Polyketide Bioderivatization Using the ... - ACS Publications

Feb 16, 2017 - Department of Chemical and Biomolecular Engineering (BK21 Plus Program), ... During polyketide biosynthesis, acyltransferases (ATs) are...
0 downloads 0 Views 2MB Size
Subscriber access provided by University of Newcastle, Australia

Letter

Polyketide bio-derivatization using the promiscuous acyltransferase KirCII Ewa Maria Musiol-Kroll, Florian Zubeil, Thomas Schafhauser, Thomas Härtner, Andreas Kulik, John B. McArthur, Irina Koryakina, Wolfgang Wohlleben, Stephanie Grond, Gavin J. Williams, Sang Yup Lee, and Tilmann Weber ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00341 • Publication Date (Web): 16 Feb 2017 Downloaded from http://pubs.acs.org on February 19, 2017

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 free 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 accessible to all readers and 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.

ACS Synthetic Biology 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 25

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

ACS Synthetic Biology

Polyketide bio-derivatization using the promiscuous acyltransferase KirCII

Ewa M. Musiol-Kroll1,2,4,# Florian Zubeil3,#, Thomas Schafhauser4, Thomas Härtner4, Andreas Kulik4, John McArthur5,7, Irina Koryakina5,8, Wolfgang Wohlleben2,4, Stephanie Grond3, Gavin J. Williams5*, Sang Yup Lee1,6* and Tilmann Weber1,2,4* #

Equal contribution

Affiliations 1

Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet B220, 2800 Kgs. Lyngby, Denmark 2

German Centre for Infection Research (DZIF), Partner site Tübingen, IMIT, Auf der Morgenstelle 28, 72076 Tübingen, Germany 3

Eberhard Karls Universität Tübingen, Institut für Organische Chemie, Auf der Morgenstelle 18, 72076 Tübingen, Germany 4

Eberhard Karls Universität Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, Mikrobiologie/Biotechnologie, Auf der Morgenstelle 28, 72076 Tübingen, Germany 5

North Carolina State University, Department of Chemistry, Raleigh, NC 27695-8204, USA 6

Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea 7

Present address: Department of Chemistry, University of California, One Shields Avenue, Davis, USA 8

Present address: Intrexon Corporation, South San Francisco, California, USA

*

Corresponding authors: Gavin Williams: [email protected], Sang Yup Lee: [email protected] and Tilmann Weber: [email protected]

1 ACS Paragon Plus Environment

ACS Synthetic 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

1

ABSTRACT:

2

During polyketide biosynthesis, acyltransferases (ATs) are the essential

3

gatekeepers which provide the assembly lines with precursors and thus

4

contribute greatly to structural diversity. Previously, we demonstrated that the

5

discrete AT KirCII from the kirromycin antibiotic pathway accesses non-

6

malonate extender units. Here, we exploit the promiscuity of KirCII to generate

7

new kirromycins with allyl- and propargyl-side chains in vivo, the latter were

8

utilized as educts for further modification by ‘click’ chemistry.

9

KEYWORDS:

10

antibiotic, kirromycin, polyketide synthase, trans-acyltransferase, engineering,

11

click chemistry

12

2 ACS Paragon Plus Environment

Page 2 of 25

Page 3 of 25

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

ACS Synthetic Biology

13

Polyketides are important secondary metabolites widely used as antibiotics,

14

antifungals, and drugs for other clinical applications1, 2. Erythromycin, mupirocin,

15

rapamycin, FK506, lovastatin and epothilone B are examples of antimicrobial,

16

immunosuppressant, hypocholesterolemic and anticancer drugs, respectively. All

17

these compounds are biosynthesized by complex multifunctional enzymes with

18

modular architecture, the polyketide synthases (PKSs).

19

In typical modular type I PKSs, acyltransferase domains (ATs) select and load

20

malonyl-CoA derived precursors onto acyl carrier proteins (ACPs). The AT domains

21

are either embedded in the PKS (cis-ATs) or encoded by distinct genes and function

22

in trans as discrete enzymes (trans-ATs)3, 4. After the AT-dependent loading step,

23

ketosynthases (KSs) catalyze the condensation of ACP-linked extender units.

24

Optional domains, such as ketoreductases (KRs), dehydratases (DHs),

25

enoylreductases (ERs) or methyltransferases (MTs) further process the polyketide

26

chain. In most cases, the product is released from the assembly line by a

27

thioesterase (TE) domain and modified by post-PKS tailoring enzymes.

28

Notably, the AT domain determines which extender unit is incorporated into the

29

growing polyketide chain5-7. Consequently, the extender unit specificity of the AT

30

cumulatively dictates large portions of the final polyketide structure, and could be

31

leveraged to improve the pharmacological properties of polyketide-based chemical

32

entities through alteration of the precursors and/or ATs8. These features make AT

33

engineering attractive for rational modification of polyketide assembly lines.

34

Directed engineering approaches such as AT domain swapping, AT site-directed

35

mutagenesis9 and cross-complementation of an AT-inactivated cis-PKS have been

36

described for various polyketides, including erythromycin, rapamycin, tylosin and

37

geldanamycin10. However, most of the previous attempts to vary AT substrate

38

specificity target cis-AT PKS pathways. Furthermore, all studies on extender unit 3 ACS Paragon Plus Environment

ACS Synthetic 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

39

variation involve non-alkyne precursors with the exception of 2-propargyl-

40

erythromycin11 and propargyl-premonensin12, which were produced by feeding N-

41

acetylcysteamine (SNAC) pre-activated substrates to a strain harboring the

42

engineered AT6DEBS and to the wild-type producing strain, respectively.

43

For trans-AT type PKSs, only a few examples of assembly lines utilizing non-malonyl-

44

CoA extender units have been reported. One of the best studied biosynthetic

45

pathways, which involves a trans-AT type PKS and incorporates a branched unit into

46

its product, is that of the antibiotic kirromycin (1) from Streptomyces collinus Tü

47

36513. The unusual and complex trans- and cis-PKS/NRPS assembly line recruits

48

two discrete ATs, KirCI and KirCII (Figure 1). In a previous study we demonstrated

49

that KirCI provides the biosynthetic machinery with malonate14. The second AT,

50

KirCII, was shown to transfer ethyl malonate onto the kirromycin ACP5 (Kirr-ACP5)

51

and thus introduces the C-28 ethyl branch into the backbone of the antibiotic5. More

52

recently, in vitro studies revealed that this enzyme accepts other non-malonyl-CoA

53

substrates such as allylmalonyl-CoA, propargylmalonyl-CoA and to a lesser extent

54

azidoethyl- and phenylmalonyl-CoA15, 16.

55

Alkyne-containing molecules, such as propargyl-derived compounds provide

56

functional groups for further derivatization by “click chemistry”. The term “click

57

chemistry” describes rapid, selective, high-yield reactions of chemical moieties under

58

mild, aqueous conditions. Copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC) is

59

the most commonly used click reaction, which fulfills those criteria. In recent years,

60

different click chemistry reactions were developed, often resulting in new chemical

61

structures and therefore, the approach become one of the most powerful tools in drug

62

discovery, chemical biology, and proteomic applications17, 18.

63

To the best of our knowledge, in vivo production of allyl- and/or propargyl-modified

64

polyketides, derived from trans-AT pathways has so far not been described. This and 4 ACS Paragon Plus Environment

Page 4 of 25

Page 5 of 25

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

ACS Synthetic Biology

65

the fact that KirCII continues to be the only characterized discrete AT with such a

66

promising spectrum of substrate selectivity, encouraged us to study the flexibility of

67

KirCII in vivo and to exploit its features for polyketide engineering.

68

In our kirromycin bio-derivatization approach, the producer strain S. collinus

69

Tü 365 was engineered to facilitate the utilization of non-natural polyketide

70

precursors. The malonyl-CoA synthetase T207G/M306I MatB16, 19, which is able to

71

activate non-natural extender units, was cloned into the vector pRM4.2 downstream

72

of the strong ermE* promoter. The resulting plasmid pRM4.2-matB was transferred

73

into S. collinus Tü 365 and an isolated S. collinus pRM4.2-matB clone was cultivated

74

for feeding experiments. Separate cultures of the engineered strain were fed with

75

allyl- and propargylmalonic acid and the production of new compounds was analyzed

76

by HPLC-MS (Supplementary Figure 1). In both the allyl- and propargyl-derived

77

extract, an ion consistent with kirromycin (1) (m/z = 795.5 [M-H]-) was present.

78

Remarkably however, additional ions at m/z = 807.5 [M-H]- and m/z = 805.4 [M-H]-

79

were detected in the allyl- and propargyl-derived samples, respectively

80

(Supplementary Figure 1). These ions corresponded to the calculated masses of

81

allyl-kirromycin (2) and propargyl-kirromycin (3) (Supplementary Table 1).

82

In order to characterize the new metabolites, fermentation and feeding experiments

83

were carried out in five liter scale. As only allylmalonic acid is commercially available

84

and propargylmalonic acid is not, the latter had to be chemically synthesized to set

85

up serial experiments with both substrates. Crude extracts obtained from these

86

feeding experiments were subjected to preparative HPLC. Notwithstanding the

87

modest structural differences between kirromycin (1) and its derivatives and, as a

88

consequence, the challenging chromatographic purification of kirromycin derivatives,

89

the compounds could be separated and highly enriched. Thereby 3.8 mg of a sample

90

containing 90% allyl-kirromycin (2) and 10% kirromycin (1) and 3.7 mg of a sample 5 ACS Paragon Plus Environment

ACS Synthetic 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

91

containing 22 % propargyl-kirromycin (3) and 78 % kirromycin (1) were obtained.

92

Interestingly, these derivative:kirromycin ratios are in better agreement with the KirCII

93

in vitro activity data compared to the MatB in vitro activity data. The engineered MatB

94

enzyme showed similar in vitro CoA-activation rates toward ethylmalonic acid

95

(91±3%), propargylmalonic acid (91±5%) and allylmalonic acid (89±2%),16 but the

96

loading efficiency of KirCII differed significantly depending on the substrate. For

97

example, both ethylmalonyl-CoA and allylmalonyl-CoA are loaded by KirCII onto the

98

cognate Kir-ACP5 with equal efficiency (~15% conversion), whereas

99

propargylmalonyl-CoA is loaded at about half of this efficiency (8% conversion)16.

100

Furthermore, the isolated yields of allyl- and propargyl-kirromycin indicate that in the

101

in vivo system, the downstream kirromycin assembly line domains are more tolerant

102

to the artificial substrate allylmalonyl-CoA than for propargyl-CoA.

103

To confirm the product identity of allyl-kirromycin (2) and propargyl-kirromycin (3), the

104

purified samples, as well as kirromycin (1) were analyzed by high resolution MS.

105

Based on the kirromycin fragments20 previously detected and described by Edwards

106

and coworkers, we proposed a fragmentation mechanism for kirromycin (1) and the

107

derivatives, allyl-kirromycin (2) and propargyl-kirromycin (3) (Supplementary Table

108

1, Supplementary Figure 3 and Supplementary Figure 4a-c). The anticipated

109

fragments A-C were detected for all three molecules, which confirms that the allyl and

110

propargyl moieties were introduced in place of the original ethyl side chain at C-28 in

111

kirromycin (1) (Supplementary Table 1, Supplementary Figure 4b-c). In addition,

112

1

113

and for the propargyl-kirromycin sample (Supplementary Figure 5I-II and

114

Supplementary Table 2). Complete NMR signal assignments of allyl-kirromycin (2)

115

substantiated the presence of the allyl moiety. In the 1H spectrum of the enriched

116

propargyl-kirromycin sample, a spin-system consisting of H-28 (δH 3.13, 1H, m), H-44

H, 13C, COSY, HSQC HMBC and NMR spectra were recorded for the allyl-kirromycin

6 ACS Paragon Plus Environment

Page 6 of 25

Page 7 of 25

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

ACS Synthetic Biology

117

(δH 2.57, 2H, m) and H-46 (δH 2.30, 1H, t, J = 2.64 Hz) was detected in addition to

118

“the collateral signals” from the residual traces of kirromycin (1). The corresponding

119

HMBC correlations unambiguously confirm the identity of propargyl-kirromycin (2)

120

(Figure 2).

121

The fact that the propargyl-kirromycin derivative (2) possesses an alkyne moiety

122

attracted our particular attention, as alkynes are highly desired chemical structures

123

for click reactions (i.e. CuAAC). Therefore, propargyl-kirromycin (2) was subjected to

124

CuAAC reaction using the fluorescent dye coumarin-343-azide. The click-reaction

125

product was purified by chromatographic methods. A total yield of 0.81 mg was

126

obtained and analyzed by HPLC-MS in negative ion mode. The analytical data

127

demonstrated the presence of a molecule ion with the sum formula C66H84N7O17,

128

which is in accordance with the coumarin-modified compound. MS/MS fragmentation

129

revealed signals corresponding to the fragments A-C of the 1,2,3-triazole compound

130

(Supplementary Table 1 and Supplementary Figure 4d). Despite the small quantity

131

of the click-reaction product, NMR analyses were carried out leading to a partial

132

spectra recording and signal assignments (Supplementary Figure 5III and

133

Supplementary Table 2). The triazole signal (δH 7.78, 1H, s) was observed and the

134

respective HMBC correlations were detected (Figure 2 and Supplementary Figure

135

5III), confirming the identity of the click-reaction product coumarin-kirromycin (4).

136

According to the HPLC-MS analysis this sample contained mainly coumarin-

137

kirromycin (4) (96 %) and only traces of kirromycin (1) (4 %). Thus, taking advantage

138

of the newly introduced propargyl-anchor, we could not only synthesize a new click

139

product, but we also exploited its structural features as a strategy to separate

140

coumarin-kirromycin (2) from kirromycin (1). The results support the application of the

141

KirCII system for synthesizing biomolecules that are provided with functional groups,

142

which can be used in highly selective click chemistry (Figure 1). This allows the 7 ACS Paragon Plus Environment

ACS Synthetic 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

143

production of further chemically modified valuable compounds as well as their

144

straightforward purification. Moreover, coupling of azidofluorescent dyes can be used

145

for fluorescent labeling of terminal alkynes in bioactive molecules, which is highly

146

sought-after for studying drug-target interactions and rational drug design.

147

The ability to generate and isolate the allyl-kirromycin (2) and coumarin-kirromycin (4)

148

raised the question of whether the modifications at C-28 affect the antibiotic activity

149

compared to kirromycin (1). The mode of action of kirromycin (1) is well understood.

150

The antibiotic binds to the elongation factor Tu (EF-Tu)21, 22 and thereby inhibits the

151

protein biosynthesis in some species of gram positive and gram negative bacteria

152

(e.g. Neisseria gonorrhoeae, Haemophilus influenzae, Streptococci and some

153

Enterococci strains). In order to examine and compare the efficiency of the new

154

derivatives, a GFP-based in vitro translation assay23, 24 was used (Supplementary

155

Figure 7). Increasing concentrations of the compounds were added to the assay

156

reactions and changes in eGFP synthesis were monitored by measuring the eGFP

157

fluorescence (Figure 3, Supplementary Tables 3a-b). In this system, allyl-

158

kirromycin (IC50 = 1.0 µM) inhibited the eGFP protein synthesis comparably to the

159

native compound kirromycin (1) (IC50 = 0.9 µM) (Figure 3). The derivative coumarin-

160

kirromycin (4) also inhibited the system (IC50= 7.3 µM), albeit to a lesser extent. The

161

data showed that the replacement of the ethyl moiety at C-28 of kirromycin (1) with

162

an allyl moiety has no negative effect on the interactions with its target. According to

163

the crystal structure of the complex of Thermus thermophiles EF-Tu with aurodox, an

164

N-methyl derivative of kirromycin (1), the antibiotic mainly interacts through the

165

pyridine, the tetrahydrofuran rings, and the aliphatic tail on the goldinoic acid with the

166

EF-Tu residues25, 26. These structural moieties do not include the branch at C-28 of

167

kirromycin (1). Thus, our results are in agreement with the binding site studies

168

obtained from the crystal structure of the EF-Tu-aurodox complex25, 26 and confirm 8 ACS Paragon Plus Environment

Page 8 of 25

Page 9 of 25

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

ACS Synthetic Biology

169

that the allyl-moiety is not directly involved in the interaction of the antibiotic with the

170

target. In case of coumarin-kirromycin (4), where the ethyl group is conjugated to the

171

relatively large coumarin moiety, the molecule is still able to inhibit protein

172

biosynthesis, but with an approximately seven-fold lower inhibitory activity, indicating

173

that the bulky coumarin residue affects the drug-target interaction. Nevertheless, our

174

results demonstrate that kirromycin (1) is robust to variations at the C-28 atom and

175

highlights that this position is suitable for further modifications.

176

The modification to propargyl-kirromycin is of particular interest as it enables the

177

“fusion” of diverse moieties to the kirromycin-backbone. The attachment of chemical

178

groups, which would alter the physicochemical properties of the molecule, could be

179

used to generate derivatives with optimized import into the cell and thereby extend

180

the spectrum of antimicrobial activity for this family of antibiotic compounds.

181

Furthermore, labelling of the structure with fluorescent agents would allow the

182

monitoring of the antibiotic penetrating the cell and provides tools for studying protein

183

biosynthesis (e.g. interactions with the elongation factor EF-Tu, which can be easily

184

visualized by fluorescence detection methods).

185

This work is the first example that leverages a tailored malonyl-CoA synthetase and a

186

trans-AT for rational and regioselective engineering of a complex polyketide antibiotic

187

in order to introduce artificial alkene and alkyne sidechains in vivo. Our results

188

demonstrate that after the extender unit substitution, which is accomplished relatively

189

early in the assembly process, the downstream NRPS and PKS modules accept the

190

un-natural sidechains and continue the product biosynthesis. These findings uncover

191

the flexibility of a mixed PKS/NRPS assembly line towards the un-natural building

192

blocks and encourage the further exploration of trans-AT-based polyketide

193

engineering. The MatB-KirCII/ACP5 system may have the potential to become a 9 ACS Paragon Plus Environment

ACS Synthetic 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

194

valuable tool, enabling the regiospecific incorporation of chemical handles into

195

polyketides, which can serve as anchors for “click” chemistry. The combination of this

196

“bio-derivatization” approach and “click” chemistry methods enables the generation of

197

novel molecular probes and analogues of compounds, which would be otherwise

198

difficult to access by using exclusively conventional organic chemistry.

199

METHODS

200

General methods and strain cultivation conditions.

201

E. coli strains, the wild type and mutants of S. collinus Tü 365 were cultivated in liquid

202

or on solid media as described previously5. Detailed information on strains, media

203

composition, antibiotic concentrations, plasmids, oligonucleotides and PCR

204

conditions can be obtained from Supporting Information.

205

Chemical synthesis of 2-(Prop-2-yn-1-yl)malonic acid.

206

Dimethyl propargylmalonate (0.94 ml, 6.2 mmol) and NaOH (2.4 g, 60 mmol) were

207

added to a round bottom flask containing 10 ml H2O and the mixture was stirred for 6

208

hours at 65°C. The solution was cooled on ice and 10 ml ice cold 12.1 N HCl was

209

added. The reaction was extracted 6x with diethyl ether. The organic extractions

210

were pooled, dried using MgSO4 and filtered. The solvent was removed under

211

vacuum to give 2-(prop-2-yn-1-yl)malonic acid as a white solid (0.298 g, 34%). 1H-

212

NMR (D2O, 300 MHz): δH 3.59 (1H, t, J = 7.2 Hz), 2.6 (3H, dd, J = 7.2, 2.7 Hz), 2.26

213

(1H, t, J = 2.7 Hz).

214

Cloning of the malonyl-CoA synthetase expression construct.

215

To activate the substrates allylmalonic acid and propargylmalonic acid, the

216

engineered malonyl-CoA synthetase (WP_011650729.1)16, 19 from Rhizobium

217

leguminosarum (MatB T207G/M306I) was transferred into S. collinus Tü 365.

218

Therefore, the engineered matB gene from the plasmid pET28a-matB, was cloned 10 ACS Paragon Plus Environment

Page 10 of 25

Page 11 of 25

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

ACS Synthetic Biology

219

into the integrative vector pRM4.2 via the restriction sites NdeI and HindIII. The

220

resulting construct pRM4.2-matB was introduced into S. collinus Tü 365 by

221

intergeneric conjugation using E. coli ET12567 pUB307. Exconjugants were

222

confirmed by control PCR with primers matB-test-up and matB-test-low (Supporting

223

Information). The obtained mutant S. collinus pRM4.2-matB was applied to feeding

224

experiments with allylmalonic acid (Fluka/Sigma-Aldrich) and the synthesized

225

propargylmalonic acid.

226

Feeding of allylmalonic acid or propargylmalonic acid to S. collinus Tü 365 and

227

S. collinus pRM4.2-matB.

228

Pre-cultures of S. collinus Tü 365 WT and S. collinus pRM4.2-matB were cultivated

229

for three days in TSB medium under antibiotic selection, if required (for antibiotic

230

concentration, see Supporting Information). A main culture of 90 ml SGG medium

231

was inoculated with 10% of the pre-culture (for media, see Supporting Information)

232

and supplemented with 50 mg of allylmalonic acid and propargylmalonic acid

233

(t0 = 0 h, final concentration 0.5 mg ml-1). After five days of incubation, each 100 ml

234

culture was extracted 1:1 with ethyl acetate (1 h, RT). The samples were centrifuged

235

(10 min, RT, 4270 g, Sorvall RC6+ Centrifuge, Thermo Scientific) and the solvent

236

fraction was concentrated to dryness under vacuum. Extracts were re-dissolved in 1

237

ml methanol and analyzed by HPLC/ESI-MS (Supporting Information).

238

Scale-up of feeding and extraction of kirromycin derivatives.

239

Four and a half liters of SGG medium, complemented with apramycin (50 µg ml-1)

240

were inoculated with 10% pre-culture of S. collinus pRM4.2-matB and fed with

241

allymalonic acid or propargylmalonic acid (final concentration, 0.5 mg ml-1). The

242

cultures were incubated in 1 L shaking flasks for five days at 29°C. After incubation

243

each allymalonic- or propargylmalonic-fed culture was extracted 1:1 with ethyl

244

acetate at RT for 1 h, using an extractor. Phase separation was achieved by 11 ACS Paragon Plus Environment

ACS Synthetic 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

245

incubation of the extract/ethyl acetate mixture over night at RT. The ethyl acetate

246

phase was collected, evaporated and the residue dissolved in ethyl acetate and

247

precipitated by addition of petroleum ether (40-60°C) and a subsequent

248

centrifugation. The precipitant was dried in a desiccator. The samples were

249

redissolved in methanol and analyzed by HPLC/ESI-MS (Supporting Information).

250

Purification of allyl-kirromycin and propargyl-kirromycin.

251

The allyl-kirromycin and propargyl-kirromycin sample were dissolved in DMSO:MeOH

252

(5:2). Multiple chromatography runs were conducted, each with a solution of 750 µl,

253

containing approximately 50 mg of the respective extract. The samples were

254

separated on a Kromasil C18, 7 µm 250 x 20 mm HPLC column (Dr. Maisch,

255

Ammerbuch, Germany) using MeCN:H2O (43:57) as mobile phase and a flow of 16

256

ml min-1. The purity of the obtained yields was determined by NMR analysis. In case

257

of allyl-kirromycin, the product of 3.8 mg contained 10% residual kirromycin and for

258

propargyl-kirromycin, the pooled fractions resulted in a mixture of 78 % kirromycin

259

and 22 % propargyl-kirromycin. The propargyl-kirromycin sample was directly utilized

260

in CuAAC reaction without further purification.

261

High Resolution Mass Spectrometry (HRMS) analysis of kirromycins.

262

The HR-MS analysis of allyl- and propargyl-kirromycin was carried out on MaXis 4G

263

instrument (Bruker Daltonics) coupled to a Ultimate 3000 HPLC (Thermo Fisher

264

Scientific). An HPLC-method was applied as follows: 0.1% formic acid in water as

265

solvent A and 0.06% formic acid in methanol as solvent B, with gradients from 10%

266

to 100% B in 20 min and 100% B for 5 min, using a flow rate of 0.3 ml min-1. The

267

separation was carried out on a Nucleoshell 2.7 µm 150 x 2mm column (Macherey-

268

Nagel).

269

The ESI source was operated at a nebulizer pressure of 2.0 bar and dry gas was set

270

to 8.0 L min-1 at 200°C. MS/MS spectra were recorded in Auto MS/MS mode 12 ACS Paragon Plus Environment

Page 12 of 25

Page 13 of 25

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

ACS Synthetic Biology

271

resulting in a collision energy stepping from 32.1 to 52.0 eV. Sodium formate was

272

used as internal calibrant and Hexakis(2,2-difluoroethoxy)phosphazene (Apollo

273

Scientific Ltd, Stockport, UK) as lock mass.

274

Nuclear Magnetic Resonance (NMR) spectroscopy of kirromycins.

275

The 1H, 13C, 1H-1H COSY, 1H-13C HSQC and 1H-13C HMBC NMR spectra were

276

recorded on Bruker Avance III HD spectrometers operating at a proton frequency of

277

600 MHz equipped with a Prodigy BBO CryoProbe or 700 MHz equipped with a TCI

278

CryoProbe, respectively (Bruker Biospin). CD3OD as solvent and an internal standard

279

were used. All spectra were recorded at room temperature. Chemical shifts were

280

expressed in parts per million (ppm, δ) relative to TMS.

281

CuAAC reaction of propargyl-kirromycin and coumarin-343-azide and product

282

purification.

283

The propargyl-kirromycin sample (3.7mg) was dissolved in 0.9 ml MeOH and

284

transferred to a Schlenk flask containing 3.75 ml t-BuOH and 3.75 ml H2O. The

285

solution was mixed with 90 µL of 50 mM coumarin-343-azid (Lumiprobe GmbH,

286

Hannover, Germany) in DMSO, 1 ml of 5 mM aqueous ascorbic acid and 1 ml of 2 M

287

aqueous triethylammmonium acetate. The reaction was flushed with argon for 5 min

288

and subsequently treated with 0.5 ml of 10 mM Cu(II)-TBTA, resolved in DMSO:H2O

289

(55:45). The solution was stirred in a sealed flask, at room temperature for 16h.

290

The reaction mixture was extracted with ethyl acetate and the solvent evaporated

291

using a rotary evaporator. The remaining crude extract was dissolved in MeOH and

292

applied to size exclusion chromatography using Sephadex LH-20 (75 x 2.6 cm,

293

Methanol, 0.5 ml min-1). Coumarin-kirromycin-containing fractions were pooled and

294

subjected to preparative HPLC using 43 % MeCN for 25 min, followed by a gradient

295

to 100% in 5 minutes. To remove the residual tris-(benzyltriazolylmethyl)amine

296

(TBTA) and kirromycin from the sample, the chromatographic separation was 13 ACS Paragon Plus Environment

ACS Synthetic 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

297

repeated using the same column and the following conditions: 12 ml min-1, gradient

298

from 40(A):60(B) to 100(A):0(B) with MeOH(A):H2O(B) within 30 minutes. The final

299

product was analyzed by LC-MS and a yield of 0.81 mg coumarin-kirromycin

300

containing 4 % kirromycin was determined.

301

In vitro activity testing of kirromycin and kirromycin derivatives.

302

The in vitro translation assay was developed based on a combination of protocols of

303

Kim et al.23, 24 and Calhoun and Swartz27. Briefly, an E. coli S12 cell extract was

304

prepared and mixed with nucleotides and amino acid as substrates for transcription

305

and translation, fructose-1,6-bisphosphate for energy supply and the reporter plasmid

306

pET28-egfp in wells of a 96 well microtiter plate to a final volume of 100 µL

307

(Supporting Information). An assay reaction contained: 57 mM Hepes-KOH (pH

308

8.2), 90 mM potassium glutamate, 80 mM ammonium acetate, 12 mM magnesium

309

acetate, 1.67 % (w/v) PEG(8000), 5 mM DTT, 1.2 mM AMP, 0.85 mM each of CMP,

310

GMP and UMP, 0.5 mM IPTG, 34 µg ml-1 L-5-formyl-THF, 2 mM each of all 20

311

proteinogenic amino acids, 33 mM fructose-1,6-bisphosphate, 25% (v/v) E. coli S12

312

extract and approximately 5 µg ml-1 of the reporter plasmid pET28-egfp (Supporting

313

Information, all chemicals were obtained from Sigma, all given values represent the

314

final concentrations). The plasmid pET28-egfp was generated by cloning of the eGFP

315

gene from pRM4.328 with NdeI and HindIII into pET28a (Novagen) to take advantage

316

of the strong inducible T7 promoter. The S-12 extract was prepared from E. coli BL21

317

strain (DE3) (Invitrogen) as described by Kim et al.29, except that 1 L of 2xYTPG

318

medium30 was used for cultivation.

319

To test the susceptibility of the in vitro translation system, a microtiter plate was pre-

320

loaded with different concentrations of kirromycin, allyl-kirromycin and coumarin-

321

kirromycin prior to the addition of the assay master mix. All measurements have been

322

performed in triplicate using the same preparation of the S12 extract. The assay was 14 ACS Paragon Plus Environment

Page 14 of 25

Page 15 of 25

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

ACS Synthetic Biology

323

incubated for approximately 4 hours at 37°C. Thereafter, the fluorescence deriving

324

from eGFP was measured at 485/520 nm excitation/emission (POLARstar Galaxy,

325

BMG) (Supporting Information). The data evaluation and IC50 calculation was

326

performed using GraphPad Prism version 7.00 for Windows, GraphPad Software, La

327

Jolla California USA, www.graphpad.com, with non-linear fit regression.

328

ASSOCIATED CONTENT

329

The Supporting Information is available free of charge on ACS Publications

330

website.

331

AUTHOR INFORMATION

332

Corresponding authors

333

*E-mail: [email protected]

334

*E-mail: [email protected]

335

*E-mail: [email protected]

336

Author Contributions

337

E.M.M-K., F.Z. contributed equally to this work.

338

S.G., G.J.W., W.W. and T.W. designed research. E.M.M-K., F.Z., T.S., T.H., and A.K.

339

performed research and analyzed data. J.M. and I.K. contributed reagent. E.M.M-K.,

340

T.S., S.Y.L, G.J.W. and T.W. wrote the paper.

341

Notes

342

The authors declare no competing financial interests.

343

ACKNOWLEDGMENTS

344

This work was supported in part by the German Center for Infection Research

345

(Deutsches Zentrum für Infektionsforschung, DZIF, TTU09.802 and TTU09.704 to

15 ACS Paragon Plus Environment

ACS Synthetic 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

346

T.W. and W.W.), the Novo Nordisk Foundation to SYL and TW (NNF15OC0016226

347

to TW), and the National Institute of Health (GM104258-01 to G.J.W).

348 349

REFERENCES

350

[1] Hertweck, C. (2009) The biosynthetic logic of polyketide diversity, Angew. Chem.

351 352 353 354 355 356 357 358

Int. Ed. 48, 4688-4716. [2] Walsh, C. T., and Wencewicz, T. A. (2014) Prospects for new antibiotics: a molecule-centered perspective, J. Antibiot. (Tokyo) 67, 7-22. [3] Musiol, E. M., and Weber, T. (2012) Discrete acyltransferases involved in polyketide biosynthesis, MedChemComm 3, 871-886. [4] Helfrich, E. J., and Piel, J. (2016) Biosynthesis of polyketides by trans-AT polyketide synthases, Nat. Prod. Rep. 33, 231-316. [5] Musiol, E. M., Härtner, T., Kulik, A., Moldenhauer, J., Piel, J., Wohlleben, W., and

359

Weber, T. (2011) Supramolecular templating in kirromycin biosynthesis: the

360

acyltransferase KirCII loads ethylmalonyl-CoA extender onto a specific ACP of

361

the trans-AT PKS, Chem. Biol. 18, 438-444.

362

[6] Zhao, C., Coughlin, J. M., Ju, J., Zhu, D., Wendt-Pienkowski, E., Zhou, X., Wang,

363

Z., Shen, B., and Deng, Z. (2010) Oxazolomycin biosynthesis in Streptomyces

364

albus JA3453 featuring an "acyltransferase-less" type I polyketide synthase that

365

incorporates two distinct extender units, J. Biol. Chem. 285, 20097-20108.

366

[7] Irschik, H., Kopp, M., Weissman, K. J., Buntin, K., Piel, J., and Muller, R. (2010)

367

Analysis of the sorangicin gene cluster reinforces the utility of a combined

368

phylogenetic/retrobiosynthetic analysis for deciphering natural product assembly

369

by trans-AT PKS, Chembiochem 11, 1840-1849.

370

[8] Mo, S., Kim, D. H., Lee, J. H., Park, J. W., Basnet, D. B., Ban, Y. H., Yoo, Y. J.,

371

Chen, S. W., Park, S. R., Choi, E. A., Kim, E., Jin, Y. Y., Lee, S. K., Park, J. Y.,

372

Liu, Y., Lee, M. O., Lee, K. S., Kim, S. J., Kim, D., Park, B. C., Lee, S. G., Kwon,

373

H. J., Suh, J. W., Moore, B. S., Lim, S. K., and Yoon, Y. J. (2011) Biosynthesis

374

of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase

375

proceeds through a dedicated polyketide synthase and facilitates the

376

mutasynthesis of analogues, J. Am. Chem. Soc. 133, 976-985. 16 ACS Paragon Plus Environment

Page 16 of 25

Page 17 of 25

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

377

ACS Synthetic Biology

[9] Koryakina, I., Kasey, C., McArthur, J. B., Lowell, A. N., Chemler, J. A., Li, S.,

378

Hansen, D. A., Sherman, D. H., and Williams, G. J. (2017) Inversion of extender

379

unit selectivity in the erythromycin polyketide synthase by acyltransferase

380

domain engineering, ACS Chem. Biol. 12, 114-123.

381

[10] Dunn, B. J., and Khosla, C. (2013) Engineering the acyltransferase substrate

382

specificity of assembly line polyketide synthases, J. R. Soc. Interface 10,

383

20130297.

384

[11] Sundermann, U., Bravo-Rodriguez, K., Klopries, S., Kushnir, S., Gomez, H.,

385

Sanchez-Garcia, E., and Schulz, F. (2013) Enzyme-directed mutasynthesis: a

386

combined experimental and theoretical approach to substrate recognition of a

387

polyketide synthase, ACS Chem. Biol. 8, 443-450.

388

[12] Bravo-Rodriguez, K., Ismail-Ali, A. F., Klopries, S., Kushnir, S., Ismail, S., Fansa,

389

E. K., Wittinghofer, A., Schulz, F., and Sanchez-Garcia, E. (2014) Predicted

390

incorporation of non-native substrates by a polyketide synthase yields bioactive

391

natural product derivatives, ChemBioChem 15, 1991-1997.

392

[13] Weber, T., Laiple, K. J., Pross, E. K., Textor, A., Grond, S., Welzel, K., Pelzer,

393

S., Vente, A., and Wohlleben, W. (2008) Molecular analysis of the kirromycin

394

biosynthetic gene cluster revealed beta-alanine as precursor of the pyridone

395

moiety, Chem. Biol. 15, 175-188.

396

[14] Musiol, E. M., Greule, A., Hartner, T., Kulik, A., Wohlleben, W., and Weber, T.

397

(2013) The AT(2) domain of KirCI loads malonyl extender units to the ACPs of

398

the kirromycin PKS, ChemBioChem 14, 1343-1352.

399

[15] Ye, Z., Musiol, E. M., Weber, T., and Williams, G. J. (2014) Reprogramming acyl

400

carrier protein interactions of an acyl-CoA promiscuous trans-acyltransferase,

401

Chem. Biol. 21, 636-646.

402

[16] Koryakina, I., McArthur, J., Randall, S., Draelos, M. M., Musiol, E. M., Muddiman,

403

D. C., Weber, T., and Williams, G. J. (2013) Poly specific trans-acyltransferase

404

machinery revealed via engineered acyl-CoA synthetases, ACS Chem. Biol. 8,

405

200-208.

406 407

[17] Haritos, V. S. (2015) Biosynthesis: A terminal triple bond toolbox, Nat. Chem. Biol. 11, 98-99. 17 ACS Paragon Plus Environment

ACS Synthetic 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

408

[18] Thirumurugan, P., Matosiuk, D., and Jozwiak, K. (2013) Click chemistry for drug

409

development and diverse chemical-biology applications, Chem. Rev. 113, 4905-

410

4979.

411

[19] Koryakina, I., and Williams, G. J. (2011) Mutant Malonyl-CoA Synthetases with

412

Altered Specificity for Polyketide Synthase Extender Unit Generation,

413

Chembiochem 12, 2289-2293.

414

[20] Edwards, D. M. F., Selva, E., Stella, S., Zerilli, L. F., and Gallo, G. G. (1992)

415

Mass spectrometric techniques for structure and novelty determination of

416

kirromycin-like antibiotics, Biol. Mass Spectrom., 51-59.

417

[21] Wolf, H., Chinali, G., and Parmeggiani, A. (1974) Kirromycin, an inhibitor of

418

protein biosynthesis that acts on elongation factor Tu, Proc. Natl. Acad. Sci.

419

USA 71, 4910-4914.

420

[22] Wolf, H., Chinali, G., and Parmeggiani, A. (1977) Mechanism of the inhibition of

421

protein synthesis by kirromycin. Role of elongation factor Tu and ribosomes,

422

Eur. J. Biochem. 75, 67-75.

423

[23] Kim, H. C., Kim, T. W., Park, C. G., Oh, I. S., Park, K., and Kim, D. M. (2008)

424

Continuous cell-free protein synthesis using glycolytic intermediates as energy

425

sources, J. Microbiol. Biotechnol. 18, 885-888.

426

[24] Kim, T. W., Keum, J. W., Oh, I. S., Choi, C. Y., Kim, H. C., and Kim, D. M. (2007)

427

An economical and highly productive cell-free protein synthesis system utilizing

428

fructose-1,6-bisphosphate as an energy source, J. Biotechnol. 130, 389-393.

429

[25] Vogeley, L., Palm, G. J., Mesters, J. R., and Hilgenfeld, R. (2001)

430

Conformational change of elongation factor Tu (EF-Tu) induced by antibiotic

431

binding. Crystal structure of the complex between EF-Tu.GDP and aurodox, J.

432

Biol. Chem. 276, 17149-17155.

433

[26] Olsthoorn-Tieleman, L. N., Palstra, R. J., van Wezel, G. P., Bibb, M. J., and Pleij,

434

C. W. (2007) Elongation factor Tu3 (EF-Tu3) from the kirromycin producer

435

Streptomyces ramocissimus is resistant to three classes of EF-Tu-specific

436

inhibitors, J. Bacteriol. 189, 3581-3590.

18 ACS Paragon Plus Environment

Page 18 of 25

Page 19 of 25

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

437

ACS Synthetic Biology

[27] Calhoun, K. A., and Swartz, J. R. (2005) An economical method for cell-free

438

protein synthesis using glucose and nucleoside monophosphates, Biotechnol.

439

Prog. 21, 1146-1153.

440

[28] Menges, R., Muth, G., Wohlleben, W., and Stegmann, E. (2007) The ABC

441

transporter Tba of Amycolatopsis balhimycina is required for efficient export of

442

the glycopeptide antibiotic balhimycin, Appl. Microbiol. Biotechnol. 77, 125-134.

443

[29] Kim, T. W., Keum, J. W., Oh, I. S., Choi, C. Y., Park, C. G., and Kim, D. M.

444

(2006) Simple procedures for the construction of a robust and cost-effective

445

cell-free protein synthesis system, J. Biotechnol. 126, 554-561.

446

[30] Kim, R. G., and Choi, C. Y. (2001) Expression-independent consumption of

447

substrates in cell-free expression system from Escherichia coli, J. Biotechnol.

448

84, 27-32.

449

19 ACS Paragon Plus Environment

ACS Synthetic 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

450

Figures

451

452 453

Figure 1. Kirromycin assembly line and the production of new kirromycin derivatives.

454 455 456 457 458

A: acyltransferase ACP: acyl carrier protein, KS: ketosynthase, DH: dehydrogenase, KR: ketoreductase, MT: methyltransferase, ER: enoylreductase, C: condensation domain, A: adenylation domain, PCP: peptidyl carrier protein, MatB: engineered malonyl-CoA synthetase (T207G/M306I), KirCII: discrete acyltransferase and R: functional group. Kirromycin (1), allyl-kirromycin (2), propargyl-kirromycin (3) and coumarin-kirromycin (4).

20 ACS Paragon Plus Environment

Page 20 of 25

Page 21 of 25

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

ACS Synthetic Biology

459 460

Figure 2. New kirromycin derivatives.

461 462

(A) Chemical structure of allyl-kirromycin (2), propargyl-kirromycin (3), coumarin-kirromycin (4). (B) Key HMBC correlations of the kirromycin derivatives.

463 464 465

466 467

Figure 3. In vitro activity assay.

468

Inhibition of eGFP translation using kirromycin and kirromycin derivatives.

21 ACS Paragon Plus Environment

ACS Synthetic 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

The promiscuity of the acyltransferase KirCII was exploited to generate new kirromycins with allyl- and propargyl-side chains in vivo, the latter were utilized as educts for further modification by click chemistry. 39x22mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 22 of 25

Page 23 of 25

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

ACS Synthetic Biology

Figure 1. Kirromycin assembly line and the production of new kirromycin derivatives. A: acyltransferase ACP: acyl carrier protein, KS: ketosynthase, DH: dehydrogenase, KR: ketoreductase, MT: methyltransferase, ER: enoylreductase, C: condensation domain, A: adenylation domain, PCP: peptidyl carrier protein, MatB: engineered malonyl-CoA synthetase (T207G/M306I), KirCII: discrete acyltransferase and R: functional group. Kirromycin (1), allyl-kirromycin (2), propargyl-kirromycin (3) and coumarinkirromycin (4).

182x180mm (300 x 300 DPI)

ACS Paragon Plus Environment

ACS Synthetic 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

Figure 2. New kirromycin derivatives. (A) Chemical structure of allyl-kirromycin (2), propargyl-kirromycin (3), coumarin-kirromycin (4). (B) Key HMBC correlations of the kirromycin derivatives.

172x150mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 24 of 25

Page 25 of 25

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

ACS Synthetic Biology

Figure 3. In vitro activity assay. Inhibition of eGFP translation using kirromycin and kirromycin derivatives.

112x60mm (300 x 300 DPI)

ACS Paragon Plus Environment