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. 2021 Mar 3;12:1411. doi: 10.1038/s41467-021-21654-x

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© The Author(s) 2021

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. 5 — a PHB-b-P4HB synthesis pathway from glucose as a sole carbon source, which is grouped into three modules including syntheses of 3-hydroxybutyrate-CoA synthesis (highlighted in light green, activated at 37 ℃), 4-hydroxubutyrate-CoA (highlighted in light orange, activated at 30 °C) and constitutive expression of PHA synthase PhaC encoded by phaC controlled by porin promoter, respectively. b Debugging the expression sensitivity of 3-hydroxybutyrate-CoA and 3-hydroxybutyrate-CoA synthesis pathways under the control of tac promoter induced by various concentrations of IPTG, respectively. 4HB synthesis pathway has more active performance in a low expression level with saturated P4HB accumulation in the presence of over 2 mg L-1 IPTG. Error bars, mean ± s.d. of three replicates. c Fed-batch fermentative production of PHB-b-P4HB was carried out for 3 times utilizing the same bioprocess under changing temperatures from 30 °C to 37 °C after 18 h growth in a 7 L bioreactor. Upper panel displays the accumulations of block PHB and P4HB in PHB-b-P4HB during the bioprocessing; middle panel shows 4HB molar fractions during the formation of PHB-b-P4HB; bottom panel displays the accumulative tendency of cell dry weights and PHA content of time course. d D-value of PHB-b-P4HB production from Batch-I determined by NMR C¹³. x-axis: chemical shift (ppm); y-axis: intensity of chemical shift. e Thermodynamics assays of PHB-b-P4HB from three repeated batches including melting temperature (T_m) and glass transfer temperature (T_g), using differential scanning calorimetry (DSC). PHB from Sigma and P4HB from Tepha were used as control for comparative analysis. x-axis: temperature range of cooling-heating cycles; y-axis: relative heat flow rate generated from DSC scanning.