Development of a Terpenoid-Production Platform in

Research Article Cite This: ACS Synth. Biol. XXXX, XXX, XXX-XXX

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Development of a Terpenoid-Production Platform in Streptomyces reveromyceticus SN-593 Ammara Khalid,‡,§ Hiroshi Takagi,† Suresh Panthee,†,⊥ Makoto Muroi,‡ Joe Chappell,∥ Hiroyuki Osada,‡,§ and Shunji Takahashi*,† †

Natural Product Biosynthesis Research Unit, RIKEN Centre for Sustainable Resource Science, Hirosawa 2-1, Wako, Saitama 351-0198, Japan ‡ Chemical Biology Research Group, RIKEN Centre for Sustainable Resource Science, Hirosawa, 2-1, Wako, Saitama 351-0198, Japan § Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan ∥ Pharmaceutical Sciences, University of Kentucky, 789 S Limestone Street, Lexington, Kentucky 40536-0596, United States S Supporting Information *

ABSTRACT: Terpenoids represent the largest class of natural products, some of which are resources for pharmaceuticals, fragrances, and fuels. Generally, mass production of valuable terpenoid compounds is hampered by their low production levels in organisms and difficulty of chemical synthesis. Therefore, the development of microbial biosynthetic platforms represents an alternative approach. Although microbial terpenoid-production platforms have been established in Escherichia coli and yeast, an optimal platform has not been developed for Streptomyces species, despite the large capacity to produce secondary metabolites, such as polyketide compounds. To explore this potential, we constructed a terpenoid-biosynthetic platform in Streptomyces reveromyceticus SN-593. This strain is unique in that it harbors the mevalonate gene cluster enabling the production of furaquinocin, which can be controlled by the pathway specific regulator Fur22. We simultaneously expressed the mevalonate gene cluster and subsequent terpenoid-biosynthetic genes under the control of Fur22. To achieve improved f ur22 gene expression, we screened promoters from S. reveromyceticus SN-593. Our results showed that the promoter associated with rvr2030 gene enabled production of 212 ± 20 mg/L botryococcene to levels comparable to those previously reported for other microbial hosts. Given that the rvr2030 gene encodes for an enzyme involved in the primary metabolism, these results suggest that optimized expression of terpenoid-biosynthetic genes with primary and secondary metabolism might be as important for high yields of terpenoid compounds as is the absolute expression level of a target gene(s). KEYWORDS: terpenoids, Streptomyces, transcriptional regulator, promoter, metabolic engineering, optimized gene expression

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deliver robust and consistent production of target compounds has been an alternative approach to overcome some of these obstacles,7 and terpenoid production platforms using Escherichia coli and yeast have been developed to obtain valuable compounds efficiently.8−13 For example, the current production titers for valuable terpenoids such as amorpha-4,11-diene in E. coli and yeast has achieved greater than 25 g/L14 and 40 g/ L,15 respectively.

he diverse and complex structures of terpenoids arise from C5 precursor units of dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP).1−3 Terpenoid compounds have attracted a lot of attention for anticancer, antimalarial drugs, antifungal agents, flavor and fragrance, rubber, and alternative biofuels.4,5 Plants and microorganism produce a diverse array of terpenoid compounds with a variety of regio- and stereochemistries of substituent groups, that are often found in low abundance in equally complex chemical mixtures. Because of their chemical complexity, their preparation by chemical synthesis is generally inefficient requiring a large number of synthetic steps with low yields.6 Hence, the development of a microbial platforms that can © XXXX American Chemical Society

Received: July 6, 2017

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DOI: 10.1021/acssynbio.7b00249 ACS Synth. Biol. XXXX, XXX, XXX−XXX

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ACS Synthetic Biology Scheme 1. Development of a Terpenoid Biosynthetic Platform in S. reveromyceticus SN-593a

a (A) Transcription of genes involved in the mevalonate gene cluster ( f ur9−f ur15) and subsequent terpenoid-biosynthetic genes (f pps, ssl1, and ssl3) are activated by Fur22. Expression of fur22 gene was controlled by intrinsic optimal promoters (IOP). Simultaneous expression of target terpenoidbiosynthetic genes was achieved under the control of the f ur1 promoter ( f ur1p). (B) Optimization of metabolite-precursor supply toward terpenoid biosynthesis.2,30 The boxes indicate precursors for polyketide and terpenoid biosynthesis. Thick blue and orange arrows indicate genes from the mevalonate cluster and target terpenoid-biosynthetic genes, respectively.

the mevalonate gene cluster and the associated target terpenoid biosynthetic genes at a late stage of growth.31 In the present study, we have focused on the potential of Streptomyces reveromyceticus SN-59332,33 because it produces 1 g/L of reveromycins (RMs) by up-regulation of a pathway specific regulator.34 In addition, the strain possesses a furaquinocin (FQ) gene cluster35 associated with a mevalonate gene cluster.36,37 Although the FQ gene cluster appears not to be expressed in S. reveromyceticus SN-593 under normal growth conditions, fusion of a constitutive gene promoter (aphII promoter) to the f ur22 gene, a specific regulatory gene, resulted in the production of 0.7 g/L of polyketide-terpenoid hybrid compounds, FQs.33 Recently, heterologous expression of codon optimized terpene synthase genes from plant sources have been reported for Streptomyces species.22,23,26 Altogether, these observations indicated that terpene biosynthetic genes are subject to transcriptional regulation by specific promoters and trans-acting factors in Streptomyces species, and that more attention to constructing a optimized expression platform might yield a more facile and higher production capacity in Streptomyces. Taking the advantage of the potential of S. reveromyceticus SN-593 as a chemical production host, we describe here the construction of a terpenoid biosynthetic platform that is widely accommodating for the functional characterization of terpene biosynthetic genes found in nature. Moreover, we demonstrate the utility of our engineered Streptomyces host to produce a well-recognized triterpene, botryococcene38 (BC), a high-value biofuel resource. We chose

With the recent advances in genome analysis, many terpene synthase genes have been discovered in Streptomyces species that may only be expressed in response to unique environmental conditions/cues or reside in silent gene clusters.16−18 Given some of the unique genetic features of Streptomyces such as their codon biases, construction of terpenoid producing platforms within Streptomyces species would expand available resources for functional characterization of putative terpene biosynthetic genes. In addition, they are well-known for their ability to produce secondary metabolites. For instance, Streptomyces avermitilis and Streptomyces albus can produce significant amount of polyketide compounds such as avermectin,19and salinomycin.20 Their productivity indicates a sufficient supply of biosynthetic precursors such as acetyl-CoA and malonyl-CoA from primary metabolism. Based on such biosynthetic potential, Streptomyces species are also attractive as terpenoid production platforms. Recently, terpenoid production in S. avermitilis 17,21−25 and S. venezuelae 26,27 by heterologous gene expression has been reported. However, the productivity of terpenoid compounds was not directly comparable to E. coli and yeast systems. All Streptomyces species utilize the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the production of primary metabolites, such as menaquinones. However, a limited number of Streptomyces strains harbor both the MEP and the mevalonate pathways,28−30 which appear to support a biological role for primary metabolism and secondary metabolism, respectively. In fact, specialized terpenoid production mainly correlated with an operation of B


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ACS Synthetic Biology

Figure 1. Metabolite analysis by LC−MS. (A) Metabolite profile of wild-type S. reveromyceticus SN-593 (i) and SR1 culture (ii). The chromatograms were monitored with a photodiode array detector, but are displayed at a wavelength of 238 nm, the absorption maximum of RM-A (1). (B) Metabolite profile of the SR1 culture (i) and SR1−1 line in which f ur22 gene expression was under the control of the aphII promoter (ii). Marked peaks correspond to the FQs shown on the right. (C) Metabolite profile of the SR2 culture (i) and SR2−1 line in which f ur22 gene expression was under the control of aphII promoter (ii). The chromatograms are displayed at 267 nm, the absorption maximum of (2). Selected ion chromatograms were obtained from SR1−1 (D) and SR2−1 line (E). Selected ion chromatogram at m/z 413.5−414.5 [M − H]− for 4 (i), m/z 414.5−415.5 [M − H]− for 3 (ii), and m/z 384.5−385.5 [M − H]− for 2 (iii).

resulting SR2 strains (ΔrevCΔrevD and Δf ur1) would exhibit increased potential to utilize primary metabolites derived from glycolysis for terpenoid production. We confirmed f ur1 gene deletion using polymerase chain reaction (PCR) primers designed inside and outside the homologous recombination sites (Figure S1), and the metabolite profiles of the SR2 strains were confirmed by high-performance liquid chromatography electrospray ionization (ESI) mass spectrometry (LC−MS). Normally, FQs are not produced in the SR1 strain, where the RM-A biosynthetic gene is disrupted. When the regulatory gene f ur22 is expressed under the control of the aphII promoter, FQD, FQ-I, and FQ-J33 accumulated (Figure 1A and B). As expected from the deletion of the f ur1 gene, the production of FQ derivatives, even when the f ur22 gene was upregulated, were abolished (Figure 1C). Loss of the FQ derivatives was also confirmed by selected ion chromatogram (Figure 1D and E). To evaluate the potential of producing terpenoids in the SR2 platform, we examined production of the native triterpene BC. Furthermore, for simultaneous expression of the mevalonate pathway gene cluster and the target BC genes, both were placed under control of the Fur22 regulator (Scheme 1A and B). In this scheme, screening and identification of promoters which

BC in part because its biosynthesis requires the coordinated activity of two enzymes, whose genes were identified from green algae Botryococcous braunii race B.39−41 In this paper, we demonstrate that the expression of terpenoid biosynthetic genes can be optimized using a transcriptional regulator controlled by suitable promoter selection, resulting in the production of BC (0.2 g/L) in S. reveromyceticus SN-593.

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RESULTS AND DISCUSSION Optimization of Precursor Supply for Terpenoid Production. Fur22 belongs to the SARP family of transcription regulators that control the FQ gene cluster and mevalonate pathway through binding to the f ur1 promoter.33 Because Fur1 expression represents the first committed step required for FQ production,42,43 we performed in-frame deletion of the f ur1 gene to abolish FQ production while maintaining the functions associated with the mevalonate gene cluster (Scheme 1A), which produces C5 precursor units of DMAPP and IPP. Moreover, to optimize acetyl-CoA and malonyl-CoA supply for the biosynthesis of C5 terpenoidprecursor units, we performed f ur1 gene deletion in the SR1 strain, which abolished the production of RMs via disruption of the revC and revD genes44 (Scheme 1B). We expected that the C


Research Article

ACS Synthetic Biology Table 1. Putative Function of Proteins Isolated from 2-D Gel Electrophoresis

a

fold ratio master no.

protein ID

score

119

RVR7933

604

207

RVR6190

901

305

RVR6682

2066

329

RVR6682

1630

358

RVR3087

1034

373

RVR6682

1338

380

RVR6190

1234

406

RVR9107

3160

440

RVR6682

933

452

RVR1215

1101

452

RVR2353

1272

452

RVR3168

1036

452

RVR3493

1209

452

RVR3991

1798

452

RVR6279

2050

452

RVR6682

1272

452

RVR7193

1024

490

RVR3650

53

494

RVR4275

940

516

RVR2030

1399

516

RVR2708

1029

516

RVR6199

1177

603

RVR3991

1919

603

RVR4941

1003

603

RVR6199

1139

603

RVR6279

1003

678

RVR1149

1925

678

RVR1226

2784

678

RVR6279

1049

688

RVR4050

1040

688

RVR6190

1049

689

RVR1226

1779

745

RVR7189

766

746

RVR6386

840

LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS MALDITOF LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS

molecular weight

pI

96998

4.84

aconitate hydratase

1.19

1.47

0.44

144875

6.94

RNA polymerase beta prime subunit

1.21

2.34***

1.60**

63855

7.62

peptide ABC transporter solute-binding protein

1.00

6.63*

16.48***

63855

7.62


0.87

1.46

19.85**

66157

5.09

protease

1.02

2.57**

1.65

63855

7.62


0.76

0.82

2.16*

144875

6.94


1.01

1.20

5.41***

51850

5.69

serine hydroxymethyltransferase

0.45***

1.50**

0.35***

63855

7.62


0.57

0.87

2.37**

78708

4.85

isocitrate dehydrogenase

0.86

0.40*

0.29**

48876

5.72

dihydrolipoamide dehydrogenase

0.86

0.40*

0.29**

52197

4.85

cell division trigger factor

0.86

0.40*

0.29**

49733

5.23

malate dehydrogenase

0.86

0.40*

0.29**

56584

4.8

class I heat-shock protein

0.86

0.40*

0.29**

56653

4.93


0.86

0.40*

0.29**

63855

7.62


0.86

0.40*

0.29**

51973

4.76

putative ATP synthase beta chain

0.86

0.40*

0.29**

45555

4.5

phosphopyruvate hydratase

1.38*

1.61**

1.25

50053

5.82

tryptophanse

1.37

3.87**

2.47.

40299

4.74

transaldolase

1.12

2.67

3.73*

44826

4.76

cytochrome P450

1.12

2.67

3.73*

43754

5.09

elongation factor EF-Tu

1.12

2.67

3.73*

56584

4.8


0.92

0.65*

0.55**

38731

5.06

histidinol-phosphate aminotransferase

0.92

0.65*

0.55**

43754

5.09

elongation factor EF-Tu

0.92

0.65*

0.55**

56653

4.93


0.92

0.65*

0.55**

32041

5.21

pyridoxine biosynthesis protein

2.25***

1.47*

1.66**

30566

5.08

3-hydroxyacyl-CoA dehydrogenase

2.25***

1.47*

1.66**

56653

4.93


2.25***

1.47*

1.66**

32128

5.52

DNA-binding protein

1.01

1.61**

1.02

144875

6.94


1.01

1.61**

1.02

30566

5.08

3-hydroxyacyl-CoA dehydrogenase

0.85

1.98**

1.44

28673

6.68

ATP synthase delta chain

0.54**

0.47***

0.52**

28983

6.24

succinate dehydrogenase iron−sulfur subunit

1.16

1.22

0.78

putative function

D

day 3

day 4

day 5


Research Article

ACS Synthetic Biology Table 1. continued

fold ratio master no.

protein ID

score

750

RVR3032

1128

774

RVR7941

1805

821

RVR2133

1030

835

RVR7845

1225

856

RVR2436

643

862

RVR7936

740

866

RVR8986

589

LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS LC−MS/ MS

molecular weight

pI

28695

6.59

secreted acid phosphatase

1.64*

2.12***

1.29

37256

7.51

ABC transporter solute-binding protein

0.80

0.90

3.45**

43366

7.78

2.25***

0.95

0.79

23827

4.96

branched-chain amino acid ABC transporter substrate-binding protein two-component system response regulator

2.13***

1.70*

1.34

32885

7.73

extracellular solute-binding protein

1.82**

0.91

0.59

50304

6.92

ABC transporter solute-binding protein

1.24

0.76

0.55*

33981

5.27

dehydrogenase

1.38.

0.76

0.61

putative function

day 3

day 4

day 5

a

The fold ratio to the expression level observed in the day 2 culture was calculated. Nonrepeated-measures analysis of variance and Dunnett’s test for post hoc analysis were performed. *P < 0.05; **P < 0.01; ***P < 0.001.

efficiently activates f ur22 gene expression would be a key for mass production of target terpenoid compounds. Screening of Optimal Promoters Necessary for Terpenoid Production. To construct an efficient terpenoidproduction platform, we focused on the selection of an optimal promoter for f ur22 gene expression. First, we performed RNAseq analysis using culture broth from culture days 1, 2, and 3 (Table S1). Because we expected that strong promoter-driven f ur22 gene expression would be effective for high-level production of terpenoid compounds, based on RNA-seq data and gene organization five putative promoters (rvr3291p, rvr3991p, rvr5758p, rvr6185p, and rvr6204p) resident in S. reveromyceticus SN-593 were selected, and incorporated upstream of the f ur22 gene. Subsequently, plasmids harboring the promoter-f ur22 gene cassettes were integrated into SR1 (Figure S2) to evaluate FQ productivity. The resulting transformants (SR1−12, −15, −18, −19, and −20) (Table S2) were cultured, and the metabolites were analyzed by LC− MS. We found that utilization of these promoters was not linked to the production of FQ (Figure S3). We then investigated whether promoters activated during late growth phase might allow optimal metabolite generation to promote terpenoid production, given that secondary metabolite biosynthesis is normally associated with the late growth phase in Streptomyces species.28 Because of the difficulty of obtaining intact RNA from S. reveromyceticus SN-593 during the late growth phase, we examined protein expression to evaluate gene-expression levels. Two-dimensional (2-D) gel electrophoresis using cell lysates from culture days 2 to 5 (Figure S4) allowed for successful identification of proteins by Matrix Assisted Laser Desorption/Ionization (MALDI)-Time of flight/mass spectrometry (TOF/MS), and high-performance liquid chromatography-MS/MS (Table 1 and Figure 2). Because we screened for proteins that significantly changed their relative abundance in late growth phase, the majority of the large spots in Figure S4 were not targeted. Putative gene promoters derived from the identified proteins were screened and incorporated upstream of the f ur22 gene, and plasmids containing promoter-f ur22 gene cassettes were integrated into SR1. The resulting transformants (Table S2) were cultured, and FQ productivity was screened by a brown color changes on SY plates (Figure S5). We selected four candidate transformants, SR1−5, −7, −14, and −27 (Table S2), harboring the promoter

Figure 2. Protein expression profiles based on 2-D gel electrophoresis analysis. A heat map was prepared for each identified spot, which were categorized by expression level at days 3, 4, and 5. Increased expression level is indicated by red, whereas decreased expression is indicated by green and relative to levels observed in the day 2 control. The plus (+) and minus (−) signs indicate screening results for FQ production on SY plates. Classes I and II proteins are categorized as those expressed during the late and early phases of growth, respectively. Nd, not determined.

of rvr2030, rvr2353, rvr3650, and rvr9107 genes, respectively, and FQ productivity in the transformants was confirmed by LC−MS (Figure 3). As shown in Figure 2, protein-expression profiles were categorized as late or early phase, and contrary to our speculation, terpenoid production was not always linked with late-stage-specific promoter activation (class I in Figure 2). Because the rvr2030, rvr2353, rvr3650, and rvr9107 genes encode transaldolase, dihydrolipoamide dehydrogenase, pyrophosphate hydratase, and serine hydroxymethyltransferase enzymes, respectively, we expected that utilization of metabolically linked promoters would be key to enabling efficient terpenoid production. Optimized Expression of Terpenoid-Biosynthetic Genes by Fur22. Using the SR2 platform and the Fur22regulatory system harboring selected promoters, we evaluated BC production. First, codon-optimized farnesyl pyrophosphate E


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IV::f ur1p-f pps-ssl1-ssl3) was integrated into the SR2 strain to create SR2-PF (platform for further transformation) (Table S2). To determine if simultaneous expression between mevalonate gene cluster and the target terpenoid synthase genes were occurring, the plasmids containing various promoters (rvr2030p, rvr2353p, rvr3650p, and rvr9107p) fused to the f ur22 gene were integrated into SR2-PF. The resulting SR2−4, 5, 6, and 7 strains were cultured, and their metabolites analyzed by gas chromatography−mass spectrometry (GC−MS) (Figure S6). The metabolite profile and strain productivity are summarized in Figure 4. The maximum production of BC (212 mg/L) and squalene (SQ; 98 mg/L) was observed for strain SR2−4, where the Fur22 regulator was controlled by the rvr2030 gene promoter related to transaldolase involved in the pentose phosphate pathway (Table 1). BC productivity in strain SR2−4 was higher than that previously reported in other microbial hosts.40,41,46 Additionally, significant amounts of BC and SQ were obtained from strain SR2−7, where Fur22 was controlled by the rvr9107 gene promoter related to serine hydroxymethyltransferase involved in serine metabolism (Table 1). Furthermore, yeast strain harboring ssl1 and ssl3 genes mainly produced BC but not SQ.40 However, the strains SR2−3−7 harboring ssl1 and ssl3 genes produced BC and SQ in different ratios (Figure 4). To elucidate the mechanism, further study will be required in future. We detected the production of 5−15 mg SQ in the SR1 platform (Figure 4). The productivity might have been caused by the presence of endogenous SQ synthases (HopA and HopB homologues),47 which are normally linked with MEP pathway in early stage of growth.48 To further optimize the production of target terpenoid compounds in S. reveromyceticus SN-593, it might be necessary to disrupt the endogenous SQ synthase genes (rvr8539 and rvr8540). Here, we have established a terpenoid-biosynthetic platform (SR2) in S. reveromyceticus SN-593. We demonstrated that the

Figure 3. LC−MS analysis of FQs. The transformants harboring promoter-fur22 cassettes were evaluated. The promoters were selected on the basis of distinct protein accumulation patterns visualized by 2-D gel electrophoresis analysis, and the following transformants were evaluated: SR1−1 (i) as a positive control, SR1−5 (ii), SR1−7 (iii), SR1−14 (iv), and SR1−27 (v), and SR1-NC (vi) as a negative control (Table S2). Marked peaks are FQ-D (2), FQ-I (3), and FQ-J (4). All of the samples were extracted and analyzed as described in the Materials and Methods. The chromatograms were monitored at 267 nm, the absorption maximum of (2).

synthase ( f pps)45 from chicken and BC synthase genes40 (ssl1 and ssl3) from B. braunii were prepared for expression in S. reveromyceticus SN-593. The f ur1 promoter was introduced upstream of f pps-ssl1-ssl3 gene cassette for the regulation by Fur22 regulator. The resulting plasmid (pKU492Acos::aac(3)-

Figure 4. Quantification of terpenoid compounds identified from transformed strains. SR1−1 and SR1−29 strains harbor the pTYM19::promoter (aphIIp and rvr2030p)-f ur22 gene cassettes in SR1, respectively. SR2−3, −4, −5, −6, and −7 strains harbor the pTYM19::promoter (aphIIp, rvr2030p, rvr2353p, rvr3650p, and rvr9107p)-f ur22 gene cassettes in SR2-PF, respectively. The presence of f ur1p-f pps-ssl1-ssl3 gene cassette in each strain was shown as (−) and (+). All strains were summarized in Table S2. After 9 days of culture, metabolites were extracted by n-hexane and analyzed by GC−MS. Product yield was quantified as described in the Materials and Methods. Black and white bars show BC and SQ accumulation, respectively. Mean values ± standard deviation were derived from three independent experiments. F


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98 °C for 10 s, 68 °C for 5 s, and 68 °C for 4 min. The amplified fragment was digested with HindIII and ligated into the HindIII site of the pIM vector44 to construct pIM-f ur1. The f ur1 gene was inactivated by PCR-targeted gene replacement according to a previously described method51 using plasmids (pKD78, pKD13, and pCP20) and E. coli BW25113. Chloramphenicol-resistant (CmR) pKD78 capable of λ-redmediated recombination was used along with pIM-f ur1. Plasmid pKD13 was used as the template for the FRT-flanked kanamycin-resistance gene cassette.52 A 1.4-kb DNA fragment with flanking sequence of 51-bp for recombination was amplified using Fur1-For-P4 and Fur1-Rev-P1 primers (Table S3) and PrimeSTAR HS DNA polymerase (TaKaRa) and the following reaction conditions: 98 °C for 10 s, followed by 25 cycles at 98 °C for 10 s, 64 °C for 5 s, and 68 °C for 1 min. To perform in-frame deletion of fur1 gene in SR1 (Table S2), pIM-Δf ur1 was transformed through conjugal gene transfer. Exoconjugates were selected on an MS plate containing 20 μg/mL Tsr and 5 μg/mL Car and cultured in SK2 medium. Upon reaching an optical density at 600 nm (OD600) of ∼8, 1 mL of culture was transferred to 70 mL of SK2 medium. After four growth cycles, spores were obtained on MS plates, and Tsr-sensitive clones were selected on SY and SY-Tsr plates (25 μg/mL Tsr). The f ur1gene deletion mutant was confirmed by PCR using primers listed in Table S3. PCR was performed using KOD-FX (TOYOBO, Osaka, Japan) and the following reaction conditions: 94 °C for 1 min, followed by 35 cycles at 98 °C for 10 s, 64 °C for 30 s, and 72 °C for 4 min. The Δf ur1 mutants were referred to as SR2 (Table S2). Vector Construction and Transformation. Target promoters were amplified from S. reveromyceticus SN-593 genomic DNA and inserted upstream of f ur22 gene using pTYM1953 (Table S2) and primers listed in Table S3. PCR was performed using PrimeSTAR HS DNA polymerase (TaKaRa) and the following reaction conditions: 98 °C for 10 s, followed by 30 cycles at 98 °C for 10 s, 65 to 70 °C for 5 s, and 68 °C for 30 s. The 765-bp fragment of the f ur1 promoter was amplified using Eco-Pfur1-F and Nde-Pfur1-R primers to construct pKU492Acos::aac(3)IV::f ur1p (Table S2). PCR was performed using PrimeSTAR HS DNA polymerase (TaKaRa) and the following reaction conditions: 98 °C for 10 s, followed by 25 cycles at 98 °C for 10 s, 60 °C for 5 s, and 68 °C for 30 s. The product was digested with EcoRI and NdeI and ligated into pKU492Acos_aac(3)IV23 using T4 DNA ligase (HC) (Promega). The codon-optimized f pps-ssl1-ssl3 gene for use in S. reveromyceticus SN-593 was synthesized (GeneArt Gene Synthesis, Thermo Fisher Scientific, Waltham, USA) (Table S4), and the f pps-ssl1-ssl3 fragment was digested with NdeI and HindIII and ligated into pKU492Acos::aac(3)IV::f ur1p to construct pKU492Acos::aac(3)IV::f ur1p-f pps-ssl1-ssl3. Conjugal gene transfer was performed as previously described,44 and the bacterial strains used are described in Table S2. Transformed S. reveromyceticus SN-593 strains were selected using appropriate antibiotics (20 μg/mL Tsr, 0.6 μg/mL Apr, and 5 μg/mL Car). Total RNA Isolation for RNA-seq Analysis. S. reveromyceticus SN-593 SR1 strain was cultured in 70 mL RM-PM using a K1 flask. Total RNA was extracted at days 1, 2, and 3 using an RNeasy protect bacteria mini kit according to manufacturer instructions (Qiagen, Hilden, Germany). Total RNA integrity and purity were assessed using an Agilent bioanalyzer (Agilent Biotechnology, Santa Clara, CA, USA). Total RNA (5 μg) was subjected to rRNA depletion using the RiboZero meta-bacteria

activation of Fur22 regulator by the suitably selected promoter was essential and simultaneous expression of terpenoid biosynthetic genes resulted in the significant yield of terpenoid compounds about 0.3 g/L (SR2−4 in Figure 4). At present, the level of terpenoid production is about 10 mg/L of bisabolene from Streptomyces venezuelae ATCC 10712.26,27 Here, we propose S. reveromyceticus SN-593 as an alternative terpenoid production platform highly applicable for synthetic biology study.

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MATERIALS AND METHODS Chemicals. Ampicillin (Amp), kanamycin (Kan), and chloramphenicol (Cm) were purchased from Nacalai (Kyoto, Japan). Streptomycin (Sm), spectinomycin (Spc), thiostrepton (Tsr), and apramycin (Apr) were purchased from SigmaAldrich (St. Louis, MO, USA). Carumonum (Car) was purchased from Takeda Pharmaceutical Co. (Osaka, Japan). All other chemicals were of analytical-grade. SQ from SigmaAldrich was used for quantification, and silica gel 60 N (Kanto Chemical Co., Inc., Tokyo, Japan) was used for column chromatography. Bacterial Strains, Plasmids, and Culture Conditions. Bacterial strains and plasmids used in this study are listed in Tables S2 and S3. E. coli strains were cultured in Luria−Bertani broth or super optimal broth supplemented with the appropriate antibiotics (Amp, 100 μg/mL; Kan, 50 μg/mL; Sm, 50 μg/mL; Spc, 100 μg/mL; or Cm, 30 μg/mL) accordingly. S. reveromyceticus SN-593 wild-type and gene disruptants were routinely grown at 28 °C on MS plates [2% (w/v) soy flour, 2% (w/v) D-mannitol, 2% (w/v) agar, and 10 mM MgCl2]. For metabolite analysis, spores were inoculated in test tubes (24φ) containing 10 mL SY medium [0.1% (w/v) yeast extract (Difco; BD Biosciences, San Jose, CA, USA), 0.1% (w/v) NZ-amine (Wako Pharmaceuticals, Osaka, Japan), and 1% (w/v) starch (pH 7)] for 2 days at 28 °C on a rotary shaker at 250 rpm. Preculture (2 mL) was used to inoculate cultures in 500 mL cylindrical flasks (K1) containing 70 mL of RM-PM [2% potato dextrose (Difco), 1% malt extract (Difco), 1% dried yeast (Asahi Brewery, Tokyo, Japan), 5% tomato juice (Table Land; Maruzen Food Industry, Chikuma City, Japan), 0.1% K2HPO4, 0.1% NaCl, 0.03% MgSO4·7H2O, 0.01% NaNO3, 0.005% ZnSO4·7H2O, and 0.005% CuSO4·5H2O (pH 6.5) before autoclaving],44 and culturing for 5 to 9 days at 28 °C on the same rotary shaker. DNA Isolation. DNA isolation and manipulation were performed as previous described.49 Genomic DNA was isolated as described in Practical Streptomyces Genetics.50 A fosmid library of S. reveromyceticus SN-593 was created using a copy control fosmid library production kit (Epicenter Biotechnology, Madison, WI, USA). Plasmid DNA was isolated using the Wizard plus SV miniprep DNA purification system (Promega, Madison, WI, USA). DNA-sequence analysis was performed using the BigDye terminator version 3.1 kit (Applied Biosystems, Foster City, CA, USA). In-Frame Deletion of f ur1 Gene. For construction of the f ur1 gene disruption plasmid (pIM-Δf ur1), a fosmid clone (PCC1FOS-16C11) was obtained from the fosmid library.44 For homologous recombination, a fragment comprising the 2678-bp upstream and 2478-bp downstream regions of fur1 gene was PCR amplified using Fur1-Hind-F and Fur1-Hind-R primers (Table S3). PCR was performed using PrimeSTAR HS DNA polymerase (TaKaRa, Shiga, Japan) and the following reaction conditions: 94 °C for 1 min, followed by 30 cycles at G


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parts of all samples in a subset of experiments were labeled with Cy2 CyDye Fluor as an internal standard. To match the common master numbers between subsets of experiments, image files, including that of the master gel, were imported into the project file, spot maps of the images were matched to the master-gel image, and the normalized volume of each spot was obtained using Progenesis SameSpots (Nonlinear Dynamics, Durham, NC, USA). For cluster analysis, the normalized volumes were calculated using the means of the uncentered correlation with centroid linkage using Cluster 3.054 and visualized using Java Treeview.55 Protein Determination. Protein extracts (500 μg) were stained with CyDye Fluor and subjected to 2-D electrophoresis. Spot maps of the images were matched to the master-gel image using Progenesis SameSpots. The protein-containing region was picked using an Ettan Spot Picker (GE Healthcare) and ingel digestion with trypsin was performed as described previously.56 Samples were analyzed using a Bruker Ultra Reflex MALDI-TOF mass spectrometer or LC−MS/MS (QExactive; Thermo Fisher Scientific). The Mascot search program (Matrix Science, Boston, MA, USA) was used to search the in-house database of S. reveromyceticus for peptide masses. Screening of Intrinsic Promoters for FQ Production. To clone strong promoter, highly expressed genes were listed by RNA-seq analysis (Table S1). Based on the gene expression profiles and gene organization obtained from genome sequence (Table S5), possible promoter regions (rvr3291p, rvr3991p, rvr5758p, rvr6185p, and rvr6204p) were selected. For screening of intrinsic promoters operating a late stage of growth, 2-D protein profiling was performed. In addition to RNA-seq analysis data, gene organization data were utilized to select candidate promoters for FQ production screening (Table S5). Each possible promoter region was cloned to include the intergenic region that is not speculated to be a part of operon. The promoters for screening were inserted upstream of fur22 gene to create the pTYM19-promoter-f ur22 plasmid (Table S2). The construct was introduced into to SR1, and transformants were cultured on SY plates containing Tsr (25 μg/mL) and Car (5 μg/mL). After 1 week, the color derived from FQ was evaluated on the SY plates. Evaluation of BC Production. SR2 strain was transformed with pKU492Acos::aac(3)IV::f ur1p-f pps-ssl1-ssl3 through conjugal gene transfer. Selection of exoconjugates was performed according to resistance to Apr and Car (final concentrations: 0.6 μg/mL and 5 μg/mL, respectively). The selected strain (SR2-PF) was transformed with pTYM19::promoter-f ur22 constructs, and exoconjugates were selected on MS plates containing Tsr (25 μg/mL) and Car (5 μg/mL). Selected strains (SR2−4, −5, −6, and −7) (Table S2) were cultured to evaluate BC production. Metabolite Extraction. Extraction of RMs and FQs was performed as previously described.33,44 Briefly, wild-type and gene disruptants were grown in 10 mL SY medium, and 2 mL of preculture (OD600 = 2) was used to inoculate 70 mL RMPM. At 5-days postinoculation, 4 mL of culture broth was extracted with an equal volume of acetone and concentrated to remove the acetone. The aqueous extract was adjusted to pH 4 by adding acetic acid and extracted twice with an equal volume of ethyl acetate. The organic layer was concentrated in vacuo and dissolved in 1.2 mL methanol. The extract was analyzed by LC−MS.

kit (Epicenter Biotechnology), and cDNA was generated from the depleted RNA using the NEBNext mRNA sample prep kit (New England Biolabs, Ipswich, MA, USA). cDNA was profiled using an Agilent bioanalyzer Agilent Technologies) and subjected to Illumina library preparation using NEBNext reagents (New England Biolabs). The quality, quantity, and size distribution of the Illumina libraries were determined using an Agilent Bioanalyzer 2100 (Agilent Technologies), and the libraries were submitted for Illumina HiSeq2000 sequencing. Paired-end 90- or 100-nucleotide reads were generated and checked for data quality using FASTQC (Babraham Institute, Cambridge, UK). The raw sequences were analyzed on CLC genomics workbench 6.0.1 (Qiagen) using the chromosome sequences of S. reveromyceticus SN-593 as a reference standard. Median normalized reads per kilobase per million mapped sequence reads was used to determine gene-expression level. The RNA-seq data were deposited in SRA database with accession code SRR5925117, SRR5925118, and SRR5925119. Protein Extraction for 2-D Gel Electrophoresis. SR1 spores were cultured in 10 mL SY medium, and after 48 h, 2 mL of the preculture was used to inoculate 70 mL RM-PM. After culture days 2, 3, 4, and 5, 20 mL of the culture was harvested by centrifugation at 5000g for 10 min (Allegra X-15R; Beckman Coulter, Brea, CA, USA). The cell pellet was resuspended in 20 mL buffer A [50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, and 5 mM imidazole] and centrifuged at 5000g for 10 min. The pellet was then resuspended in 50 μL buffer B (buffer A containing 0.5 mg/ mL lysozyme) and sonicated (TOMY UR-20P; TOMY Digital Biology, Tokyo, Japan) 10 times for 5 s each. The supernatant was collected by centrifugation at 5000g for 30 min, treated with 2.5 μL benzonase (Merck, Billerica, MA, USA) on ice for 1 h, and centrifuged at 11,300g for 3 min at 4 °C. Protein concentration was measured using a protein assay kit (Bio-Rad, Hercules, CA, USA). To confirm reproducibility, SR1 was cultured three independent times, prepared cell lysates, and measured protein concentration. Protein Labeling and 2-D Gel Electrophoresis. Proteome analysis of the protein extract was performed by 2D difference gel electrophoresis (2D-DIGE) system (GE Healthcare, Pittsburgh, PA, USA). Protein (50 μg) was labeled with 200 pmol CyDye DIGE Fluor minimal dyes (GE Healthcare) according to manufacturer instructions (Cy3 and Cy5 for samples and Cy2 for internal control, consisting of equal parts of all samples in an experiment). Samples were applied by rehydration onto immobilized pH-gradient (IPG) strips (24 cm, pH 3−10, nonlinear gradient; GE Healthcare) and subjected to isoelectrofocusing using an IPGphor IEF system (GE Healthcare). Strips were incubated in equilibration buffer [6 M urea, 30% glycerol, 2% SDS, 50 mM Tris-HCl (pH 8.8)] containing 1% dithiothreitol for 15 min, followed by incubation in the same buffer containing 2.5% iodoacetamide for 15 min. Strips were transferred to the tops of 11% polyacrylamide gels and electrophoresed overnight in a DALT Twelve apparatus (GE Healthcare). After electrophoresis, gels were scanned using a Typhoon 9400 imager at 100 dpi resolution (GE Healthcare). Experimental Design and Data Processing for 2-D Profiling. To perform visual 2-D protein profiling, experiments were divided into subsets. We used the spot number of the master gel as the common master number for all the experiments. The gel containing the most spots was chosen as the master gel. All samples were prepared in triplicate. Equal H


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ACS Synthetic Biology Present Address

For the extraction of SQ and BC, wild-type and transformed strains were cultured as described. After 5 to 9 days, 10 mL of culture broth from K1 flask (70 mL) was mixed with an equal volume of acetone. After sonication for 1 min, the acetone broth was extracted with 10 mL of n-hexane three times. The nhexane extract was concentrated under nitrogen flow, dissolved in 2 mL n-hexane, applied to a silica column (5 × 40 mm), and washed with three column volumes of n-hexane. The passing fraction was concentrated under a gentle nitrogen flow and dissolved in 1 mL n-hexane for GC−MS analysis. LC−MS Analysis. RM and FQ were analyzed by LC−MS using an ACQUITY UPLC system (Waters Corporation, Milford, MA, USA) equipped with an API-3200 mass spectrometer (SCIEX, Foster City, CA, USA). The UPLC system used an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm; Waters Corporation) with a flow rate of 0.7 mL/ min. Solvent A consisted of 0.05% aqueous formic acid, and solvent B consisted of acetonitrile. After column equilibration with 30% solvent B, 1 μL of the extracted sample was injected onto the column, which was developed using a linear gradient of 30% to 100% solvent B for 1.9 min, followed by maintenance at 100% solvent B for 1 min. Mass spectra were collected in ESI-negative mode. GC−MS Analysis. BC and SQ were analyzed using an Agilent 7890 GC/5975 MSD (Agilent Technologies) equipped with an HP-5 ms UI column (30 m × 250 μm × 0.25 μm). Extract (1 μL) was injected into the GC−MS apparatus in splitless mode at a front-inlet temperature of 260 °C. The initial oven temperature was maintained at 120 °C for 1 min, followed by increases of 10 °C/min to 260 °C and maintenance for 10 min. Helium was used as the carrier gas at a flow rate of 1 mL/ min. Metabolite Quantification. The amount of RM-A-E in the culture broth was calculated using RM-A as a standard. After applying ranges of 1 ng to 140 ng of RM-A for LC−MS analysis, the area of absorbance unit (AU) at 238 nm was obtained to create the calibration curve. The amount of FQ-A-J in culture broth was calculated using FQ-D as the standard. After applying ranges of 200 ng to 1600 ng of FQ-D for LC− MS analysis, the area of AU at 267 nm was obtained to create the calibration curve. The amount of SQ and BC in culture broth was calculated using SQ as the standard. The calibration curve was obtained after injection of 100 ng to 500 ng SQ into the GC−MS apparatus.

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⊥

Teikyo University Institute of Medical Mycology, 359 Otsuka, Hachioji, Tokyo, 192−0395, Japan.

Author Contributions

A.K., H.T., S.P., and M.M. performed the experiments. S.T. designed the experiments. A.K. and S.T. wrote the manuscript. H.O., J.C., and S.T. integrated the research. All authors discussed the results and commented on the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank M. Tanaka for conducting 2-D gel electrophoresis and MALDI-TOF/MS analysis, and Dr. N. Dohmae and T. Suzuki for LC−MS/MS. We are grateful to Dr. S. Okada for providing botryococcene standard. We thank Dr. H. Ikeda and Dr. H. Onaka for pKU492Acos and pTYM19 vectors, respectively. We thank Dr. J. Ishikawa and Dr. A. Toyoda for genome sequence analysis of S. reveromyceticus SN-593. This work was supported by JSPS KAKENHI Grant Number 17H05455 and “Project focused on developing key technologies for discovering and manufacturing drugs for nextgeneration treatment and diagnosis” from the Japan Agency for Medical Research and Development (AMED).

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ABBREVIATIONS CDP-ME, 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol; CDP-ME2P, 2-phospho-4-(cytidine 5′-diphospho)-2-C-methylD-erythritol; DMAPP, dimethylallyl pyrophosphate; DPMVA, diphosphomevalonate; DXP, 1-deoxy-D-xylulose 5-phosphate; FPP, farnesyl pyrophosphate; GAP, glyceraldehyde 3-phosphate; GGPP, geranylgeranyl pyrophosphate; GPP, geranyl pyrophosphate; HMBDP, (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; IPP, isopentenyl pyrophosphate; MECDP, 2-C-methyl-Derythritol 2,4-cyclodiphosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; PMVA, phosphomevalonate

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.7b00249. Tables S1−S5 and Figures S1−S6 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: +81-48-467-4044. Fax: +81-48-462-4669. E-mail: [email protected]. ORCID

Ammara Khalid: 0000-0002-3120-604X Suresh Panthee: 0000-0003-4021-7936 Hiroyuki Osada: 0000-0002-3606-4925 I


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