Heterologous Biosynthesis of Spinosad: An Omics-Guided Large

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Heterologous biosynthesis of spinosad: an omics-guided large polyketide synthase gene cluster reconstitution in Streptomyces Gao-Yi Tan, Kunhua Deng, Xinhua Liu, Hui Tao, Yingying Chang, Jia Chen, Kai Chen, Zhi Sheng, Zixin Deng, and Tiangang Liu ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00330 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017

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ACS Synthetic Biology

1

Submitted to ACS Synthetic Biology as research article

2 3

Heterologous biosynthesis of spinosad: an omics-guided large

4

polyketide synthase gene cluster reconstitution in Streptomyces

5 6

Gao-Yi Tan a, c, Kunhua Deng a, b, Xinhua Liu a, b, Hui Tao a, b, Yingying Chang a, b, Jia

7

Chen a, b, Kai Chen d, Zhi Sheng d, Zixin Deng a, b, e, Tiangang Liu a, b, *

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a

Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan

10

University), Ministry of Education, and Wuhan University School of Pharmaceutical

11

Sciences, Wuhan, 430071, China

12

b

13

Biotechnology, Wuhan, 430075, China

14

c

15

China University of Science and Technology, 130 Meilong Road, Shanghai 200237,

16

China

17

d

18

Shengyang 110021, China

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e

20

Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai

21

200240, China

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* Email: [email protected]; Telephone: 086-27-68755086; Fax: 086-27-68755086

Hubei Engineering Laboratory for Synthetic Microbiology, Wuhan Institute of

State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East

Shenyang Research Institute of Chemical Industry Ltd., Co., SINOCHEM Group,

State Key Laboratory of Microbial Metabolism, and School of Life Sciences &

ACS Paragon Plus Environment


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Abstract

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With the advent of the genomics era, heterologous gene expression has been used

25

extensively as a mean of accessing natural products (NPs) from environmental DNA

26

samples. However, the heterologous production of NPs often has very low efficiency

27

or is unable to produce targeted NPs. Moreover, due to the complicated transcriptional

28

and metabolic regulation of NP biosynthesis in native producers, especially in the

29

cases of genome mining, it is also difficult to rationally and systematically engineer

30

synthetic pathways to improved NPs biosynthetic efficiency. In this study, various

31

strategies ranging from heterologous production of a NP to subsequent application of

32

omics-guided synthetic modules optimization for efficient biosynthesis of NPs with

33

complex structure have been developed. Heterologous production of spinosyn in

34

Streptomyces spp has been demonstrated as an example of the application of these

35

approaches. Combined with the targeted omics approach, several rate-limiting steps of

36

spinosyn heterologous production in Streptomyces spp have been revealed.

37

Subsequent engineering work overcame three of selected rate-limiting steps, the

38

production of spinosad was increased step by step and finally reached 1460 µg/L,

39

which is about 1,000-fold higher than the original strain S. albus J1074 (C4I6-M).

40

These results indicated that the omics platform developed in this work was a powerful

41

tool for guiding the rational refactoring of heterologous biosynthetic pathway in

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Streptomyces host. Additionally, this work lays the foundation for further studies

43

aimed at the more efficient production of spinosyn in a heterologous host. And the

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strategy developed in this study is expected to become readily adaptable to high

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efficient heterologous production of other NPs with complex structure.

46

Keywords: Spinosyn, Streptomyces, Heterologous production, Metabolomics,

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Proteomics, Module optimization


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Introduction

49

Natural products (NPs) or secondary metabolites, obtained from the microbial, plant

50

and marine worlds, were important sources for the development of new

51

pharmaceuticals

52

number of NPs pathways were discovered in microbes, making it became one of the

53

most promising sources for drug discovery 5. However, many biosynthetic genes are

54

derived from microbes that are uncultivable or not amenable to genetic manipulation

55

microbes 6. Accordingly, heterologous gene expression has been used extensively as a

56

mean of accessing target NPs in genome mining. Streptomyces spp. is a rich source of

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NPs, in which different kinds of cellular intermediates, including sugars, amino acids,

58

fatty acids, terpenes, shikimate, etc., could be used for the biosynthesis of

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pharmaceutical compounds with complex structure

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Streptomyces spp, including S. avermitilis, S. coelicolor, S. venezuelae, S. lividans, S.

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albus etc., have been developed as potential hosts and used widely for heterologous

62

expression of NPs 9-16.

1-4

. Moreover, with the advent of the post-genomic era, a large

7, 8

. Consequently, many

63 64

To date, many kinds of NPs have been biosynthesized in heterologous Streptomyces

65

hosts

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metabolites biosynthetic genes clusters (4 – 75 kb) have been introduced into the

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heterologous host S. avermitilis by protoplast transformation, and all the

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corresponding metabolites were produced 7. A 67 kb lipopeptide antibiotic taromycin

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A

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Saccharomonospora sp. CNQ-490 by transformation-associated recombination and

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efficiently expressed in S. coelicolor

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kb), such as type I polyketide compound, the bacterial artificial chromosome (BAC)

17

. For example, as reported by Komatsu et al, more than 20 secondary

biosynthetic gene cluster was cloned from the marine actinomycete

18

. For large gene clusters encoding NPs (> 80



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library could be used as an efficient approach for gene cluster cloning and subsequent

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heterologous expression 19, 20.

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However, it should be noted that occasionally possibly owing to the different genetic

76

distances (native producer vs heterologous host) and complex native regulation, the

77

heterologous production of NPs has very low efficiency or is even unable to produce

78

targeted metabolites

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mining and heterologous expression. It is often difficult to rationally and

80

systematically identify obstacles which hinder the production of NPs. The emergence

81

of the innovative systems metabolic engineering concepts23, which integrate systems

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and synthetic biology with metabolic engineering24, has contribute to developing

83

efficient strategies to improve NPs biosynthetic efficiency on a global scale.

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Therefore, as one of the main approaches in system biology, the omics approach such

85

as transcriptomics, proteomics and metabolomics

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systematic analysis to identify the rate-liming steps or synthetic modules

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information provided by omics analysis could guide heterologous expression of NPs

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in several aspects, such as transcription regulation, module optimization, and host

89

improvement, etc (Figure 1).

7, 21, 22

. And such qualities are prevalent in the cases of genome

25-27

, which can be applied to 8, 28

. The

90 91

As a proof of concept, this study used the heterologous biosynthesis of spinosad (a

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mixture of spinosyn A and spinosyn D) in Streptomyces host as an example for

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several reasons. Firstly, as a typical large type I polyketide antibiotic produced by

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Saccharopolyspora spinosa NRRL 18395 29, spinosyn is a novel environment-friendly

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insecticide, which has been used widely for insect control in agriculture 30. Secondly,

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the biosynthesis of spinosyn was very complicated and needed many participating

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genes 31 (Figure S1). In the 80-kb spinosyn biosynthetic gene cluster, 14 of 19 genes


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are responsible for spinosyn post-modification, which include complicated

99

intramolecular cycloaddition 35-39

32, 33

and cross-bridging

and methylation

34

, as well as multiple steps of

40

100

glycosylation

. Lastly, but most importantly, probably due to

101

such a complicated and delicate post-modification, previous attempts on heterologous

102

production of spinosyn in Streptomyces were ended in failure 41.

103 104

In this work, a set of strategies involved in BAC library construction, efficient

105

conjugation and integration of a large plasmid into a Streptomyces host, identification

106

of rate-limiting steps by omics analysis, and rational synthetic module optimization

107

for production of targeted products in a heterologous host have been developed. This

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resulting approach is likewise expected to become adaptable to highly efficient

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heterologous production of valuable NPs with complex structures.

110 111

Result and Discussion

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Construction of Sa. spinosa NRRL 18395 BAC library and screening of spinosyn

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biosynthesis gene cluster.

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With the emerging of synthetic biology, significant advances have been achieved in

115

the heterologous biosynthesis of NPs 42. In order to clone the whole biosynthetic gene

116

cluster of a targeted secondary metabolite, there are many available technologies and

117

methodological

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Red/ET-mediated recombination 43-45, transformation-associated recombination 18 and

119

the Gibson assembly method

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however, because they can accommodate large secondary metabolic pathways

121

spanning at least 100 kb.

approaches,

including

cosmid/fosmid

library

construction,

46

. It appears BAC vectors are usually more versatile,

122



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To obtain the intact spinosyn biosynthetic gene cluster (≈ 80 kb), the genomic DNA

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of Sa. spinosa NRRL 18395 was used for BAC library construction (containing 2304

125

clones: six plates × 384 clones/plate). In all, 39 clones were selected at random for

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BAC library characterization and evaluation; nearly all the insert sizes of BAC clones

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ranged from 97 – 145 kb (Figure S2A). PCR screening was used to identify the clone

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containing the intact spinosyns biosynthetic gene cluster. Three different primer pairs

129

F/R; (lib-screen-up F/R, lib-screen-md F/R and lib-screen-dn Table S2) for regions in

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the middle and in the left/right boundary of the gene cluster were used in the assay.

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As a result, one BAC clone (C4I6) was obtained by library screening (Figure S2B).

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In addition, the three PCR products FI, FII and FIII were amplified by using C4I6

133

plasmid as template, suggesting C4I6 contains the intact spinosyn biosynthetic gene

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cluster. In addition, plasmid C4I6 has been confirmed by end sequencing with primers

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pIndigoF/R (Table S2), and the size of the C4I6 insert was greater than 100 kb

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(Figure S3C). Here, using the pBeloBAC11-derived vector pIndigoBAC536-S,

137

which can largely facilitate the construction and characterization of BAC libraries 47,

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the 80 kb spinosyn biosynthetic gene cluster was easily cloned and screened. In our

139

experience, BAC library construction is a more straightforward and efficient approach

140

to obtaining large DNA fragments, although it is marginally time-consuming for

141

library construction.

142 143

Introduction of spinosyn biosynthetic gene cluster into Streptomyces by

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triparental conjugation

145

By introducing of the aac(3)IV-oriT-attP(ΦC31)-int(ΦC31) cassette into the BAC

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plasmid, we were able to efficiently integrate pIndigoBAC536-S and its inserts into

147

the chromosome of the Streptomyces spp. via conjugation. In this study, plasmid


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C4I6-M was generated by replacing the chloramphenicol-resistance gene of the C4I6

149

with the aac(3)IV-oriT-attP(ΦC31)-int(ΦC31) cassette (Figure S3A&B).

150 151

It is well known that E.coli ET12567 (pUZ8002) has been widely used for biparental

152

conjugation

153

ET12567(pUZ8002) was initially applied for integration of the C4I6-M, nearly all the

154

conjugant contains the incomplete spinosyn biosynthetic gene cluster (Table 1). As an

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alternative solution, E.coli ET12567(pUB307)-mediated triparental conjugation was

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used for integration of the C4I6-M into the chromosome of S. albus J1074 or S.

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lividans TK24 49. The exconjugant frequency was calculated by growing a number of

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conjugants on the ISP Medium 4 plate and counting the total conjugants on the plates,

159

which yielded rough estimates of 10–5 – 10–6 (Table 1). Consequently, eleven random

160

conjugants were selected for further verification. When the primer pair lib-screen-up

161

F/R were used for PCR analysis, DNA fragment FI could be amplified in nine of the

162

11 conjugants (Figure S5). These results indicated BAC plasmid C4I6-M could be

163

introduced efficiently into chromosomes of the Streptomyces host. Subsequently, the

164

second round of PCR verification showed all the 14 post-modification genes (spnF,

165

spnG, spnH thru spnR and spnS) could be amplified from six randomly selected

166

conjugants (date not shown). After two rounds of screening and verification, several

167

conjugants were selected for additional analysis.

in

Streptomyces

48

.

In

this

study,

however,

when

E.coli

168 169

It worth to be noted that, as an RK2 derivative, pUZ8002 is not self-transmissible but

170

can mobilize other plasmids 48. Therefore, before conjugation with Streptomyces, the

171

BAC plasmid should be introduced into ET12567(pUZ8002) by electroporation, and

172

the ET12567 donor cell has to hold the > 50 kb helper plasmid pUZ8002 and the 110



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kb BAC plasmid C4I6-M. In contrast, pUB307 is an RPI-derived self-transmissible

174

plasmid

175

oriT-containing BAC plasmid, and DH10B(BAC) and ET12567(pUB307) could be

176

used directly for conjugation. According to the results of this study, triparental

177

conjugation was more suitable for the conjugation of a large BAC plasmid.

49

, which provides in trans the function for the mobilization of the

178 179

Transcriptional and translational analysis of biosynthetic genes in Streptomyces

180

and identification of heterologous production of spinosyn

181

The transcription of genes involved in biosynthesis served as the prerequisite for the

182

heterologous production of spinosyn. In this study, the transcription of spinosyn

183

biosynthetic genes in Streptomyces host was analyzed by RT-PCR using cDNA

184

generated from RNA isolated after fermentation for 48 h. According to our results

185

(data not shown), the 14 post-modification genes were located in different operons

186

(Figure 2A). Genes from different operons have been selected for transcriptional

187

analysis, as shown in Figure 2B, which revealed that the transcription of all the

188

selected genes could be detected. Under identical PCR conditions, the intensity of

189

amplified products indicated transcription activity of genes. In this study, the

190

transcription activity of spnI and spnP were lower compared to the others. Similar to

191

the detection of the post-modification genes, all five PKS gene fragments were

192

amplified using cDNA as template, which suggested that the spinosyn biosynthetic

193

genes could be successfully transcribed in Streptomyces spp. However, transcription

194

activity of spnE was lower compared to the others PKS genes.

195 196

The translation of spinosyn biosynthetic genes in Streptomyces host was also analyzed

197

by targeted proteomics approach. The 48 h fermentation broth was collected for


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targeted proteomics analysis of Sa. spinosa NRRL 18395, S. albus J1074 (C4I6-M)

199

and S. lividans TK24 (C4I6-M). Nearly two thousand proteins were detected in this

200

study (Table S3) and the 23 spinosyn biosynthetic proteins were analyzed. As given

201

in Table 2, most of the spinosyn biosynthetic proteins were present in Sa. spinosa

202

NRRL 18395 and S. albus J1074 (C4I6-M). However, approximately half of the

203

targeted proteins were not detected in S. lividans TK24 (C4I6-M). In addition, likely

204

because the expression levels of the proteins were below the detection limit of our

205

method, all the NDP-L-rhamnose biosynthetic proteins (or homologues), some of the

206

PKS proteins, methyltransferase SpnI and forosamine biosynthetic protein SpnO,

207

SpnN were not found in S. albus J1074 (C4I6-M) or S. lividans TK24 (C4I6-M).

208 209

Anyway, the above results suggested the possibility of heterologous production of

210

spinosyn in Streptomyces, especially in S. albus J1074. Therefore, the heterologous

211

production of spinosyn was qualitatively analyzed by high-resolution MS. In Figure 3,

212

as expected, the spinosyn A was detected in both S. albus J1074 (C4I6-M) and S.

213

lividans TK24 (C4I6-M). LC-MS/MS analysis confirmed spinosyn A could be

214

produced in Streptomyces spp. in a very tiny amount (a few microgram per liter)

215

(Figure S6). As a complex polyketide compound, the heterologous production of

216

spinosyn indicated both these Streptomyces hosts could provide all the essential

217

"building blocks" for spinosyn synthesis. The whole spinosyn biosynthetic pathway

218

was demonstrated in both Streptomyces hosts; however, as the last unsettled question

219

in the biosynthesis of spinosyn, the auxiliary protein for SpnP is remain unknown 39.

220

The lack of this protein hampers transformation of the forosamine moiety to spinosyn

221

50

222

(C4I6-M) and S. lividans TK24 (C4I6-M) have counterparts of the unknown auxiliary

223

protein.

. Upon the basis of the present results, it is rational to believe both S. albus J1074



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Omics analysis revealed the rate-limiting steps in heterologous production of

226

spinosyn

227

The precursor supply and cofactor availability for the biosynthesis of spinosyn, as

228

well as the biosynthetic intermediate of spinosyn (or kinds of spinosyn components or

229

intermediates, Figure S7), were analyzed quantitatively in the metabolomics analysis.

230

As shown in Table 3, compared with Sa. spinosa NRRL 18395 and S. lividans TK24

231

(C4I6-M), S. albus J1074 (C4I6-M) has a more abundant supply of acyl-CoA

232

substrate, and has a very large accumulation of acetyl-CoA. Compared to Sa. spinosa

233

NRRL 18395, S. lividans TK24 (C4I6-M) and S. albus J1074 (C4I6-M) have more

234

cellular SAM and UDP-glucose. In addition, the different redox potential of three

235

kinds of bacterial cells have been investigated by relative quantitative analysis of

236

nicotinamide adenine dinucleotide hydrate (NADH), NAD+, nicotinamide adenine

237

dinucleotide phosphate (NADPH) and NADP+. As indicated in Table 3, the ratio of

238

NAD+NRRL18395/ NAD+J1074(C4I6-M)/NAD+TK24(C4I6-M) was 1:9:5, while the ratio of

239

NADHNRRL18395/ NADHJ1074(C4I6-M)/NADHTK24(C4I6-M) was 1:11:5. At the same time,

240

the

241

NADPHNRRL18395/ NADPHJ1074(C4I6-M)/NADPHTK24(C4I6-M) were 1:3:1 and 1:8:1,

242

respectively. These results suggested that both the supply of NADH and NADPH in S.

243

albus J1074(C4I6-M) was higher than Sa. spinosa NRRL 18395 and, S. lividans

244

TK24(C4I6-M). S. lividans TK24 and S. albus J1074 are universal Streptomyces hosts

245

used widely for heterologous expression of NPs. However, several prior studies

246

involved in the heterologous expression of NPs showed S. albus J1074 has a better

247

yield compared to S. lividans or other Streptomyces spp

248

naturally minimized size of 6.8 Mb, the genome of S. albus J1074 is the smallest

ratios

of

NADP+NRRL18395/NADP+J1074(C4I6-M)/NADP+TK24(C4I6-M)


and

51-54

. In addition, with a

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249

among the completely sequenced species of the genus Streptomyces 15. In this study,

250

about half of the targeted proteins have not been detected in S. lividans TK24 (Table

251

2). As indicated by the results of metabolomics analysis, S. albus J1074 has

252

considerably larger precursors (acyl-CoA, UDP-glucose) and cofactors (NADPH,

253

SAM) compared to S. lividans TK24 (Table 3). This suggests S. albus J1074 might

254

have better potential for the increased production of targeted products.

255 256

Both the rhamnose and forosamine moieties are essential for the biosynthesis of

257

spinosyn. It is suggested highly efficient biosynthesis of TDP-L-rhamnose could

258

dramatically improve production of spinosyns by duplication of gtt, gdh, epi and kre

259

55-57

260

lividans TK24 (C4I6-M) are undetectable (Table 2). Therefore, the insufficient

261

catalytic activity (or low enzyme amount) of rhamnose biosynthesis likely indicated a

262

rate-limiting step for biosynthesis of spinosyn. Further, it was found that the PKS

263

protein SpnE, methyltransferase SpnI and the forosamine biosynthetic protein SpnO

264

and SpnN were not detectable by targeted proteomics analysis in S. albus J1074

265

(C4I6-M). Based on metabolomics analysis, it appeared that the accumulation of

266

spinosyn-CH2 also indicated an insufficient enzymatic activity of methyltransferase

267

SpnI

268

biosynthesis of spinosyn in S. albus J1074. In addition, the accumulation of other

269

intermediates or components, including spinosyn B, E, L3 and P-CH2, also indicated

270

an insufficient amount (or low activity) of the corresponding enzymes in S. albus

271

J1074 (C4I6-M) (Table 4). And the biosynthetic pathway of spinosyn requires

272

systematically refactoring to improve the synthetic efficiency in S. albus J1074.

273

Combined with the targeted metabolomics and the translational analysis of spinosyn

274

biosynthetic genes in Streptomyces host, it was clearly indicated that the biosynthesis

. However, the expression of these proteins in both S. albus J1074 (C4I6-M) and S.

40

. This is likely another important rate-limiting step in the heterologous



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275

of sugar ligands and the insufficient enzymatic activity of methyltransferase and

276

polyketide synthase were probably the rate-limiting steps of heterologous production

277

of spinosyn in S. albus J1074 (C4I6-M) (Table S4).

278 279

Overexpression of sugar biosynthetic module, methyltransferase and polyketide

280

synthase to promote spinosyn biosynthesis

281

Under the guidance of the above information, the subsequent synthetic modules

282

optimization were employed in attempt to overcome these rate-limiting steps in S.

283

albus J1074 (C4I6-M). The high-efficiency Streptomyces promoters, such as kasOp*

284

58

285

ermE promoter in S. albus (data not shown), were used to overexpress the rhamnose

286

biosynthetic genes and methyltransferase gene spnI (Figure 4A). Facilitated by the E.

287

coli-Streptomyces shuttle plasmid pJTU1278, the overexpression cassette was

288

introduced stably into the chromosome of S. albus J1074 (C4I6-M) by double

289

crossover (Figure 4B). The engineered strain S. albus J1074 (C4I6-M)-OE1 were

290

screened and verified by PCR (Figure 4C & Figure 5A). The engineered strain was

291

used in the fermentation study. As shown in Figure 5B, the ratio of spinosyn P-CH2

292

(relative abundance): spinosyn A (relative abundance) was decreased from 100% to

293

10% in S. albus J1074 (C4I6-M)-OE1. Consequently, compared to S. albus J1074

294

(C4I6-M), the production of spinosad could be easily detected (383µg/L) in S. albus

295

J1074 (C4I6-M)-OE1 (Figure 5C). By using the same strategy as described above,

296

the whole TDP-D-forosamine biosynthetic module has been subsequently

297

overexpressed in S. albus J1074 (C4I6-M)-OE2 (Figure S8 & Figure 5A). As shown

298

in Figure 5B, by overexpression of forosamine biosynthetic modules, the

299

heterologous efficiency of spinosad in S. albus J1074 (C4I6-M)-OE2 has been

rpsL-TP and rpsL-cf 59, of which the strengths are ten-fold higher compared to the


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300

significantly improved and the production reached 686µg/L. Lastly, the native

301

promoter of PKS gene spnE was also replaced with kasOp* in S. albus J1074

302

(C4I6-M)-OE3 (Figure S9 & Figure 5A), the production of spinosad finally reached

303

1460 µg/L in 48 hours (Figure S10), which is about 1,000-fold higher than the

304

original strain S. albus J1074 (C4I6-M) (Figure 5B). Accordingly, under the guidance

305

of omics analysis, the heterologous biosynthetic efficiency of spinosad could be

306

increased step by step after several rounds of synthetic pathway refactoring.

307 308

By the way, it also has to realize that, compared to Sa. spinosa NRRL 18395 and Sa.

309

erythraea

310

However, the heterologous production of spinosyn in this work has opened a new

311

route for future modification or production of targeted product in Streptomyces host,

312

due to their rapid proliferation and readily genetic manipulation. And in Streptomyces,

313

the spinosad could be efficiently produced in a very short period compared with

314

Saccharopolyspora (Figure S10). More importantly, in this study, various strategies

315

ranging from heterologous production of a NP to subsequent application of

316

“omics”-guided metabolic engineering for efficient biosynthesis of NPs with complex

317

structure have been developed. Heterologous production of spinosyn in Streptomyces

318

spp has been demonstrated as an example of the application of these approaches.

319

Although the biosynthetic efficiency of spinosyn in Streptomyces app. has been

320

increased dramatically, in this study, the current results should serve as the start point

321

for further studies aimed at the more efficient production of spinosyn in a

322

heterologous host. Importantly, this approach could be readily adaptable to highly

323

efficient heterologous production of other valuable NPs.

60

, the current production of spinosad in Streptomyces was relatively low.

324



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

325

Methods

326

Microorganisms, fermentation and analysis

327

The plasmids and strains used in this study are given in Table S1. Streptomyces and

328

its derivatives were cultivated on soybean flour-mannitol agar plates (2% (w/v)

329

soybean flour, 2% (w/v) mannitol and 2% (w/v) agar) and International Streptomyces

330

Project (ISP) Medium 4 agar plates (BD Biosciences, San Jose, CA, USA) for

331

sporulation and conjugation, respectively. Spores were collected, suspended in 20%

332

(v/v) glycerol and stored at –80°C. In fermentation experiments, seeds were grown in

333

trypticase soy broth and the fermentation medium was 4% (w/v) glucose, 1% (w/v)

334

glycerol, 3% (w/v) soluble starch, 1.5% (w/v) Difco soytone, 1% (w/v) beef extract,

335

0.65% (w/v) peptone, 0.05% (w/v) yeast extract, 0.1% (w/v) MgSO4, 0.2% (w/v)

336

NaCl and 0.24% (w/v) CaCO3. Spinosyn was characterized and quantified using a

337

liquid chromatography (LCQ) Fleet™ ion-trap mass spectrometer or a high-resolution

338

Q-Exactive™ Hybrid Quadrupole- OrbitrapTM mass spectrometer (Thermo Scientific™,

339

Waltham, MA). Unless special instruction, all the spinosyn samples were analyzed at

340

the end of 5-day fermentation.

341 342

BAC library construction

343

Sa. spinosa NRRL 18395 was cultivated on trypticase soy broth supplemented with

344

0.5% (w/v) glycine for 48 h. The mycelium was collected and used for the preparation

345

of chromosomal DNA plugs. A pBeloBAC11-derived BAC vector pIndigoBAC536-S

346

was used for BAC library construction essentially as described 47, 61. The BAC library

347

was evaluated by measuring the migration of plasmid during pulsed-field gel

348

electrophoresis. The BAC plasmid that contained the intact spinosyns biosynthetic

349

gene cluster was screened and verified by polymerase chain reaction (PCR).


Page 14 of 42

Page 15 of 42

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350 351

Conjugation of BAC plasmid to Streptomyces

352

The aac(3)IV-oriT-attP(ΦC31)-int(ΦC31) cassette was amplified from plasmid

353

pSET152 using a primer (Table S2). The cassette was used to replace the

354

chloramphenicol-resistance gene of the BAC plasmid by Red/ET recombination

355

And aac(3)IV gene could be used as a selectable marker (apramycin resistance) in

356

subsequent study. The modified BAC plasmid was introduced into the Streptomyces

357

chromosome by triparental conjugation

358

Escherichia coli DH10b(C4I6-M) and E. coli ET12567(pUB307) were grown to an

359

absorbance at 600 nm (A600) of 0.4 – 0.6. Cells were pelleted by centrifugation at 4000

360

rpm for 4 min, washed twice in Luria–Bertani (LB) broth, and finally suspended in a

361

smaller volume of LB. The fresh or frozen (stored at –40°C) purified Streptomyces

362

spores (S. albus J1074 or S. lividans TK24) were washed twice in LB, suspended in

363

TES (2-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino] ethanesulfonic acid) buffer

364

(0.05 M, pH 8.0) and incubated at 50°C for 10 min to activate germination. Then an

365

equal volume of double-strength germination medium (1% (w/v) Oxoid yeast extract,

366

1% (w/v) Difco Casamino acids and 0.01 M CaCl2) was added and the mixture

367

incubated at 37°C with shaking (220 rpm) for 2 – 3 h. The germinated spores were

368

pelleted by centrifugation, collected and suspended in TES buffer. Approximately 108

369

E. coli cells (DH10b(C4I6-M) : ET12567(pUB307) ≈ 1 : 1) were added to the

370

prepared spores (not less than 108 spores/conjugation) and the mixture was spread

371

onto an ISP Medium 4 agar plate. The conjugation plates were incubated for 11 – 14 h

372

at 30°C and then the surface of the plates was overlaid with 1 mL of sterile water

373

(containing 600 µg trimethoprim and 1 mg apramycin). The plates were then

49

62

.

(Figure S4). Briefly, the plasmid donors



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Page 16 of 42

374

incubated for an additional 3 – 5 days at 30°C, until the transconjugants could be

375

picked for subsequent assay.

376 377

RNA isolation and Reverse Transcription-PCR (RT-PCR)

378

Cells in fermentation broth were collected after incubation for 48 h. Total RNA

379

extraction and reverse transcription were perormed as described

380

performed with cDNA as template and primers designed in this study (Table S2) to

381

verify the transcription of spinosyn biosynthetic genes.

63

. PCR was

382 383

Targeted Proteomics analysis

384

Cells collected from fermentation broth were pelleted by centrifugation (12,000 rpm

385

for 5 min at 4ºC, collected and washed three times in cold wash buffer (100 mM NaCl,

386

25 mM Tris–HCl, pH 7.5). The washed pellet was ground thoroughly under liquid

387

nitrogen and 1 g of the cell powder was suspended in 2 mL pre-chilled lysis buffer (8

388

M

389

3-[(3-cholamido-propyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 75 mM

390

NaCl, 50 mM Tris–HCl (pH 8.0) and 1 complete EDTA-Free protease inhibitor

391

cocktail tablet (Roche, Indianapolis, IN) per 10 mL of buffer). The suspended sample

392

was vortex-mixed for 30 s at 20 min intervals for 2 h; the supernatant materials from

393

the lysed cells was collected by centrifugation (13,000 rpm for 50 min at 4°C).

394

Proteins from the cell lysates were measured using a non-interference protein assay

395

kit (Sangon Biotech, Shanghai, China) then adjusted to 3 µg/µL using lysis buffer. An

396

equal volume of 100 mM ammonium bicarbonate buffer (pH 8.0) was subsequently

397

added and the sample was reduced at 37°C for 1 h by adding 3 mM TCEP

398

(tris(2-carboxyethyl)-phosphine) then alkylated by adding 15 mM iodoacetamide and

urea,

2

M

thiourea,


4%

(w/v)

Page 17 of 42

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399

incubating the sample in darkness for 1 h. The excess iodoacetamide was quenched by

400

adding TCEP to 15 mM final concentration and incubating for 15 min. The sample

401

was diluted with ammonium bicarbonate buffer to 1 M urea final concentration before

402

the addition of trypsin (enzyme/substrate ratio 1:50, w/w) and incubation for 12 h at

403

37°C. The detergent and salt in the digested peptide sample were removed by passage

404

through a Pierce™ detergent removal spin column (Thermo Scientific, Rockford, IL)

405

and then a Sep-Pak™ C18 cartridge (Waters Corp., Milford, MA), respectively. The

406

purified peptides were freeze-dried (–80°C at reduced pressure) for subsequent liquid

407

chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

408 409

The peptide samples were analyzed with a hybrid quadrupole-time-of-flight (TOF)

410

liquid chromatography (LC) tandem mass (MS/MS) spectrometer (TripleTOFTM

411

5600+, AB Sciex, Foster City, CA) equipped with a nanospray ion source. The

412

samples were firstly loaded onto a C18 trap column (5 µm, 5 × 0.3 mm, Agilent

413

Technologies) and eluted into a C18 analytical column (75 µm × 150 mm, 3 µm

414

particle size, 100 Å pore size, Eksigent). Elution of the peptides were conducted using

415

a stepwise gradient with mobile phase A (3% (v/v) dimethyl sulfoxide, 97% (v/v)

416

water and 0.1% (v/v) formic acid) and mobile phase B (3% (v/v) dimethyl sulfoxide,

417

97% (v/v) acetonitrile and 0.1% (v/v) formic acid). Peptides were identified via

418

database searching of raw data using ProteinPilotTM v4.5 software (AB SCIEX)

419

against the designated proteome database.

420 421

Targeted metabolomics analysis

422

For rapid sampling and quenching, 10 mL of fermentation broth was quenched

423

quickly by adding 40 mL of chilled (–80°C) 80% (v/v) methanol and the pellet was



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

Page 18 of 42

64-66

424

collected by centrifugation (8000 rpm for 5 min at 4°C)

. For metabolite

425

extraction, 5 mL of hot (95°C) ethanol was added, vortex-mixed for 30 s, incubated at

426

95°C for 3 min followed by freezing at –40°C for 15 min. The supernatant was

427

collected by centrifugation (5000 rpm for 5 min at 4°C) and freeze-dried at –80°C

428

under reduced pressure.

429 430

The metabolite sample was analyzed using an ultra-high-performance liquid

431

chromatography (UHPLC) system (Shimadzu LC20ADXR) equipped with a hybrid

432

quadrupole-TOF LC/MS/MS mass spectrometer (TripleTOFTM 5600+, AB Sciex).

433

Samples

434

chromatography (HILIC) (Acquity UPLC BEH Amide column; mobile phase A

435

containing 10 mM ammonium formate and 0.1% formic acid in water and mobile

436

phase B containing 95% acetonitrile and 5% water) and a C18 reversed-phase

437

chromatography (Kinetex C18 column, mobile phase A containing 2 mM ammonium

438

formate and 0.05% formic acid in water and mobile phase B containing 50%

439

acetonitrile and 50% methanol) in both positive and negative ionization modes.

440

Conditions for the TOF survey scan were: scan range 100 – 1600 Da, accumulation

441

time 0.1 s. TOF MS was followed by an eight product ion scan, accumulation time 50

442

ms/scan (m/z 50 – 1250).

were

analyzed

separately

using

both

a

hydrophilic

interaction

443 444

Gene overexpression plasmid construction

445

Genes gtt, epi, gdh and kre were amplified from NRRL18395, promoter kasOp* was

446

amplified from pN1. Then, a 1.6 kb kasOp*-controlled gtt-epi DNA fragment and

447

other 2.0 kb kasOp*-controlled gdh-kre fragment were clone to pSET152 to generate

448

a 3.6 kb NDP-L-rhamnose biosynthetic gene overexpression cassette by restriction


Page 19 of 42

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449

enzyme digestion and ligation. The rpsLp-cf –controlled spnI gene (promoter

450

rpsLp-cf was amplified from pN2, spnI was amplified from NRRL18395) and

451

selection marker (neo, amplified from pK18mob) were also ligated by restriction

452

enzyme digestion and ligation to generate a 3.0 kb fragment. This 3.0 kb fragment

453

was subsequently introduced into former pSET152 derivate by Red/ET to generate a

454

6.6 kb overexpression cassette (as shown in Figure 4A). Meanwhile, a 1.54 kb

455

HindIII-XbaI left arm containing the 5ʹ germinus of XNR_5867 (a biosynthetic gene

456

located in Candicidin gene cluster of S. albus J1074) and 1.54 kb EcoRV-HindIII

457

right arm containing the 3ʹ germinus of XNR_5867 were ligated with HindIII digested

458

pUC57ʹ (removed the multiple cloning sites) to generate pUC57ʹ-LR. The 6.6 kb

459

overexpression cassette was cut from the pSET152 derivate by XbaI and EcoRV, and

460

then ligated to XbaI-EcoRV digested pUC57ʹ-LR to generate pUC57ʹ-LR-gegk-I. The

461

9.6 kb DNA fragment was cut from pUC57ʹ-LR-gegk-I by HindIII and finally ligated

462

with HindIII digesged pJTU1278 to generate the Streptomyces overexpression

463

plasmid pJTU1278-gegk-I-OE.

464 465

The TDP-D-forosamine biosynthetic gene spnO, spnN, spnQ, spnR and spnS and PKS

466

gene spnE were overexpressed using kasOp*, rpsLp-cf and rpsLp-TP. And the

467

corresponding plasmid pJTU1278-ONQRS-OE and pJTU1278-spnE-OE were

468

constructed by DNA fragments assembly in yeast 67. All the primers used for plasmids

469

construction were shown in Table S2.

470 471

Statistical analysis

472

Statistical analyses were carried out using Microsoft Excel 2013. All the data were

473

expressed as mean ± standard error of mean (SEM) and analyzed by unpaired



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

474

two-tailed Student’s t-test unless specified in the figure legend. P < 0.05 was used as a

475

standard criterion of statistical significance.

476 477

Supporting Information

478

Table S1, the strains and plasmids used in this study; Table S2, the sequences of PCR

479

primer pairs; Table S3, summary statistics of the proteins detected in S.albus J1074

480

(C4I6-M) and S. lividans TK24 (C4I6-M); Table S4, the transcriptional and

481

translational profiling of spinosyn biosynthetic genes and corresponding metabolites

482

in S. albus J1074(C4I6-M); Figure S1, spinosyn biosynthetic pathway; Figure S2,

483

screening of the intact spinosyn biosynthetic gene cluster from the Sa. spinosa NRRL

484

18395 BAC library; Figure S3, modification of the C4I6 plasmid; Figure S4,

485

flowchart of triparental and bioparental conjugation; Figure S5, triparental

486

conjugation and preliminary screening of conjugants by PCR; Figure S6, qualitative

487

analysis of spinosyn A in S. albus J1074 (C4I6-M) by LC-MS/MS; Figure S7,

488

chemical structures of spinosyn components and intermediates; Figure S8,

489

overexpression of the TDP-D-forosamine biosynthetic module in S. albus

490

J1074(C4I6-M)-E2; Figure S9,

491

J1074(C4I6-M)-OE3; Figure S10. time course of spinosad fermetation in the S. albus

492

J1074(C4I6-M)-OE3.

overexpression of the PKS gene spnE in S. albus

493 494

Acknowledgments

495

We thank Prof. Meizhong Luo (National Key Laboratory of Crop Genetic

496

Improvement, Huazhong Agricultural University) for his help in the construction of

497

the BAC library. We also thank Shuai Fu (J1 Biotech Co. Ltd, Wuhan, China) and Dr.

498

Xiaoyan Xu (AB Sciex Pte. Ltd) for her help in proteomics and metabolomics


Page 20 of 42

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499

analysis, respectively. This study was supported by National 973 Program of China

500

(No. 2012CB721000), the Open Project of State Key Laboratory of Microbial

501

Metabolism funded this work (No. MMLKF15-13), the Science and Technology

502

Investment Program of SINOCHEM Group (No. 2015ADSW0039), and the Young

503

Talents Program of National High-level Personnel of Special Support Program (The

504

“Ten Thousand Talent Program”) to T. Liu. The authors also specially acknowledge

505

the support from the National Natural Science Foundation of China (No. 31500072),

506

the Natural Science Foundation of HuBei Province (No. 2015CFB415), and China

507

Postdoctoral Science Foundation Grant (No. 2014M562052).

508 509

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33. Fage, C. D., Isiorho, E. A., Liu, Y., Wagner, D. T., Liu, H.-w., and Keatinge-Clay,

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611

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biosynthesis of spinosyn in Saccharopolyspora spinosa: synthesis of the

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cross-bridging precursor and identification of the function of SpnJ, Journal of

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35. Zhao, Z., Hong, L., and Liu, H.-w. (2005) Characterization of protein encoded by

617

spnR from the spinosyn gene cluster of Saccharopolyspora spinosa:

618

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619

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36. Hong, L., Zhao, Z., and Liu, H.-w. (2006) Characterization of SpnQ from the

621

spinosyn biosynthetic pathway of Saccharopolyspora spinosa: mechanistic

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and evolutionary implications for C-3 deoxygenation in deoxysugar

623

biosynthesis, Journal of the American Chemical Society 128, 14262-14263.

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37. Hong, L., Zhao, Z., Melançon, C. E., Zhang, H., and Liu, H.-w. (2008) In vitro

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characterization of the enzymes involved in TDP-D-forosamine biosynthesis

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in the spinosyn pathway of Saccharopolyspora spinosa, Journal of the

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American Chemical Society 130, 4954-4967.

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38. Chen, Y.-L., Chen, Y.-H., Lin, Y.-C., Tsai, K.-C., and Chiu, H.-T. (2009)

629

Functional

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spinosyn

630

rhamnosyltransferase by in vitro reconstitution of spinosyn biosynthetic

631

enzymes, Journal of Biological Chemistry 284, 7352-7363.

632

39. Isiorho, E. A., Jeon, B.-S., Kim, N. H., Liu, H.-w., and Keatinge-Clay, A. T.

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(2014) Structural studies of the spinosyn forosaminyltransferase, SpnP,

634

Biochemistry 53, 4292-4301.

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40. Kim, H. J., White-Phillip, J. A., Ogasawara, Y., Shin, N., Isiorho, E. A., and Liu,

636

H.-w. (2010) Biosynthesis of spinosyn in Saccharopolyspora spinosa:

637

synthesis of permethylated rhamnose and characterization of the functions of

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SpnH, SpnI, and SpnK, Journal of the American Chemical Society 132,

639

2901-2903.



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41. Zhang, R., Xia, H., Xu, Q., Dang, F., and Qin, Z. (2013) Recombinational cloning

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of the antibiotic biosynthetic gene clusters in linear plasmid SCP1 of

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Streptomyces coelicolor A3 (2), FEMS Microbiology Letters 345, 39-48.

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and Yuan, Y.-J. (2015) Engineered biosynthesis of natural products in

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heterologous hosts, Chemical Society Reviews 44, 5265-5290.

646 647

43. Gust, B. (2009) Chapter 7 Cloning and analysis of natural product pathways, In Methods in Enzymology, pp 159-180, Academic Press.

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44. Tu, Q., Herrmann, J., Hu, S., Raju, R., Bian, X., Zhang, Y., and Müller, R. (2016)

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Genetic engineering and heterologous expression of the disorazol biosynthetic

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gene cluster via Red/ET recombineering, Scientific reports 6.

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45. Wang, H., Li, Z., Jia, R., Hou, Y., Yin, J., Bian, X., Li, A., Muller, R., Stewart, A.

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F., Fu, J., and Zhang, Y. (2016) RecET direct cloning and Redalphabeta

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recombineering of biosynthetic gene clusters, large operons or single genes for

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heterologous expression, Nature Protocols 11, 1175-1190.

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46. Li, L., Zhao, Y., Ruan, L., Yang, S., Ge, M., Jiang, W., and Lu, Y. (2015) A

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stepwise increase in pristinamycin II biosynthesis by Streptomyces

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pristinaespiralis through combinatorial metabolic engineering, Metabolic

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47. Shi, X., Zeng, H., Xue, Y., and Luo, M. (2011) A pair of new BAC and BIBAC

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vectors that facilitate BAC/BIBAC library construction and intact large

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genomic DNA insert exchange, Plant methods 7, 1.

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48. Paget, M. S., Chamberlin, L., Atrih, A., Foster, S. J., and Buttner, M. J. (1999)

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Evidence that the extracytoplasmic function sigma factor sigmaE is required

664

for normal cell wall structure in Streptomyces coelicolor A3 (2), Journal of

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Bacteriology 181, 204-211.

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49. Flett, F., Mersinias, V., and Smith, C. P. (1997) High efficiency intergeneric

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conjugal transfer of plasmid DNA from Escherichia coli to methyl

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DNA-restricting streptomycetes, FEMS Microbiology Letters 155, 223-229.

669

50. Kim, H. J., Choi, S. h., Jeon, B. s., Kim, N., Pongdee, R., Wu, Q., and Liu, H. w.

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(2014) Chemoenzymatic synthesis of spinosyn A, Angewandte Chemie

671

International Edition 53, 13553-13557.

672

51. Wendt-Pienkowski, E., Huang, Y., Zhang, J., Li, B., Jiang, H., Kwon, H.,

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Hutchinson, C. R., and Shen, B. (2005) Cloning, sequencing, analysis, and

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heterologous expression of the fredericamycin biosynthetic gene cluster from

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Streptomyces griseus, Journal of the American Chemical Society 127,

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52. Chen, Y., Wendt-Pienkowski, E., and Shen, B. (2008) Identification and utility of

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FdmR1 as a Streptomyces antibiotic regulatory protein activator for



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fredericamycin production in Streptomyces griseus ATCC 49344 and

680

heterologous hosts, Journal of Bacteriology 190, 5587-5596.

681 682 683 684

53. Bibb, M. J. (2005) Regulation of secondary metabolism in streptomycetes, Current opinion in microbiology 8, 208-215. 54. Bibb, M., and Hesketh, A. (2009) Analyzing the regulation of antibiotic production in streptomycetes, Methods in Enzymology 458, 93-116.

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55. Pan, H.-X., Li, J.-A., He, N.-J., Chen, J.-Y., Zhou, Y.-M., Shao, L., and Chen,

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D.-J. (2011) Improvement of spinosad production by overexpression of gtt and

687

gdh controlled by promoter PermE* in Saccharopolyspora spinosa

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SIPI-A2090, Biotechnology letters 33, 733-739.

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56. Madduri, K., Waldron, C., and Merlo, D. J. (2001) Rhamnose biosynthesis

690

pathway supplies precursors for primary and secondary metabolism in

691

Saccharopolyspora spinosa, Journal of Bacteriology 183, 5632-5638.

692

57. Xue, C., Duan, Y., Zhao, F., and Lu, W. (2013) Stepwise increase of spinosad

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production in Saccharopolyspora spinosa by metabolic engineering,

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Biochemical Engineering Journal 72, 90-95.

695

58. Wang, W., Li, X., Wang, J., Xiang, S., Feng, X., and Yang, K. (2013) An

696

engineered strong promoter for streptomycetes, Applied and Environmental

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Microbiology 79, 4484-4492.

698

59. Bai, C., Zhang, Y., Zhao, X., Hu, Y., Xiang, S., Miao, J., Lou, C., and Zhang, L.

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elements to unlock the microbial natural products in Streptomyces,

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Proceedings of the National Academy of Sciences 112, 12181-12186.

702

60. Huang, J., Yu, Z., Li, M.-H., Wang, J.-D., Bai, H., Zhou, J., and Zheng, Y.-G.

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Saccharopolyspora erythraea, Applied and Environmental Microbiology 82,

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5603-5611.

706

61. Luo, M., Wang, Y.-H., Frisch, D., Joobeur, T., Wing, R. A., and Dean, R. A.

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(2001) Melon bacterial artificial chromosome (BAC) library construction

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using improved methods and identification of clones linked to the locus

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conferring resistance to melon Fusarium wilt (Fom-2), Genome 44, 154-162.

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62. Zhang, Y., Muyrers, J. P., Testa, G., and Stewart, A. F. (2000) DNA cloning by

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homologous recombination in Escherichia coli, Nature Biotechnology 18,

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1314-1317.

713

63. Tan, G. Y., Bai, L., and Zhong, J. J. (2013) Exogenous 1, 4‐butyrolactone

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Streptomyces hygroscopicus 5008, Biotechnology and bioengineering 110,

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717

64. Canelas, A. B., Ras, C., Ten Pierick, A., van Dam, J. C., Heijnen, J. J., and Van

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Gulik, W. M. (2008) Leakage-free rapid quenching technique for yeast

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720

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721

Nicastro, R., and Nielsen, J. (2015) Expanded metabolite coverage of

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Saccharomyces cerevisiae extract through improved chloroform/methanol

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extraction and tert-butyldimethylsilyl derivatization, Analytical Chemistry

724

Research 6, 9-16.

725

66. Zhao, F., Xue, C., Wang, M., Wang, X., and Lu, W. (2013) A comparative

726

metabolomics analysis of Saccharopolyspora spinosa WT, WH124, and

727

LU104 revealed metabolic mechanisms correlated with increases in spinosad

728

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729

67. Noskov, V. N., Karas, B. J., Young, L., Chuang, R.-Y., Gibson, D. G., Lin, Y.-C.,

730

Stam, J., Yonemoto, I. T., Suzuki, Y., and Andrews-Pfannkoch, C. (2012)

731

Assembly of large, high G+C bacterial DNA fragments in yeast, ACS

732

Synthetic Biology 1, 267-273.

733 734 735 736 737 738 739 740 741 742 743


Page 32 of 42

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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


744 745 746 747 748 749 750

Table 1. Comparison of ET12567 (pUB307) and ET12567 (pUZ8002) as helpers in

751

intergeneric conjugation experiments with S.albus J1074 and S. lividans TK24.

Conjugation

S. albus J1074 True Exconjugant positive frequency rate *

S. lividans TK24 True Exconjugant positive frequency rate *

752

Biparental, < 2% 10-5 ~ 10-6 < 2% 10-5 ~ 10-6 ET12567(pUZ8002) Triparental, 10-5 ~ 10-6 > 70% 10-5 ~ 10-6 > 70% ET12567(pUB307) * True positive rate: the percentage of conjugants which contains the intact spinosyn

753

biosynthetic gene cluster. The right conjugants were preliminarily verified by PCR by

754

using the lib-screen-up F/R, lib-screen-mid F/R, or lib-screen-dn F/R primer pairs.

755 756 757 758 759 760 761 762 763 764 765 766 767 768



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

769 770

Table 2. Analysis the expression of targeted proteins involved in spinosyns

771

biosynthesis in S. spinosa NRRL 18395, S. lividans TK24 (C4I6-M) and S. albus

772

J1074 (C4I6-M) by presenting the number of peptides hits.

773

S. spinosa NRRL S. lividans TK24 S. albus J1074 18395 (C4I6-M) (C4I6-M) Peptides % Cov Peptides % Cov Peptides Proteins % Cov (95) (95%) (95) (95%) (95) (95%) SpnA 0 0 ND ND 0.8 2 SpnB 4.6 5 ND ND 2.3 3 SpnC 1.1 2 ND ND 1.6 3 SpnD 3.2 7 2.9 6 3.1 8 SpnE 2.8 7 1.8 4 ND ND 9.5 2 21.5 4 SpnF 4.4 1 SpnG 3.1 1 3.3 1 25.1 7 SpnH 10.8 1 30 4 52 9 SpnI 10.6 3 ND ND ND ND SpnJ 3.3 1 17.6 5 27.6 9 SpnK 23.2 5 44.6 10 52.1 12 SpnL ND ND 3.2 1 14.5 3 SpnM ND ND 3.1 1 26.6 5 SpnN 3.3 1 ND ND ND ND SpnO ND ND ND ND 0 0 SpnP 5.7 2 ND ND 19.8 7 SpnQ 12.3 4 ND ND 8.4 3 SpnR 4.2 1 ND ND 4.2 1 SpnS 13.3 2 ND ND 32.5 6 Gtt 0 0 / / / / / / Epi 10.4 1 / / Gdh 6.4 1 / / / / Kre 3.0 1 / / / / % Cov(95): peptide coverage with 95% confidence; Peptides (95%): number of

774

identified specific peptides with 95% confidence; ND: NOT detected; /: NOT existed;

775

In S. albus J1074, the expression of XNR_0593, XNR_0594, XNR_0573 and

776

XNR_2077 (homologs of Gtt, Epi, Gdh and Kre, respectively) were too low to detect.

777 778 779 780 781


Page 34 of 42

Page 35 of 42

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


782 783

Table 3. Relative quantitative analysis of precursors and cofactors involved in

784

spinosyn biosynthesis in Sa. spinosa NRRL 18395, S. lividans TK24 (C4I6-M) and S.

785

albus J1074 (C4I6-M). Relative abundance Metabolites

Mode / Adduction

NRRL 18395

J1074 (C4I6-M)

TK24 (C4I6-M)

HILIC- / [M-H]ˉ

Precursors #

Acetyl-CoA

Malonyl-CoA Methylmalonyl-CoA #

Propionyl-CoA

#

UDP-Glucose

45.4 ± 7.2

381.2 ± 21.1*

42.5 ± 3.3

C18+ / [M+H]

+

92.8 ± 55.3

375.3 ± 37.1*

362.0 ± 150.6*

C18+ / [M+H]

+

222.3 ± 39.7

817 ± 282*

438.5 ± 81.7*

HILIC+ / [M+H]+

8.7 ± 2.4

15.6 ± 0.6*

6.9 ± 1.7

HILIC- / [M-H]ˉ

1532.3 ± 337.5

3261.6 ± 464.7*

112.2 ± 10.9

HILIC+ / [M+H]+

4.0 ± 1.0

9527.9 ± 343.4*

20423.3 ±543.1*

79.4 ± 24.6

750.2 ± 75.6*

370.8 ± 17.6*

Cofactors # #

786 787

# # #

SAM

HILIC+ / [M+H]

+

NADH

HILIC+ / [M+H]

+

2.9 ± 0.8

32.8 ± 4.2*

14.0 ± 0.4*

NADP+

HILIC- [M-H]ˉ

15.3 ± 0.6

48.7 ± 5.1*

14.5 ± 0.5

NADPH

HILIC- [M-H]ˉ

1.2 ± 0.1

9.8 ± 0.9*

1.3 ± 0.3

NAD

+

HILIC-: HILIC column in negative ionization mode; HILIC+: HILIC column in

positive ionization mode; C18+: C18 column in positive ionization mode.

788

Due to the different orders of magnitude, the metabolites with the symbol

789

octothorpe (#) indicate the adjustment of the corresponding relative abundance. The

790

actual relative abundance was 1000-fold higher than presented in the table.

791 792

Values are mean ± SEM of 4 replications. * P < 0.05 compared to Sa. Spinose NRRL 18395.

793 794 795 796 797 798 799



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

Page 36 of 42

800

Table 4. Relative quantitative analysis of spinosyn components (or intermediates) in

801

Sa. spinosa NRRL 18395, S. lividans TK24 (C4I6-M) and S. albus J1074 (C4I6-M). Metabolites

Relative abundance

Mode /

#

Adduction

NRRL 18395

J1074 (C4I6-M)

TK24 (C4I6-M)

Spinosyn K

C18+ / [M+H]+

22.9 ± 7.0

ND

ND

Spinosyn J

C18+ / [M+H]

+

50.9 ± 11.4

ND

ND

C18+ / [M+H]

+

50.2 ± 13.2

ND

ND

C18+ / [M+H]

+

406.9 ± 99.1

104

35.0 ± 7.6

C18+ / [M+H]

+

2.1 ± 1.3

109.0 ± 16.6

ND

+

Spinosyn H Spinosyn B Spinosyn E Spinosyn L

C18+ / [M+H]

15.1 ± 2.7

ND

ND

Spinosyn A

C18+ / [M+H]+

5965.5 ± 1245.7

653.3 ± 518.3

145.0 ± 42.9

Spinosyn A-iso

C18+ / [M+H]+

213.2 ± 48.9

ND

ND

C18+ / [M+H]

+

3.8 ± 1.5

123.2 ± 19.4

ND

C18+ / [M+H]

+

2862.4 ± 677.9

197.8 ± 172.4

73.8 ± 18.9

C18+ / [M+H]

+

19.1 ± 2.6

ND

ND

Spinosyn P

C18+ / [M+H]

+

ND

249.0 ± 36.4

ND

Spinosyn P-CH2

C18+ / [M+H]+

1.4 ± 0.8

534.1 ± 154.8

ND

Spinosyn L3 Spinosyn D Spinosyn D2

802

C18+: C18 column in positive ionization mode;

803

The chemical structures of each spinosyn component (or intermediate) could be

804 805

found in Figure S7; Due to the different orders of magnitude, the sample with the symbol octothorpe

806

(#) indicate the adjustment of the corresponding relative abundance. The actual

807

relative abundance was 1000-fold higher than presented in the table.

808

Values are mean ± SEM of 4 replications; ND: NOT detected.


Page 37 of 42

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 1. The workflow of the omics analysis guided natural product heterologous production in genome mining. 184x141mm (300 x 300 DPI)



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. The transcription of spinosyn biosynthetic genes in S. albus J1074. (A) The spinosyn biosynthetic genes are located in different operons. Each red arrow indicates the gene(s) in the same operon. (B) Transcription analysis of spinosyn post-modification genes and PKS genes. These selected postmodification genes were located in different operons. PKS genes spnA, B, C, D and E belong to the same operon. C4I6 plasmid DNA and S. albus J1074 (C4I6-M) cDNA were used as templates for PCR. In negative control, randomly selected spnS gene could not be detected by using total RNA samples. From left to right, the templates used in PCR reactions are the RNA samples of J1074(C4I6) (1), TK24(C4I6) (2) and NRRL18395 (3), ddH2O (-) and C4I6 (+). Primer sequences are given in Table S2). 33x28mm (300 x 300 DPI)


Page 38 of 42

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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 3. High-resolution mass spectrum of spinosyn A. The 96 h fermentation broth (1liter) extracted twice with EtOAc (2 liters) and concentrated in vacuo, the extracts were redissolved in 0.5 mL HPLC methanol. After passage through a 0.45 µm filter, these concentrated EtOAc extracts were used for MS detection. In S. albus J1074 (C4I6-M) and S. lividans TK24 (C4I6-M), a mass of 754,4495 Da (or 754,4488 Da) is consistent with the sodium adduct of spinosyn A (Sigma standard, expected mass, 754,4500 Da). 101x61mm (300 x 300 DPI)



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 4. Overexpression of the rhamnose biosynthetic module and methyltransferase for spinosad overproduction in S. albus J1074. (A) Construction of the gene overexpression cassette. gtt, epi, gdh, kre and spnI were amplified from NRRL18395. (B) Schematic of gene overexpression strategy in S. albus J1074 (C4I6-M). (C) PCR verification of S. albus J1074 (C4I6-M)-OE. FIV and FV are the PCR products that were amplified using up-ck-F/R and dn-ck-F/R, respectively (primer sequences are given in Table S2). In S. albus J1074 (C4I6-M), FIV and FV cannot be amplified under the same conditions. 135x72mm (300 x 300 DPI)


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Figure 5. Effects of gene overexpression on the spinosad biosynthesis in S. albus J1074. (A) Biosynthetic modules optimized strains. (B) The ratio (relative abundance) of spinosyn P-CH2 : spinosyn A in S. albus J1074 (C4I6-M) and S. albus J1074 (C4I6-M)-OE. (C) The production of spinosad in S. albus J1074 (C4I6-M)-OE1/2/3. 121x82mm (300 x 300 DPI)



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

125x87mm (300 x 300 DPI)


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Heterologous Biosynthesis of Spinosad: An Omics-Guided Large

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