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      Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications

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          Abstract

          The use of Bacillus subtilis in synthetic biology and metabolic engineering is highly desirable to take advantage of the unique metabolic pathways present in this organism. To do this, an evaluation of B. subtilis’ intrinsic biological parts is required to determine the best strategies to accurately regulate metabolic circuits and expression of target proteins. The strengths of promoter candidates were evaluated by measuring relative fluorescence units of a green fluorescent protein reporter, integrated into B. subtilis’ chromosome. A total of 84 predicted promoter sequences located upstream of different classes of proteins including heat shock proteins, cell-envelope proteins, and proteins resistant against toxic metals (based on similarity) and other kinds of genes were tested. The expression levels measured ranged from 0.0023 to 4.53-fold of the activity of the well-characterized strong promoter P43. No significant shifts were observed when strains, carrying different promoter candidates, were cultured at high temperature or in media with ethanol, but some strains showed increased activity when cultured under high osmotic pressure. Randomly selected promoter candidates were tested and found to activate transcription of thermostable β-galactosidase (bgaB) at a similar level, implying the ability of these sequences to function as promoter elements in multiple genetic contexts. In addition, selected promoters elevated the final production of both cytoplasmic bgaB and secreted protein α-amylase to about fourfold and twofold, respectively. The generated data allows a deeper understanding of B. subtilis’ metabolism and will facilitate future work to develop this organism for synthetic biology.

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          Most cited references48

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          FACS-optimized mutants of the green fluorescent protein (GFP).

          We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67. We then used fluorescence-activated cell sorting (FACS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima. In addition, when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.
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            Tuning genetic control through promoter engineering.

            Gene function is typically evaluated by sampling the continuum of gene expression at only a few discrete points corresponding to gene knockout or overexpression. We argue that this characterization is incomplete and present a library of engineered promoters of varying strengths obtained through mutagenesis of a constitutive promoter. A multifaceted characterization of the library, especially at the single-cell level to ensure homogeneity, permitted quantitative assessment correlating the effect of gene expression levels to improved growth and product formation phenotypes in Escherichia coli. Integration of these promoters into the chromosome can allow for a quantitative accurate assessment of genetic control. To this end, we used the characterized library of promoters to assess the impact of phosphoenolpyruvate carboxylase levels on growth yield and deoxy-xylulose-P synthase levels on lycopene production. The multifaceted characterization of promoter strength enabled identification of optimal expression levels for ppc and dxs, which maximized the desired phenotype. Additionally, in a strain preengineered to produce lycopene, the response to deoxy-xylulose-P synthase levels was linear at all levels tested, indicative of a rate-limiting step, unlike the parental strain, which exhibited an optimum expression level, illustrating that optimal gene expression levels are variable and dependent on the genetic background of the strain. This promoter library concept is illustrated as being generalizable to eukaryotic organisms (Saccharomyces cerevisiae) and thus constitutes an integral platform for functional genomics, synthetic biology, and metabolic engineering endeavors.
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              Design, construction and characterization of a set of insulated bacterial promoters

              We have generated a series of variable-strength, constitutive, bacterial promoters that act predictably in different sequence contexts, span two orders of magnitude in strength and contain convenient sites for cloning and the introduction of downstream open-reading frames. Importantly, their design insulates these promoters from the stimulatory or repressive effects of many 5′- or 3′-sequence elements. We show that different promoters from our library produce constant relative levels of two different proteins in multiple genetic contexts. This set of promoters should be a useful resource for the synthetic-biology community.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                5 July 2016
                2016
                : 11
                : 7
                : e0158447
                Affiliations
                [1 ]Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P. R. China
                [2 ]Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P. R. China
                [3 ]National Engineering Research Center for Non-food Biorefinery, State Key Laboratory of Non-food Biomass Energy and Enzyme Technology, Nanning 5300074, P. R. China
                [4 ]Guangxi Biomass Industrialization Engineering Institute, Guangxi Academy of Sciences, Nanning 530007, P. R. China
                Imperial College London, UNITED KINGDOM
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: YS GF DZ. Performed the experiments: YS JN GF JC. Analyzed the data: YS JN GF JC QL NX. Wrote the paper: YS JN JC PZ JS DZ.

                Article
                PONE-D-16-08445
                10.1371/journal.pone.0158447
                4933340
                27380260
                156baa7c-c85d-4965-9972-fe84cfdafdd1
                © 2016 Song et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 27 February 2016
                : 16 June 2016
                Page count
                Figures: 8, Tables: 2, Pages: 18
                Funding
                Funded by: National Nature Science Foundation of China
                Award ID: 31370089
                Award Recipient :
                Funded by: National Nature Science Foundation of China
                Award ID: 21506244
                Award Recipient :
                Funded by: State Key Development 973 Program for Basic Research of China
                Award ID: 2013CB733601
                Award Recipient :
                Funded by: National Nature Science Foundation of China
                Award ID: 31400079
                Award Recipient :
                Funded by: the Guangxi Science and Technology Development Project
                Award ID: 14125008-2-22
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100006606, Natural Science Foundation of Tianjin City;
                Award ID: 16JCYBJC23500
                Award Recipient :
                Funded by: Nature Science Foundation of Tianjin City (CN)
                Award ID: 15JCQNJC09500
                Award Recipient :
                This work was supported by National Nature Science Foundation of China (31370089 to DZ, 21506244 to GF, 31400079 to NX; http://www.nsfc.gov.cn/), State Key Development 973 Program for Basic Research of China (2013CB733601 to DZ, http://www.most.gov.cn/), Nature Science Foundation of Tianjin City (CN) (16JCYBJC23500 to DZ, 15JCQNJC09500 to DZ; http://www.tstc.gov.cn/) and the Guangxi Science and Technology Development Project (14125008-2-22 to NX; http://www.gxsti.net.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Bacteria
                Bacillus
                Bacillus Subtilis
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
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                Medicine and Health Sciences
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                Research and Analysis Methods
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                Spectrum Analysis Techniques
                Spectrophotometry
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