Rational Design of Hybrid Natural Products by ... - ACS Publications

Subscriber access provided by IDAHO STATE UNIV

Article

Rational design of hybrid natural products by utilizing the promiscuity of an amide synthetase Jing Zhu, Songya Zhang, David L. Zechel, Thomas Paululat, and Andreas Bechthold ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00351 • Publication Date (Web): 16 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 3

ACS Chemical Biology

Rational design of hybrid natural products by utilizing the promiscuity of an amide synthetase

4

Running Title. New natural products by genome editing

5 6

Jing Zhu1, Songya Zhang2 , David L. Zechel3, Thomas Paululat4, Andreas Bechthold1*

7 8 9 10 11

1. Pharmaceutical Biology and Biotechnology, Institute of Pharmaceutical Sciences, Albert-Ludwigs University, Stefan-Meier-Str. 19, Freiburg, Germany 2. Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 1068 Xueyuan Avenue, Shenzhen, P.R.China

12

3. Department of Chemistry, Queen's University, 90 Bader Lane, Kingston, Ontario, K7L 3N6, Canada

13

4. Department of Organic Chemistry, University of Siegen, Adolf-Reichwein-Str. 2, Siegen, Germany

14

*Corresponding Authors. AB: Email: [email protected], Tel. +49 761 203

15

8371.

16 17 18 19 20 21 22 23 24 25 26 27

1 ACS Paragon Plus Environment

ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

28

ABSTRACT

29

WS9326A and annimycin are produced by Streptomyces asterosporus DSM 41452. WS9326A is a

30

nonribosomal peptide synthetase-(NRPS-) derived depsipeptide containing a cinnamoyl moiety, while

31

annimycin is a linear polyketide bearing a 2-amino-3-hydroxycyclopent-2-enone (C5N) group. Both gene

32

clusters have been sequenced and annotated. In this study we show that the amide synthetase Ann1,

33

responsible for attaching the C5N unit during annimycin biosynthesis. has the ability to catalyse fortuitous

34

side reactions to polyenoic acids in addition to its main reaction. Novel compounds were generated by

35

feeding experiments and in vitro studies. We also rationally designed a hybrid natural product consisting

36

of the cinnamoyl moiety of WS9326A and the C5N moiety of annimycin by creating a mutant of S.

37

asterosporus that retains genes encoding biosynthesis of the C5N unit of annimycin and the cinnamoyl

38

group of WS9326A. The promiscuity of Ann1 also proved useful for trapping compounds that arise from

39

acyl-ACP intermediates, which occur in the biosynthesis of the cinnamoyl moiety of WS9326A, by

40

hydrolysis. In this pathway we postulate that sas27 and sas28 genes are involved in the biosynthesis of the

41

cinnamoyl moiety in WS9326A.

42


Page 2 of 26

Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


43

INTRODUCTION

44

Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are large enzyme complexes

45

that synthesize a variety of natural products. Biosynthesis of these compounds processes through loading

46

of building blocks, C-N and C-C bond formation and chain translocation, and finally thioester lysis to

47

release the product. The final products are often modified and these activities are the province of specialized

48

enzymes 1. Compounds may also contain moieties which are not as widely spread. Examples are unsaturated

49

2-amino-3-hydroxy-cyclopent-2-enone moieties (C5N moieties)

50

are components of moenomycins 3, asukamycin 4, colabomycin E 5, bafilomycins 6 and annimycin 7 (Figure

51

S1), while cinnamoyl moieties are components of skyllamycin 8, coprisamide A 9, atratumycin

52

pepticinnamin

53

(Figure S2). The biosynthetic gene clusters of WS9326s and annimycins were recently identified 17. The

54

biosynthesis of C5N units and their attachment is well understood from in vitro studies with purified

55

enzymes

56

biosynthesis of the C5N unit include Ann2, a pyridoxal phosphate-dependent 5-aminolevulinate synthase,

57

Ann3, an acyl-CoA ligase, and Ann1, a putative ATP-dependent amide synthetase responsible for the

58

ligation of C5N unit to a polyenoic acid (Figure 1). In contrast to the C5N unit, the biosynthesis of the

59

cinnamoyl moiety has not been studied in detail.

, NC-1

11

18 19 20.

12,

WS9326A 13, mohangamide A

14,

2

and cinnamoyl moieties. C5N moieties

atrovimycin

15,

and kitacinnamycins A

,

10

16

In the case of 4-Z-annimycin, the homologous enzymes predicted to be involved in the

60

The C5N and cinnamoyl containing natural products are of pharmaceutical interest. Asukamycin and

61

moneomycin exhibit strong antibacterial activitiy 3 4 , colabomycin E acts as an anti-inflammatory agent 5

62

and bafilomycins exhibit a wide range of biological activity, including anti-tumor, anti-parasitic,

63

immunosuppressant and antifungal activity 21. Skyllamycins as well as WS9326s also exhibit a broad

64

range of pharmaceutical activities

65

factor signaling pathway

66

displays significant anti-parasitic activity

67

Staphylococcus aureus 24, and WS9326H exhibites antiangiogenic activity 25.

22,

13 22 23 24 25,

skyllamycin A inhibits the platelet-derived growth

WS9326A is a potent tachykinin receptor antagonists 13, WS9326D 23,

WS9326B has shown to be active against



68

In this study we show that the substrate promiscuity of the amide synthetase Ann1 can be used to generate

69

new C5N containing molecules. Previously, while studying the biosynthesis of annimycin A in S.

70

asterosporus DSM41452 we created a mutant which produced a new polyketide, annimycin B. The mutant

71

was created by integrating a plasmid into ann5 which most probably was leading to an non-functional

72

polyketide synthase gene and not to module skipping as discussed in 17. Annimycin B is a truncated form

73

of annimycin consisting of a polyketide chain and a C5N unit. It exhibits modest inhibitory activity against

74

Plasmodium falciparum

75

that a so far unknown PKS-gene cluster is responsible for its formation. The accumulation of annimycin B

76

indicates that Ann1 has certain substrate flexibility. In this study we describe the heterologous expression

77

of three genes (ann1, ann2 and ann3) responsible for the formation of the C5N unit during annimycin

78

biosynthesis in Streptomyces lividans TK24. We show through feeding experiments that the amide

79

synthetase Ann1 can catalyze the transfer of the C5N unit to unsaturated fatty acids with widely variable

80

structures. We exploited this promiscuity to genetically engineer cross-reactivity between the WS9326A

81

and annimycin biosynthetic pathways that are present in S. asterosporus DSM41452. Additionally,

82

mutagenesis has revealed the identity of 4 genes (sas27, sas28, sas30, and sas32) that encode enzymes

83

involved in the biosynthesis of the cinnamoyl group of WS9326A.

17.

The origin of the polyketide-chain of annimycin B is not known. We belive

84 85 86 87 88

Keywords NRPS, cinnamoyl moiety, C5N unit, polyketide, WS9326A, annimycin


Page 4 of 26

Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


90

RESULTS AND DISCUSSION

91

Studies on the function of Ann1

92

To show that ann1 is involved in the biosynthesis of annimycin, S. asterosporus DSM 41452Δann1 was

93

generated. Annimycin production was abolished in this mutant and its precursor polyenoic acid was

94

accumulating instead. In complementation studies annimycin production could be restored (data not

95

shown). In order to express ann1, ann2 and ann3 in E. coli genes from S. asterospours DSM 41452 were

96

amplified by PCR and ligated into pET28a(+) to generate expression plasmid pET28a(+)-Ann1,

97

pET28a(+)-Ann2 and pET28a(+)-Ann3. Plasmids were introduced into E. coli BL21(DE)star for protein

98

expression. The recombinant proteins of Ann1 (58.05 kD), Ann2 (47.22 kD) and Ann3 (56.84 kD) were

99

produced with a N-terminal His6 tag in E. coli BL21 (DE3)star, and purified by Ni-NTA affinity

100

chromatography and size exclusion chromatography (Figure S3).

101

Ann1, Ann2 and Ann3 were incubated with Mg2+, ATP, CoA, pyridoxal phosphate, glycine, succinyl-

102

CoA (substrates for the biosynthesis of the C5N unit) and 2,4,6-octatrienoic acid (OTEA) or caffeic acid at

103

28 ºC for 2 h. As shown by HPLC-MS analysis OTEA-C5N and caffeic acid-C5N were successfully

104

produced. No product appeared when any of the enzymes was missing in this reaction, confirming that all

105

three enzymes are needed for the formation and attachment of the C5N unit (Figure S4 and S5).

106

For the formation of the C5N moiety of annimycin in the surrogate host Streptomyces lividans TK24,

107

ann1, ann2 and ann3 gene cassette was cloned into the replicative vector pUWL-H. The resulting plasmid

108

pUWL-H-ann1-3 was transferred into S. lividans TK24 by intergeneric conjugation. The correct mutant

109

was named S. lividans TK24::pUWL-H-ann1-3. When OTEA was added to a culture of S. lividans

110

TK24::pUWL-H-ann1-3 a novel compound was produced (Figure S6). UV spectra and the mass of the

111

compound indicate that the compound OTEA-C5N consists of OTEA and the C5N unit.

112



113

Generation and characterization of J1 (1), J5 (2), J3 (3), J4 (4) and J6 (5)

114

For the generation of a mutant with deletions in the gene clusters of WS9326A and annimycin A, S.

115

asterosporus DSM41452ΔNmet

116

non-producing mutant in which the gene encoding the embedded methyltransferase domain in the NRPS

117

gene-sas17 was deleted. Analogous to the mutagenesis performed in S. calvus ATCC 133827 plasmid

118

pUC19Δ3100spec was integrated into ann5 of the annimycin cluster to yield the annimycin and WS9326A

119

non-producing mutant S. asterosporus DSM 41452ΔNmet::pUC19Δ3100spec. When this mutant was

120

grown in SG+ medium beside annimycin B five new products named J1 (1), J5 (2), J3 (3), J4 (4) and J6 (5)

121

(Figure 2) were detected by LC-MS analysis. The compounds were purified by HPLC and their structures

122

determined (Figure 3) by high resolution MS and NMR spectroscopy (Figures S7-S13, summarized in

123

Tables S5-S11).

26

was used as host. S. asterosporus DSM 41452ΔNmet is a WS9326A-

124

The molecular ion observed for 1 by HRESI-MS corresponded to a molecule with the formula C19H23NO3

125

and 9 degrees of unsaturation (obsvd: m/z = 312.1604 [M-H]-, calcd for C19H22NO3, m/z = 312.1605 [M-

126

H]-). The 1H NMR spectrum of 1 (Figure S7b) exhibited signals corresponding to the a pair of cis olefinic

127

protons at δH 6.54 dt (11.6, 1.7) and 5.77 dt (11.6, 7.3), an ortho-substituted aromatic ring at δC 130.5

128

suggested that this pentene moiety is linked with the 3-phenylpropanoic fragment at the ortho-position. The

129

1H

130

2.37 (2H, m) and 2.49 (2H, m). HMBC spectrum shown that this methylene pair have correlations with

131

three olefinic carbons C-1’ (δC 115.6), C-2’(δC 173.7), and a carbonyl carbon C-5’(δC 197.8). Those typical

132

signals suggested the presence of a C5N moiety in the molecule. In addition, the HMBC correlations of

133

hydroxyl hydrogen 2’-OH (δH 13.08) with carbonyl carbon C-1 (δC 174.8) indicated that the C5N ring

134

moiety and the substituted 3-phenylpropanoic chain are connected together by an amino bond to form the

135

structure as shown in Figure 3.

NMR and COSY spectra for 1 show signals corresponding to another pair of methylene protons at δH

136

The molecular ion observed for 2 by HRESI-MS corresponded to a molecule with the formula C19H21NO3

137

and 10 degrees of unsaturation (obsvd: m/z = 310.1446 [M-H]-, calcd for C19H20O3N, m/z = 310.1449). The


Page 6 of 26

Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


138

1D and 2D NMR data of 2 (Figure S10) resemble that of 1, including the signals for the pentene moiety,

139

the C5N ring, and the ortho-substituted aromatic ring. In contrast to 1, the 1H NMR spectrum of 2 exhibits

140

additional signals at δH 6.84 (d, J = 15.5Hz) and 7.83 (d, J = 15.5Hz), which was assigned to a trans olefin

141

at C2-C3. The final assigned structure of 2 (Figure 3) suggests that a dehydrogenation reaction occurs at

142

the α-β position of the 3-phenylpropanoic chaing to produce the cinnamoyl moiety.

143

The NMR spectra for 3, 4, and 5 lacked aromatic signals, indicating that these compounds were distinct

144

from 1 and 2. The molecular ion observed for 3 by HRESI-MS corresponded to a molecule with a molecular

145

formula C17H27NO3 (obsvd: m/z = 292.1917 [M-H]-, calcd for C17H26NO3, m/z = 292.1918). In comparison

146

to 1 the 1H NMR spectrum is lacking aromatic signals. The corresponding proton and carbon signals were

147

assigned by 2D NMR (COSY, HSQC and HMBC). The spectra revealed the existence of an aliphatic chain

148

containing one cis double bond. Interestingly, the aliphatic chain terminates with an isopropyl group.

149

Finally, the aliphatic chain contains carbonyl carbon C-1 (δC 175.4) that links to the C5N unit (Table S6,

150

Figure S8a-f). The molecular ion observed for 4 by HRESI-MS corresponded to a molecule with a

151

molecular formula C17H27NO3 (obsvd: m/z = 292.1917 [M-H]-, calcd for C17H26NO3, m/z = 292.1918). The

152

NMR spectra revealed 4 was a structural isomer of 3, terminating with a linear aliphatic chain (Table S7a-

153

b, Figure S9a-k). The molecular ion observed for 5 by HRESI-MS corresponded to a molecule with a

154

molecular formula C18H29NO3 (obsvd: m/z = 306.2072 [M-H]-, calcd for C18H28NO3, m/z = 306.2075). The

155

NMR spectra for 5 resembled that of 3 and 4, with the exception of additional signals corresponding to a

156

terminal isopropyl group (Figure S11a-k). Overall, the structure of 2 is consistent with a condensation

157

reaction between the C5N unit and the cinnaomyl-moiety of WS9326A, while 1 is a derivative of 2 via

158

saturation of the C2-C3 alkene. In contrast, the fatty acid chains of 3, 4, and 5 seem to originate from

159

another source.

160

Gene disruption of sas32 and sas33

161

Fourteen genes (sas24-sas37) of the WS9326 biosynthetic gene cluster have been predicted to be involved

162

in the biosynthesis of the cinnamoyl moiety (Table S4). Sas32 and Sas33 are predicted to act as

163

ketosynthases as part of a reducing type II polyketide synthase system. To ascertain that Sas32 and Sas33 7 ACS Paragon Plus Environment


164

are involved in the biosynthesis of 1 and 2, the corresponding genes were deleted. Extracts of both mutants

165

(S. asterosporus DSM 41452ΔNmet:: pUC19Δ3100specΔsas32::aac(3)IV and S. asterosporus DSM

166

41452ΔNmet::pUC19Δ3100spec::pKC1132-Insas33) were analyzed by LC-MS. Both mutants failed to

167

produce 1 and 2, indicating that sas32 and sas33 are essential for the biosynthesis of both compounds.

168

However, compounds 3, 4 and 5 were still produced by both mutants.

169

Gene disruption of sas24, sas28, and sas27

170

To further narrow the scope of the genes encoding the biosynthesis of 1 and 2 the genes sas24, sas27, and

171

sas28 were individually inactivated. The sas24 sequence is predicted to encode a ferredoxin or FAD-

172

dependent oxidoreductase, sas27 an isomerase, and sas28 a phytoene dehydrogenase or NAD(P)/FAD-

173

dependent oxidoreductase. Sas27 and Sas28 share 67% and 57% amino acid identity with Sky27 and Sky28

174

from the skyllamycin pathway, respectively 27. Sky27 was proposed to be an isomerase involved in the

175

biosynthesis of an intermediate with a specific (4E, 6Z, 8E)-configuration and Sky28 was proposed to be

176

an oxidoreductase involved in the formation of the benzene ring of the cinnamoyl moiety of skyllamycin

177

27.

178

sas28 led to the production of novel compounds named J8 (6) and J9 (7), respectively (Figure 2). Gene

179

complementation experiment was carried out to verify the function of gene sas27. The integrative

180

plasmid pTESbz-sas27 was constructed and transformed into the sas27 mutant. It was observed

181

that the production of 1 and 2 were restored in mutant S. asterosporus DSM 41452Δsas27:: pTESbz-sas27

182

(Figure 2 and 4). This finding indicates Sas27 is essential for the formation of the cinnamoyl moiety.

The deletion of sas24 still led to the production of 1 and 2 (Figure 2). In contrast, deletion of sas27 and

183 184

Isolation and characterization of J8 (6)

185

6 was isolated from the fermentation broth of S. asterosporus DSM 41452ΔNmet::pUCΔ3100specΔsas27,

186

as described in the experimental section. Structure determination was achieved by MS analysis and NMR

187

(Figure S12a-f, summarized in Table S10). The molecular formula of J8 (6) is predicted to be C17H21NO3

188

based on a molecular ion of m/z = 286.1448 [M-H]- in HRESI-MS (calculated m/z = 286.1449 [M-H]-). The

189

chemical structure of 6 was elucidated based on 1D and 2D NMR data (Table S10). The 1H NMR spectrum 8 ACS Paragon Plus Environment

Page 8 of 26

Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


190

showed the typical NMR signals for the exchangeable amide NH (δH 7.96) and hydroxyl protons (δH 13.3)

191

in the C5N scaffold of annimycin.7 Deduced from 1H NMR spectrum together with COSY an all trans

192

tetraene chain was identified due to the typical double bond chemical shifts (δH 7.3-5.9) and their typical

193

vicinal 1H-1H coupling constants range from 14.5 Hz to 15 Hz (Table S10). Consecutive COSY correlations

194

from H2 (δH 6.23) to H12 (δH 0.89) revealed those conjugated olefins are flanked with two methylene group

195

(C10-C11) and a terminal methyl group (C12), forming a coupling system. The other side of the tetraene is

196

connected to an amide carbonyl (C-1, δC 167.3), established by HMBC correlations C-1/2-H and C-1/3-H.

197

Attached to the carbonyl is the C5N unit showing typical carbon chemical shifts of C1’ (δC 115.8), C2’ (δC

198

176.3), C3’ (δC 26.3) and C4’ (δC 35.9). Taken together, the chemical structure of 6 was determined to be

199

that shown in Figure 5.

200

Isolation and characterization of J9 (7)

201

7 was obtained as yellow amorphous powder. Due to the small amount we were not able to get a pure

202

compound so the structure elucidation was done using an impure sample. Moreover the amount did not

203

allow to measure a carbon NMR spectrum so all carbon data was taken from HSQC and HMBC.

204

Comparison to the other derivatives allow us to prove the presence of C5N unit due to typical signals. The

205

chain attached via the amide (C-1, δC 166.0) could be identified as an all trans pentaene with a methyl group

206

on the other side. The all trans pentaene could be established via alternating coupling constants of arroud

207

15 Hz and around 11 Hz supported by informations from H2BC and HMBC (Tabele S11). To prove the

208

pentaene signals in an impure sample a 1D TOCSY with excitation on 7-H signal (δH 6.45, Figure S13g)

209

and band selective TOCSY spectra with different mixing times (150, 50, 15ms, excitation of double bond

210

signals, Figures S13h-13j) were measured to establish the complete signal assignment of J9 (Figures S13a-j,

211

summarized in Table S11) resulting in the structure shown in Figure 5. The final structure agrees with the

212

observed high resolution mass spectrum of 7, which exhibits a molecular ion of m/z = 284.1292 [M-H]- that

213

matches the predicted molecular formula of C17H19NO3 (calculated m/z = 284.1292 [M-H]-) (Figure S13a ).

214

The mass of 7 is two Da smaller than that of 6, which agrees with the trans configured double bond between

215

C10 and C11 that is observed by 1H and 2D NMR. A longer absorbance maximum wavelength (383 nm) is 9 ACS Paragon Plus Environment


216

also observed for 7 relative to 6 (Figure S14), which agrees with the greater degree of conjugation of the

217

former.

218

CONCLUSION

219

S. asterosporus DSM 41452 is the producer of WS9326s and annimycins. The NRPS derivatives WS9326s

220

contain a cinnamoyl-moiety at their N-terminus and the PKS derived annimycins own a C5N moiety. Their

221

gene clusters located at different locus. In 2016, Thong et al. isolated two compounds from a Streptomyces

222

strain consisting of a C5N unit and a methylbenzene moiety, and their biosynthetic genes also were predicted

223

to be located at different position of the genome32. We demonstrated that Ann1 is flexible towards the

224

polyketide substrate by in vivo- and in vitro-studies. When biosynthetic genes of the NRPS of WS9326A

225

and the PKS of annimycin were deleted the C5N unit of annimycin was transferd to the cinnamoyl moiety

226

of WS9326A leading to compounds J1 (1) and J5 (2) (Figure 6). While compound 2 contains the entire

227

cinnamoyl moiety of WS9326A compound 1 contains a modified cinnamoyl moiety with a reduced bond

228

between C2 and C3. Compounds J3 (3), J4 (4) and J6 (5) were also produced by the strain. They consist of

229

the C5N moiety and unsaturated fatty acid moieties sharing a double bond between C5 and C6.

230

Recently the Ohnishi group reconstituted the Iga PKS in vitro and successfully led to the formation of a

231

polyene, which demonstrated that type II PKS posess the ability to biosynthesize highly reduced polyketides

232

28.

233

Iga12, the acyl carrier protein Iga10, and the ketoreductase (KR) Iga13 were found in the gene clusters of

234

WS9326A, skyllamycin (Table S4) and a number of other compounds belonging to the manumycin family

235

(e.g. asukamycins and colabomycins). Very recently, Bode and colleagues reported that aryl polyene

236

pigments (APE) are biosynthesized by this new type II PKS. X-ray crystallography analysis demonstrated

237

that KS, CLF, DH and KR undergo protein-protein interactions during APE biosynthesis 29. As WS9326A

238

is a NRPS-derivative to which the cinnamoyl moiety is attached, it was hypothesized that the cinnamoyl

239

moiety might be biosynthesized by the highly reducing type II PKS and by a so far unknown cyclase. The

240

mutants S. asterosporus DSM 41452ΔNmet::pUCΔ3100spec::pKC1132-Insas33 and S. asterosporus DSM

241

41452ΔNmet::pUCΔ3100specΔsas32::aac(3)IV fail to produce 1 and 2. Instead the accumulation of the

The corresponding homologs of the beta-ketoacyl synthase (KS) Iga11, the chain length factor (CLF)


Page 10 of 26

Page 11 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


242

polyenes J8 (6) and J9 (7) was observed. These observations confirm that the substituted cinnamoyl moiety

243

originates from a highly reduced PKS pathway. Interestingly, the carbon chain length of the acyl moiety of

244

products accumulating in the sas27- and sas28-mutant is different from 1 and 2 (Figure 6). This may

245

indicate that certain reactions catalyzed by Sas27 and Sas28 are required for further chain elongation. Ge

246

and coworkers found homologes of sas27 coexist with this special highly reducing type II PKS-encoding

247

genes in many other gene clusters. It was discussed that the isomerases Kcn15 and Kcn33 are involved in

248

the cyclization of this substituted cinnamoyl moiety during kitacinnamycins biosynthesis 16. Our reults may

249

indicate that Sas28 acts as reductase involved in the reduction of a terminal double bond.

250

Fatty acids and linear polyketides are often very difficult to detect as they are either instable or not well

251

visible using UV detectors. The promiscuity of Ann1 is striking. By attaching the C5N unit to fatty acids

252

novel compounds are produced. Ann1 together with Ann2 and Ann3 act like a trap capturing the

253

compounds. Eventually it will also accept intermediates of the biosynthesis of the cinnamoyl moiety of

254

WS9326A and this will open us the opportunity to study this interesting pathway in more detail.

255 256

METHODS

257

General

258

All antibiotics, medium components used in this study were purchased from Roth and Sigma-Aldrich.

259

Restriction enzymes, T4 DNA ligase, NEBuilder® HiFi DNA Assembly Master Mix were bought from

260

NEB Biotechnology Co. Ltd.. Plasmid, gel purification and cycle-pure kits were acquired from Promega.

261

Primer synthesis and DNA sequencing were performed by Eurofins Co., Ltd..

262

Strains, plasmids and culture conditions

263

Primers, plasmids and strains used in this study are described in Table S1-S3. E. coli DH10B were cultivated

264

in liquid or on solid LB medium at 37 °C, overnight. E. coli ET12567 (pUZ8002) or ET12567(pR9406)

265

were used for conjugation

266

cultivated in liquid TSB medium and fermented in SG+ medium. The working concentration of antibiotics

267

was used as follows: ampicillin (100 µg mL-1), apramycin (50 µg mL-1), chloramphenicol (25 µg mL-1),

30.

BW25113/pKD46 was used for λ Red-recombination. Streptomyces was



268

kanamycin (50 µg mL-1), fosfomycin (200 µg mL-1), spectinomycin (100 µg mL-1).

269

Generation of S. lividans containg ann1, ann2 and ann3

270

Ann1, ann2 and ann3 gene cassette was amplified from S. asterosporus DSM 41452 using primers listed

271

in Table S1. The PCR product was ligated to pUWL-H to generate pUWL-H-ann1-3, which was introduced

272

into S. lividans TK24 by intergeneric conjugation. Correct exconjugants were selected by their ability to

273

grow on hygromycin-supplemented MS agar plates.

274

Feeding of OTEA to S. lividans TK24::pUWL-H-ann1-3

275

1 mL of a 24-h-old seed culture of S. lividans TK24::pUWL-H-ann1-3 was used to inoculate 100 mL of

276

SG ＋ medium. The strain was cultivated in a rotary shaker for 24 h. The culture was then supplemented

277

with 0.3 mg of the test compound OTEA dispersed in dimethyl sulfoxide (DMSO) (30 mg mL-1) every

278

time. The feeding procedure was repeated three times every 24 h. After 4 days of cultivation, the pH of the

279

culture was adjusted to 4, the culture was centrifuged, the supernatant was extracted with ethyl acetate, and

280

the received crude extract was analyzed for biotransformation products by LC-MS.

281

Cloning, overexpression, and purification of Ann1, Ann2 and Ann3

282

Ann1, ann2 and ann3 were amplified by PCR using using primers listed in Table S1, and genomic DNA of

283

S. asterospours DSM 41452 as template. PCR products were ligated into pET28a(+) to generate plasmid

284

pET28a(+)-Ann1, pET28a(+)-Ann2 and pET28a(+)-Ann3. E. coli BL21(DE)star was used as host for

285

protein expression. 20 ml of overnight cultures were used to inoculate 2 L LB medium containing 50 µg

286

mL-1 kanamycin. Cells were gown at 37 °C to an OD600 of 0.6. After induction with 0.1 mM isopropyl-β-

287

D-thiogalactopyranoside (IPTG) cell were grown at 20 °C for 16 h. Cells were harvested by centrifugation

288

at 4 °C (5000 rpm, 10 min), resuspended in 30 mL Buffer A (50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 5 mM

289

imidazole) and lysed by sonication on ice. Cell debris were removed by ultracentrifugation at 4 °C (10,000

290

rpm, 40 min). 2ml of Ni-NTA agarose resin was added and incubated with the supernatant at 4 °C for 1 h.

291

Then the mixture was loaded on Ni-NTA column, and proteins were eluted by gradient Buffer B (50 mM

292

Tris-HCl pH 8.0, 0.5 M NaCl, 50-500 mM imidazole). Purified proteins were concentrated by Vivaspin®


Page 12 of 26

Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


293

20 Ultrafiltration Unit. Gel filtration was performed on a Sephadex G-25 column connected to an ÄKTA

294

FPLC system at 4 °C (50 mM HEPES, 150 mM NaCl, pH 8.0). Finally, concentrated proteins were flash-

295

frozen in liquid nitrogen and stored at −80 °C.

296

Generation of S. asterosporus DSM 41452∆Nmet::pUC19∆3100spec

297

To disrupt ann5 gene from the annimycin gene cluster in S. asterosporus DSM 41452ΔNmet 26, plasmid

298

pUC19∆3100spec was introduced into S. asterosporus DSM 41452∆Nmet by intergeneric conjugation. The

299

correct exconjugant was selected as a spectinomycin-resistant mutant and verified by PCR verification

300

using primers listed in Table S1 (Figure S15b).

301

Generation of S. asterosporus DSM41452ΔNmet::pUC19Δ3100spec::pKC1132-Insas33

302

For inactivation of gene sas33 in S. asterosporus DSM 41452ΔNmet::pUC19∆3100spec, a partial gene

303

sas33 fragment was ligated into plasmid pKC1132 to yield plasmid pKC1132-Insas33. It then was

304

conjugated into S. asterosporus DSM41452ΔNmet::pUC19Δ3100spec. One apramycin-resistant

305

exconjugant was analyzed by PCR using primers listed in Table S1 (Figure S15c). It was named S.

306

asterosporus DSM 41452ΔNmet::pUC19Δ3100spec::pKC1132-Insas33.

307

Generation of plasmid for gene deletion

308

The vector pEpiFOS was used to clone part of sas gene cluster for gene mutagenesis in S. asterosporus

309

DSM 41452ΔNmet::pUC19Δ3100spec. The DNA sequence covering sas25-40 genes were separated into

310

two fragments and amplified from the genomic DNA of S. asterosporus DSM 41452 using primers listed

311

in Table S1 and then assembled into pEpiFOS using Gibson Assembled Master Mix following the

312

commercial protocol. The gene cassette consists of genes encoding kanamycin, OriT and C31 integrase,

313

was then integrated into the plasmid by λ Red-recombination, generating the plasmid pEpiFOS-sas-int.

314

Gene deletion of sas27, sas28, sas32 and sas24

315

The gene disruption was carried out by PCR targeting method 31. To delete sas27, sas28, and sas32 an

316

apramycin resistance gene flanked by two loxP sites (aac(3)IV-loxp) was amplified from plasmid pLERECJ

317

for each gene using primers listed in Table S1. Each cassette was then introduced into



Page 14 of 26

318

BW25113/pKD46/pEpiFOS-sas-int for target gene replacement by λ Red-recombination. The resulting

319

plasmids pEpiFOS-sas-intΔsas27, pEpiFOS-sas-intΔsas28 and pEpiFOS-sas-intΔsas32 were individually

320

transferred into S. asterosporus DSM 41452ΔNmet::pUC19Δ3100spec by triparental conjugation. Positive

321

mutants were screened for their kanamycin sensitivity and apramycin-resistence phenotypes, and they were

322

further verified by PCR using primers listed in Table S1. Finally, S. asterosporus DSM

323

41452ΔNmet::pUC19Δ3100specΔsas27::

324

41452ΔNmet::pUC19Δ3100specΔsas28::aac(3)IV,

325

41452ΔNmetpUC19Δ3100specΔsas32::aac(3)IV were constructed (Figure S15 d-g). Through Cre-loxp site

326

specific recombination, aac(3)IV cassette was excised from the chromosome of S. asterosporus DSM

327

41452ΔNmet::pUC19Δ3100specΔsas27::aac(3)IV

328

41452ΔNmet::pUC19Δ3100specΔsas27 (Figure S15e).

aac(3)IV, and

to

generate

S. S.

mutant

asterosporus asterosporus

S.

asterosporus

DSM DSM

DSM

329

To delete sas24, a 4890 bp PCR product containing sas24 was ligated into the pBluescript SK(-) to yield

330

pBSK-sas24. A aac(3)IV-loxp cassette was amplified using primers listed in Table S1. The resulting

331

cassette was used to replace gene sas24 in pBSK-sas24 by λ Red-recombination, yielding BW25113/pBKS-

332

sas24::aac(3)IV. Then, the fragment containg the aac(3)IV cassette was amplified using primers listed in

333

Table S1. The resulting fragment was cloned into the pKGLP2-gusA to generate plasmid pKGLP2-gusA-

334

sas24::aac(3)IV. It was transferred into S. asterosporus DSM 41452ΔNmet::pUC19Δ3100spec by

335

intergeneric conjugation. Hygromycin sensitive, apramycin-resistence colonies were selected, and were

336

confirmed by PCR using primers listed in Table S1. The correct mutant was designated as S. asterosporus

337

DSM 41452ΔNmet::pUC19Δ3100specΔsas24::aac(3)IV (Figure S15d).

338 339

Generation of S. asterosporus DSM41452Δann1

340

A 5711 bp PCR product containing ann1 was ligated into the pBluescript SK(-) to yield pBSK-ann1.

341

aac(3)IV-loxp cassette amplified from pLERECJ was used to replace ann1 in pBSK-ann1 by λ Red-

342

recombination, yielding BW25113/pBSK-ann1::aac(3)IV. Then, the fragment containing aac(3)IV was

343

amplified using primers listed in Table S1. The resulting fragment was cloned into the pKGLP2-gusA to


Page 15 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


344

generate plasmid pKGLP2-gusA-ann1::aac(3)IV. The plasmid was introduced into S. asterosporus DSM

345

41452 by conjugation and S. asterosporus DSM 41452Δann1::aac(3)IV was obtained after screenning for

346

apramycin resistance and hygromycin sensitive colonies. To eliminate aac(3)IV the Cre recombinase

347

expression plasmid pALCre was introduced into the mutant. The mutant S. asterosporus DSM 41452Δann1

348

was confirmed by PCR using primers listed in Table S1 (Figure S15a).

349 350

Complementation of Sas27 and Ann1

351

To construct plasmid pTESbz-sas27 and pTESbz-ann1, sas27 (798 bp) and ann1 (1568 bp) were amplified

352

from S. asterosporus DSM 41452 using primers listed in Table S1. The resulting fragments were ligated

353

into pTESbz and transferred into S. asterosporus DSM 41452ΔNmet::pUC19Δ3100specΔsas27 and S.

354

asterosporus DSM 41452Δann1 respectively by conjugation. Apramycin resistant colonies were analyzed

355

for production.

356 357

LC-MS analysis of secondary metabolites from mutants

358

Strains were cultivated in TSB medium supplemented with corresponding antibiotics for 2 days at 28 °C,

359

then 1% of the seed culture was inoculated into SG+ medium for 4 days at 28 °C. After fermentation, 10

360

mL supernatant of fermentation broth was extracted with an equal volume of ethyl acetate. The organic

361

phase was evaporated, then the extracts were resuspended in MeOH (1mL), and filtered through syringe

362

filters (LLG, PVDF, 0.45um) prior to LC-MS analysis. Samples were analyzed by an Agilent 1100 LC/MS

363

system with electrospray ionization (ESI). The HPLC system was equipped with a XBridge C18 column

364

(3.5 μm; 100 mm × 4.6 mm) and an diode array detector. The mobile system is composed of solvent A

365

(CH3CN with 0.1% HAc, vol/vol) and solvent B (H2O with 0.1% HAc, vol/vol). A 10 μL aliquot of the

366

MeOH-soluble extract was injected for analysis. A gradient elution method was used (5% A over 4 min, 5–

367

95% A from 4 to 20 min, 95% A from 20 to 22 min, 95-5% A from 22 to 23 min, and 5% A from 23 to 30

368

min; and a flow rate of 0.5 mL/min). MSD settings were as follows: acquisition mass range m/z 150–1000,

369

MS scan rate 1s-1, MS/MS scan rate 2s-1, fixed collision energy 20 eV; ion source drying gas temperature 15 ACS Paragon Plus Environment


370

350 °C, drying gas flow 10 L/min; Nebulizer pressure 35 psig; ion source mode API-ES; capillary voltage

371

3000. LC (DAD) and MS data were analyzed with ChemStation software (Agilent).

372

Purification of compound 1, 2, 3, 4, 5, 6 and 7

373

After fermentation in SG+ medium compound 1, 2, 3, 4 and 5 were isolated from S. asterosporus DSM

374

41452ΔNmet::pUC19Δ3100spec, 6 from S. asterosporus DSM 41452ΔNmet::pUC19Δ3100specΔsas27

375

and 7 from S. asterosporus DSM41452ΔNmet::pUC19Δ3100specΔsas28::aac(3)IV. Crude extracts were

376

dissolved in 20 mL MeOH and fractionated by C18 SPE column (Oasis® HLB 20 / 35cc) using a stepwise

377

MeOH gradient. Fractions afforded from the SPE column were analyzed by LC-MS. Among those

378

fractions, fractions containing the target molecule were subsequently used for purification by semi-

379

preparative HPLC. The machine was equipped with a Waters ZORBAX SB-C18 column (9.4 x 150 mm, 5

380

µm) and a Zorbax 80SB-C8 guard column (9.4 x 15 mm, 7 µm). Compounds were eluted from the column

381

using a isocratic method (70% CH3CN +0.5% acetic acid; flow rate 1 mL/min). Compounds 1 (3.5 mg), 2

382

(1.5 mg), 3 (10 mg), 4 (5.4 mg), 5 (5 mg), 6 (2.7mg) and 7 (1 mg) were collected and used for structure

383

elucidation.

384

Structure elucidation of compound 1, 2, 3, 4, 5, 6 and 7

385

NMR spectra are recorded using a Varian VNMR-S 600 spectrometer equipped with 3mm triple resonance

386

inverse and 3mm dual broadband probes in CD3CN at T = 25 °C (in addition J4 was measured at T = -30

387

°C, J6 at T = -20 °C). Residual solvent signals were used as an internal standard (for CD3CN: δH = 1.93, δC

388

= 1.28). In addition, their high-resolution MS were measured on a LTQ Orbitrap XL (Thermo Scientific).

389

ASSOCIATED CONTENT

390

The Supporting Information is available free of charge via the Internet. It contains information about strains,

391

bacteria, NMR-data of all new compounds and describtions of mutants generated during this study.


Page 16 of 26

Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


392

ACKNOWLEDGMENTS

393

We thank C. Warth from the OC-MS service of Institute of Organic Chemistry in the University of Freiburg

394

for HR-ESI-MS measurements. We also thank D. Figurski from Columbia University for providing plasmid

395

pR9406, and M. Bibb from John Innes Centre for providing strain ET12567.

396

FUNDING

397

This study was supported in part by the DFG (RTG 1976-235777276) granted to A.B., we also express

398

our gratitude to the China Scholarship Council for the scholarship to J. Zhu.

399

REFERENCES

400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433

1. Walsh, C.-T. (2004) Polyketide and nonribosomal peptide antibiotics: modularity and versatility, Science 303, 1805-1810. 2. Petříčková, K., Chroňáková, A., Zelenka, T., Chrudimský, T., Pospíšil, S., Petříček, M., and Krištůfek, V. (2015) Evolution of cyclizing 5-aminolevulinate synthases in the biosynthesis of actinomycete secondary metabolites: outcomes for genetic screening techniques, Front. Microbiol. 6, 814. 3. Ostash, B., and Walker, S. (2010) Moenomycin family antibiotics: chemical synthesis, biosynthesis, and biological activity, Nat. Prod. Rep. 27, 1594-1617. 4. Omura, S., Kitao, C., Tanaka, H., Oiwa, R., and Takahashi, Y. (1976) A new antibiotic, asukamycin, produced by Streptomyces, J. Antibiot. 29, 876. 5. Petříčková, K., Pospíšil, S., Kuzma, M., Tylová, T., Jágr, M., Tomek, P., Chroňáková, A., Brabcová, E., Anděra, L., Krištůfek, V., and Petříček, M. (2014) Biosynthesis of colabomycin E, a new manumycin-family metabolite, involves an unusual chain-length factor, Chembiochem 15, 13341345. 6. Nara, A., Hashimoto, T., Komatsu, M., Nishiyama, M., Kuzuyama, T., and Ikeda, H. (2017) Characterization of bafilomycin biosynthesis in Kitasatospora setae KM-6054 and comparative analysis of gene clusters in Actinomycetales microorganisms, J. Antibiot. 70, 616. 7. Kalan, L., Gessner, A., Thaker, M. N., Waglechner, N., Zhu, X., Szawiola, A., Bechthold, A., Wright, G. D., and Zechel, D.-L. (2013) A cryptic polyene biosynthetic gene cluster in Streptomyces calvus is expressed upon complementation with a functional bldA gene, Chem. Biol. 20, 1214-1224. 8. Toki, S., Agatsuma, T., Ochiai, K., Saitoh, Y., Ando, K., Nakanishi, S., Lokker, N. A., Giese, N. A., and Matsuda, Y. (2001) RP-1776, a novel cyclic peptide produced by Streptomyces sp., inhibits the binding of PDGF to the extracellular domain of its receptor, J. Antibiot. 54, 405-414. 9. Um, S., Park, S. H., Kim, J., Park, H. J., Ko, K., Bang, H. S., Lee, S. K., Shin, J., and Oh, D. C. (2015) Coprisamides A and B, new branched cyclic peptides from a gut bacterium of the dung beetle Copris tripartitus, Org. Lett. 17, 1272-1275. 10. Bae, M., Kim, H., Moon, K., Nam, S. J., Shin, J., Oh, K. B., and Oh, D. C. (2015) Mohangamides A and B, new dilactone-tethered pseudo-dimeric peptides inhibiting Candida albicans isocitrate lyase, Org. Lett. 17, 712-715. 11. Omura, S., Van der Pyl, D., Inokoshi, J., Takahashi, Y., and Takeshima, H. (1993) Pepticinnamins, new farnesyl-protein transferase inhibitors produced by an actinomycete. I. Producing strain, fermentation, isolation and biological activity, J. Antibiot. (Tokyo) 46, 222-228. 12. Liu, M., Liu, N., Shang, F., and Huang, Y. (2016) Activation and Identification of NC‐1: A Cryptic Cyclodepsipeptide from Red Soil‐Derived Streptomyces sp. FXJ1. 172, Eur. J. Org. Chem. 2016, 3943-3948. 17 ACS Paragon Plus Environment


434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484

13. Hayashi, K., Hashimoto, M., Shigematsu, N., Nishikawa, M., Ezaki, M., Yamashita, M., Kiyoto, S., Okuhara, M., Kohsaka, M., and Imanaka, H. (1992) WS9326A, a novel tachykinin antagonist isolated from Streptomyces violaceusniger no. 9326. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities, J. Antibiot. 45, 1055. 14. Bae, M., Kim, H., Moon, K., Nam, S.-J., Shin, J., Oh, K.-B., and Oh, D.-C. (2015) Mohangamides A and B, new dilactone-tethered pseudo-dimeric peptides inhibiting Candida albicans isocitrate lyase, Org. Lett. 17, 712-715. 15. Liu, Q., Liu, Z., Sun, C., Shao, M., Ma, J., Wei, X., Zhang, T., Li, W., and Ju, J. (2019) Discovery and Biosynthesis of Atrovimycin, an Antitubercular and Antifungal Cyclodepsipeptide Featuring Vicinal-dihydroxylated Cinnamic Acyl Chain, Org. Lett. 21, 2634-2638. 16. Shi, J., Liu, C. L., Zhang, B., Guo, W. J., Zhu, J., Chang, C.-Y., Zhao, E. J., Jiao, R. H., Tan, R. X., and Ge, H. M. (2019) Genome mining and biosynthesis of kitacinnamycin as a STING activator, Chem Sci. 10, 4839-4846. 17. Zhang, S., Zhu, J., Zechel, D.-L., Jessen, T.- C., Eastman, R. T., Paululat, T., and Bechthold, A. (2018) New WS9326A Derivatives and One New Annimycin Derivative with Antimalarial Activity are Produced by Streptomyces asterosporus DSM 41452 and Its Mutant, Chembiochem 19, 272-279. 18. Tsantrizos, Y. S., Yang, X., and McClory, A. (1999) Studies on the Biosynthesis of the Fungal Metabolite Oudenone. 2. Synthesis and Enzymatic Cyclization of an α-Diketone, Open-Chain Precursor into Oudenone in Cultures of Oudemansiella r adicata, J. Org. Chem. 64, 6609-6614. 19. Petříček, M., Petříčková, K., Havlíček, L., and Felsberg, J. (2006) Occurrence of two 5-aminolevulinate biosynthetic pathways in Streptomyces nodosus subsp. asukaensis is linked with the production of asukamycin, J. Bact. 188, 5113-5123. 20. Zhang, W., Bolla, M. L., Kahne, D., and Walsh, C. T. (2010) A Three Enzyme Pathway for 2-Amino3-hydroxycyclopent-2-enone Formation and Incorporation in Natural Product Biosynthesis, J. Am. Chem. Soc. 132, 6402-6411. 21. Pan, H.-Q., Yu, S.-Y., Song, C.-F., Wang, N., Hua, H.-M., Hu, J.-C., and Wang, S.-J. (2015) Identification and characterization of the antifungal substances of a novel Streptomyces cavourensis NA4, J. Microbial. Biotechnol. 25, 353-357. 22. Schubert, V., Di Meo, F., Saaidi, P.-L., Bartoschek, S., Fiedler, H.-P., Trouillas, P., and Süssmuth, R. D. (2014) Stereochemistry and Conformation of Skyllamycin, a Non-Ribosomally Synthesized Peptide from Streptomyces sp. Acta 2897, Chem. Eur. J. 20, 4948-4955. 23. Yu, Z., Vodanovic-Jankovic, S., Kron, M., and Shen, B. (2012) New WS9326A congeners from Streptomyces sp. 9078 inhibiting Brugia malayi asparaginyl-tRNA synthetase, Org. Lett. 14, 49464949. 24. Desouky, S.-E., Shojima, A., Singh, R.-P., Matsufuji, T., Igarashi, Y., Suzuki, T., Yamagaki, T., Okubo, K.-i., Ohtani, K., and Sonomoto, K. (2015) Cyclodepsipeptides produced by actinomycetes inhibit cyclic-peptide-mediated quorum sensing in Gram-positive bacteria, FEMS Microbiol. Lett. 362, pii fnv 109. 25. Bae, M., Oh, J., Bae, E.-S., Oh, J., Hur, J., Suh, Y.-G., Lee, S.-K., Shin, J., and Oh, D.-C. (2018) WS9326H, an antiangiogenic pyrazolone-bearing peptide from an intertidal mudflat actinomycete, Org. Lett. 20, 1999-2002. 26. Zhang, S. Genomics, Proteomics and Secondary Metabolites Biosynthesis Research on Streptomyces asterosporus DSM 41452, Doctoral dissertation, Freiburg University, Freiburg, Germany, 2018. 27. Pohle, S., Appelt, C., Roux, M., Fiedler, H.-P., and Süssmuth, R.-D. (2011) Biosynthetic gene cluster of the non-ribosomally synthesized cyclodepsipeptide skyllamycin: deciphering unprecedented ways of unusual hydroxylation reactions, J. Am. Chem. Soc. 133, 6194-6205. 28. Du, D., Katsuyama, Y., Shin-Ya, K., and Ohnishi, Y. (2018) Reconstitution of a Type II Polyketide Synthase that Catalyzes Polyene Formation, Angew. Chem. 57, 1954-1957. 29. Grammbitter, G. L.-C., Schmalhofer, M., Karimi, K., Schöner, T.-A., Tobias, N.-J., Morgner, N., Groll, M., and Bode, H.-B. (2019) An Uncommon Type II PKS Catalyzes Biosynthesis of Aryl Polyene Pigments, J. Am. Chem. Soc. doi: 10.1021/jacs.8b10776 18 ACS Paragon Plus Environment

Page 18 of 26

Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

485 486 487 488 489 490 491 492 493 494 495 496


30. Flett, F., Mersinias, V., and Smith, C.-P. (1997) High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes, FEMS Microbiol. Lett. 155, 223-229. 31. Gust, B., Challis, G.-L., Fowler, K., Kieser, T., and Chater, K.-F. (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin, Proc. Natl. Acad. Sci. USA 100, 1541-1546. 32. Thong, W.-L., Sin-ya, K., Nishiyama, M., and Kuzuyama, T. (2016) Methylbenzene-containing polyketides from a Streptomyces that spontaneously acquired rifampicin resistance: Structural elucidation and biosynthesis, J. Nat. Prod. 79, 857-864.



Page 20 of 26

498

Legends of Figures

499 500

Figure 1: Proposed biosynthetic pathway of 4-Z-annimycin and WS9326A: ACP: acyl carrier protein,

501

KS: ketosynthease, CLF: chain length factor, DH: dehydrase, KR: ketoreductase

502 503

Figure 2: HPLC chromatogram of secondary metabolites from S. asterosporus DSM 41452ΔNmet (I), S.

504

asterosporus

505

41452ΔNmet::pUCΔ3100specΔsas24::aac(3)IV

506

41452ΔNmet::pUCΔ3100specΔsas27 (IV), S. asterosporus DSM 41452ΔNmet::pUCΔ3100specΔsas27 x

507

pTESbz-sas27 (V), S. asterosporus DSM 41452ΔNmet::pUCΔ3100specΔsas28::aac(3)IV (VI), and S.

508

asterosporus DSM 41452ΔNmet::pUCΔ3100specΔsas32::aac(3)IV (VII)

509

Figure 3: Structures of J1 (1), J5 (2), J3 (3), J4 (4), and J6 (5)

510

Figure 4. LC-MS extracted ion chromatogram (EICs) for the [M-H]- ions corresponding to J1 and J8 in

511

ethyl acetate extracts of S. asterosporus DSM 41452∆Nmet::pUC19Δ3100spec (A); S. asterosporus DSM

512

41452∆Nmet::pUC19Δ3100specΔsas27

513

41452∆Nmet::pUC19Δ3100specΔsas27::pTESbz-sas27 (C)

DSM

41452ΔNmet::pUC19Δ3100spec

(II),

(III),

(B);

and

S.

S.

S.

asterosporus asterosporus

asterosporus

DSM DSM

DSM

514 515

Figure 5. Chemical structures of J8 (6) and J9 (7)

516 517

Figure 6: Generation of novel compounds in different mutants by utilizing the promiscuity of an amide

518

synthetase (the annimycin- and WS9326A-gene cluster are not not shown as a whole)

519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534


Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Sas32 Sas33 O CLF SCoA KS R Acetyl-CoA

O

O HO

ACP

CoA

O

O

HO

S-CoA

KS CLF

S-ACP

O

O S-ACP

KR OH

Sas32 Sas33

R

S-ACP DH

O OH

O

R

S-ACP n times

O

H N O

H N

N O

O

H N

O

O

HO

O

N H

H 2N

O

O S-ACP

O O

n

NH

Sas27 Sas28

NH OH

S-ACP

WS9326A

O

O H 2N

OH

Glycine

Ann2, Ann3

HO

NH2 O

+

Ann1

O CoA

S

O OH

OH Succinyl-CoA

535 536 537

O

O N H

4-Z-annimycin

Figure 1

538 539 540


OH


Page 22 of 26

541 542

Figure 2 11

9

7

13

O 3

O J1 (1)

1 NH 1' 5

4'

2'

3

7

O

12 1 NH 1'

5

3

543 544 545

O

2'

J3 (3)

HO

7

5' 4' 3'

9

1 NH 1' 5

3

O J4 (4)

2'

HO

11

9

7

O

11

3'

HO

11 9

13 1 NH 1'

5' 4' 3'

Figure 3


5' 4'

2'

O J5 (2)

3'

HO

13

O

5'

1 NH 1' 5

11

9

7

5

3

O

J6 (5)

2'

HO

O 5' 4' 3'

Page 23 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


546 547

Figure 4

548 O 11

9

7

5

3

1 NH 1'

5'

2'

3'

O

HO

J8 (6)

4'

O 11

549 550 551 552

9

7

5

3

J9 (7)

1 NH 1'

5'

2'

3'

O

HO

4'

Figure 5

553 554 555 556 557 558



559 560

Figure 6

561 562 563


Page 24 of 26

Page 25 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


564 565 566 567 OH

568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583

H N O

O

O

O

O H N

O

HO H 2N

H N

N

N H O

O

O

OH NH

O

O N H

O NH

OH

OH

O

H N O

HO

Table of content graphic



118x105mm (150 x 150 DPI)

ACS Paragon Plus Environment

Page 26 of 26

Rational Design of Hybrid Natural Products by ... - ACS Publications

Recommend Documents