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. 2020 Oct 23;11:5385. doi: 10.1038/s41467-020-19171-4

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© The Author(s) 2020

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. 3 — a, b Analysis of P. putida galETKM multi-gene engineered strains and a control strain (P. putida galETKM, empty vector plasmid) for production of indigoidine using glucose (a) or galactose (b) as the sole carbon source in M9 minimal medium. The culture format assessed is indicated above each panel. A fed-batch mode of cultivation was implemented in the ambr® 250 cultivation format. Glucose feeding is indicated by the gray shaded area. Control samples indicated with black outlined bars or black dots. The multi-gene engineered strains are indicated with blue bars or blue dots. c Analysis of indigoidine yield across cultivation formats for both glucose-fed and galactose-fed strains. Yield from the control strain is indicated with black bars, and the multi-gene engineered strain is indicated with green bars. For the deep-well plate and shake flask experiments, data are presented as mean ± SD with an overlay of corresponding data points from n = 3 independent experiments. When the engineered strain was tested in the deep-well plate format and fed glucose, n = 6 independent experiments. For the industrially relevant formats (2 L and ambr250®) data are presented as mean ± SD with an overlay of corresponding data points from biological duplicate and sampled each in technical triplicate. d Computed production envelope using genome scale model and constraint-based methods represented as theoretical yields of indigoidine as a function of biomass yields. Possible yield space for wild-type P. putida (gray fill), predicted 16-gene cMCS (yellow fill), down-selected and implemented 14 gene set (hashed green fill). The range of observed yield space for either the control (black fill) or engineered strain (teal fill) across different production formats is represented. The realized production yield vs. biomass yield in the shake flask format for engineered and control strain are represented by a red and white dot respectively. The phase shift in production from stationary phase to exponential is not depicted. Source data are provided as a Source Data file.