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. 2018 May 11;9:1858. doi: 10.1038/s41467-018-04211-x

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© The Author(s) 2018

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. 2 — Sequential optimization of different carotenoids. The promoter library (T7, TM1, TM2 and TM3) was used to optimize the mevalonate pathway and carotenoid production. a The whole pathway (the mevalonate pathway and lycopene biosynthetic pathway) was divided into 3 modules (AHT, MPPI and EBIA in Fig. 1). Each module was controlled by 4 different promoters, thus obtaining 64 different combinations. b Agar plates with 0.1 mM IPTG, we selected distinguished phenotypes (colonies with intense red color) and reversed engineered the colonies. Validation of the chosen genotypes in liquid media: c lycopene titers and d cell density, OD₆₀₀. On top of lycopene strain 322, we first compared two different lycopene cyclases (crtY). e The cyclase from Pantoea ananatis LMG20103 (crtY1) performed better than that from Uncultured marine bacterium HF10_19P19 (crtY2). f Improving β-carotene production by tuning the promoter strength of crtY1. g Correlation between β-carotene production and the promoter strength. Error bars, mean ± s.d., n = 3. Statistically significant difference of carotenoids produced by two cyclases was denoted *P < 0.05 (two-tailed Student’s t-test)