RimA PKS loading module for rimocidin (Figure 1) from S. diastaticus var. 108 101
capable of loading the extender module with either acetate or butyrate (210, 245, Figure 2), as 102
well as the AT1 from NysB with specificity for methylmalonyl CoA. Although our attempts, 103
which included module swaps, hybrid construction, and site-specific mutagenesis, were 104
unsuccessful in terms of producing new nystatin analogues, important information regarding 105
the initiation of polyene macrolide biosynthesis, as well as interplay between certain amino 106
acid residues in the loading module was obtained. Heterologous expression of certain NysA-107
RimA hybrids satrongly suggestedrevealed relaxed substrate specificity of NysA AT domain, 108
which, keeping in mind our previous attempts of loading module engineering (6), implies a 109
gate-keeping function of the first extender module in the nystatin PKS. These data will provide 110
important support for rational design of future PKS manipulations aimed at production of new 111
macrolides. 112
113
MATERIALS AND METHODS 114
115
Bacterial strains, media, and growth conditions 116
Bacterial strains and plasmids used and constructed during this study are listed in Table S1 117
(Supplementary data). S. noursei strains were maintained on ISP2 agar medium (Difco, USA), 118
S. diastaticus var. 108 strains were grown in SYM2 medium (276) and Escherichia coli strains 119
were grown in L broth or L agar (243). S. diastaticus var. 108 strains were manipulated for by 120
protoplasting, transfection and transformation as described elsewhere (15). Intraspecific 121
conjugation was carried out as described previously (265). 122
Conjugal plasmid transfer from E. coli ET 12567 (pUZ8002) and the gene replacement 123
procedure in S. noursei were performed as described previously elsewhere (287). 124
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Transformation of E. coli was performed as described elsewhere in Sambrook et al. (243). 125
Agar and liquid media, when where appropriate, were supplemented with antibiotics using the 126
following concentrations: chloramphenicol, 20 μg/ml; kanamycin, 50 μg/ml; thiostrepton, 50 127
μg/ml; erythromycin, 100 μg/ml; ampicillin, 100 μg/ml; apramycin, 50 μg/ml. Analysis of the 128
production of polyene macrolides by S. noursei strains was done by liquid chromatography-129
mass spectroscopy (LC-TOF) of dimethyl sulfoxide (DMSO) extracts of the 5 days–old 130
cultures from well plate cultivations according to Bruheim et al. (8). The well plate cultivation 131
was performed in semi-defined 0.5xSAO-23 medium according to the protocol described 132
previously (2930). For feeding experiments with either crotonic or butyric acids, stock 133
solutions (1 M, pH 7.0) were added in equal portions at 48, 60 and 72 hours during 134
fermentations to a final concentration of 15 mM. Production of polyene macrolides from S. 135
diastaticus var. 108 were tested by HPLC analysis of methanol extracts from the 5 days-old 136
cultures grown on the SYM2 plates as previously described (276). 137
138
DNA manipulations, DNA sequencing, and PCR 139
General DNA manipulations were performed as described in Sambrook et al. (240). DNA 140
fragments from agarose gels were purified using the Qiaex II kit (Qiagen, Germany) or 141
PerfectPrep Gl Cleanup kit (Eppendorf, Germany). Oligonucleotide primers were purchased 142
from Qiagen (Germany), and primers used for Quickchange (site-directed mutagenesis) were 143
PAGE-purified. DNA sequencing was performed using the ABI Prism Big Dye Terminator 144
Cycle sequencing kit and the Genetic Analyzer 3100 (Applied Biosystems, Inc., USA). The 145
PCRs were performed with the Expand High Fidelity PCR System (Roche Molecular 146
Biochemicals, USA) using the Eppendorf Mastercycler (Eppendorf, Germany) as described 147
previously (6). DNA sequence encompassing the regions used in this study has been reported 148
previously by Brautaset et al. (5). 149
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150
Construction of the RimA expression vector 151
The rimA gene was excised from the pSM736 plasmid as a 5.9 kb BglII fragment, treated with 152
the Klenow fragment of DNA polymerase, and cloned into the HincII site of pGEM3Zf(-). 153
After verification of the correct orientation, the 5.9 kb XbaI-HindIII rimA-containing fragment 154
from the resulting construct was cloned, together with the HindIII-XbaI fragment with 155
containing the nysAp promoter (2829) into the pSOK804 vector. The resulting plasmid, 156
pRIM1, was used for in complementation experiment. 157
158
Replacement of AT0 with AT1 in NysA using gene SOEing 159
Replacements of the AT0 in NysA with the methylmalonyl-specific AT1 in from the nystatin 160
PKS NysB were conducted both with and without the inter-domain linker region from module 161
1 of NysB using gene SOEing technique (12). The acetyltransferase AT1 coding region in nysB 162
was fused with nysA using PCR. The fusion points used for the assembly procedure were in all 163
cases positioned in the inter-domain linker regions and the fusions were sited in regions were 164
the DNA gave done within overlapping DNA fragments, and ensuringed there were no gaps in 165
the amino acid alignments. This resulted, giving in a seamless hybrids, an thereby avoiding any 166
unwarranted structural effects for on the resulting hybrid proteins. The vector plasmid pl76B11 167
(5) served as a template for the amplified fragments. Fusion-primers and flanking-primers with 168
restriction sites are given Table S2 (Supplementary data). All the resulting constructs were 169
verified by DNA sequencing. 170
Without linkers 171
As a primary step, the AT1 coding region and the KSS and DH0 coding regions with C-terminal 172
and N-terminal linkers, respectively, were amplified separately. A region containing KSS 173
coding region with the C-terminal linker was amplified with a flanking primer mluI-F 174
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containing MluI site located upstream ks0 and a fusion primer ks0link-R, yielding the 175
ks0linkup DNA fragment (0.73 kb). The AT1 coding region was amplified as two parts with 176
hybrid primers, at1-F and at1-R and primers on both sides of NruI , nruI-F and nruI-R yielding, 177
respectively, DNA fragments at1casup (0.90 kb) and at1casdown (0.11kb). The DNA fragment 178
encoding DH0 domain with N-terminal linker was amplified with a fusion-primer, dh0-F and a 179
flanking primer, pstI-R, containing PstI recognition site located downstream DH0 coding 180
sequence, yielding dh0link fragment (0.82-kb). A secondary PCR fused ks0linkup with 181
at1casup and at1casdown with dh0link resulting in at1casup (1.6 kb) and at1casdown (0.90 kb), 182
respectively. These fragments were respectively cloned into pLIT38NruI(-) MluI/NruI and 183
pLIT38NruI(-) NruI/PstI resulting in pAT1casup and pAT1casdown plasmids. The MluI/nruI 184
and NruI/PstI fragments from the latter constructs were excised and ligated into the 185
pLITMUS38 MluI/PstI yielding the pAT1cassette. Subsequently, the MluI/NcoI fragment (2.42 186
kb) from pAT1cassette was ligated into the pl76ES4.6 MluI-NcoI fragment (5.40 kb) resulting 187
in pNysA-AT1cas. Finally, the 4.67 kb HindIII-EcoRI fragment from pNysA-AT1cas was 188
excised and ligated into the HindIII/EcoRI sites of vector pSOK804, resulting in the integrative 189
vector pSOKAT1cassette for expression of this hybrid. 190
With linkers 191
Similar procedure was employed for replacing the intrinsic AT0 domain of NysA with N-and 192
C-terminal linkers by the corresponding part of NysB protein encompassing AT1 domain and 193
linkers. First, the ks0up DNA fragment (0.44 kb) was amplified using primers mluI-F and ks0-194
R. The AT1 coding region with interdomain linker regions was amplified first as two different 195
fragments with hybrid primers ks1link-F and dh1link-R, and primers on both sides of NruI 196
nruI-R and nruI-F, giving PCR fragments at1linkup (1.21 kb) and at1linkdown (0.16 kb), 197
respectively. Next, the dh0down fragment (0.76kb) was amplified using primers dh0link-F and 198
pstI-R. A second PCR subsequently resulted in at1briup (0.44 kb) and at1bridown (0.16 kb) 199
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fragments, which were cloned into the pLIT38NruI(-) MluI/NruI and pLIT38NruI(-) NruI/PstI 200
yielding the pAT1briup and pAT1bridown constructs, respectively. The latter vectors were 201
processed in the same manner as the corresponding vectors in the previous section (see above), 202
yielding the pAT1bridge plasmid. The 2.4 kb MluI-NcoI fragment from the latter vector was 203
inserted into pl76ES4.6 MluI-NcoI fragment (5.40-kb) resulting in pNysA-AT1bri. Finally, the 204
4.6 kb HindIII-EcoRI fragment from pNysA-AT1bri was excised and ligated into the 205
HindIII/EcoRI sites of vector pSOK804, resulting in the integrative vector pSOKAT1bridge. 206
207
Construction of the NysA-RimA hybrids 208
Fusion of nysAp promoter to rimA CoL: DNA fragments of 250 bp and 239 bp were amplified 209
by PCR using primers NR-F1-U (5’-CGGGATCCACCATGATTACGCCAAGCTATTTAGG-210
3’) and NR-F1-M-r (5’-GTGTGGACGGGCACCATGCCTTGCTCACCCCTG-3’) on 211
template pL76ES4.6, and primers NR-F1-M (5’-CAGGGGTGAGCAAGGCATGGTGCC 212
CGTCCACAC-3’) and NR-F1-D (5’-ACCGCTCGAGGACGCCCGATTCCTGGAG-3’) on 213
template pRIM1, respectively. The latter plasmid harbours the rimA gene of S. diastaticus var. 214
108. These PCR products were purified and fused in a new PCR together with primers 215
NR-F1-U and NR-F1-D to yield a PCR product of 456 bp, which was cloned as the BamHI-216
XhoI fragment into pGEM-7zf(+) to yield plasmid pF1BX. The latter construct was sequenced 217
to confirm correct fusion. 218
Fusion of rimA ACP to nysA KS: DNA fragments of 309 bp and 870 bp were amplified by PCR 219
using primers NR-F3-U (5’-CAGGATCCCAGGTCCTCGGCAGCAG-3’) and NR-F3-M-r 220
(5’-CGACGACCACCACCGGATCGGCCTCG-3’) on template pRIM1, and primers 221
NR-F3-M (5’-CGAGGCCGATCCGGTGGTGGTCGTCG-3’) and NR-F3-D (5’-AGACCTC 222
GAGTGACGCGTTCCTGGGC-3’) on template pL76ES4.6, respectively. These PCR products 223
were purified and fused in a new PCR together with primers NR-F3-U and NR-F3-D to yield a 224
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PCR product of 1153 bp, which was cloned into the vector pPCR-Script Amp SK(+) with the 225
PCR-Script™ Amp Cloning Kit (Promega) to yield plasmid pF3BX’ and sequenced to confirm 226
correct fusion. 227
Fusion of rimA AT to nysA DH: DNA fragments of 208 bp and 227 bp were amplified by PCR 228
using primers NR-F4-U (5’-ATGGATCCGGAGGAGCGCGTCACC-3’) and NR-F4-M-r (5’-229
CGGGCCAGTCGACGGGTGCG-3’) on template pRIM1, and primers NR-F4-M (5’-CGCA 230
CCCGTCGACTGGCCCG-3’) and NR-F4-D (5’-ACCACTCGAGGTGGGTGCGGAGGG 231
AG-3’) on template pL76ES4.6, respectively. These PCR products were purified and fused in a 232
new PCR together with primers NR-F4-U and NR-F4-D to yield a PCR product of 415 bp, 233
which was cloned as the BamHI-XhoI fragment into pGEM-7zf(+) to yield plasmid pF4BX and 234
sequenced to confirm correct fusion. 235
Fusion of rimA KS to nysA AT: A DNA fragment of 1164 bp was amplified from template 236
pL76ES4.6 with primers NR-F5-U (5’-CGGGATCCGTCTTCTCCGGACAGGGCAGCC-3’) 237
and NR-F4-D and cloned directly into dephosphorylated (Calf Intestinal Phosphatase, New 238
England Biolabs) vector pGEM-3zf(+) to yield plasmid pF5BX*, which was sequenced to 239
confirm correct fusion. 240
Construction of complementation plasmid for nysA-rimA hybrid C: A 3760 bp HindIII-BspEI 241
fragment from pLit28-F1-RIM, a 1037 bp BspEI-ApaI fragment from pF5BX*, a 1845 bp 242
ApaI-EcoRI fragment from pL76ES4.6 and pSOK804 cut EcoRI-HindIII were ligated to yield 243
complementation plasmid pNR-C. 244
Construction of complementation plasmid for nysA-rimA hybrid D: A 4690 bp HindIII-NotI 245
fragment from pLit28-F1-RIM, a 139 bp NotI-ApaI fragment from pF4BX, a 1845 bp ApaI-246
EcoRI fragment from pL76ES4.6 and pSOK804 cut EcoRI-HindIII were ligated to yield 247
complementation plasmid pNR-D. 248
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Cloning of rimJ under control of nysRIVp promoter into the plasmids for expression of nysA-249
rimA hybrids: A 1435 bp fragment containing rimJ from pSM765 was amplified using primers 250
RimJ-f: 5’-ATATTCTAGACGCCTTTTCCGGAGGCTC-3’ and RimJ-r: 5’– ATAACACCGG 251
TGAAGCTTGCGTCCCGCAAGGGC-3’. The use of these primers introduced XbaI site at the 252
5’ end, and HindIII and SgrAI restriction sites at the 3’ end of the rimJ PCR product. A 3.5 kb 253
plasmid pNR4pHX (containing nysRIV promotor cloned as a HindIII-XbaI fragment in 254
pGEM3Zf(-)) was linearized through digestion with XmaI and XbaI, and and ligated with the 255
XbaI + SgrAI-digested rimJ PCR product, resulting in pNR4rimJ. In the latter plasmid rimJ 256
appeared under control of the nysRIVp promoter. A 1751 bp HindIII cassette from pNR4rimJ 257
was cloned into the HindIII site of the nysA-rimA hybrid expressing plasmids constructed as 258
described above. 259
260
Site-directed mutagenesis of the nysA AT0 coding region 261
To prepare a suitable template for site-directed mutagenesis, the 1.76 kb MluI-ApaI fragment 262
from the plasmid pl76ES4.6 was excised and ligated into pLITMUS38, resulting in plasmid 263
plit38-AT0. Site-directed mutagenesis using of QuickChange (Stratagene, USA) were was 264
doneexecuted on plit38-AT0 template and, successively, on the mutants thereof (from now on 265
called plit38-mutX). Stepwise exchange of specific amino acids in AT0 were conducted by 266
altering the codons for the following amino acids in the nystatin PKS loading 267
acetyltransferaseAT0 domain: T56V, G57D, W58V, V89Q, S184D, H185Y and F187S, 268
M117I. The primers used in the site-directed mutationsmutagenesis are given in Table S3 269
(Supplementary data). 270
The mutant plasmids plit38-mutX were isolated, and all mutations were verified by DNA 271
sequencing. Thnis was, followed by isolation of the 1.76 kb MluI-ApaI fragment in from 272
plit38-mutX and ligated ligation into the 6.04 kb MluI-ApaI fragment from of pl76ES4.6, 273
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resulting yieldingin pl76ES4.6AT0-mutX consdtructs. A 4.65 kb HindIII-EcoRI fragments 274
from pl76ES4.6AT0_mutX was were excised and ligated into the HindIII/EcoRI sites of vector 275
pSOK804, resulting in the nysA integration vectors pAT0_mutX. 276
277
Combining mutation in AT0 with KSC and KSQ 278
The mutations in the NysA KS-like domain’s “active site” were constructed previously (6). 279
These constructs were combined with some of the mutations in AT0 using conventional cloning 280
techniques. A 1.05-kb MluI-HindIII fragment from the plasmids pKSCAT0 and pKSQAT0 were 281
respectively excised and ligated into the 6.75-kb MluI-HindIII fragment from certain 282
§pl76ES4.6AT0_mutX in questionconstructs, resulting in pKSCAT0-mutX and pKSQAT0-283
mutX. The latter plasmids were used to complement S. noursei nysA mutant NDA59.. 284
285
RESULTS 286
287
Expression of the rimocidin PKS loading module restores nystatin biosynthesis in S. 288
noursei nysA mutant 289
The RimA PKS loading module ofin S. diastaticus var. 108 is capable of loading both acetate 290
and butyrate onto the first rimocidin PKS extension module, resulting in production of CE-108 291
and rimocidin, respectively. We have hypothesized that RimA might also initiate nystatin 292
biosynthesis, and is able to prime the NysB PKS with butyrate, which would result in the 293
production of a new nystatin analogue. The rimA gene was placed under control of the nysAp 294
promoter, yielding the pRIM1 vector, which was introduced into the nysA-deficient S. noursei 295
NDA59 mutant deficient for nysA (6). Testing of the transconjugants for polyene macrolide 296
production revealed nystatin as the final product, and no new nystatin analogues could be 297
detected. Keeping in mind that precursor supply might be a limiting factor for the biosynthesis 298
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of new analogues, feeding experiments were performed according to Liu and Reynolds (17). 299
However, supplementation of the fermentation medium with either crotonic or butyric acid did 300
not result in the production of new nystatin analogues. Thus, despite that the rimA gene seemed 301
to be functional in complementing the nysA mutation, the biosynthetic pathway for nystatin 302
could successfully proceed only when primed with acetate. 303
Complementation of the S. diastaticus var. 108 rimA disruption mutant with the nysA gene was 304
also attempted. For that purpose, nysA was cloned under the control of the xysA promoter 305
(xysAp) of the xylanase gene from S. halstedii JM8 (2223). A BstEII-BglII fragment carrying 306
nysA gene, was fused to the xysAp promoter and cloned in several steps described in Table S1, 307
Supplementary data. The resulting recombinant plasmid pSM767 was introduced into S. 308
diastaticus var. 108 (wild type) and then transferred by intraspecific conjugation into the rimA-309
deficient gene disruptant strainmutant (S. diastaticus var. 108/PM1-500). No tetraene 310
production was observed in the fermentation broth of the latter recombinant strain, suggesting 311
the inability of NysA to prime rimocidin biosynthesis, most probably due to the lack of a 312
proper interaction with RimB. Together, these results suggest imply that NysB has a more 313
flexible docking domain compared to RimB, allowing it to interact with both NysA and RimA. 314
315
Replacements of the AT0 domain in NysA with AT1 of NysB using gene SOEing 316
Next, as an attempt to change the specificity of NysA toward methylmalonyl CoA, we have 317
replaced AT0 with AT1 from NysB (providing the first extender unit) using restriction sites 318
created through PCR. AT1 domain of NysB has a clear specificity toward methylmalonyl CoA, 319
as it ensures incorporation of a propionate extender during the nystatin biosynthesis (5). Such 320
hybrids proved to be non-functional, as in a sense that they did not provide forcould not initiate 321
biosynthesis of nystatin or any other nystatin-related polyene macrolides (6). This could be 322
attributed to the changes in amino acid sequences within the KSS-AT0 and AT0-DH0 linkers in 323
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NysA, more specifically introduction of sequences coding for additional amino acid residues 324
during the hybrid assembly. Relatively little is known about the linker regions and it can be 325
anticipated that these regions affect how the domains are functioning within the module (3334). 326
Keeping this in mind, we employed a method of gene SOEing, which ensures accurate fusion 327
of virtually any DNA sequences at any nucleotide using PCR-based technique (12). The 328
strategy for gene SOEing was chosen for precise splicing of the AT1 domain into the NysA 329
either with or without its original KS1-AT1 and AT1-DH1 linkers. The pSOK804-based 330
plasmids pSOKAT1 cassette and pSOKAT1bridge expressing recombinant NysA containing 331
AT1 with or without its original linkers (Figure 3), respectively, were constructed. Both 332
plasmids were introduced by conjugation into the nysA- deficient S. noursei NDA59 mutant, 333
which was unable to produce nystatin. It has previously been shown (6) that complementation 334
of this strain with nysA expressed in trans restores nystatin production almost to the wild-type 335
levels. Analysis of metabolites produced by the above pSOKAT1cassette- and 336
pSOKAT1bridge-containing recombinant strains revealed that neither of the constructs could 337
restore nystatin production or initiate synthesis of novel nystatin analogues in the NDA59 338
mutant. 339
340
Certain hybrids between NysA and rimocidin PKS loading module RimA are able to 341
initiate nystatin biosynthesis 342
In the next attempt to change the starter unit specificity of the NysA, the gene SOEing 343
technique was applied for construction of hybrid loading modules between NysA and the 344
initiator protein of the rimocidin/CE-108 PKS RimA. The latter protein is composed of two 345
modules, one containing carboxylic acid-CoA ligase (CoL) and ACP, and another containing 346
KSS, AT, and ACP domains (2425). RimA was shown to be responsible for the choice of 347
starting unit in rimocidin and CE-108 biosynthesis, incorporating either acetate (CE-108) or 348
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butyrate (rimocidin) (2425). The exact mechanism by which RimA selects the starter unit is not 349
presently clear, and it was not possible to predict whether the CoL or AT domain in RimA 350
could be responsible for the incorporation of butyrate. Although the AT domain in RimA was 351
originally proposed to be acetate-specific, re-examination of the sequence revealed that two 352
residues critical for specificity residues, the His185 and Phe187 highly conserved in acetate-353
specific domains (10), are replaced with Val and Ala, respectively. 354
Four hybrid loading module genes were constructed based on rimA and nysA using gene 355
SOEing (Figure 3), and placed under control of the nysAp promoter, which was shown to 356
function efficiently during previous complementation experiments (6, 2829). The C-terminal 357
part of the hybrids was always represented by the NysA counterpart in order to ensure proper 358
interaction with the NysB protein during polyketide assembly. To facilitate the biosynthesis of 359
butyryl CoA, the rimJ gene encoding a putative crotonyl CoA reductase, which catalyzes the 360
reduction of crotonyl CoA to butyryl CoA, was placed under control of the nysRIVp promoter 361
(2829) and cloned, together with the rimA-nysA hybrids into the pSOK804 integrative vector. 362
The recombinant plasmids were then introduced into the S. noursei NDA59 mutant, and the 363
recombinant strains were tested for the production of polyene macrolides. The recombinant 364
plasmids expressing hybrid A along with rimJ was able to restore nystatin biosynthesis in the 365
NDA59 mutant almost to the wild-type level (data not shown), while no polyene macrolide 366
production could be detected in the case of hybrids B, C and D. No new nystatin analogues 367
could be detected in the extracts of the mutants harbouring the A hybrid. 368
369
Hybrid RimA/NysA proteins carrying AT domain of NysA are able to restore rimocidin 370
and CE-108 biosynthesis in the rimA disruption mutant 371
The hybrids C and D were tested for restoring restoration of the production of polyene 372
macrolides production in the rimA-deficient disrupted mutant of S. diastaticus. Both hybrid 373
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proteins only differ in the AT domain, which is originate from NysA or RimA in case of 374
hybrids C and D, respectively (Figure 3). The corresponding hybrid genes were placed under 375
the control of the ermEP* promoter in several steps described in Table S1 (Supplementary 376
data). The resulting plasmids (pLEC104C and pLEC106D for the hybrids C and D, 377
respectively) were used to transform protoplasts of a new rimA disruptant (S. diastaticus var. 378
108::PM1/pLEC101). Several candidates were selected and confirmed by direct plasmid 379
extraction (for the presence of recombinant plasmid) and by Southern blot for the correct 380
integration of PM1/pLEC101 phage into the chromosome. The HPLC analysis of the 381
fermentation broth of the recombinant strains showed restoration of CE-108D, CE-108, and 382
rimocidin D and rimocidin production in both cases (Figure 4C, D), as evident from the 383
appearance of peaks eluting, respectively, at ca 9.5 min, 10.5 min, 11.5 min and 12.3 min. The 384
identities of these metabolties have previously been confirmed by LC-MS analyses of the 385
extract from the wild type S. diastaticus var. 108 ((Seco et al., unpublished data). Some 386
additional peaks appeared on the chromatogram within the ca 2-8 min elution time after upon 387
complementation with hybrid C within the ca 6-8 min elution time. However, we could not 388
correlate these peaks with polyene UV spectra, and do not know at present what they represent. 389
In any case, successful complementation of S diastaticus rimA mutant with C and D hybrids 390
strongly suggests that AT0 domain of NysA under heterologous conditions is able to recognize 391
and recruit both malonyl CoA and butyryl CoA. Moreover, the C and D hybrids could 392
functionally interact with the RimB PKS, which was surprising considering the presence of 393
intact NysA C-terminus on both hybrids and the lack of such interaction between NysA and 394
RimB (see above). 395
396
The loading module of pimaricin PKS is not able to prime RimB with a butyryl starter 397
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PimS0 is a loading PKS module responsible for the priming of pimaricin biosynthesis in S. 398
natalensis (1). The loading modules RimA and PimS0 share high degree of homology (70%), 399
and have identical architectural organization with the catalytic domains CoL, ACP, KSS, AT 400
and a second ACP similarly arranged (Figure 2). Despite this similarity, PimS0 seems to be 401
able to prime the pimaricin PKS only with acetyl CoA in S. natalensis. However, this could 402
also be due to the strict specificity of the accepting extender module of pimaricin PKS for the 403
acetate primer. Considering the apparent relaxed specificity of RimB towards priming units, we 404
used the S. diastaticus var. 108 rimA mutant as a host for analysing the substrate specificity of 405
AT domain in PimS0. For this propose, the 6,229 bp XmnI-EcoRI fragment from pimaricin 406
biosynthetic cluster (containing the 3´ end of pimG, pimF, pimS0 and 255 bp downstream the 407
stop codon of pimS0) was rescued excised from the Cos37 cosmid (1) and cloned under the 408
control of the constitutive promoter ermE*P in several steps described in Table S1 409
(Supplementary data). The resulting plasmid pLEC108 was introduced by transformation into 410
S. diastaticus var. 108::PM1/pLEC101 protoplasts. As a control, the plasmid pSM783 carrying 411
rimA gene under control of ermE*P was also introduced into the rimA mutant. Plasmid 412
extraction and Southern blot confirmed that the rimA-disrupted mutant carries, as expected, the 413
corresponding plasmids pLEC108, pSM783 and pSM780, giving rise to the S. diastaticus var. 414
108::PM1/pLEC101-pLEC108, S. diastaticus var. 108::PM1/pLEC101-pSM783 and S. 415
diastaticus var. 108::PM1/pLEC101-pSM780, respectively. 416
The HPLC analysis of the fermentation broth of the corresponding recombinant strains 417
confirmed that the rimA mutant, carrying pimS0 under the control of ermE*P produces CE-108 418
but not rimocidin (Figure 4B), as evident from the absence of rimocidin D- and rimocidin-419
characteristic peaks eluting at ca 11.5 min and 12.3 min, respectively. while tThe same mutant 420
transformed with the rimA gene restores production ofced both CE-108 and rimocidin (Figure 421
4A). These results strongly suggestclearly indicate that AT domain of PimS0 has a strict 422
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substrate specificity for either malonyl CoA or acetyl CoA, and is not able to prime the 423
rimocidin pathway with butyryl CoA. 424
425
Site-specific mutagenesis of certain AT0 amino acid residues differentially affects the 426
ability of recombinant NysA to initiate nystatin biosynthesis 427
Several reports on the site-specific mutagenesis of the AT domains, along with the structural 428
data for the Streptomyces coelicolor malonyl CoA:ACP transacylase (14, 16), and recently 429
published crystal structure of erythromycin PKS fragment (3132) suggest that only a limited 430
number of amino acids play a critical role in determining specificity of these domains. The 431
study by Del Vecchio et al. (9) demonstrated that changing only 2 amino acids (namely, those 432
corresponding to His185 and Phe187) in the AT of the erythromycin PKS can drastically 433
influence the choice of the substrate by this domain. Guided by these observations, we 434
designed a strategy for step-wise mutagenesis of the AT0 domain in NysA aimed at changing 435
its specificity from malonyl CoA to methylmalonyl CoA. We have focused on six amino acid 436
residues marked in the alignment of the two AT domain regions supposedly important for 437
substrate selectivity (Figure S1, Supplementary data). The DNA fragment encoding these 438
regions of AT0 was excised from the nysA gene, subjected to site-specific mutagenesis, and 439
cloned back into the original plasmid expressing NysA (see Materials and Methods). The 440
recombinant plasmids expressing the NysA mutants (Table S1, Supplementary datal) were 441
introduced into S. noursei NDA59, and the resulting strains were analysed for polyene 442
macrolide production in order to monitor the effect the that particular substitutions had on the 443
biosynthesis of nystatin and, eventually, new polyene macrolides. 444
The single and combined site-specific mutations in AT0 and their effects on nystatin 445
biosynthesis are presented in Table 1. Although none of the recombinant strains were shown to 446
produce novel nystatin analogues, most of the mutants were able to successfully initiate 447
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nystatin biosynthesis, albeit at different levels. Substitutions of several amino acids in region A 448
of AT0 (see Figure S1, Supplementary data), in particular T56V, G57D, W58V, and V89Q, 449
apparently had a mildly negative effect on the ability of NysA to initiate nystatin biosynthesis. 450
No new polyene macrolides besides those already described for the wild-type S. noursei (8) 451
could be detected in the extracts of the recombinant strains expressing the abovementioned 452
mutants. 453
Next, the His185 and Phe187 residues in the region B of the AT0 domain (Figure S1, 454
Supplementary data), which were shown to be important for substrate specificity of AT domain 455
from erythromycin PKS (9), were changed to Tyr and Ser, respectively. Both single mutations 456
and combination thereof had a negative effect on nystatin biosynthesis by the NDA59 457
expressing these mutants, reducing nystatin production level by ca 25 % and 80 %, respectively 458
(Table 1). Introduction of the V89Q mutation into the H185Y F187S double mutant have 459
drastically (by ca 98 %) reduced the nystatin production, while having only moderate effect 460
when introduced into either of the single mutants. Next, the H185Y and F187S mutations were 461
introduced on the T56V G57D W58V V89Q background. All such mutants were shown to 462
provide for nystatin biosynthesis at the levels ranging from 3 % to 37 % of the control. 463
Detailed analysis of the extracts from all the abovementioned recombinant strains failed to 464
detect production of any new nystatin analogues. 465
Finally, we have constructed the T56V G57D W58V V89Q M117I F187S NysA AT0 mutant 466
based on the suggestion by Keatinge-Clay et al. (14), who implied that the Met and Phe 467
residues (corresponding to Met117 and Phe187) provide a selectivity filter for the malonyl-468
CoA:ACP transacylase of S. coelicolor. As in all other cases, however, this NysA mutant 469
failed to initiate synthesis of new nystatin analogues, while being able to restore the level of 470
nystatin production to ca 11 % compared to the wild-type NysA (Table 1). 471
472
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Combination of the AT0 and KS-like domain mutations reveals interplay between certain 473
residues in these domains in initiation of nystatin biosynthesis 474
Next, we examined a possibility that there might exist an interplay between certain residues in 475
AT0 and KS-like domain of NysA in terms of ability of this protein to initiate nystatin 476
biosynthesis. We then combined several AT0 mutations with the mutations in the KS-like 477
domain of NysA constructed previously (6). The original KS-like domain of NysA harbours a 478
Ser residue instead of Cys in it’s active site, and it was suggested by others that amino acid 479
residue in this position might be involved in decarboxylation during condensation with the first 480
extender unit. As it was previously shown, however, changing this Ser residue in the NysA KS-481
like domain to either Glu or Cys does not significantly affect the ability of this protein to 482
initiate nystatin biosynthesis. The KSQ and KSC mutations were first introduced into the 483
H185Y and F187S single AT0 mutants, and the recombinant strains were tested for nystatin 484
production. The data obtained clearly demonstrated that the KSC mutation completely 485
abrogates initiation of nystatin biosynthesis by the H185Y AT0 mutant, while the KSQ 486
mutation has no effect on this background (Table 1). The situation was different on the F187S 487
mutant background, where the KSQ mutation had no effect, while the KSC mutation had a 488
moderate negative effect. The KS mutations were then introduced into the H185Y F187S 489
double mutant. The KSC H185Y F187S mutant was unable to initiate nystatin biosynthesis, 490
while the KSQ H185Y F187S restored the nystatin production in NDA59 to ca 17 % (Table 1). 491
Taken together, these results suggest reveal a complex interplay between the KS-like and AT 492
domains in the nystatin PKS loading module during initiation of the polyketide assembly (see 493
Discussion). 494
495
DISCUSSION 496
497
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Multiple attempts on changing the starter unit specificity of the nystatin PKS loading module 498
NysA using domain swaps, heterologous expression of loading module with wider substrate 499
specificities, construction of hybrids, and site-specific mutagenesis of the AT domain in this 500
protein have failed to produce novel nystatin analogues. Since cross-complementation of S. 501
noursei nysA mutant withBecause RrimA (known to have substrate flexibility) heterologously 502
expressed in S. noursei is able to complement the nysA mutation yieldinged only nystatin, we 503
suggest that the failure to produce new nystatin derivatives analogues ismust be due to the 504
inability of the first extender module of NysB PKS to accept the propionate or butyrate as 505
starter units, rather than to the absence of flexibility of AT0 domain of NysA in recognizing 506
other carboxylic acids. This suggestion is strongly supported by the fact that hybrids C and D 507
(both carrying the AT0 domain of NysA) can restore both rimocidin and CE-108 production in 508
S. diastaticus var. 108 rimA mutant. Considering these results, and the fact that S. noursei is 509
capable of producing nystatin and its analogues in substantial quantities (8), most probably, the 510
key factor for generating new nystatin derivatives lies within the KS1 domain of NysB. The 511
latter domain, which, for some reason that could not be deduced from the amino acid sequence 512
of this domain, probably has a strong preference for processing an acetate unit as a biosynthetic 513
primer. However, since our attempts on manipulating replacement of the KS1 domain in NysB 514
with another KS domain from the nystatin PKS have so far been unsuccessful, we could not 515
confirm this hypothesis experimentally by replacing KS1 domain with a heterologous one. 516
From the results obtained we can also suggest that the AT0 domain of the NysA, unlike that of 517
PimS0, shares with RimA similar flexibility for substrate recognition, at least for butyryl CoA 518
and malonyl (or/and acetyl) CoA. In addition, the RimA was recently shown to accept 519
methylmalonyl CoA, priming the rimocidin PKS with a propionate starter (Seco et al., 520
unpublished data). Possible alternative strategy for engineering NysB towards greater 521
flexibility with regard to the priming could be replacement of KS1 with a heterologous KS 522
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domain known to accept a large number of different acyl CoAs. From this point of view, KS 523
domain from the first module of avermectin PKS could be a good candidate, as it was shown to 524
accept and extend over 40 starter units (19). If successful, such replacement can also be 525
combined with the replacement of AT0 domain in NysA with AT domain from avermectin PKS 526
loading module, providing opportunity for production of a wide range of nystatin analogues 527
with alternative C-37 side chains. 528
Interestingly, while yielding no new nystatin analogues, one of the hybrids between the 529
rimocidin/CE-108 PKS loading module RimA and NysA was able to efficiently initiate 530
nystatin biosynthesis. This ability was shown only for the hybrid containing the original NysA 531
KSS – AT0 domain assembly. Despite the fact that KS-like domains in the NysA and RimA are 532
quite similar (62 % identity), and KS-like domain of RimA also contains Ser in the “active 533
site”, the presence of the NysA KS, AT, and the KS-AT linker seems to be crucial for the 534
functionality of the hybrid. 535
From the data on erythromycin PKS (9), it could have been expected that the H185 F187S 536
double mutant shall utilize methylmalonyl CoA as a starter, facilitating production of 537
methylnystatin. The facts that methylnystatin was not detected in the extract of NDA59 538
expressing the latter mutant, and that nystatin yield was substantially reduced could be 539
explained by a shift of specificity towards methylmalonyl CoA combined with reduced ability 540
of the NysB to be primed with propionate starter. 541
Results obtained after combination of the AT0 mutations with the alterations of the “active site” 542
residue of the NysA KS-like domain provided a clear evidence of cooperation of these domains 543
in the process of initiation of nystatin biosynthesis. Previously, based on the results obtained 544
upon site-specific mutagenesis of the KS-like domain of NysA (6), we have demonstrated that 545
the residue in the “active site” of the KS-like domain in NysA does not affect the functionality 546
of this protein with the wild-type AT0. The latter implied that the “active site” residue in KS-547
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like domain of NysA, in contrast to the KSQ domains in other PKS loading modules (4), is not 548
crucial for decarboxylase activity required for utilization of malonyl CoA as a starter. The fact 549
that the KSQ H185Y mutant is non-functional, in contrast to the H185Y, KSC H185Y, KSC 550
F187S and KSQ F187S mutants, strongly suggests an interplay between the “active site” 551
residue of KS-like domain of NysA and His185 in AT0. From these data, one would expect the 552
KSQ H185Y F187S mutant to be non-functional as well in terms of initiation of nystatin 553
biosynthesis. Unexpectedly, this mutant provided for reduced but significant nystatin 554
biosynthesis, while the KSC H185Y F187S completely failed to do so. These results suggest 555
indicate that the “active site” residue in the KS-like domain of NysA cooperates with both 556
His185 and Phe187 in AT0 during recruitment of a starter unit, and that the composition of this 557
“triad” is crucial for successful initiation of nystatin biosynthesis. 558
559
ACKNOWLEDGEMENTS 560
561
This work was supported by the Research Council of Norway and the grant BIO2008-03683 562
from the Spanish MICINN. 563
564
REFERENCES 565
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3029. Sletta, H., Borgos, S.E., Bruheim, P., Sekurova, O.N., Grasdalen, H., Aune, R., 656
Ellingsen, T.E., and S.B. Zotchev. 2005. Nystatin biosynthesis and transport: nysH and 657
nysG genes encoding a putative ABC transporter system in Streptomyces noursei ATCC 658
11455 are required for efficient conversion of 10-deoxynystatin to nystatin. Antimicrob 659
Agents Chemother. 49:4576-4583. 660
310. Schwecke, T., Aparicio, J.F., Molnari, I., Konig, A., Khaw, E., Haydock, S.F., 661
Oliynyk T.M., Caffrey. P., Cortes. J., Lester.J.B., Bohmt, G.A, Staunton.J., and 662
P.F. Leadlay. 1995. The biosynthetic gene cluster for the polyketide immunosuppressant 663
rapamycin. Proc. Natl. Acad. Sci. USA 92:7839-7843. 664
321. Tang, Y., Kim, C.Y., Mathews, I.I., Cane, D.E., and C. Khosla. 2006. The 2.7-A 665
crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B 666
synthase. Proc Natl Acad Sci U S A. 103:11124-11129. 667
332. Tsuji, S.Y., Cane, D.E., and C. Khosla. 2001. Selective protein-protein interactions 668
direct channeling of intermediates between polyketide synthase modules. Biochemistry. 669
40:2326-2331. 670
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343. Weissman, K.J. 2004. Polyketide biosynthesis: understanding and exploiting 671
modularity. Philos Transact A Math Phys Eng Sci. 362:2671-2690. 672
673
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Table 1. Effect of certain amino acid substitutions in the AT0 domain of NysA on the ability 674
of the recombinant protein to restore nystatin biosynthesis in the S. noursei nysA- mutant. 675
676
AT0 mutations KS mutations Nystatin yield (% of WT) T56V G57D W58V V89Q H185Y F187S M117I KSC KSQ
x x 46±2
x x x 46±4
x x x x 51±5
x 82±6
x x 55±5
x 72±5
x x 62±5
x x 22±2
x x x 2±1
x x x x x 37±4
x x x x x 10±2
x x x x x x 3±1
x x x x x x 11±2
x x 41±4
x x 72±4
x x 72±5
x x 0
x x x 0
x x x 17±1
x x x 93±6
677
NB: The data represent an average from 3 parallel experiments. NDA59 mutant complemented 678
with wild-type nysA gene was used as a reference. 679
680
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Figure legends 681
682
Figure 1. Chemical structures of nystatin A1 (acetate starter), rimocidin/CE-683
108 (acetate/butyrate starter), and pimaricin (acetate starter). 684
685
Figure 2. Current modles for initiation of polyene macrolide biosynthesis in S. 686
noursei (nystatin producer) and S. diastaticus var. 108 (rimocidin/CE-108 687
producer). 688
689
Figure 3. Schematic representation of the NysA and RimA loading modules, and their 690
hybrids. Solid boxes and thin lines represent RimA enzymatic domains and interdomain 691
linkers, respectively. 692
693
Figure 4. HPLC analysis of the culture extracts of the S. diastaticus rimA mutant 694
complemented with (A) rimA; (B) pimS0; (C) nysA-rimA hybrid C; (D) nysA-rimA hybrid D. 695
696
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Figure 1. 697
698
699
700
701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739
Nystatin A1
COOH
CH3
OH CH3
CH3
O O H O H O H
O H
O H
O HO H
O
O
O CH3O H
NH2O H
O
Rimocidin/CE-108R = -CH3, CE-108R = -CH2-CH2-CH3, rimocidin
COOHOO H
C H3
O
R
O HO H
O
C H3O H
N H2O H
OO
O H
Pimaricin
COOHCH3
O HO H
OO HO
CH3O H
NH2O H
OO
O
O
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Figure 2. 740 741
742
Formatted: Numbering: Continuous
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Figure 3. 743
744
KSS DH0 ACP0
ATRimA
KSS AT0 DH0 ACP0
CoLRimA ACPRimA
AT0 DH0 ACP0
CoLRimA ACPRimA KSRimA
Hybrid B
Hybrid C
Hybrid D
CoLRimA ACPRimA KSRimA ATRimA
DH0 ACP0
KSS AT0 DH0 ACP0
CoL ACP KSS AT ACP
RimA
Hybrid A
NysA
Formatted: Numbering: Continuous
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Figure 4. 745
746
Formatted: Numbering: Continuous
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