Technical Note Cite This: ACS Synth. Biol. 2019, 8, 1469−1472
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Characterization of a Panel of Strong Constitutive Promoters from Streptococcus thermophilus for Fine-Tuning Gene Expression Ling-Hui Kong,†,§ Zhi-Qiang Xiong,†,§ Xin Song,† Yong-Jun Xia,† Na Zhang,‡ and Lian-Zhong Ai*,† †
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Shanghai Engineering Research Center of Food Microbiology, School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China ‡ Key Laboratory of Food Science and Engineering of Heilongjiang Province, Harbin University of Commerce, Harbin 150076, China S Supporting Information *
ABSTRACT: Streptococcus thermophilus, one of the most important probiotic lactic acid bacteria (LAB), is widely used in the dairy industry and attracts a lot of attention in metabolic engineering and synthetic biology. However, the available well-characterized constitutive promoters are rather limited to modulate gene expression in S. thermophilus. Here, a pool of constitutive promoters was screened by RNA-seq analysis and characterized in S. thermophilus, Lactobacillius casei, and Escherichia coli using the reporter red fluorescent protein. To assess their application potential, six constitutive promoters were selected for the expression of superoxide dismutase and significantly improved enzyme activity in the above three bacteria. Moreover, two strong constitutive promoters were used to construct a dual-expression vector for overexpressing epsA and epsE, key proteins of exopolysaccharide (EPS) biosynthesis, resulting in the change of molecular weight and the titer of EPS. Taken together, this is the first well-characterized constitutive promoter library from S. thermophilus, which could be used as a basic toolbox for various applications in LAB and other bacteria. KEYWORDS: Streptococcus thermophilus, constitutive promoters, RNA-seq, exopolysaccharide biosynthesis
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that the available constitutive promoters are rather limited in S. thermophilus, the transcriptional profiling of all 1911 genes in the genome of S. thermophilus S-37 (a high EPS-producing strain isolated from Chinese traditional dairy products by our group) was performed via RNA-seq analysis, and the promoters were sorted by their transcript levels to discover strong constitutive promoters based on the two target culturing conditions (GM17 liquid medium and LM17 liquid medium, the common media for cell growth of S. thermophilus) (Figure 1A and S1, Supporting Information). For all strains, primers, and methods in this study, see Supporting Information, Tables S1 and S2. The expression of 36 genes were higher than that of the housekeeping gene (DNA-directed RNA polymerase subunit omega) under two conditions (Figure 1B). Among these 36 genes, there are 8 genes in the same operon. Therefore, there were only 28 candidate genes to choose for cloning of their promoters (Table S3). Most of the 28 candidate promoters were 100−300 bp (Table S4). Real time qPCR analysis was also used to assess the activity of the 28 selected promoters in S-3 cultivated at 12, 24, 36, and 48 h. Compared to the expression of the housekeeping gene at the transcriptional level, most of the promoters except #27 and #28 showed highly and stably transcribed at different time with
treptococcus thermophilus is one of the most important “generally recognized as safe” lactic acid bacteria (LAB) for yogurt and cheese production.1 It has probiotic function and produces value-added metabolic products, for example, exopolysaccharide (EPS) for improving food viscosity and texture, which attracts large amounts of attention in metabolic engineering and synthetic biology. To study the function and metabolic pathway of S. thermophilus, transcriptional finetuning and regulation are essential for gene expression. Promoters are some of the most important DNA parts for gene regulation. To date, only several promoters such as the strong constitutive promoters P23 and P32 have been applied in S. thermophilus for gene expression. Thus, one of the key limitations is lack of a panel of well-characterized promoters to fine-tune gene expression in S. thermophilus. More recently, a method based on RNA-seq has been developed to conveniently obtain native promoters with different transcriptional strength.2−5 Using this method, Luo et al.5 discovered and identified 32 candidate strong constitutive promoters from Streptomyces albus J1074. The strengths of the identified constitutive promoters varied from 200 to 1300% of the strength of the well-known strong constitutive promoter ermE*p in actinomycete. Li et al.6 identified and evaluated a set of constitutive promoters with different strengths by RNA-seq analysis, enriching the available promoter library in streptomycetes. To address the problem © 2019 American Chemical Society
Received: February 3, 2019 Published: May 22, 2019 1469
DOI: 10.1021/acssynbio.9b00045 ACS Synth. Biol. 2019, 8, 1469−1472
Technical Note
ACS Synthetic Biology
Figure 1. Screening and Identification of constitutive promoters from S. thermophilus via transcriptome. Scatter-plot of the expression level of genes or transcripts at the two conditions GM17 and LM17 medium by RNA-seq (A). The red dots indicate significantly up-regulated genes; the blue dots indicate significantly down-regulated genes; black spots indicate nonsignificant genes; FPKM value: fragments per kilobase per million mapped reads. Venn diagram of the number of genes expressed higher than the housekeeping gene under two conditions by RNA-seq (B). Transcriptional change of different genes in GM17 (C) and LM17 (D) medium, respectively, at 12, 24, 36, and 48 h; the gene recA as an internal reference gene. RFP expression by different constitutive promoters in S. thermophilus S-3 (E) and L. casei LC2W (F), respectively, using promoter P32 as the control. The error bars indicate the standard deviations from three independent replicates.
To assess the application potential of our screened constitutive promoters, six strong constitutive promoters (#9, #11, #12, #13, #14, and #18) were selected for the expression of superoxide dismutase (SOD) from L. casei. Except #9 promoter, the SOD activities by other 5 constitutive promoters varying from 4880 to 18 805 U were 1.8 to 6.1-fold higher than that (2705 U) of P32 in S. thermophilus (Figure 2A). Five constitutive promoters except #13 promoter were also higher than P32 in L. casei (Figure 2B). SOD could also be expressed by these constitutive promoters in E. coli (Figure S4) Furthermore, two of these six strong constitutive promoters, #11 and #14, were used to construct a novel dual-expression shuttle vector, pKLH118 (Figure S5). RFP and green fluorescence protein (GFP) were simultaneously expressed using pKLH118 in S-3 (Figure 2C and S6), suggesting that these constitutive promoters can be used for dual-expression. pKLH118 was also used to express two key proteins of exopolysaccharide (EPS) biosynthesis, EpsA (Pathway-specific regulator) and EpsE (Controlling EPS repeat unit).7 Compared with the control, EPS titer increased 22% and molecular weight of EPS decreased 486 kDa with no change of monosugar constituent of EPS9 (Figure 2D, see Supporting Information for the methods of EPS experiments). Most EPS
two culturing conditions, suggesting that they would be constitutive promoters (Figure 1C and 1D). To further verify these putative constitutive promoters, promoter activity was evaluated by determining fluorescence intensity of the reporter red fluorescence protein (RFP) in S-3 with a common LAB strong constitutive promoter P32 as the reference. Only 17 promoters were successfully ligated with RFP and transformed into S. thermophilus (Figure S2, see Supporting Information for the construction of plasmids). The strength of 17 promoters were similar at different culture time (Figure 1E), confirming that they are constitutive promoters. Seven constitutive promoters #6, #9, #11, #12, #13, #14, and #18 showed high activities enhanced 1.2-fold to 5-fold compared with that of P32 in S-3 (Figure 1E). Interestingly, most of these promoters also had high activities in Lactobacillius casei (Figure 1F) and Escherichia coli (Figure S3), suggesting that these promoters have the potential of broad use in LAB and other bacteria. Although Slos et al.8 reported on the screening of native promoters from S. thermophilus, only six promoters were obtained but not well-characterized. To our knowledge, this is the first well-characterized constitutive promoter library in S. thermophilus. 1470
DOI: 10.1021/acssynbio.9b00045 ACS Synth. Biol. 2019, 8, 1469−1472
Technical Note
ACS Synthetic Biology
Figure 2. Application of constitutive promoters from S. thermophilus for protein expression. SOD expression by the selected 6 promoters in S. thermophilus S-3 (A) and L. casei LC2W (B), respectively, using promoter P32 as the control. One unit (U) of enzyme was defined as the amount of SOD per mg of protein in 1 mL of reaction solution. The error bars indicate the standard deviations from three independent replicates; Coexpression of RFP (A) and GFP (B) by constitutive promoters #11 and #14 in S-3, imaged by confocal laser scanning microscope (C); Effect of coexpression of epsA and epsE on EPS titer, molecular weight, and monosaccharide composition in S-3 (D). The error bars indicate the standard deviations from three independent replicates.
genes were significantly up-regulated by overexpression of epsA and epsE (Figure S7). Our result suggested that these constitutive promoters have the potential for the application in natural product synthesis. In conclusion, native constitutive promoters with various strengths were isolated by RNA-seq analysis in S. thermophilus. These promoters can be used for fine-tuning the expression of various proteins and biosynthesis of valuable natural products. Therefore, our result provided a panel of well-characterized constitutive promoters for metabolic engineering and synthetic biology in S. thermophilus and other prokaryotes.
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Materials and Methods, Figures S1−S7, Tables S1−S4 (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +86-21-55273786. E-mail: ailianzhong@hotmail. com. ORCID
Lian-Zhong Ai: 0000-0002-6681-9102 Author Contributions §
L. Kong and Z. Xiong contributed equally to this work. Conceived the concept of the study and sequence design: L.K., Z.X., L.A. Planned and performed the experiments: L.K., Z.X. Analyzed and interpreted the data: L.K., Z.X., X.S., Y.X., N.Z., L.A. Wrote the paper: L.K., Z.X., L.A.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.9b00045.
Notes
The authors declare no competing financial interest. 1471
DOI: 10.1021/acssynbio.9b00045 ACS Synth. Biol. 2019, 8, 1469−1472
Technical Note
ACS Synthetic Biology
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 31771956 and 31871776), National Key R&D Program of China (Grant No. 2018YFD0502306), Natural Science Foundation of Shanghai (Grant No. 18ZR1426800), and Shanghai Engineering Research Center of Food Microbiology (Grant No. 19DZ2281100).
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REFERENCES
(1) Bolotin, A., Quinquis, B., Renault, P., Sorokin, A., Ehrlich, S. D., Kulakauskas, S., Lapidus, A., Goltsman, E., Mazur, M., Pusch, G. D., Fonstein, M., Overbeek, R., Kyprides, N., Purnelle, B., Prozzi, D., Ngui, K., Masuy, D., Hancy, F., Burteau, S., Boutry, M., Delcour, J., Goffeau, A., and Hols, P. (2004) Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus. Nat. Biotechnol. 22, 1554−1558. (2) Trapnell, C., Hendrickson, D. G., Sauvageau, M., Goff, L., Rinn, J. L., and Pachter, L. (2013) Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat. Biotechnol. 31, 46−53. (3) Mitschke, J., Georg, J., Scholz, I., Sharma, C. M., Dienst, D., Bantscheff, J., Voss, B., Steglich, C., Wilde, A., Vogel, J., and Hess, W. R. (2011) An experimentally anchored map of transcriptional start sites in the model cyanobacterium Synechocystis sp. PCC6803. Proc. Natl. Acad. Sci. U. S. A. 108, 2124−2129. (4) Mahr, R., von Boeselager, R. F., Wiechert, J., and Frunzke, J. (2016) Screening of an Escherichia coli promoter library for a phenylalanine biosensor. Appl. Microbiol. Biotechnol. 100, 6739−6753. (5) 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. l4, 1001−1010. (6) Li, S., Wang, J., Li, X., Yin, S., Wang, W., and Yang, K. (2015) Genome-wide identification and evaluation of constitutive promoters in streptomycetes. Microb. Cell Fact. 14, 172. (7) Xiong, Z. Q., Kong, L. H., Lai, P. F., Xia, Y. J., Liu, J. C., Li, Q. Y., and Ai, L. Z. (2019) Genomic and phenotypic analyses of exopolysaccharide biosynthesis in Streptococcus thermophilus S-3. J. Dairy Sci. 102, 4925−4934. (8) Slos, P., Bourquin, J. C., Lemoine, Y., and Mercenier, A. (1991) Isolation and characterization of chromosomal promoters of Streptococcus salivarius subsp. thermophilus. Appl. Environ. Microbiol. 57, 1333−1339. (9) Xu, Z. Y., Guo, Q. B., Zhang, H., Wu, Y., Hang, X. M., and Ai, L. Z. (2018) Exopolysaccharide produced by Streptococcus thermophiles S-3: molecular, partial structural and rheological properties. Carbohydr. Polym. 194, 132−138.
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