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. 2019 Jun 13;10:2615. doi: 10.1038/s41467-019-10224-x

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — Diversity of β-ionone and NG production from a randomized ATF/BS library. a Schematic overview of the multi-locus integration of β-ionone and NG pathway genes. The McrtI-, CrtE-, McrtYB-, RiCCD1-, PhCHI-, HaCHS-, At4CL-2-, AtPAL-2-, and AtC4H-ATR2-CDS donors were integrated into the X-4, XII-5, XI-3, ura3.a, X-3, XI-2, X-2, XI-5, XII-2 loci of MXFde0.2, respectively, via CRISPR/cas9-mediated multi-locus integration. Plating cells on SC-Ura/-Leu/-His/-Trp/-Lys media containing G418, hygromycin B, phleomycin, bleomycin, and nourseothricin allows maintaining the Cas9/sgRNA encoding plasmids and screening integrated cassettes. Subsequently, ATFs are expressed from an IPTG-inducible promoter in the presence of inducers and target their BSs upstream of pathway genes. β-Ionone production is quantified by HPLC, while the production of NG is detected using the FdeR biosensor. This results in yEGFP expression is detectable by flow cytometry. Black/white-striped squares, homology regions. Black asterisk, BTS1 or GGPPSbc. Orange arrow, β-ionone pathway genes. Brown arrow, naringenin pathway genes. Gray arrows, nine ATF/BS units. Black arrow, driver and effector-based biosensor. Green arrow, yEGFP fluorescence reporter. For simplicity, the IPTG-inducible promoter, terminator, and cleavage sites are not included in the figure. b Sequencing results of ATF/BS units present upstream of each CDS. The color code is given in Supplementary Fig. 1. Beige boxes indicate that an alternative gene was integrated into the genome. Black/white striped boxes indicate that no valid sequencing results were obtained. c Screening of NG production. Twenty colonies were pre-cultured in SC medium with appropriate selection markers and subsequently used to monitor yEGFP output in the absence (YPDA, 2% (w/v) glucose) and presence of inducer (YPDA, 2% (w/v) galactose and 20 µM IPTG). Gray, noninduction medium; green, induction medium. Data are geometric means ± SD (n = 9) of the fluorescence intensity obtained from three cultures, each derived from an independent yeast colony and determined in three technical replicates. AU arbitrary units. Full data are given in Supplementary Data 13. d HPLC analysis for β-ionone production. Data are means ± SD from three biological replicates. Full data are given in Supplementary Data 13