Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces Chaoxian Baia,b,1, Yang Zhanga,b,1, Xuejin Zhaoa,1, Yiling Hua,c, Sihai Xianga, Jin Miaoa, Chunbo Loua,2, and Lixin Zhanga,2 a Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology and Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; bUniversity of Chinese Academy of Science, Beijing 100149, China; and cSchool of Life Sciences, Anhui University, Hefei 230601, China
There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.
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synthetic biology natural product flow cytometry single-cell resolution modular regulatory elements
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treptomycetes are well known as the most abundant source of bioactive secondary metabolites (1), including medically important antimicrobial agents [e.g., chloramphenicol from Streptomyces venezuelae (2)], agricultural chemicals [e.g., avermectin from Streptomyces avermitilis (3)], and anticancer agents and immunosuppressants [e.g., rapamycin from Streptomyces hygroscopicus (4)]. However, the increasing difficulty of discovering novel drugs via traditional high-throughput screening and the “one strain many compounds” approach is frustrating pharmaceutical productivity (5, 6). Deciphering the genome sequences of Streptomyces surprisingly established the presence of a plethora of gene clusters encoding for yet-unobserved molecules, even in intensively investigated Streptomyces coelicolor A3 (2), revealing a much higher potential of novel bioactive agent production than originally anticipated (7, 8). Therefore, the enormous number of natural products that have been obtained likely represent only a tiny portion of the repertoire of bioactive compounds that can possibly be produced. This has brought about extensive research into applied genomics aimed at investigating these new gene clusters, generally referred to as “cryptic,” “silent,” or “orphan” (9–11). With data on more than 12,000 in-house draft bacterial genomes, the potential for the discovery of a number of novel chemicals encrypted in silent biosynthetic gene clusters has been detected by genome mining. Many new strategies have been documented for “awakening” poorly expressed and/or silent gene clusters in Streptomyces, enabling the discovery of new bioactive agents. Two main substantially overlapped ways are used: physiological triggers (12, 13) www.pnas.org/cgi/doi/10.1073/pnas.1511027112
and synthetic or genetic manipulations (14–16). Even though the genome size of Streptomyces is significantly smaller than that of Saccharomyces cerevisiae, it has many more ORFs than its eukaryotic counterparts, a substantial part of which regulate the transcriptional and translational machineries of gene clusters responsible for the biosynthesis of secondary metabolites (7, 17, 18). Thus, most activation approaches aim to stimulate the transcription of gene clusters. Nevertheless, a convenient and precise approach for characterization of the relevant transcriptional and translational elements in Streptomyces has become a bottleneck in the effort to activate the cryptic gene clusters. Although antibiotic resistance genes [e.g., the neomycin/kanamycin resistance gene (19) and chloramphenicol resistance gene (20)] were broadly used as qualitative reporters in Streptomyces, they are unable to do quantitation. Luciferase assay [e.g., luxBA operon (21) from Vibrio harveyi and luxCDABE operon (22) of the bioluminescent bacterium Photorhabdus luminescens], as well as chromogenic assay [e.g., xylE gene (23, 24) from Pseudomonas putida and gusA gene (25, 26) from Escherichia coli], have wide application in quantitation. However, both of these assays are based on enzymatic reaction, and further activity normalization for dry cell weight is required, which decreases accuracy and is time-consuming. A GFP-based reporter system is another approved strategy to qualitatively monitor spatial and temporal trafficking of proteins and other proteinrelated physiological processes (27–30). Although different from the unicellular bacterium E. coli, Streptomyces grow by hyphal extension and exponential branching and ultimately form multicellular network-structured pellets with diameters of up to Significance To meet the increasing demands of drug discovery and biosynthetic studies, we established a precise quantitative method based on flow cytometry at single-cell (protoplast) resolution in Streptomyces for the identification of regulatory elements. A series of native or synthetic promoters and ribosomal binding sites has been characterized. Moreover, an insulator was demonstrated to eliminate element–element interference. As a proof of concept, a native silent gene cluster was activated by the synthetic modular regulatory elements in a predictable manner. The universality of these elements is of high value to the synthetic biology of Streptomyces. Author contributions: C.L. and L.Z. designed research; C.B., Y.Z., X.Z., and Y.H. performed research; C.B., Y.Z., X.Z., Y.H., S.X., and J.M. analyzed data; and C.B., C.L., and L.Z. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
C.B., Y.Z., and X.Z. contributed equally to this work.
2
To whom correspondence may be addressed. Email:
[email protected] or louchunbo@ gmail.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1511027112/-/DCSupplemental.
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2,000 μm (31). The network structure of the pellets appears as a mixture of multiple layers of cells on a microscope slide, with the sizes of the pellets being too large to pass through the nozzle of a commercial FACS machine (32). This led to no description of the GFP-based quantitative measurement in Streptomyces. The work explained here was performed to overcome these problems. We developed a quantitative method for gene expression based on flow cytometry of protoplasts in Streptomyces. The quantitative accuracy was improved by introducing propidium iodide (PI) as another reporter to differentiate between viable and dead cells, and was further validated by quantitative PCR. We demonstrated the convenience and compatibility of this approach by characterizing a large number of native or synthetic promoters and RBSs. We also used the method to characterize the combination of seven promoters and nine RBSs and to quantify the predictability after introducing the RiboJ insulator (33). Thus, a universal toolbox of synthetic modular regulatory elements has been developed to systematically replace the indigenous promoter and RBS sequences to activate the expression of the cryptic gene clusters at various levels. The feasibility and efficiency of the universal cassettes were confirmed by the overproduction of lycopene in S. avermitilis.
stabilizing the osmotic pressure of the protoplasts, it might damage the fluidic system of the FACS machine. To conquer this problem, we sought an alternative buffer that is not only less sticky but also capable of maintaining the viability of the protoplasts. In addition, the membrane-impermeable dye, PI, was introduced to recognize the cell death in the dead hyphae, of which the protoplasts can be stained and analyzed by confocal laser-scanning fluorescence microscopy (Fig. 2 A and B). As a negative control, PBS buffer or water did not maintain the osmotic pressure of the protoplasts; these lysed protoplasts can be stained by PI (Fig. 1B). Appropriate concentration (0.4∼0.8 M) of NaCl or KCl in the buffer successfully maintained the osmotic pressure of the protoplasts, resulting in ∼20% viable cells being obtained. Further optimization showed that the use of the PBS buffer (pH 7.4) supplemented with 0.5 M NaCl can achieve more than 50% viability, which is almost twice that achieved with the original P10 buffer (Fig. 1C). We thus not only solved the problem accompanied by the morphogenesis of Streptomyces for FACS processing but also developed a quantitative strategy at single-cell resolution for Streptomyces. Eight promoters were selected, based on extensive literatures (SI Appendix, Table S3), to measure their GFP fluorescence by flow cytometry. To validate the accuracy of the new method, their cognate mRNA level was also detected by real-time quantitative PCR. The results showed that the fluorescence of the protoplasts is consistent with the mRNA level of their hyphae (R2 = 0.90), indicating that the flow cytometry-based GFP reporter correctly represented the activities of the promoters in Streptomyces (Fig. 3). Moreover, flow cytometry can process tens of thousands of individual cells within a few seconds and can simultaneously monitor up to 20 different parameters [e.g., side scatter (SSC), forward scatter (FSC), and up to 18-color fluorescence]. These advantages of flow cytometry can facilitate further
Results and Discussion A Flow Cytometry-Based Quantitative Method for Streptomyces. For
the multicellular Streptomyces, we first developed a quantitative method at single-cell resolution (Fig. 1A). After introducing a strong promoter (kasOp*) with the superfolder green fluorescent protein (sfGFP) gene into the S. venezuelae genome via sitespecific recombination, the hyphae were treated with lysozyme to release protoplasts into P10 buffer containing 10% (wt/vol) sucrose, which is very sticky. Although the sticky sucrose was important in
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Fig. 1. (A) Workflow of single-cell quantification for gene expression in Streptomyces by flow cytometry. Effect of different buffers on the viability of protoplasts, released from the mycelium of S. venezuelae, indicated by scatter plot (B) and histogram (C), with the population of viable cells being gated. RFP, red fluorescent protein.
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Fig. 2. Distinguishing viable individuals from dead ones by PI staining. Images of the PI-stained mycelium (A) and protoplasts (B) of S. venezuelae harboring kasOp*-driven sfGFP were taken by a confocal laser-scanning microscope. (C and D) The dead individuals were separated from the viable ones by PI staining. A weak promoter (ermEp*) (C) and a strong promoter (kasOp*) (D) were both demonstrated to show the advantage of PI staining.
analysis of heterogeneity in a population of the multicellular Streptomyces. Meanwhile, PI was enrolled to exclude the interference of dead cells, as a single parameter (GFP) cannot distinguish the low GFP-expressing viable cells from the dead ones (Fig. 2 C and D), which therefore greatly improved the reliability of our approach and the accuracy of the results. In contrast, the most widely used quantitative methods in Streptomyces, such as xylE, gusA, and luxCDABE, are based on enzymatic reactions that require further normalization of their activities to dry cell weight, thereby compromising the accuracy of the measurement. However, dead cells are inevitably present within dry cells of Streptomyces mycelium and can further reduce the accuracy, as programmed cell death is occurring during the developmental process (34, 35). Therefore, these traditional methods are far from satisfying the sophisticated elucidation of the regulatory elements for the biosynthesis of secondary metabolites.
other library was made by mutating the spacer sequence between the −10 and −35 regions of kasOp* to increase the diversity of the promoters (SI Appendix, Fig. S3A). In the two libraries, six of 180 synthetic promoters displayed stronger activity than that of kasOp*, with five of them from the first library. In comparison with the original kasOp* promoter, the activities of the synthetic promoters varied from 0.95 to 187.5%. Then we sequenced 44 of the promoters with gradually increased strength to facilitate further application (Fig. 4A and SI Appendix, Fig. S3B). Similarly, 15 native and 174 synthetic RBSs were characterized in this study at the same time. Among the 15 native RBSs, the RBS of capsid protein from phage ϕC31 (37) showed the highest activity (Fig. 4B and SI Appendix, Table S3). Thus, four libraries originated from the RBS of capsid protein were established to elicit a variety of RBSs. To obtain stronger RBSs, we firstly randomized the sequence either up- or downstream of the ShineDalgarno sequence (SI Appendix, Fig. S3A). As a result, 66 of 130 RBSs were stronger than the template RBS, thereby revealing the potential of optimizing the local structure of mRNA to improve translational activity. Second, we partially mutated the core Shine-Dalgarno sequence to degenerate the strength of the RBS to obtain weaker RBSs. On the whole, we acquired 177 synthetic RBSs with activity covering a 200-fold range (SI Appendix, Fig. S3C) and sequenced 41 RBSs with varying strength among them (Fig. 4B). Moreover, the result of the time-course experiment (SI Appendix, Fig. S4) reconfirmed that the engineered short and strong promoter was less likely to interfere with the global regulatory factors (36), in comparison with the previously reported regulatory elements, thereby providing a great opportunity to rationally design the pathway-specific regulatory system for a gene cluster. Therefore, the characterized synthetic promoter and RBS libraries are of high application value for activation and optimization of cryptic secondary metabolic pathways. The RiboJ Insulator Enables Predictable Combination of Promoters and RBSs in S. venezuelae. As regulatory elements, promoters
and RBSs are responsible for the transcriptional and translational activities of their downstream genes, respectively. However, the two functional elements might interfere with each other by the promoter escaping process (38) or by the formation of some structures inaccessible to ribosomes on the messenger RNA. Different insulators have been reported to eliminate the unanticipated regulatory
Characterization of Native or Synthetic Promoters and RBSs in Streptomyces. Apart from the advantage of single-cell resolu-
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tion, the flow cytometry-based method has the potential to combine with high-throughput manipulation for Streptomyces. For high-throughput application, 24-well plates were adopted for culturing and 96-well plates were used for treating the hyphae with lysozyme and harvesting protoplasts. Considering it has faster growth than other Streptomyces, S. venezuelae was preferential as host for the verification of the procedure. In fact, we also confirmed the application of the strategy in other Streptomyces such as S. avermitilis and S. coelicolor (SI Appendix, Fig. S2). To demonstrate the convenience of this method, we identified 195 native or synthetic promoters and 192 RBSs. In total, 15 native or engineered promoters were chosen and inserted upstream of the sfGFP gene. As shown in Fig. 4A, the kasOp* promoter, originally obtained by Wang and coworkers (36), exhibited the strongest activity among all the promoters. Its activity is 20-fold higher than that of the widely used ermEp* promoter. For the purpose of acquiring stronger and versatile promoters, the kasOp* promoter was used as a template to construct two randomly mutated libraries. The first library was to randomize the nucleotides downstream of the −10 sequences of kasOp*, and the
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Fig. 3. Correlation of GFP expression with mRNA abundance, measured by quantitative RT-PCR. Open circle, gapdhp (SG); solid circle, rpsLp (SA); open square, rpsLp (RE); solid square, ermEp*; solid triangle, gapdhp (KR); solid upside-down triangle, rpsLp (TP); open triangle, rpsLp (CF); open upsidedown triangle, kasOp*. Error bars, data are presented as mean ± SD obtained from at least three experiments performed on different days.
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75-nt RiboJ was inserted between the pairwise combined seven promoters and nine RBSs, resulting in 63 promoter–RiboJ–RBS regulatory cassettes upstream of sfGFP (Fig. 5B). As for control, the RiboJ sequence was removed from these 63 combinatorial sequences (Fig. 5A). Moreover, a full factorial ANOVA model was applied to quantify the contribution of the interfered promoter::RBS (interaction term), the promoter, and the RBS to the GFP expression (40). ANOVA revealed that the contribution of promoter::RBS interaction without RiboJ (31%) (Fig. 5C) is much larger than that observed with RiboJ (3%) (Fig. 5D), indicating that the RiboJ insulator can also effectively eliminate the unpredictable interaction between the promoter and RBS. For instance, antagonistic interaction was observed in the combination of a strong promoter (P6) and a strong RBS (R9) without RiboJ (Fig. 5E), whereas the GFP expression exhibited a modest correlation between promoters and RBSs with the presence of RiboJ (Fig. 5F). The contributions of other elements were comparable in the two circumstances (promoter: 58% and 47% with and without RiboJ, respectively; RBS: 38% and 21% with and without RiboJ, respectively) (Fig. 5 C and D). Because the remaining experimental error contributes to only 1% in both cases, it was taken as a negligible factor. These findings demonstrated that the RiboJ insulator could further improve the designability of the activation and optimization of secondary metabolic pathways in Streptomyces.
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Fig. 4. Relative strength of native and synthetic promoters and RBSs evaluated in S. venezuelae ISP5230. (A) Relative strength of 15 native or engineered promoters (gray) and 44 sequenced synthetic promoters (white) with kasOp* (*) as the reference. The widely used promoter ermEp* is shown in red. (B) The relative strength of 15 native RBSs (gray) and 41 sequenced synthetic RBSs (white) with the RBS of capsid protein from phage ϕC31 (*) as the reference. Error bars, data are presented as mean ± SD obtained from at least three experiments performed on different days.
Application of the Synthetic Modular Regulatory Elements for Lycopene Overproduction in S. avermitilis. Lycopene synthase pro-
teins are highly conserved but rarely expressed in most Streptomyces. We therefore put effort into activating the silent lycopene biosynthetic gene cluster in S avermitilis to explore the potential of the universal synthetic modular regulatory elements for activating cryptic gene clusters. Considering that there is enough supply of the universal acyclic precursors GPP, FPP, and GGPP in S. avermitilis, a set of synthetic promoters with RiboJ were inserted upstream of the lycopene biosynthetic cluster in S. avermitilis. As expected, we observed a correlation between lycopene production
element interaction in E. coli (33, 39). Here, we introduced the well-elucidated RiboJ insulator between promoter and RBS to improve the predictability of the combination. To test the modularity and predictability of the insulated promoter and RBS, the
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Fig. 5. Performance of the RiboJ insulator in Streptomyces to eliminate interferences between the promoter and RBS. (A and B) A scheme of the combined seven promoters and nine RBSs to form a full combinatorial library of expression control elements without RiboJ (A) or with RiboJ (B). Heat maps show GFP fluorescence for all combinations of promoter (P, columns) and RBS (R, row) elements driving the expression of GFP reporter gene for the with-RiboJ (F) and without-RiboJ (E) cases. Each value was obtained by flow cytometry with three experimental duplications. Analysis of variances by full factorial ANOVA was performed for the without-RiboJ (C) and with-RiboJ library data (D). Promoter: GSV, gapdhp (SV); GSA, gapdhp (SA); GSG, gapdhp (SG); RTP, rpsLp (TP); GCF, rpsLp (CF); REL, gapdhp (EL); KSC, kasOp*. RBS: TER, terminase (ϕC31); TAP, tape measure protein (ϕC31); KSC, kasO (SC); TAI, tail protein (ϕC31); NUK, nucleotide kinase (ϕC31); HEL, helicase (ϕC31); CAP, capsid protein (ϕC31); GSG, GAPDH (SG); RCF, 30s ribosomal protein S12 (CF).
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the consistent expression of the same promoters in not only Streptomyces lividans TK24 and Streptomyces albus J1074 but also rare actinomycetes such as Salinispora tropica CNB-440 and Saccharothrix espanaensis DSM 44229. Therefore, the success of activating lycopene overproduction in S. avermitilis indicates that our regulatory elements could be applied as well in other actinomycetes. In conclusion, the universal synthetic modular regulatory elements we constructed will be beneficial to the synthetic biology community and will facilitate the drug discovery process in streptomycetes as well as actinomycetes in general.
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Fig. 6. Lycopene production in S. avermitilis under the control of different promoters with the presence of RiboJ. Promoter strength is shown by the relative strength to the kasOp* promoter. Solid circle, SP12; open circle, SP18; solid square, SP23; open square, SP26; open triangle, kasOp*; open diamond, SP43; open upside-down triangle, SP44. Data are expressed as mean ± SD of the results of three parallel studies. Promoter sequences are listed in SI Appendix, Table S3.
and promoter strength, and the highest production reached 82 mg/g dry cell weight (Fig. 6 and SI Appendix, Table S7). The extent of overproduction validated the strength of our synthetic promoters, and the correlation between lycopene titer and promoter strength highlighted the predictive superiority endowed by the addition of the insulator. Indeed, these synthetic promoters and insulator circumvent the stringent endogenous regulatory system and not only activate the gene cluster of lycopene but also facilitate overproduction. Insulation by RiboJ enables rational combination of promoters and RBSs, which will make the precise control of protein expression level and the production of desirable metabolites possible. Conclusion Quantitative characterization of regulatory elements in Streptomyces is very important to secondary metabolic pathway reconstitution. The flow cytometry-based method has improved the resolution of the quantification of gene expression in the filamentous bacteria; that is, from the mycelium level to the singlecell level. The quantitative PCR experiments indicated that the mRNA abundance was consistent with the cognate GFP expression level. For further exploitation, we developed a versatile toolbox of regulatory elements in Streptomyces by elucidation of 195 native or synthetic promoters and 192 RBSs. Moreover, an insulator was introduced to further improve the modularity and predictability of the regulatory elements. Indisputably, the quantitative method developed here and the modular promoter and RBS libraries have tremendous potential on the activation of more complex cryptic gene clusters enabling the discovery of new bioactive natural products and promote the rational design of heterologous pathways in Streptomyces. Siegl and colleagues developed a synthetic promoter library for actinomycetes based solely on the −10 and −35 consensus sequences of the widely used ermEp* promoter (26). Through a quantitative method, we can improve the modularity and greatly increase the dynamic range of these regulatory elements by up to 1,000-fold. As a whole, synthetic promoters are advantageous over native promoters because they are less likely to underlie intracellular regulation. Moreover, Siegl’s work demonstrated Bai et al.
Strains, Media, and Growth Conditions. E. coli strains were cultivated at 37 °C in Luria-Bertani medium or on Luria-Bertani agar plates. DH5α and ET12567 (pUZ8002) were used as E. coli hosts for plasmid construction and E. coli– Streptomyces conjugation, respectively. Mannitol soya flour medium was used for conjugation, and malt extract–yeast extract–maltose medium was used for liquid inoculation of the spores of S. venezuelae ISP5230. The media were supplemented with different antibiotics at appropriate concentrations as follows: ampicillin at 100 μg/mL, apramycin at 50 μg/mL, nalidixic acid at 25 μg/mL, kanamycin at 25 μg/mL, and chloramphenicol at 25 μg/mL Spores of S. avermitilis WT transformants were used to inoculate a 250-mL flask containing 25 mL seed medium [glucose (5 g), soy flour (15 g), and yeast extract (5 g) per liter, at pH 7.2], and the culture was allowed to grow with shaking (220 rpm) at 30 °C for 2 d. A 1-mL aliquot of the culture was used to inoculate a 250-mL flask containing 50 mL production medium [glucose (60 g), (NH4)2SO4 (2 g), MgSO4•7H2O (0.1 g), K2HPO4 (0.5 g), NaCl (2 g), FeSO4•7H2O (0.05 g), ZnSO4•7H2O (0.05 g), MnSO4•4H2O (0.05 g), CaCO3 (5 g), and yeast extract (2 g) per liter at pH 7.0] (41). The samples were incubated with shaking (220 rpm) at 28 °C for 5 d. Preparation of Protoplasts from Streptomyces. Broth culture (1 mL) containing the mycelia was spun down and washed by 10% (wt/vol) sucrose once and resuspended in 1 mL of the P10 buffer supplemented with 5 mg/mL of lysozyme. After incubation at 37 °C for 1 h, protoplasts were filtered through cotton wool to remove the mycelia. For high-throughput manipulation, 24square deep well microtiter plates were used for the cultivation of Streptomyces, and 96-well filter plates with cotton wool were used for preparation of protoplasts. PI Staining. Before the FACS test, dilute 100 μL protoplasts suspension with 900 μL PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 at pH 7.4) supplemented with 0.5 M NaCl, 1 mg/mL kanamycin, and 5 μg/mL PI. The samples were kept in the dark at room temperature (25 °C) for 5 min. The PI-stained protoplasts were measured by flow cytometry with an excitation wavelength of 488 nm and an emission wavelength of 585 nm. Confocal Laser-Scanning Fluorescence Microscopy Analysis of S. venezuelae. The PI-stained samples of liquid mycelium and protoplasts were observed under Leica SP8 confocal laser-scanning microscope at excitation wavelengths of 488 and 561 nm and emission wavelengths of 500∼550 nm (green) and 570∼650 nm (red), respectively. Images were merged using the Leica confocal software. Quantitative Measurement of GFP Expression by Flow Cytometry. The protoplasts were analyzed by BD FACSCalibur Flow Cytometer with a 488-nm excitation laser and the FL1 (530/30 nm band-pass filter) detector. Each sample collected 50,000 events, and the data were further acquired using BD FACSuite software and analyzed by FlowJo 9.3.2 software (Tree Star, Inc.). The parameters of the FACS setting were as follows: FSC-E00, SSC-650, FL1-400, FL2-400; threshold: FSC-50, SSC-400. The fluorescence of each sample was the geometric mean of all of the measured cells and was normalized to the corresponding FSC value, which indicates the size of the cells. Statistical Analysis. A heat map was generated from FACS data reflecting GFP expression by MATLAB R2011b (MathWorks, Inc.). ANOVA was performed by SPSS v. 22. ACKNOWLEDGMENTS. We thank Keqian Yang and Huarong Tan for the helpful discussions and comments on the manuscript, and thank Guoqing Niu, Xiaolan Zhang, Pei Huang, Jiaqian Cao, and Jingjing Xu for the technical help. This work was supported by funding in part from the Ministry of Science and Technology of China (Grants 2013CB734000 and 2011CBA00805) and the National Natural Science Foundation of China (Grant 31470818). L.Z. is an awardee of the National Distinguished Young Scholar Program in China.
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Lycopene producon (mg/g dry cell weight)
80
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