Construction and Characterization of Broad-Spectrum Promoters for

Characterization of genetic circuits and biosynthetic pathways in different hosts always requires promoter substitution and redesigning. Here, a stron...
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Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology Sen Yang,† Qingtao Liu,† Yunfeng Zhang,† Guocheng Du,†,‡ Jian Chen,*,†,‡ and Zhen Kang*,†,‡ †

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China ‡ Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China S Supporting Information *

ABSTRACT: Characterization of genetic circuits and biosynthetic pathways in different hosts always requires promoter substitution and redesigning. Here, a strong, broad-spectrum promoter, Pbs, for Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae was constructed, and it was incorporated into the minimal E. coli−B. subtilis−S. cerevisiae shuttle plasmid pEBS (5.8 kb). By applying a random mutation strategy, three broad-spectrum promoters Pbs1, Pbs2, and Pbs3, with different strengths were generated and characterized. These broad-spectrum promoters will expand the synthetic biology toolbox for E. coli, B. subtilis, and S. cerevisiae. KEYWORDS: broad-spectrum promoter, shuttle plasmid, Bacillus subtilis, Saccharomyces cerevisiae, Escherichia coli

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a promoter library with 128 members variants of the PHO5 promoter was generated rapidly by modifying both Pho4 binding sites of the PHO5 promoter.17 To facilitate operational processes and minimize unexpected background interactions, lists of short, synthetic core promoters with different activities have been provided for S. cerevisiae,18,19 Pichia pastoris,20,21 Yarrowia lipolytica,22,23 and mammalian cells.24 More recently, by applying a computational, multifactor design approach, Portela et al. successfully generated universal core promoters for different yeast species.22 With the rapid development of synthetic biology, many novel enzyme-encoding genes,25 genetic circuits,26 and biosynthetic pathways27 that were designed for a specific organism may require comparative investigations in conventional model microorganisms, for instance, E. coli, B. subtilis, and S. cerevisiae. Additionally, identification and characterization of natural unknown DNA fragments or gene clusters in different contexts and hosts is required frequently.28 However, in most cases, many existing promoters are incompatible in different species.29 As a result, the gene clusters or biosynthetic pathways have to

romoters, which are the basic transcriptional regulatory elements, have been used widely for gene expression and pathway engineering.1,2 To date, many naturally occurring promoters, especially for model organisms such as Escherichia coli,3,4 Bacillus subtilis,5 and Saccharomyces cerevisiae,6 have been identified and characterized. The structure of natural prokaryotic promoter motifs is well understood.7 Thus, many random,8 semirational,9 and rational10 approaches have been developed to engineer prokaryotic promoters in the past decades. As a result, numerous constitutive or inducible promoters with desirable properties have been constructed and applied for enzyme expression, metabolic engineering, and synthetic biology.11−13 In contrast, because of their comparatively more complex structure, only a few promoter-engineering studies of yeast and mammalian cells have been reported in the past decade.14 To realize gene expression across a full continuum of possible expression levels, Nevoigt et al.15 constructed 11 promoters whose activities ranged from 8% to 120% of the wild-type TEF1 promoter. By applying a synthetic hybrid-promoter approach, Blazeck et al.16 created strong promoter libraries for metabolic engineering and synthetic biology in S. cerevisiae. After analyzing the promoter architecture and applying the Gibson DNA assembly method, © XXXX American Chemical Society

Received: July 19, 2017 Published: October 23, 2017 A

DOI: 10.1021/acssynbio.7b00258 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Technical Note

ACS Synthetic Biology

Figure 1. Design and verification of the broad-spectrum promoter Pbs. (A) Schematic representation of the design of the broad-spectrum promoter Pbs based on the minimal yeast promoter Pmin. The 5′-TTGAAA-3′ sequence in the Pmin UAS region and the 5′-TTAAT-3′ sequence in the AT-rich region were modified to 5′-TTGACA-3′ (−35 box, underlined) and 5′-TATAAT-3′ (−10 box, underlined), respectively. An adenine and a thymine (indicated with dots) in the UAS2 region were removed to shorten the distance between the −35 box and −10 box to 17 bp. A guanine downstream of the 5′-TATAAT-3′ sequence was substituted with an adenine (underlined) to improve transcriptional initiation. In addition, a universal ribosomebinding site sequence, 5′-AGGAGGAAAAA-3′ (underlined), was added downstream of the modified promoter Pbs. Various elements of yeast promoters and their corresponding sequences are drawn in the same colors. (B) Evaluation of the broad-spectrum promoter Pbs in E. coli, B. subtilis, and S. cerevisiae. The Pbs-gf p expression cassette was inserted into an E. coli−S. cerevisiae shuttle plasmid and a B. subtilis plasmid to generate pY26Pbs-gf p and pUCP-Pbs-gf p, respectively. Fluorescent microscopy images of E. coli, B. subtilis, and S. cerevisiae harboring the corresponding GFP expression plasmids are shown.

To investigate promoter activity, the green fluorescent protein (GFP) was used as a reporter. Clearly, GFP was expressed successfully in E. coli, B. subtilis, and S. cerevisiae using Pbs (Figure 1B). In contrast, the initial promoter Pmin failed to express GFP in E. coli (data not shown). On this basis, the activity of Pbs was evaluated quantitatively and compared with that of the strong promoters PJ23119 (http://parts.igem.org/ Part:BBa_J23119) (E. coli), Pcdd (B. subtilis), and PGPD (S. cerevisiae). In E. coli, Pbs was much stronger than PJ23119 (Supplementary Figure S1A), while the strength of Pbs was approximately 75% of that of Pcdd in B. subtilis (Supplementary Figure S1B). In S. cerevisiae, Pbs exhibited a similar activity compared to Pmin, while its activity was lower than that of the strong promoter PGPD (Supplementary Figure S1C). Taken together, these results indicate that Pbs (113 bp) should be classified as a strong constitutive promoter in E. coli, B. subtilis, and S. cerevisiae. In bacteria, a long 5′-untranslated region (UTR) might alter the translation rate of target genes.30 Thus, the 70-bp 5′-UTR transcribed by Pbs could be cleaved by introducing ribozyme32 or RNase E sites33 when necessary. Construction of Shuttle Vectors with the BroadSpectrum Promoter. In most cases, shuttle plasmids for two hosts harbor at least two screening markers with respective promoters. The large size of such plasmids not only results in difficulties during subcloning, it also increases the metabolic burden on host cells. Thus, based on the construction of the broad-spectrum promoter Pbs, the shuttle plasmids pEB (4.0 kb, E. coli and B. subtilis) (Supplementary Figure S2A), pES (4.2 kb, E. coli and S. cerevisiae) (Supplementary Figure S2B), and pEBS (5.8 kb, E. coli, B. subtilis, and S. cerevisiae) (Supplementary Figure S2C) were developed, in which the expression of the Geneticin resistance gene kanR was driven by Pbs. In detail, kanamycin was used for plasmid selection in E. coli and B. subtilis, while Geneticin was used for S. cerevisiae. To confirm the successful transformation and replication of the constructed

be reconstructed with specific promoters and plasmids in different hosts, which results in a tedious construction process. Consequently, the development of a broad-spectrum promoter library comprising promoters of varying strengths is attractive and meaningful. In the present study, our aim was to construct broad-spectrum promoters and a small shuttle plasmid for E. coli (a Gram-negative bacterium), B. subtilis (a Gram-positive bacterium), and S. cerevisiae (a eukaryote).

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RESULTS Design and Characterization of the Broad-Spectrum Promoter Pbs. In E. coli and B. subtilis, the sequences of the conserved −35 box and −10 box that are recognized by the housekeeping σ70 and σ43 factors are identical, and their sequences are 5′-TTGACA-3′ and 5′-TATAAT-3′, respectively.7 Thus, to obtain an ideal broad-spectrum promoter, the core regions of constitutive promoters from E. coli, B. subtilis, and S. cerevisiae were combined. Here, a strong S. cerevisiae synthetic minimal promoter, Pmin, comprising comprehensive promoter elements,18 was chosen as a platform because it contains a 5′-TTGAAA-3′ sequence in its upstream activating sequence (UAS) region, as well as a 5′-TTAAT-3′ sequence in its AT-rich region. Thus, the 5′-TTGAAA-3′ sequence in the Pmin UAS region and the 5′-TTAAT-3′ sequence in the AT-rich region were modified to 5′-TTGACA-3′ and 5′-TATAAT-3′, respectively. In consideration of an ideal 17-bp interval between the −35 box and −10 box, an adenine and a thymine in the UAS2 region were removed. Moreover, a guanine downstream of the 5′-TATAAT-3′ sequence was substituted with an adenine to improve transcriptional initiation (Figure 1A). In addition, a universal ribosome-binding site sequence, 5′AGGAGGAAAAA-3′, which is supposed to be effective in both E. coli and B. subtilis, was designed (RBS Calculator 2.0 (www.denovodna.com)30,31 and added downstream of the modified promoter Pbs. B

DOI: 10.1021/acssynbio.7b00258 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Technical Note

ACS Synthetic Biology

Figure 2. Design and evaluation of broad-spectrum promoters with different strengths. (A) Sequences of broad-spectrum promoters with different strengths. Broad-spectrum promoters were designed to contain three conserved regions (the −35 box [5′-TTGACA-3′], UAS3 [5′-TAGCATGTGA3′], and the −10 box [5′-TATAAT-3′]), which are shown in red, and 24 random mutation sites (N5, N7, and W3NW8), which are shown in black. The upstream and downstream sequences of the modified region are shown in green. (B) Single-cell analysis of E. coli, B. subtilis, and S. cerevisiae strains harboring pEBS-Pbsx-gf p. Cells were collected at the midexponential phase (6 h of growth for E. coli and B. subtilis and 8 h of growth for S. cerevisiae), washed twice in phosphate-buffered saline, and diluted 1:100 with 0.01 M phosphate-buffered saline. For each assay, 120 000 cells were recovered. Relative gf p mRNA levels in E. coli (C), B. subtilis (D), and S. cerevisiae (E) strains harboring pEBS-Pbsx-gf p. The gf p mRNA levels transcribed by Pbs in E. coli, B. subtilis, and S. cerevisiae were set as 1. Data are presented as the mean ± standard deviation (n = 3).

different activities were constructed and analyzed by flow cytometry. Specifically, the activity of Pbs in E. coli, B. subtilis, and S. cerevisiae was set as 1. As shown in Figure 2B, the activities of Pbs1, Pbs2, and Pbs3 were 12.0%, 29.8%, and 55.7% that of Pbs in E. coli, respectively; 8.3%, 17.2%, and 48.6% that of Pbs in B. subtilis, respectively; and 11.4%, 22.5%, and 44.3% that of Pbs in S. cerevisiae, respectively. In parallel, a quantitative real-time polymerase chain reaction (qRT-PCR) was also performed to directly investigate the transcriptional strength of Pbs1, Pbs2, Pbs3, and Pbs in E. coli, B. subtilis, and S. cerevisiae. As shown in Figures 2C, 2D, and 2E, the activities of Pbs1, Pbs2, and Pbs3 were 5.5%, 12.8%, and 34.4% that of Pbs in E. coli, respectively; 5.8%, 18.7%, and 36.4% that of Pbs in B. subtilis, respectively; and 10.5%, 19.8%, and 41.3% that of Pbs in S. cerevisiae, respectively. The results confirmed that the activities of the shuttle promoters are in the order of Pbs1 < Pbs2 < Pbs3 < Pbs in E. coli, B. subtilis, and S. cerevisiae. Therefore, these broad-spectrum promoters could be used for optimizing genetic circuits or biosynthetic pathways in different hosts, which can simplify or shorten the operational process.

plasmid pEBS in the three hosts, a Pbs-gf p expression cassette was constructed and inserted into pEBS to generate pEBS-Pbsgf p (Supplementary Figure S2D). In consideration of the potential for homologous recombination, the kanR gene was placed between the two Pbs promoters. Thus, kanR would be removed after homologous recombination, guaranteeing the suppression of recombinant strains that contain mutated plasmids. As expected, obvious fluorescence was observed (Figure 2B), demonstrating the successful replication of the shuttle plasmid pEBS in E. coli, B. subtilis, and S. cerevisiae. Compared with the frequently used plasmids pP43NMK and pY26TEF-GPD (6.7 kb and 7.4 kb, respectively), the smaller sizes of pEB, pES, and pEBS should benefit subcloning and transformation procedures. Construction of Broad-Spectrum Promoters with Different Strengths. Precise regulation and optimization of genetic circuits or biosynthetic pathways is always required for synthetic biology. Thus, the development of broad-spectrum promoters with different strengths is meaningful and attractive. Here, based on the construction of the strong promoter Pbs in E. coli, B. subtilis, and S. cerevisiae, and its corresponding vector pEBS, the broad-spectrum promoter Pbs was engineered using a random mutation strategy. As shown in Figure 2A, the partial UAS 1 and UAS 2 fragments CCTCC and CTGAATT, respectively, that are close to the −35 box, as well as the partial AT-rich region TAACTTAATATT that is close to the −10 box, was randomly mutated to construct the library. After constructing a mutant library and analyzing GFP fluorescence, three new broad-spectrum promoters, Pbs1, Pbs2, and Pbs3, with

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CONCLUSIONS A small E. coli−B. subtilis−S. cerevisiae shuttle plasmid was constructed, in which the expression of the sole selection marker, a kanamycin resistance gene, was driven by the broadspectrum promoter Pbs. After random mutation and screening, three new broad-spectrum promoters, Pbs1, Pbs2, and Pbs3, were constructed, and their activities, which were in the order of Pbs1 < Pbs2 < Pbs3 < Pbs in E. coli, B. subtilis, and S. cerevisiae, were C

DOI: 10.1021/acssynbio.7b00258 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Technical Note

ACS Synthetic Biology

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characterized. These broad-spectrum promoters will expand the synthetic biology toolbox for E. coli, B. subtilis, and S. cerevisiae.

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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.7b00258. Materials and Methods, Supplementary Tables, and Supplementary Figures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +86-510-85918307. Fax: +86-510-85918309. E-mail: [email protected]. *Phone: +86-510-85918307. Fax: +86-510-85918309. E-mail: [email protected]. ORCID

Zhen Kang: 0000-0003-1479-3075 Author Contributions

Z.K. designed the research. S.Y., Q.T.L. and Y.F.Z. performed the experiments. Z.K., S.Y., G.C.D. and J.C. analyzed the data and wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (31670092), the Fundamental Research Funds for the Central Universities (JUSRP51707A), and the Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT_15R26), and the 111 Project.

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DOI: 10.1021/acssynbio.7b00258 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Technical Note

ACS Synthetic Biology site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Res. 42, 2646−2659. (31) Salis, H. M. (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946− 950. (32) Lou, C., Stanton, B., Chen, Y. J., Munsky, B., and Voigt, C. A. (2012) Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nat. Biotechnol. 30, 1137−1142. (33) Pfleger, B. F., Pitera, D. J., Smolke, C. D., and Keasling, J. D. (2006) Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat. Biotechnol. 24, 1027−1032.

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DOI: 10.1021/acssynbio.7b00258 ACS Synth. Biol. XXXX, XXX, XXX−XXX