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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

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Submitted to ACS Synthetic Biology as research article

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Heterologous biosynthesis of spinosad: an omics-guided large

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polyketide synthase gene cluster reconstitution in Streptomyces

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

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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

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Sciences, Wuhan, 430071, China

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b

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Biotechnology, Wuhan, 430075, China

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c

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

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China

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d

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Shengyang 110021, China

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e

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

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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 &

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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

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spinosyn heterologous production in Streptomyces spp have been revealed.

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

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

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

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

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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

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

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

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pharmaceuticals

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

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

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

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

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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,

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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

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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

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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

70

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

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

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

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targeted metabolites

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

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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

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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

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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

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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

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intramolecular cycloaddition 35-39

32, 33

and cross-bridging

and methylation

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, as well as multiple steps of

40

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glycosylation

. Lastly, but most importantly, probably due to

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

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production of spinosyn in Streptomyces were ended in failure 41.

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

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

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

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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.

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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

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

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

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methodological

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

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the Gibson assembly method

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however, because they can accommodate large secondary metabolic pathways

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spanning at least 100 kb.

approaches,

including

cosmid/fosmid

library

construction,

46

. It appears BAC vectors are usually more versatile,

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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

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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

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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

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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,

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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

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

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

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library construction.

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Introduction of spinosyn biosynthetic gene cluster into Streptomyces by

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triparental conjugation

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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

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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

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

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

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conjugation

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

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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,

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

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

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

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

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

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

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

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

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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

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

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

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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

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plasmid

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

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

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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

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and identification of heterologous production of spinosyn

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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)

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

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

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

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

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

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

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

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PKS proteins, methyltransferase SpnI and forosamine biosynthetic protein SpnO,

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

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

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

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

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

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

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

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

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spinosyn indicated both these Streptomyces hosts could provide all the essential

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

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

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

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

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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

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spinosyn

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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.

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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

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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

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

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

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

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

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

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the

241

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

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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

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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

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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)

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51-54

. In addition, with a

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

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

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

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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

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have better potential for the increased production of targeted products.

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

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

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

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55-57

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

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

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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.

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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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of sugar ligands and the insufficient enzymatic activity of methyltransferase and

276

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

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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

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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

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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

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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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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

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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).

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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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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,

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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

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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

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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

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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

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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

References

510

1. Newman, D. J., and Cragg, G. M. (2012) Natural products as sources of new drugs

511

over the 30 years from 1981 to 2010, Journal of Natural Products 75,

512

311-335.

513

2. Kinghorn, A. D., Pan, L., Fletcher, J. N., and Chai, H. (2011) The relevance of

514

higher plants in lead compound discovery programs, Journal of Natural

515

Products 74, 1539-1555.

516 517 518 519

3. Berdy, J. (2005) Bioactive microbial metabolites, The Journal of Antibiotics 58, 1-26. 4. Blunt, J. W., Copp, B. R., Keyzers, R. A., Munro, M. H., and Prinsep, M. R. (2015) Marine natural products, Natural Product Reports 32, 116.

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Page 22 of 42

520

5. Luzhetskyy, A., Pelzer, S., and Bechthold, A. (2007) The future of natural products

521

as a source of new antibiotics, Current Opinion in Investigational Drugs

522

(London, England: 2000) 8, 608-613.

523

6. Galm, U., and Shen, B. (2006) Expression of biosynthetic gene clusters in

524

heterologous hosts for natural product production and combinatorial

525

biosynthesis, Expert Opinion on Drug Discovery 1, 409-437.

526

7. Komatsu, M., Komatsu, K., Koiwai, H., Yamada, Y., Kozone, I., Izumikawa, M.,

527

Hashimoto, J., Takagi, M., Omura, S., Shin-ya, K., Cane, D. E., and Ikeda, H.

528

(2013) Engineered streptomyces avermitilis host for heterologous expression

529

of biosynthetic gene cluster for secondary metabolites, ACS Synthetic Biology

530

2, 384-396.

531 532

8. Tan, G. Y., and Liu, T. (2017) Rational synthetic pathway refactoring of natural products biosynthesis in actinobacteria, Metabolic Engineering 39, 228-236.

533

9. Liu, Q., Xiao, L., Zhou, Y., Deng, K., Tan, G., Han, Y., Liu, X., Deng, Z., and Liu,

534

T. (2016) Development of Streptomyces sp. FR-008 as an emerging chassis,

535

Synthetic and Systems Biotechnology 1, 207-214.

536

10. Li, L., Zheng, G., Chen, J., Ge, M., Jiang, W., and Lu, Y. (2017) Multiplexed

537

site-specific genome engineering for overproducing bioactive secondary

538

metabolites

539

10.1016/j.ymben.2017.01.004.

in

actinomycetes,

Metabolic

ACS Paragon Plus Environment

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doi:

Page 23 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

ACS Synthetic Biology

540

11. Kim, E. J., Yang, I., and Yoon, Y. J. (2015) Developing Streptomyces venezuelae

541

as a cell factory for the production of small molecules used in drug discovery,

542

Archives of Pharmacal Research 38, 1606-1616.

543

12. Komatsu, M., Uchiyama, T., Ōmura, S., Cane, D. E., and Ikeda, H. (2010)

544

Genome-minimized Streptomyces host for the heterologous expression of

545

secondary metabolism, Proceedings of the National Academy of Sciences 107,

546

2646-2651.

547

13. McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., and Khosla, C. (1993)

548

Engineered biosynthesis of novel polyketides, Science 262, 1546-1550.

549

14. Brady, S. F., Simmons, L., Kim, J. H., and Schmidt, E. W. (2009) Metagenomic

550

approaches to natural products from free-living and symbiotic organisms,

551

Natural Product Reports 26, 1488-1503.

552

15. Zaburannyi, N., Rabyk, M., Ostash, B., Fedorenko, V., and Luzhetskyy, A. (2014)

553

Insights into naturally minimised Streptomyces albus J1074 genome, BMC

554

Genomics 15, 97.

555

16. Yin, S., Li, Z., Wang, X., Wang, H., Jia, X., Ai, G., Bai, Z., Shi, M., Yuan, F., Liu,

556

T., Wang, W., and Yang, K. (2016) Heterologous expression of

557

oxytetracycline biosynthetic gene cluster in Streptomyces venezuelae

558

WVR2006 to improve production level and to alter fermentation process,

559

Applied Microbiology and Biotechnology 100, 10563-10572.

ACS Paragon Plus Environment

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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

560

17. Ongley, S. E., Bian, X., Neilan, B. A., and Müller, R. (2013) Recent advances in

561

the heterologous expression of microbial natural product biosynthetic

562

pathways, Natural Product Reports 30, 1121-1138.

563

18. Yamanaka, K., Reynolds, K. A., Kersten, R. D., Ryan, K. S., Gonzalez, D. J.,

564

Nizet, V., Dorrestein, P. C., and Moore, B. S. (2014) Direct cloning and

565

refactoring of a silent lipopeptide biosynthetic gene cluster yields the

566

antibiotic taromycin A, Proceedings of the National Academy of Sciences 111,

567

1957-1962.

568

19. Sosio, M., Giusino, F., Cappellano, C., Bossi, E., Puglia, A. M., and Donadio, S.

569

(2000) Artificial chromosomes for antibiotic-producing actinomycetes, Nature

570

Biotechnology 18, 343-345.

571

20. Nah, H.-J., Woo, M.-W., Choi, S.-S., and Kim, E.-S. (2015) Precise cloning and

572

tandem integration of large polyketide biosynthetic gene cluster using

573

Streptomyces artificial chromosome system, Microbial Cell Factories 14, 1.

574

21. Martin, V. J., Pitera, D. J., Withers, S. T., Newman, J. D., and Keasling, J. D.

575

(2003) Engineering a mevalonate pathway in Escherichia coli for production

576

of terpenoids, Nature Biotechnology 21, 796-802.

577 578

22. Galanie, S., Thodey, K., Trenchard, I. J., Interrante, M. F., and Smolke, C. D. (2015) Complete biosynthesis of opioids in yeast, Science 349, 1095-1100.

ACS Paragon Plus Environment

Page 24 of 42

Page 25 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

ACS Synthetic Biology

579

23. Lee, J. W., Na, D., Park, J. M., Lee, J., Choi, S., and Lee, S. Y. (2012) Systems

580

metabolic engineering of microorganisms for natural and non-natural

581

chemicals, Natural Chemical Biology 8, 536-546.

582

24. Cho, C., Choi, S. Y., Luo, Z. W., and Lee, S. Y. (2015) Recent advances in

583

microbial production of fuels and chemicals using tools and strategies of

584

systems metabolic engineering, Biotechnology Advances 33, 1455-1466.

585

25. Bantscheff, M., Lemeer, S., Savitski, M. M., and Kuster, B. (2012) Quantitative

586

mass spectrometry in proteomics: critical review update from 2007 to the

587

present, Analytical and Bioanalytical Chemistry 404, 939-965.

588

26. Patti, G. J., Yanes, O., and Siuzdak, G. (2012) Innovation: Metabolomics: the

589

apogee of the omics trilogy, Nature Reviews Molecular Cell Biology 13,

590

263-269.

591 592

27. Lee, S. Y., Lee, D.-Y., and Kim, T. Y. (2005) Systems biotechnology for strain improvement, Trends in Biotechnology 23, 349-358.

593

28. Tan, G.-Y., Zhu, F., Deng, Z., and Liu, T. (2016) In vitro reconstitution guide for

594

targeted synthetic metabolism of chemicals, nutraceuticals and drug precursors,

595

Synthetic and Systems Biotechnology 1, 25-33.

596

29. Mertz, F. R., and Yao, R. C. (1990) Saccharopolyspora spinosa sp. nov. isolated

597

from soil collected in a sugar mill rum still, International Journal of

598

Systematic Bacteriology 40, 34-39.

ACS Paragon Plus Environment

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599

30. Thompson, G. D., Dutton, R., and Sparks, T. C. (2000) Spinosad–a case study: an

600

example from a natural products discovery programme, Pest Management

601

Science 56, 696-702.

602

31. Waldron, C., Matsushima, P., Rosteck, P. R., Broughton, M. C., Turner, J.,

603

Madduri, K., Crawford, K. P., Merlo, D. J., and Baltz, R. H. (2001) Cloning

604

and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora

605

spinosa, Chemistry & Biology 8, 487-499.

606

32. Kim, H. J., Ruszczycky, M. W., Choi, S.-h., Liu, Y.-n., and Liu, H.-w. (2011)

607

Enzyme-catalysed [4+2] cycloaddition is a key step in the biosynthesis of

608

spinosyn A, Nature 473, 109-112.

609

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

610

A. T. (2015) The structure of SpnF, a standalone enzyme that catalyzes [4+2]

611

cycloaddition, Nature Chemical Biology 11, 256-258.

612

34. Kim, H. J., Pongdee, R., Wu, Q., Hong, L., and Liu, H.-w. (2007) The

613

biosynthesis of spinosyn in Saccharopolyspora spinosa: synthesis of the

614

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

615

the American Chemical Society 129, 14582-14584.

616

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

mechanistic implications for forosamine biosynthesis, Journal of the American

619

Chemical Society 127, 7692-7693.

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620

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

621

spinosyn biosynthetic pathway of Saccharopolyspora spinosa: mechanistic

622

and evolutionary implications for C-3 deoxygenation in deoxysugar

623

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

624

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

625

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

626

in the spinosyn pathway of Saccharopolyspora spinosa, Journal of the

627

American Chemical Society 130, 4954-4967.

628

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

629

Functional

characterization

and

substrate

specificity

of

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.

633

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

634

Biochemistry 53, 4292-4301.

635

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

638

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

639

2901-2903.

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640

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

641

of the antibiotic biosynthetic gene clusters in linear plasmid SCP1 of

642

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

643

42. Luo, Y., Li, B.-Z., Liu, D., Zhang, L., Chen, Y., Jia, B., Zeng, B.-X., Zhao, H.,

644

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

645

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.

648

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

649

Genetic engineering and heterologous expression of the disorazol biosynthetic

650

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

651

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

652

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

653

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

654

heterologous expression, Nature Protocols 11, 1175-1190.

655

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

656

stepwise increase in pristinamycin II biosynthesis by Streptomyces

657

pristinaespiralis through combinatorial metabolic engineering, Metabolic

658

Engineering 29, 12-25.

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659

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

660

vectors that facilitate BAC/BIBAC library construction and intact large

661

genomic DNA insert exchange, Plant methods 7, 1.

662

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

663

Evidence that the extracytoplasmic function sigma factor sigmaE is required

664

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

665

Bacteriology 181, 204-211.

666

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

667

conjugal transfer of plasmid DNA from Escherichia coli to methyl

668

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.

670

(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.,

673

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

674

heterologous expression of the fredericamycin biosynthetic gene cluster from

675

Streptomyces griseus, Journal of the American Chemical Society 127,

676

16442-16452.

677

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

678

FdmR1 as a Streptomyces antibiotic regulatory protein activator for

ACS Paragon Plus Environment

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679

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.

685

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

686

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

687

gdh controlled by promoter PermE* in Saccharopolyspora spinosa

688

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

689

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

693

production in Saccharopolyspora spinosa by metabolic engineering,

694

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

697

Microbiology 79, 4484-4492.

698

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

699

(2015) Exploiting a precise design of universal synthetic modular regulatory

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elements to unlock the microbial natural products in Streptomyces,

701

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.

703

(2016) High level of spinosad production in the heterologous host

704

Saccharopolyspora erythraea, Applied and Environmental Microbiology 82,

705

5603-5611.

706

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

707

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

708

using improved methods and identification of clones linked to the locus

709

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

710

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

711

homologous recombination in Escherichia coli, Nature Biotechnology 18,

712

1314-1317.

713

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

714

stimulates A ‐ factor ‐ like cascade and validamycin biosynthesis in

715

Streptomyces hygroscopicus 5008, Biotechnology and bioengineering 110,

716

2984-2993.

717

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

718

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

719

metabolomics, Metabolomics 4, 226-239.

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720

65. Khoomrung, S., Martinez, J. L., Tippmann, S., Jansa-Ard, S., Buffing, M. F.,

721

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

722

Saccharomyces cerevisiae extract through improved chloroform/methanol

723

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

yield, Bioscience, Biotechnology, and Biochemistry 77, 1661-1668.

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

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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

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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

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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

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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.

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

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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)

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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)

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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)

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