Enhanced heterologous spinosad production from a 79-kb synthetic

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Enhanced heterologous spinosad production from a 79-kb synthetic multi-operon assembly

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Subscriber access provided by FONDREN LIBRARY, RICE UNIVERSITY Chaoyi Song, Ji Luan, Qingwen Cui, Qiuyue Duan, Zhen Li, Yunsheng Gao, Ruijuan Li, Aiying Li, Yue-Mao Shen, Yue-Zhong Li, Adrian Francis Stewart, Youming Zhang, Jun Fu, and Hailong Wang

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Enhanced heterologous spinosad production from a 79-kb synthetic multi-operon assembly

Page 1Song, of 29Ji Luan, Qingwen Cui, Qiuyue Duan,ACS Synthetic Biology Chaoyi Zhen Li, Yunsheng Gao, Ruijuan Li, Aiying Li, Yuemao Shen, Yuezhong Li, A. Francis Stewart, Youming Zhang, Jun Fu, & Hailong Wang 1

Promoters Rhamnose genes 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

The original spinosad gene cluster

Direct cloning, DNA assembly and Recombineering The artificial spinosad gene cluster with synthetic multiple operons

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

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Enhanced heterologous spinosad production from a 79-kb synthetic multi-operon assembly Chaoyi Song,†,‖ Ji Luan,†,‖ Qingwen Cui,† Qiuyue Duan,† Zhen Li,† Yunsheng Gao,† Ruijuan Li,† Aiying Li,† Yuemao Shen,† Yuezhong Li,† A. Francis Stewart‡,§, Youming Zhang,*,† Jun Fu,*,† & Hailong Wang *,† † Shandong

University–Helmholtz Institute of Biotechnology, State Key Laboratory of

Microbial Technology, School of Life Science, Shandong University, Binhai Road 72, 266237 Qingdao, People’s Republic of China ‡ Genomics,

Biotechnology Center, Technische Universität Dresden, Tatzberg 47-51, 01307

Dresden, Germany § GenArc ‖

GmbH, BioInnovationsZentrum, Tatzberg 47, Dresden 01307, Germany

These authors contributed equally

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ACS Synthetic Biology

ABSTRACT Refactoring biosynthetic pathways for enhanced secondary metabolite production is a central challenge for synthetic biology. Here we applied advanced DNA assembly methods and a uniform overexpression logic using constitutive promoters to achieve efficient heterologous production of the complex insecticidal macrolide spinosad. We constructed a 79-kb artificial gene cluster in which 23 biosynthetic genes were grouped into 7 operons, each with a strong constitutive promoter. Compared with the original gene cluster, the artificial gene cluster resulted in a 328-fold enhanced spinosad production in Streptomyces albus J1074. To achieve this goal, we applied the ExoCET DNA assembly method to build a plasmid from 13 GC-rich fragments with high efficiency in one step. Together with our previous direct cloning and recombineering tools, we present new synthetic biology options for refactoring large gene clusters for diverse applications. KEYWORDS: biosynthesis, gene clusters, synthetic operons, DNA engineering, secondary metabolites, heterologous expression.

Secondary metabolites are an important source of antibiotics, drugs, insecticides and other commercially important bioactive compounds. DNA sequencing of bacterial genomes has revealed thousands of unknown secondary metabolite pathways, most of which presumably encode bioactive molecules. 1,2 Unlocking this bio-treasure trove is a major challenge for synthetic biology. Polyketide synthase (PKS) pathways are central to the biosynthesis of secondary metabolites in prokaryotes. Simple carboxylic acids are elegantly assembled by PKS into a polyketide chain which is further modified by additional enzymes to form compounds with great structural diversity and complexity. The complex molecular structure of polyketide compounds hampers their chemical synthesis on a large scale and cell-based biosynthesis

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is usually preferred because production in scalable fermenters can be used. Recent advances in understanding and engineering of polyketide biosynthesis have greatly accelerated the production optimization of target compounds by manipulating the expression level of biosynthetic genes. Because genetic tools for modifying original producing organisms are often unavailable, cloning and heterologous expression of biosynthetic genes in genetically tractable hosts offer important alternatives for novel product discovery, 3-5 yield improvement, 6 pathway elucidation, 7-9 and new derivative generation.

10,11

The ever-increasing options in the synthetic biology toolbox, which

includes promoter libraries and DNA engineering methods, have greatly facilitated the artificial construction of complex polyketide biosynthetic pathways. A great number of promoters have been identified to be functional in Streptomyces which is an outstanding producer of antibiotics. To activate silent gene clusters in Streptomyces by decoupling gene expression from endogenous regulation, Luo et al. 8 and Shao et al. 12 employed the DNA assembler method to construct synthetically active versions of silent gene clusters by inserting a constitutive or inducible promoter upstream of each biosynthetic gene. To enhance this strategy, Luo et al. 13 subsequently characterized 32 candidate constitutive promoters from S. albus J1074 and found that 10 of them are stronger than the widely used constitutive promoter ermE*p under the chosen culturing condition. The 38-kb nor gene cluster consisting of 10 genes was the most complex one they refactored. 12 In addition to in vivo DNA assembly methods, a range of in vitro methods suitable for gene cluster refactoring have been developed. SLIC (sequence- and ligationindependent cloning) 14,15 and Gibson 16,17 use exonuclease to generate single-stranded overhangs in DNA fragments and assemble them in vitro by overlapping annealing. DiPaC (direct pathway cloning) 18,19 combines long-amplicon PCR and Gibson or SLIC to assemble biosynthetic gene clusters. CPEC (circular polymerase extension cloning) 20 is based on polymerase extension to join overlapping DNA fragments into a circular molecule.

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AFEAP (assembly of fragment ends after PCR) 21 utilizes the asymmetric PCR to generate overlapping single-stranded overhangs and anneal multiple DNA fragments. These methods have revolutionized the natural products research. However, development of new DNA assembly methods with high efficiency and accuracy is still needed to refactor complex secondary metabolite biosynthetic gene clusters, especially those containing multiple massive PKS genes. Spinosad, a macrolide insecticide with exceptional environmental and mammalian safety profiles, is the mixture of spinosyns A and D produced by the soil actinomycete Saccharopolyspora spinosa. 22,23 It is comprised of a 21-carbon tetracyclic lactone backbone to which forosamine and tri-O-methylrhamnose are attached. 24 Spinosyns A and D are the most abundant spinosyns from S. spinosa fermentation broth. Biosynthesis of spinosad involves 23 genes in Saccharopolyspora spinosa. The nineteen genes in the 74kb gene cluster include PKS genes spnA, spnB, spnC, spnD and spnE; 25 polyketide bridging genes spnF, spnJ, spnL and spnM; 26,27 forosamine biosynthesis genes spnO, spnN, spnQ and spnR; 28 the forosamine methytransferase gene spnS; 28 the rhamnosyl transferase gene spnG; 29 the forosamyl transferase gene spnP 30 and rhamnose methylation genes spnH, spnI and spnK. 31 Four genes (gtt, gdh, epi and kre) for TDP-Lrhamnose biosynthesis are outside the gene cluster and located in three different regions on the chromosome. These genes provide rhamnose for both secondary metabolite production and cell wall synthesis. 32 Additionally, the products of gtt and gdh genes also catalyze the first two steps in TDP-forosamine biosynthesis. 28 In this study, the spinosad gene cluster, a representative of the complex polyketide gene clusters, was refactored to enhance the spinosad production in the heterologous host Streptomyces albus J1074 by boosting expression of all 23 biosynthetic genes. We constructed a 79-kb artificial gene cluster in which the 23 biosynthetic genes were divided into 7 operons and each operon is under control of a strong constitutive promoter.

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Compared with the original gene cluster, the artificial gene cluster resulted in a 328-fold enhanced spinosad production. The synthetic multi-operon strategy, requiring only a limited number of promoters, is particularly suitable to refactor complex gene clusters with many genes. RESULTS AND DISCUSSION Direct Cloning and Heterologous Expression of the Native Spinosad Biosynthetic Pathway in Streptomyces The 73.2-kb spinosad gene cluster of S. spinosa DSM44228 was retrieved in two pieces into pBeloBAC11 (a 71.3-kb XbaI+HpaI piece) and pBR322 (the adjacent 19.4-kb XbaI+HpaI piece) respectively using the ExoCET method 33 (Figure 1A). The four genes (gtt, gdh, epi and kre) responsible for biosynthesis of TDP-L-rhamnose were amplified by PCR, joined together by Gibson assembly 16 and then inserted downstream of the spnE gene in the pBR322-19.4kb clone by Red recombineering 7,34-36 (Figure 1A). Upstream of each rhamnose biosynthetic gene, 500 bp was included in the PCR amplification to keep these four genes under control of their native promoters. The complete spinosad gene cluster was rebuilt by fusing the partial spnE gene together with the rhamnose biosynthetic genes from the pBR322 vector into the pBeloBAC11 vector by Red recombineering (Figure 1A). The product was converted into an E. coli-Streptomyces shuttle vector by inserting a cassette containing an apramycin resistance gene, the origin for conjugal transfer (oriT), the phiC31 integrase encoding gene and its recombination target site (Figure 1A).

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Figure 1. Cloning and heterologous expression of full set of spinosad biosynthetic genes. (A) Construction of the E. coli-Streptomyces shuttle vector carrying all spinosad biosynthetic genes. The corresponding DNA analyses are shown in Figure S1. (B) HPLC-MS analysis (extracted ion chromatogram) of spinosad (spinosyns A and D) production in S. albus J1074. STD, the spinosad standard; spn-SA, S. albus J1074 with all spinosad genes. (C) The MS2 fragmentation patterns of spinosyn A (top) and spinosyn D (down) produced in S. albus J1074 and the spinosad standard. The fragment ion at m/z 142.1 and 189.1 are the characteristic forosamine and trimethylrhamnose fragments respectively. 37 6



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This assembly of biosynthetic genes were transformed into three different Streptomyces hosts (S. coelicolor CH999 38, S. lividans K4-114 39 and S. albus J1074 40) by conjugation and integrated onto their phiC31 attB sites on the chromosome. Correct mutants with intact gene clusters were confirmed by PCR using sentinel primers from different regions of the gene cluster (Table S1). Streptomyces hosts with and without the spinosad gene cluster were cultured in the M1 medium for 12 days before analysis by high performance liquid chromatography (HPLC)-mass spectrometry (MS). Only spinosyns A and D were detected in the fermentation broth from all Streptomyces hosts with the spinosad gene cluster (Figure 1B and 1C). The yield of spinosad in S. albus J1074 was highest among these three Streptomyces hosts in the M1 medium. Then, we checked the spinosad production in S. albus J1074 using the fermentation medium recently published by Tan et al. 41 and found a higher yield (3.4 ± 1.4 g L-1) than the M1 medium. Consequently, we chose S. albus J1074 as the host to express refactored spinosyn gene clusters and the fermentation medium published by Tan et al. 41 Enhanced Spinosad Production by Overexpressing PKS genes To increase spinosad production in S. albus J1074 by overexpressing PKS genes, the cassette containing homology arms, the hygromycin resistance gene and a strong constitutive promoter (the ermE*p, kasOp* or SF14p promoter 42) was inserted upstream of spnA, the first PKS gene, on pBAC-spn-phiC31-apra by Red recombineering to generate pBAC-ermEp-spn, pBAC-kasOp-spn and pBAC-SF14p-spn (Figure 2A). Then, pBACermEp-spn, pBAC-kasOp-spn and pBAC-SF14p-spn were transformed into S. albus J1074 respectively and the spinosad gene cluster containing the PKS operon under the control of the ermE*p, kasOp* or SF14p promoter was integrated onto the phiC31 attB site in the chromosome. Metabolites of correct S. albus mutants with the spinosad gene clusters were extracted from the fermentation broth and analyzed via HPLC-MS. Compared with the original construct, insertion of ermE*p and kasOp*resulted in a 2.1- and 1.3-fold enhanced 7


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spinosad production respectively (Figure 2B). Insertion of the SF14p promoter had little effect on spinosad production. This modest result indicated that the yield of spinosad could be improved by enhancing transcription. However, substantial improvements probably require major changes in design. Consequently we aimed for drastically increased spinosad production by enhancing the transcription of all spinosad genes.

Figure 2. Enhanced spinosad production by overexpressing PKS genes. (A) Schematic illustration of the spinosad gene cluster containing the PKS operon under control of the ermE*p, kasOp* or SF14p promoter. The corresponding DNA analyses are shown in Figure S2. (B) Yield of spinosad in S. albus J1074 with the spinosad gene clusters. Spn, the original promoter of the PKS operon. Each fermentation was performed in triplicate (n = 3) and error bars represent standard deviation (s.d).

Construction of the Artificial Spinosad Gene Cluster and ExoCET Multi-piece DNA Assembly We reconstructed an artificial spinosad gene cluster using strong constitutive promoters identified from S. albus J1074 by Luo et al. 13 by dividing the 23 genes into five groups according to their functions in spinosad biosynthesis: (i) The PKS operon including spnAspnE genes; (ii) polyketide cross-bridging genes spnJ, spnM, spnF and spnL; (iii) rhamnose 8



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biosynthesis (gtt, gdh, epi and kre) and transferase (spnG) genes; (iv) rhamnose methylation (spnI, spnK and spnH) and forosamine transferase (spnP) genes; (v) forosamine biosynthesis (spnO, spnN, spnQ and spnR) and methylation (spnS) genes. A strong constitutive promoter was placed upstream of each gene group (Figures 3A and 4A). In each synthetic operon, the genes were ordered to be collinear with their biosynthetic sequence. Each gene except PKS genes was amplified by PCR to generate 40 bp overlaps between adjacent fragments and stitched together by DNA assembly. Because the ribosomal binding sites (RBSs) in SA6p (640 bp upstream of the heat shock protein 60 family chaperone GroEL, XNR_2393), SA2p (695 bp upstream of the heat shock protein 60 family cochaperone GroES, XNR_3799) and SA13p (614 bp upstream of the 30S ribosomal protein S10, XNR_3728) are 16, 18 and 24 bp upstream of the start codon respectively, to avoid the incorrect assembly caused by these long RBS intervals, we incorporated short RBS (12 bp) in SA15p (517 bp upstream of the peptide transport system secreted peptidebinding protein, XNR_1700) directly upstream of start codon of spnM, spnF and spnL in the SA6p-spnJMFL group; gdh, epi, kre and spnG in the SA2p-rhaspnG group; spnN, spnQ, spnR and spnS in the SA13p-spnONQRS group. In above three groups, the first gene downstream of each promoter keeps the RBS of the corresponding promoter. The RBS in SA31p (414 bp upstream of the chaperone protein DnaK, XNR_3170) is 12 bp upstream of the start codon, therefore, its RBS was incorporated upstream of each gene in the SA31pspnIKHP group. Also, the start codon of all genes was changed to ATG in the PCR oligonucleotide design. Assembly of 24 fragments (18 biosynthetic genes, 5 promoters and a chloramphenicol resistance gene) in a single reaction is a daunting challenge, so we combined the 24 PCR products into 12 fragments with overlap extension PCR (Figure 3A). But we failed to stitch these 12 fragments (1.5-2.5 kb) into a pBR322 linear vector (2.1 kb) using 40-bp overlaps between adjacent fragments, in a single Gibson assembly reaction 16. All of the Gibson

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assembly products were incorrect recombinant plasmids showing deletion of many fragments (Figure 3B).

Figure 3. Multi-piece DNA assembly and comparison of different methods. (A) Schematic illustration of the 13-piece assembly. The promoters: p2, SA2p; p6, SA6p; p13, SA13p; p15, SA15p; p31, SA31p are shown in black. (B) Colony numbers per ml and accuracy (correct/number checked by restriction digestion) of indicated methods in the 13-piece assembly. Each assembly experiment was performed in triplicate (n = 3). The corresponding DNA analyses are shown in Figure S3A. (C) Schematic illustration of the 7-piece assembly. (D) Colony numbers per ml and accuracy (correct/number checked by restriction digestion) of indicated methods in the 7-piece assembly. Each assembly experiment was performed in triplicate (n = 3). The corresponding DNA analyses are shown in Figure S3B.

Recently, we reported a breakthrough in direct cloning of specified DNA segments from mammalian genomes, 33 which also greatly extends the capacity to directly clone specified segments from bacterial genomes. ExoCET (Exonulcease combined with RecET recombination) direct cloning can retrieve target DNA segments larger than 50 kb directly into E. coli vectors from bacterial and mammalian genomes. ExoCET combines an in vitro

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annealing step using the 3’ exonuclease of T4 polymerase (T4pol) with the remarkable property of full length RecE (with RecT) to recombine short homology regions. Here we used ExoCET to successfully join 12 PCR products with a plasmid to build a synthetic gene cluster in one round (Figure 3A). The recombinant pBR322 plasmids were evaluated by restriction digestion to determine as success rate of 58% (Figures 3B and S3A). We then tested whether the combination of Gibson assembly and RecET would be effective in stitching the 13 DNA fragments. Compared with ExoCET (T4pol+RecET), Gibson+RecET delivered a 20-fold increase in colony number, however, the success rate evaluated by restriction digestions was less at 25% (Figure 3B). As we found with the Gibson method alone, most of the incorrect plasmids from Gibson+RecET were missing fragments (Figure S3A). We also compared ExoCET with Gibson using a 7 piece multi-assembly exercise in which PCR amplified fragments containing SA13p (614 bp), spnO (1552 bp), spnN (1011 bp), spnQ (1493 bp), spnR (1158 bp) and spnS (854bp), each with 40-bp terminal homologies, were joined together with a pBR322 vector (2130 bp) to form a 8.5-kb plasmid (Figure 3C). ExoCET was efficient whereas Gibson failed and Gibson+RecET was less successful (Figure 3D). In this case, Gibson promoted a huge number of circularized empty vector with or without RecET. On the pBeloBAC11 vector carrying the original spinosad gene cluster, the biosynthetic genes except the PKS genes (spnA-E) were replaced by ampicillin and hygromycin resistance genes via Red recombineering (Figure 4A). The fragment containing the refactored, multi-promoter, spinosad biosynthetic genes was released by PacI digestion and inserted upstream of spnA via Red recombineering to complete the construction of the artificial spinosad gene cluster (Figure 4A).

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Figure 4. Construction and heterologous expression of the artificial spinosad gene cluster. (A) Spinosad genes spnF to spnS were deleted from the BAC assembled in Figure 1A and replaced with the product of Figure 3A. The corresponding DNA analyses are shown in Figure S4. (B) Time course of spinosad yield resulted from S. albus J1074 harboring the original gene cluster from Figure 1A or the artificial gene cluster. Each fermentation was performed in triplicate (n = 3) and error bars represent standard deviation (s.d). (C) Real-time PCR analysis of spinosad biosynthetic genes in the original gene cluster and the artificial gene cluster in S. albus J1074. Each reaction was performed in triplicate (n = 3) and error bars represent standard deviation (s.d).

Enhanced Spinosad Production by the Artificial Gene Cluster in S. albus J1074 For expression analysis, the artificial spinosad gene cluster was transformed into S. albus J1074 and integrated onto the phiC31 attB site in its chromosome. Correct S. albus mutants 12



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with intact gene clusters were confirmed by PCR using sentinel primers from different regions of the gene cluster (Table S1). Metabolites extracted from the fermentation broth were analyzed via HPLC-MS. Compared with the original gene cluster, the artificial gene cluster resulted in a 328-fold enhancement of spinosad production in 12 days (1116.4 ± 88.1 g L-1 vs 3.4 ± 1.4 g L-1) (Figure 4B). Besides spinosyns A and D, N-demethyl spinosyn A (spinosyn B) and N-demethyl spinosyn D 43 were also detected according to the mass spectrometry data (Table S2), whereas only spinosyns A and D were detected in the fermentation broth of S. albus J1074 harboring the native gene cluster. This is the first report that N-demethyl spinosyn D, a semisynthetic spinosyn analogue, 43 was isolated from fermentation broths. Therefore, N-demethyl spinosyn D can be produced by biosynthesis. The spnS gene for forosamine methylation was verified by Sanger sequencing. The Ndemethyl spinosyn A and N-demethyl spinosyn D may be produced when the forosamine dimethyltransferase activity of SpnS is insufficient to methylate all forosamine synthesized when the forosamine biosynthesis genes (spnO, spnN, spnQ and spnR) are overexpressed and SpnP transfers N-demethyl forosamine to the macrolide. The first and the last genes from each operon in the artificial gene cluster, i.e., spnA and spnE, spnJ and spnL, gtt and spnG, spnI and spnP, spnO and spnS, were selected for transcriptional analysis using real-time PCR. S. albus cells containing the original or artificial gene cluster were grown in fermentation broth for 10 days and RNA isolated at 4, 6, and 10 days. Real-time PCR analysis revealed that transcription levels of all selected spinosad biosynthetic genes in the artificial gene cluster were consistently higher than in the original gene cluster (Figure 4C). It is noteworthy that transcriptions of spnJ and spnO were increased 14.7 and 12.8 times respectively in the artificial gene cluster at 4 days, and transcriptions of spnA, spnJ and spnI were increased 10.4, 25 and 22.6 times respectively in the artificial gene cluster at 6 days.

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Figure 5. Reverse transcription PCR analysis of the operon organization of the artificial spinosad gene cluster. (A) The artificial spinosad gene cluster and operon organization detected via reverse transcription PCR. (B) Gel electrophoresis of reverse transcription PCR products.

The transcription level of the first gene was higher than the last gene in operons spnJMFL, rhaspnG and spnIKHP. However, this was not the case for spnA/spnE and spnO/spnS. The operon organization of the artificial gene cluster was then assessed by reverse transcription PCR (RT-PCR). Transcriptional coupling of genes, spnJMFL, gtt-gdhepi-kre-spnG and spnIKHP was confirmed using appropriate primers (Table S3) and RTPCR. Although spnD and spnE were cotranscribed, spnA, spnB and spnC are transcribed separately as a single mRNA (Figure 5). Therefore there may be a terminator downstream of spnC and a promoter upstream of spnD, which decouple the control of SA15p to spnDE and splits spnABCDE into two operons. Similarly, co-transcription of spnO, spnN and spnQ was detected but spnR and spnS are cotranscribed as a separate mRNA (Figure 5). Therefore, spnONQRS is also split into two operons with spnONQ expressed from SA13p and spnRS expressed from a promoter upstream of spnR. Consequently, the artificial spinosad gene cluster is organized into 7 operons, spnABC, spnDE, spnJMFL, gtt-gdh-epikre-spnG, spnIKHP, spnONQ and spnRS. The increased transcription of spnE and spnS in the artificial gene cluster in S. albus J1074 may be caused by the higher concentration and accumulation of intermediates synthesized by genes in the upstream biosynthetic pathway.

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Low or undetectable production of the target compound is usually caused by inadequate expression of the gene cluster in heterologous hosts. To obtain sufficient amount of compound for structure elucidation, bioactivity assay or commercial purpose, the gene cluster needs to be refactored to improve the expression. Luo et al. 8 and Shao et al. 12

developed a de novo assembly strategy to fully reconstruct the gene cluster by inserting

a set of well characterized promoters upstream of biosynthetic genes. However, for the very complex gene cluster containing multiple PKS genes, such as the spinosad gene cluster, the high sequence identity among PKS domains makes de novo assembly of the whole gene cluster for artificial architecture very challenging. In this study, we retrieved the entire spinosad gene cluster from the chromosome into a vector and removed non-PKS genes. Then the non-PKS genes were refactored and inserted back into the vector together with a number of relevant DNA elements, to form an artificial gene cluster in which 23 biosynthetic genes were grouped into 7 operons. This work required efficient direct cloning, precise modification of large episome and reliable DNA assembly techniques. In addition to the recombineering methods we have previously developed, 3,34,36,44-46 we show here that the logic of our recently described ExoCET direct cloning, which efficiently recombines a linear vector and a specified region from genomic DNA, 33 can be extended to multi-piece DNA assembly. Furthermore, scarless assembly of multiple GC-rich fragments is very challenging. The GC content of polyketide gene clusters from actinomycetes is usually higher than 60% and can be higher than 70%. GC content of the spinosad gene cluster is 65%. Nevertheless we obtained high efficiencies with a challenging 13-way assembly that exceeded the success rate obtained with Gibson assembly. Among other DNA assembly methods, the DNA assembler method 47

relying on yeast homologous recombination can also splice more than 10 fragments.

However this method takes much longer and is more laborious than ExoCET, which takes only a few days because it uses E. coli as the host. Another advantage of ExoCET is that

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DNA assembly and downstream analytical, engineering and preparative work can be done in the same E. coli host. 45 The production of secondary metabolites can be enhanced by overexpressing the ratelimiting biosynthetic genes. Tan et al. 41 obtained the whole spinosad gene cluster by BAC library construction. Using Streptomyces hosts for heterologous expression, they identified three rate-limiting steps of spinosad biosynthesis to obtain a spinosad yield of 1.5 mg L-1 in Streptomyces albus J1074 after step by step overexpression of deoxysugar biosynthetic genes, the rhamnose 2’-O-methyltransferase gene spnI and the PKS gene spnE. 41 However, the identification of rate-limiting steps is often tedious and can require systematic analyses of complicated transcriptomics, proteomics and metabolomics data. These issues are particularly apt when considering polyketide synthase pathways. In this study, we achieved a similar yield of 1.1 mg L-1 in the same host and fermentation medium by a brute force approach using the strong constitutive promoters to boost expression of the entire pathway. The powerful DNA engineering toolbox including ExoCET direct cloning and multipiece assembly as well as Red recombineering bypassed the major limitations inherent in the retrieval and refactoring of complex gene clusters larger than 50 kb. Here we present a powerful and broadly applicable strategy to fully reconstruct complex gene clusters for diverse applications, such as enhancing production and producing desirable derivatives or intermediates. METHODS Bacteria strains, plasmids and growth conditions Escherichia coli strains were cultured at 30 ºC (harboring the pSC101-BAD-ETgA plasmid) or 37 ºC in LB medium supplemented with appropriate antibiotics: tetracycline, 5 g ml-1; chloramphenicol, 15 g ml-1; kanamycin, 15 g ml-1; ampicillin, 100 g ml-1; apramycin, 20 g ml-1; hygromycin, 30 g ml-1. S. spinosa DSM44228 was maintained in tryptic soy broth

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or on brain heart infusion agar plates at 30 ºC. S. albus J1074 40, S. coelicolor CH999 38 and S. lividans K4-114 39 were cultured on mannitol soya flour agar plates for spore preparation and conjugation. Exconjugants were streaked onto brain heart infusion agar plates containing 50 g ml-1 apramycin and 25 g ml-1 nalidixic acid and incubated at 30 °C. Direct Cloning of the Spinosad Gene Cluster Genomic DNA of S. spinosa DSM44228 was recovered from lysed cells by phenolchloroform-isoamyl alcohol (25:24:1, pH 8.0) extraction and ethanol precipitation. Ten micrograms of XbaI+HpaI digested genomic DNA was used for ExoCET cloning 33. The pBeloBAC11 vector used to clone the 71.3-kb fragment released with XbaI+HpaI digestion was constructed using oligonucleotides Spn71-1, Spn71-2, Spn71-3 and Spn71-4 (Table S4) with the protocol described by Wang et al. 45. One microgram of the BAC vector linearized with BamHI digestion was used for ExoCET cloning. The pBR322-amp vector used to clone the 19.4-kb fragment released with HpaI digestion was amplified by overlap extension PCR using PrimeSTAR Max DNA Polymerase (Takara): Products of the first round PCR using oligonucleotides Spn19-2 and Spn19-3 purified with gel electrophoresis was used as the template for the second round PCR using oligonucleotides Spn19-1, Spn19-4 (Table S4). After these two rounds of PCR, homology arms for direct cloning and the 50-bp overlap with the HpaI terminus of the 71.3-kb XbaI+HpaI fragment was attached to the pBR322-amp vector. Two hundred nanograms of the pBR322-amp PCR product purified with the Universal DNA Purification Kit (Tiangen, Beijing China) after agarose gel electrophoresis was used for ExoCET cloning. The mixture of digested genomic DNA and linear cloning vector was treated with 0.02 U l-1 T4pol at 25 C for 1h, 75 C for 20 min, 50 C for 30 min and then held at 4 C. The in vitro assembly products were electroporated into L-arabinose induced E. coli GB05-dir harboring pSC101-BAD-ETgA-tet 33 after desalting by drop dialysis against ddH2O using Millipore Membrane Filters (Merck-Millipore, cat. no. VSWP01300). The recombinant BAC 17


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containing the 71.3-kb XbaI+HpaI fragment (pBAC-spn71XH; Fig. 1A) and the recombinant pBR322 plasmid containing the 19.4-kb HpaI fragment (pBR322-spn19H) were identified with PstI restriction analysis (Figure S1) of colonies selected on LB plates containing appropriate antibiotics. Cloning of Rhamnose Biosynthetic Genes and Construction of the Spinosad Expression Vector Carrying All Biosynthetic Genes The pBR322-amp vector, gtt, gdh-kre, epi, and the kanamycin resistance gene (neo) were amplified by PCR to generate 40 bp overlaps between adjacent fragments using the PrimeSTAR HS DNA Polymerase with GC Buffer (Takara, cat. no. R044A). Templates and oligonucleotides are listed in Table S4. The PCR products were purified with the Universal DNA Purification Kit (Tiangen, Beijing China, cat. no. DP214) after agarose gel electrophoresis. These five fragments (150 ng each) were assembled with the Gibson assembly method 16 and then electroporated into E. coli GB2005 34 to form the plasmid pBR322-Rhaneo in which the gtt-gdh-kre-epi-neo cassette is flanked by BstZ17I sites and 40-bp overlaps with pBR322-spn19H. pBR322-Rhaneo was identified with PvuI restriction analysis (Figure S1) of colonies selected on LB plates containing kanamycin and ampicillin. Two hundred nanograms of the gtt-gdh-kre-epi-neo cassette released with BstZ17I digestion and 500 ng pBR322-spn19H digested with NotI+NheI were co-electroporated into L-arabinose induced E. coli GB05-dir 3 expressing the full length RecET to form the plasmid pBR322-spnERhaneo in which the gtt-gdh-kre-epi-neo cassette is downstream the spnE gene and the spnE-gtt-gdh-kre-epi-neo cassette is flanked by BstZ17I sites and 50-bp overlaps with pBAC-spn71XH. pBR322-spnERhaneo was identified with BamHI+SphI restriction analysis (Figure S1) of colonies selected on LB plates containing kanamycin and ampicillin. Five hundred nanograms of the spnE-gtt-gdh-kre-epi-neo cassette (Figure 1A) was electroporated into L-arabinose induced Red expressing E. coli GB05-Red 44 containing 18



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pBAC-spn71XH to form the 104.2-kb recombinant BAC carrying all of the spinosad biosynthetic genes (pBAC-spn; Figure 1A). Then 200 ng of the apra-oriT-attP-phiC31int cassette 45 was electroporated into L-arabinose induced Red expressing GB05-Red containing pBAC-spn to form the E. coli-Streptomyces shuttle vector pBAC-spn-phiC31apra (Figure 1A). Correct recombinant plasmids pBAC-spn and pBAC-spn-phiC31-apra were identified with PstI restriction analysis (Figure S1) of colonies selected on LB plates containing chloramphenicol and apramycin. Insertion of Promoters ermE*p, kasOp* and SF14p Upstream of spnA The ermE*p, kasOp* and SF14p promoters and 50-bp homology arms for Red recombineering were attached to hygromycin resistance gene by overlap extension PCR using oligonucleotides listed in Table S5. In the first round PCR, the template p15Ahyg-ccdB and oligonucleotide pairs hyg-pro-spnA-1 and hyg-ermEp-spnA-2, hyg-pro-spnA1 and hyg-KasOp-spnA-2, hyg-pro-spnA-1 and hyg-SF14-spnA-2 were used. The first round PCR products were purified with the Universal DNA Purification Kit (Tiangen) after agarose gel electrophoresis and used as the template for the second round PCR. In the second round PCR, oligonucleotide pairs hyg-pro-spnA-1 and hyg-ermEp-spnA-3, hyg-prospnA-1 and hyg-KasOp-spnA-3, hyg-pro-spnA-1 and hyg-SF14-spnA-3 were used. The second round PCR products were purified with the Universal DNA Purification Kit (Tiangen) and eluted in ddH2O. Two hundred nanograms of PCR products were used for Red recombineering. Correct recombinant plasmids were identified with SphI restriction analysis (Figure S2) of colonies selected on LB plates containing hygromycin. Multi-piece DNA Assembly spnJ, spnM, spnF, spnL, gtt, gdh, epi, kre, spnG, spnI, spnK, spnH, spnP, spnO, spnN, spnQ, spnR, spnS, five strong constitutive promoters (SA15p, SA2p, SA6p, SA31p and SA13p) from S. albus J1074, the chloramphenicol resistance gene (cm) and the pBR322-

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amp-ccdB vector were amplified with PCR to generate 40 bp overlaps between adjacent fragments using the PrimeSTAR HS DNA Polymerase with GC Buffer (Takara). Templates and oligonucleotides are listed in Table S6. Two adjacent fragments were then joined together by overlap extension PCR with oligonucleotides listed in Table S6. The PCR products were purified with the Universal DNA Purification Kit (Tiangen) after agarose gel electrophoresis. In ExoCET assembly, the 20 l mixture containing 150 ng of each fragment, 1  NEBuffer 2.1 and 0.02 U l-1 T4pol (NEB, cat. no. M0203) was prepared in a 0.2 ml PCR tube and reacted at 25 C for 1h, 75 C for 20 min, 50 C for 30 min and then held at 4 C in a thermocycler. Reaction mixtures were dialyzed against ddH2O on Millipore Membrane Filters (Merck-Millipore, cat. no. VSWP01300) at room temperature for 30 min to remove salts, then electroporated into arabinose induced E. coli GBdir-gyrA462 45 harboring pSC101-BAD-ETgA-tet 3 which expresses full length RecE/RecT from both the chromosome and the pSC101 plasmid. In Gibson assembly, the 20 l mixture containing 150 ng of each fragment and 1  Gibson Assembly Master Mix (NEB, cat. no. E2611) was reacted at 50 C for 1 h and then held at 4 C in a thermocycler. Desalted reaction mixtures were electroporated into E. coli GBdir-gyrA462 harboring pSC101-BAD-ETgA-tet with (Gibson+RecET) or without arabinose induction. The number of colonies was counted after overnight incubation and the colony number per ml (c.f.u. ml-1) was calculated. Recombinant plasmids generated with DNA assembly were analyzed with restriction digestion (Figure S3) of colonies selected on LB plates containing ampicillin to identify the correct assembled plasmid pBR322-spnFSRhaNEW (Figure 3A). Construction of the Artificial Spinosad Gene Cluster

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In pBAC-spn-phiC31-para, the spnF-spnS genes and rhamnose biosynthetic genes together with the chloramphenicol resistance gene were replaced with ampicillin and hygromycin resistance genes via Red recombineering (Figure 4A). The ampicillin and hygromycin resistance genes were amplified with PCR using the PrimeSTAR Max DNA Polymerase (Takara, cat. no. R045A) to attach homologous arms for Red recombineering. Templates and oligonucleotides are listed in Table S6. PCR products were purified with the Universal DNA Purification Kit (Tiangen) and eluted into ddH2O. Two hundred nanograms of PCR products were used for Red recombineering. Correct recombinant plasmids pBAC-spnAE-hyg and pBAC-spnAE-hyg-amp were identified with PstI restriction digestion (Figure S4) of colonies selected on LB plates containing hygromycin or ampicillin. pBR322-spnFSRhaNEW generated by ExoCET assembly (Figure 3A) was digested with PacI to release the fragment containing refactored spinosad biosynthetic genes. After digestion, the DNA was precipitated with ethanol and 500 ng was used to for Red recombineering. Refactored spinosad biosynthetic genes were inserted upstream spnA in pBAC-spnAE-hyg-amp to generate pBAC-spnNEW containing the artificial spinosad gene cluster (Figure 4A). Correct pBAC-spnNEW were identified with XmnI restriction analysis (Figure S4) of colonies selected on LB plates containing chloramphenicol and apramycin. Fermentation and High Performance Liquid Chromatography–Mass Spectrometry Analysis of Metabolites from Wild Type and Engineered Streptomyces Strains Wild type and engineered Streptomyces strains were inoculated from a plate into 30 ml tryptic soy broth in 250-ml flasks as the seed culture and incubated at 30 C with 220-rpm shaking for 3-4 days until achieving high particle density or turbidity. Five hundred microliters (1:100 dilution) of the seed culture was inoculated into 50 ml M1 broth (1% starch, 0.4% yeast extract, 0.2% peptone), or Fermentation broth (4% glucose, 1% glycerol, 3% soluble starch, 1.5% soytone, 1% beef extract, 0.65% peptone, 0.05% yeast 21


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extract,0.1% MgSO4, 0.2% NaCl, 0.24% CaCO3) published by Tan et al. 41 in 250-ml flasks, and incubated at 30 C with 220-rpm shaking for 10 days. One milliliter (2%, v/v) of the adsorber resin Amberlite XAD-16 was added and the mixture was incubated for another 2 days. The cells and XAD-16 were harvested with centrifugation at the maximum speed for 10 min in an Eppendorf 5810R centrifuge. The pellet was extracted with 50 ml methanol. Then, the extract was evaporated and redissolved in 1 ml methanol. Three microliters of the concentrated extract was used for HPLC-MS analysis. The high-resolution mass spectrometry was done on a Bruker Impact HD micrOTOF Q III mass spectrometer (BrukerDaltonics, Bremen, Germany) using the standard ESI source equipped with a Acclaim RSLC 120, C18, 2.2 µm, 2.1 x 100 mm (Thermo Scientific). Mass spectra were acquired in centroid mode ranging from 100 to 1500 m/z with positive-mode electrospray ionization and auto MS2 fragmentation. HPLC parameters were as follows: solvent A, H2O with 0.1 % (v/v) formic acid; solvent B, 0.1 % (v/v) formic acid in acetonitrile; gradient at a constant flow rate of 0.3 ml/min, 5% B for 5 min, 5% to 55% B in 5 min, maintain at 55% B for 5 min, 55% to 95% B in 5 min, and finally, maintain at 95% B for 5 min; detection by UV spectroscopy at 200-600 nm. To quantify the amount of spinosad produced in S. albus J1074, a calibrated curve was generated using the spinosad standard (Sigma-Aldrich, cat. no. 33706). Real-time PCR and reverse transcription PCR Cells from 1 ml culture were collected at 4, 6, and 10 days. Total RNA was prepared using the RNAprep pure Kit (Tiangen, cat. no. DP430). DNA elimination and reverse transcription was performed with the PrimeScript RT reagent Kit with gDNA Eraser (Takara, cat. no. RR047A). Real-time PCR was performed on StepOnePlus Real-Time PCR System (Applied Biosystems) using SYBR Premix Ex Taq GC (Takara, cat. no. RR071A) according to the manufacturer’s protocol. Oligonucleotides are listed in Table S3. The endogenous gene 22



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hrdB, encoding RNA polymerase sigma factor, was used as the internal control. Expression levels of spinosad biosynthetic genes were normalized by the expression of the internal control. Data were analysed using StepOne Software v2.3 (Applied Biosystems). The operon organization of the artificial spinosad gene cluster was examined by PCR with LA Taq with GC Buffer (Takara, cat. no. RR02AG) using cDNA as the template and oligonucleotides listed in Table S3. ASSOCIATED CONTENT Supporting Information Figure S1: Restriction analysis of recombinant BACs and plasmids in Figure 1; Figure S2: SphI restriction analysis of pBAC-ermEp-spn, pBAC-kasOp-spn and pBAC-SF14p-spn obtained in Redαβ recombineering; Figure S3: BamHI restriction analysis of colonies obtained in Figure 3; Figure S4: Restriction analysis of recombinant BACs in Figure 4; Table S1: Primers for PCR detection of Streptomyces mutant strain harboring the spinosad gene cluster; Table S2: Spinosyns detected in fermentation broth of S. albus J1074 harboring the spinosad gene clusters; Table S3: Primers used for real-time PCR and reverse transcription PCR; Table S4: Oligonucleotides for cloning of spinosad biosynthetic gene; Table S5: Oligonucleotides used for insertion of promoters ermE*p, kasOp* and SF14p upstream of spnA. Table S6: Oligonucleotides used for construction of the artificial spinosad gene cluster. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. Tel: +86-532-67720918. *E-mail: [email protected]. Tel: +86-532-67721908. *E-mail: [email protected]. Tel: +86-532-67722928. ORCID

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Hailong Wang: 0000-0001-9874-5892 Author Contributions HW, YS, YL, AFS, YZ and JF designed the experiments. HW, CS, JL, QC, QD, RL, ZL, YG, and AL conducted the experiments. HW, JF, YZ and AFS wrote the paper. Notes AFS and YZ are shareholders in Gene Bridges GmbH, which holds exclusive commercial rights to recombineering. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 31700073, 31570094 and 31670097), the International S&T Cooperation Program of China (No. ISTCP 2015DFE32850), a Major Project of Science and Technology of Shandong Province (No. 2015ZDJS04001), a Shandong Demonstration Base Program of International S&T Cooperation (No. 2017JHZ009), the 111 Project (No. B16030), the Recruitment Program of Global Experts in Shandong University to Y.Z., the Qilu Youth Scholar Startup Funding of SDU to H.W., and the Taishan Scholar Program of Shandong Province to J.F and H.W. REFERENCES (1)

O'Brien, R.V., Davis, R.W., Khosla, C. and Hillenmeyer, M.E. (2014) Computational

identification and analysis of orphan assembly-line polyketide synthases. J. Antibiot. (Tokyo) 67, 89-97. (2)

Shen, B. (2015) A new golden age of natural products drug discovery. Cell 163,

1297-1300. (3)

Fu, J., Bian, X., Hu, S., Wang, H., Huang, F., Seibert, P.M., Plaza, A., Xia, L., Müller,

R., Stewart, A.F. and Zhang, Y. (2012) Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat. Biotechnol. 30, 440-446. (4)

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

Dorrestein, P.C. and Moore, B.S. (2014) Direct cloning and refactoring of a silent

24



Page 26 of 29

lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A. Proc. Natl. Acad. Sci. U.S.A. 111, 1957-1962. (5)

Montiel, D., Kang, H.S., Chang, F.Y., Charlop-Powers, Z. and Brady, S.F. (2015)

Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters. Proc. Natl. Acad. Sci. U.S.A. 112, 8953-8958. (6)

Huang, J., Yu, Z., Li, M.H., Wang, J.D., Bai, H., Zhou, J. and Zheng, Y.G. (2016)

High level of spinosad production in the heterologous host Saccharopolyspora erythraea. Appl. Environ. Microbiol. 82, 5603-5611. (7)

Chai, Y., Shan, S., Weissman, K.J., Hu, S., Zhang, Y. and Muller, R. (2012)

Heterologous expression and genetic engineering of the tubulysin biosynthetic gene cluster using Red/ET recombineering and inactivation mutagenesis. Chem. Biol. 19, 361-371. (8)

Luo, Y., Huang, H., Liang, J., Wang, M., Lu, L., Shao, Z., Cobb, R.E. and Zhao, H.

(2013) Activation and characterization of a cryptic polycyclic tetramate macrolactam biosynthetic gene cluster. Nat. Commun. 4, 2894. (9)

Ongley, S.E., Bian, X., Zhang, Y., Chau, R., Gerwick, W.H., Muller, R. and Neilan,

B.A. (2013) High-titer heterologous production in E. coli of lyngbyatoxin, a protein kinase C activator from an uncultured marine cyanobacterium. ACS Chem. Biol. 8, 1888-1893. (10)

Arslanian, R.L., Tang, L., Blough, S., Ma, W., Qiu, R.G., Katz, L. and Carney, J.R.

(2002) A new cytotoxic epothilone from modified polyketide synthases heterologously expressed in Myxococcus xanthus. J. Nat. Prod. 65, 1061-1064. (11)

Sugimoto, Y., Ding, L., Ishida, K. and Hertweck, C. (2014) Rational design of

modular polyketide synthases: morphing the aureothin pathway into a luteoreticulin assembly line. Angew. Chem. Int. Ed. Engl. 53, 1560-1564. (12)

Shao, Z., Rao, G., Li, C., Abil, Z., Luo, Y. and Zhao, H. (2013) Refactoring the silent

spectinabilin gene cluster using a plug-and-play scaffold. ACS Synth. Biol. 2, 662-669. (13)

Luo, Y., Zhang, L., Barton, K.W. and Zhao, H. (2015) Systematic identification of a

panel of strong constitutive promoters from Streptomyces albus. ACS Synth. Biol. 4, 10011010. (14)

Li, M.Z. and Elledge, S.J. (2007) Harnessing homologous recombination in vitro to

generate recombinant DNA via SLIC. Nat. Methods 4, 251-256. (15)

Kushnir, S., Sundermann, U., Yahiaoui, S., Brockmeyer, A., Janning, P. and Schulz,

F. (2012) Minimally invasive mutagenesis gives rise to a biosynthetic polyketide library. Angew. Chem. Int. Ed. Engl. 51, 10664-10669.

25


Page 27 of 29 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

(16)


Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison, C.A., 3rd and Smith,

H.O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343-345. (17)

Temme, K., Zhao, D. and Voigt, C.A. (2012) Refactoring the nitrogen fixation gene

cluster from Klebsiella oxytoca. Proc. Natl. Acad. Sci. U.S.A. 109, 7085-7090. (18)

D'Agostino, P.M. and Gulder, T.A.M. (2018) Direct Pathway Cloning combined with

Sequence- and Ligation-Independent Cloning for fast biosynthetic gene cluster refactoring and heterologous expression. ACS Synth. Biol. 7, 1702-1708. (19)

Greunke, C., Duell, E.R., D'Agostino, P.M., Glockle, A., Lamm, K. and Gulder, T.A.M.

(2018) Direct Pathway Cloning (DiPaC) to unlock natural product biosynthetic potential. Metab. Eng. 47, 334-345. (20)

Cimermancic, P., Medema, M.H., Claesen, J., Kurita, K., Wieland Brown, L.C.,

Mavrommatis, K., Pati, A., Godfrey, P.A., Koehrsen, M., Clardy, J., Birren, B.W., Takano, E., Sali, A., Linington, R.G. and Fischbach, M.A. (2014) Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters. Cell 158, 412-421. (21)

Zeng, F., Zang, J., Zhang, S., Hao, Z., Dong, J. and Lin, Y. (2017) AFEAP cloning: a

precise and efficient method for large DNA sequence assembly. BMC Biotechnol. 17, 81. (22)

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

soil collected in a sugar mill rum still. Int. J. Syst. Evol. Microbiol. 40, 34-39. (23)

Sparks, T.C., Crouse, G.D. and Durst, G. (2001) Natural products as insecticides:

the biology, biochemistry and quantitative structure-activity relationships of spinosyns and spinosoids. Pest Manag. Sci. 57, 896-905. (24)

Crouse, G.D., Sparks, T.C., Schoonover, J., Gifford, J., Dripps, J., Bruce, T., Larson,

L.L., Garlich, J., Hatton, C., Hill, R.L., Worden, T.V. and Martynow, J.G. (2001) Recent advances in the chemistry of spinosyns. Pest Manag. Sci. 57, 177-185. (25)

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

Madduri, K., Crawford, K.P., Merlo, D.J. and Baltz, R.H. (2001) Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa. Chem. Biol. 8, 487-499. (26)

Kim, H.J., Ruszczycky, M.W., Choi, S.H., Liu, Y.N. and Liu, H.W. (2011) Enzyme-

catalysed [4+2] cycloaddition is a key step in the biosynthesis of spinosyn A. Nature 473, 109-112. (27)

Kim, H.J., Pongdee, R., Wu, Q., Hong, L. and Liu, H.W. (2007) The biosynthesis of

spinosyn in Saccharopolyspora spinosa: synthesis of the cross-bridging precursor and identification of the function of SpnJ. J. Am. Chem. Soc. 129, 14582-14584. 26



(28)

Page 28 of 29

Hong, L., Zhao, Z., Melancon, C.E., 3rd, Zhang, H. and Liu, H.W. (2008) In vitro

characterization of the enzymes involved in TDP-D-forosamine biosynthesis in the spinosyn pathway of Saccharopolyspora spinosa. J. Am. Chem. Soc. 130, 4954-4967. (29)

Isiorho, E.A., Liu, H.W. and Keatinge-Clay, A.T. (2012) Structural studies of the

spinosyn rhamnosyltransferase, SpnG. Biochemistry 51, 1213-1222. (30)

Isiorho, E.A., Jeon, B.S., Kim, N.H., Liu, H.W. and Keatinge-Clay, A.T. (2014)

Structural studies of the spinosyn forosaminyltransferase, SpnP. Biochemistry 53, 42924301. (31)

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

(2010) Biosynthesis of spinosyn in Saccharopolyspora spinosa: synthesis of permethylated rhamnose and characterization of the functions of SpnH, SpnI, and SpnK. J. Am. Chem. Soc. 132, 2901-2903. (32)

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

supplies precursors for primary and secondary metabolism in Saccharopolyspora spinosa. J. Bacteriol. 183, 5632-5638. (33)

Wang, H., Li, Z., Jia, R., Yin, J., Li, A., Xia, L., Yin, Y., Muller, R., Fu, J., Stewart, A.F.

and Zhang, Y. (2018) ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes. Nucleic Acids Res. 46, e28. (34)

Fu, J., Wenzel, S.C., Perlova, O., Wang, J., Gross, F., Tang, Z., Yin, Y., Stewart,

A.F., Muller, R. and Zhang, Y. (2008) Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition. Nucleic Acids Res. 36, e113. (35)

Wenzel, S.C., Gross, F., Zhang, Y., Fu, J., Stewart, A.F. and Muller, R. (2005)

Heterologous expression of a myxobacterial natural products assembly line in pseudomonads via red/ET recombineering. Chem. Biol. 12, 349-356. (36)

Zhang, Y., Buchholz, F., Muyrers, J.P. and Stewart, A.F. (1998) A new logic for DNA

engineering using recombination in Escherichia coli. Nat. Genet. 20, 123-128. (37)

Lewer, P., Hahn, D.R., Karr, L.L., Duebelbeis, D.O., Gilbert, J.R., Crouse, G.D.,

Worden, T., Sparks, T.C., Edwards, P.M. and Graupner, P.R. (2009) Discovery of the butenyl-spinosyn insecticides: novel macrolides from the new bacterial strain Saccharopolyspora pogona. Bioorg. Med. Chem. 17, 4185-4196. (38)

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

biosynthesis of novel polyketides. Science 262, 1546-1550. 27


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

(39)


Ziermann, R. and Betlach, M.C. (1999) Recombinant polyketide synthesis in

Streptomyces: engineering of improved host strains. Biotechniques 26, 106-110. (40)

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

Insights into naturally minimised Streptomyces albus J1074 genome. BMC Genomics 15, 97. (41)

Tan, G.Y., Deng, K., Liu, X., Tao, H., Chang, Y., Chen, J., Chen, K., Sheng, Z., Deng,

Z. and Liu, T. (2017) Heterologous biosynthesis of spinosad: an omics-guided large polyketide synthase gene cluster reconstitution in Streptomyces. ACS Synth. Biol. 6, 9951005. (42)

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

strong promoter for streptomycetes. Appl. Environ. Microbiol. 79, 4484-4492. (43)

Sparks, T.C., Crouse, G.D., Dripps, J.E., Anzeveno, P., Martynow, J., Deamicis, C.V.

and Gifford, J. (2008) Neural network-based QSAR and insecticide discovery: spinetoram. J. Comput. Aided Mol. Des. 22, 393-401. (44)

Wang, H., Bian, X., Xia, L., Ding, X., Müller, R., Zhang, Y., Fu, J. and Stewart, A.F.

(2014) Improved seamless mutagenesis by recombineering using ccdB for counterselection. Nucleic Acids Res. 42, e37. (45)

Wang, H., Li, Z., Jia, R., Hou, Y., Yin, J., Bian, X., Li, A., Müller, R., Stewart, A.F., Fu,

J. and Zhang, Y. (2016) RecET direct cloning and Red recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression. Nat. Protoc. 11, 1175-1190. (46)

Muyrers, J.P., Zhang, Y., Testa, G. and Stewart, A.F. (1999) Rapid modification of

bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 27, 1555-1557. (47)

Shao, Z. and Zhao, H. (2014) Manipulating natural product biosynthetic pathways via

DNA assembler. Curr. Protoc. Chem. Biol. 6, 65-100.

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