Fig. 4: Combinatorial optimization to increase 3HP production.

a Genome engineering strategy and genetic constructs used for genomic integration. b Genome-overexpression and deletion of enzymes in the 1,3-propanediol to 3HP biosynthetic pathway enhanced 3HP production. Gene dddA and PHB synthesis operon phaCAB encoding 3HP degradation enzyme and PHB synthesis enzymes were deleted, respectively. 1,3-propanediol to 3HP biosynthetic pathway of P. putida KT2440 and H. bluephagenesis were inserted into H. bluephagenesis TDΔdddA and H. bluephagenesis ΔdddAΔphaCAB, respectively. Cells were grown in the defined minimal medium supplemented with 20 g L⁻¹ glucose, 20 g L⁻¹ 1,3-propanediol, and 3 g L⁻¹ acetic acid. c Characterization of 3HP and PHB produced by metabolically engineered H. bluephagenesis TD27 cultured at gradient concentrations of acetic acid to balance the redox state. Cells were grown in the defined minimal medium containing 20 g L⁻¹ glucose, 20 g L⁻¹ 1,3-propanediol, and gradient concentrations of acetic acid (g L⁻¹) 0, 3, 6, and 9, respectively. The pH was adjusted to 9 after incubation for 24 h. d Characterization of 3HP and PHB produced by metabolically engineered H. bluephagenesis TD27 cultured at a modified minimal medium containing different phosphate buffers to balancing the pH fluctuations. Cells were grown with 20 g L⁻¹ glucose, 20 g L⁻¹ 1,3-propanediol, and 6 g L⁻¹ acetic acid. All titers were obtained after 48 h cultivation at 200 r.p.m. and 37 °C. e 5, 10, 15, and 20 g L⁻¹ glucose (5G, 10G, 15G, and 20G) were co-fed with 20 g L⁻¹ 1,3-propanediol (20PDO) or 30 g L⁻¹ 1,3-propanediol (30PDO), respectively, to cultures of H. bluephagenesis TD27 grown in a modified minimal medium containing 6 g L⁻¹ acetic acid. f Time profiles of cell growth (DCM), PHB content, and concentrations of carbon sources (glucose, 1,3-propanediol, acetic acid), and 3HP production during the fed-batch culture of H. bluephagenesis TD27. 60 g L⁻¹ 1,3-propanediol and 6 g L⁻¹ acetic acid were added at 24, 32 and 38 h during fed-batch culture, respectively. For the shake-flask cultivation, The initial pH of all shake-flask studies was 9. All data represent the mean of n = 3 biologically independent samples and error bars show s.d.