Hyperproduction of 3-hydroxypropionate by Halomonas bluephagenesis

Abstract

3-Hydroxypropionic acid (3HP), an important three carbon (C3) chemical, is designated as one of the top platform chemicals with an urgent need for improved industrial production. Halomonas bluephagenesis shows the potential as a chassis for competitive bioproduction of various chemicals due to its ability to grow under an open, unsterile and continuous process. Here, we report the strategy for producing 3HP and its copolymer poly(3-hydroxybutyrate-co-3-hydroxypropionate) (P3HB3HP) by the development of H. bluephagenesis. The transcriptome analysis reveals its 3HP degradation and synthesis pathways involving endogenous synthetic enzymes from 1,3-propanediol. Combing the optimized expression of aldehyde dehydrogenase (AldD_Hb), an engineered H. bluephagenesis strain of whose 3HP degradation pathway is deleted and that overexpresses alcohol dehydrogenases (AdhP) on its genome under a balanced redox state, is constructed with an enhanced 1.3-propanediol-dependent 3HP biosynthetic pathway to produce 154 g L^-1 of 3HP with a yield and productivity of 0.93 g g^-1 1,3-propanediol and 2.4 g L^-1 h^-1, respectively. Moreover, the strain could also accumulate 60% poly(3-hydroxybutyrate-co-32-45% 3-hydroxypropionate) in the dry cell mass, demonstrating to be a suitable chassis for hyperproduction of 3HP and P3HB3HP.

Fig. 1. Metabolic engineering of H. bluephagenesis for 3HP production.

a Overall strategies for 3HP production involving a combination of marker-free dddA deletion and plasmid-expressing dhaT_Pp, aldD_Pp. Red color “X” indicates inactivation of metabolic pathways. Gene dhaT_Pp encodes P. putida KT2440 alcohol dehydrogenase, aldD_Pp encodes P. putida KT2440 aldehyde dehydrogenase, adhP encodes H. bluephagenesis alcohol dehydrogenase, aldD_Hb encodes H. bluephagenesis aldehyde dehydrogenase. DddA putative 3-hydroxypropionate dehydrogenase in H. bluephagenesis, DddC CoA-acylating methylmalonate-semialdehyde dehydrogenase in H. bluephagenesis, PhaA 3-ketothiolase, PhaB acetoacetyl-CoA reductase, PhaC PHA synthase. b 3HP production by E. coli and H. bluephagenesis harboring plasmid p30, respectively. c PHB production by H. bluephagenesis cultured on 5 g L⁻¹ 3HP as a sole carbon source. d Influence of 3HP on cell metabolism at the transcriptional level in H. bluephagenesis. Volcano plot and fold change of gene expression of differentially expressed gene (DEG) distribution. A corrected P-value of 0.05 and log2 (fold change) of 1 were set as the thresholds for significantly differential expression. Red, upregulated genes; blue, downregulated genes. qRT-PCR analysis of several putative upregulated dehydrogenases expression levels in the 3HP containing cultures compared to that of the glucose-containing ones. e 3HP and PHB production by H. bluephagenesis TDΔdddA harboring plasmid p30. Cells were grown in a defined minimal medium supplemented with 30 g L⁻¹ glucose and 10 g L⁻¹ 1,3-propanediol. All titers were obtained after 48 h cultivation at 200 r.p.m. at 37 °C. 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. Two-tailed Student’s t-tests were performed to determine the statistical significance for two-group comparisons. DCM dry cell mass, PHB polyhydroxybutyrate.

Fig. 2. Selection of endogenous enzymes catalyzing 1,3-propanediol to 3HP biosynthesis.

a Influence of 1,3-propanediol on cell metabolism at the transcriptional level in H. bluephagenesis TDΔdddA. Volcano plot and fold change of gene expression of differentially expressed gene (DEG) distribution. A corrected P-value of 0.05 and log2 (fold change) of 1 were set as the thresholds for significantly differential expression. Red, upregulated genes; blue, downregulated genes. b 3HP production by H. bluephagenesis TDΔdddA overexpressing different biosynthetic genes using 1,3-propanediol as a carbon source. Plasmid pKS and p30 were used as the negative and positive controls, respectively. Gene dhaT_Pp encodes P. putida KT2440 alcohol dehydrogenase, aldD_Pp encodes P. putida KT2440 aldehyde dehydrogenase, aldD_Hb encodes H. bluephagenesis aldehyde dehydrogenase, GME_RS01345 encodes NAD(P)-dependent alcohol dehydrogenase; GME_05160 encodes zinc-binding alcohol dehydrogenase, GME_RS01585 encodes zinc-binding dehydrogenase, GME_RS00365 encodes alcohol dehydrogenase AdhP. c 3HP production and d cell growth (DCM) and PHB content under optimal expression levels of aldehyde dehydrogenase. A stronger RBS was cloned instead of the original construct p58 to form plasmid p59. A stronger promoter Pporin replaced the original construct p58 to generate plasmid p60 (Supplementary Table 5). Cells were grown in the defined minimal medium supplemented with 30 g L⁻¹ glucose, 10 g L⁻¹ 1,3-propanediol, and 3 g L⁻¹ acetic acid. All titers were obtained after 48 h cultivation at 200 r.p.m. and 37 °C. Initial pH of all shake-flask studies was 9. Two-tailed Student’s t-tests were performed to determine the statistical significance for two-group comparisons. e The enzyme activity of AldD_Hb was assayed using the recombinant H. bluephagenesis TDΔdddA harboring p58-p60 deleted with adhP gene cultivated in 60-LB medium for 24 h. The comparison of activities was conducted using a crude extract. All data represent the mean of n = 3 biologically independent samples and error bars show s.d.

Fig. 3. Selection of efficient enzymes for biosynthesis of 3HP in H. bluephagenesis.

a Construction of the plasmids to express enzymes from different species. Following plasmids were constructed: plasmid p90 expressing adhP gene with a His-tag on C-terminal, p91 expressing aldD_Hb gene with a His-tag on C-terminal, p92 expressing dhaT_Pp gene with a His-tag on C-terminal and p98 expressing aldD_Pp gene with a His-tag on N-terminal. The aldH gene, with a His-tag on N-terminal, was amplified via PCR using E. coli genomic DNA as a template and cloned into the p58 by replacing the aldD_Hb gene. This plasmid is labeled as p99. The dhaT_Kp or puuC gene, with a His-tag, was amplified via PCR using K. pneumoniae DSMZ 2026 genomic DNA as a template and cloned into the p58 by replacing the adhP or aldD_Hb gene. These plasmids were labeled as p95 and p100. 3HP production of different alcohol dehydrogenases (b), and different aldehyde dehydrogenases (c). Cells were grown in the defined minimal medium containing 20 g L⁻¹ glucose, 20 g L⁻¹ 1,3-propanediol, and 3 g L⁻¹ acetic acid. All titers were obtained after 48 h cultivation at 200 r.p.m. and 37 °C. The initial pH of all shake-flask studies was 9. All data represent the mean of n = 2 biologically independent. The comparation of the various alcohol dehydrogenases oxidative activities (d), and various aldehyde dehydrogenases activities (e) in a purified enzyme. All data represent the mean of n = 3 biologically independent samples and error bars show s.d.

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.

Fig. 5. Enzyme screening for improving biosynthesis of P3HB3HP by engineered H. bluephagenesis TD27.

a Strategies for microbial co-production of 3HP and P3HB3HP involving a combination of genome-based gene expression and plasmid-based gene expression. Red color “X” indicates the inactivation of metabolic pathways. Gene adhP encodes 1,3-propanediol dehydrogenase from H. bluphagenesis, aldD_Hb encodes aldehyde dehydrogenase from H. bluphagenesis, pcs encodes coenzyme A ligase domain from Chloroflexus aurantiacus, pduP encodes propionaldehyde dehydrogenase from Salmonella typhimurium, prpE encodes propionyl-CoA synthase from E. coli, acoE encodes acetyl-CoA synthase from Ralstonia entropha. Genes were expressed under the control of constitutive promoters in a multiple-copy episomal plasmid pKS. b P3HB3HP contents and monomer compositions generated by engineered H. bluephagenesis TD27 overexpressing different 3-hydroxypropionyl-CoA biosynthetic genes. Cells were grown in the modified minimal medium containing 20 g L⁻¹ glucose, 10 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. 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. c ¹H NMR of P(3HB-co-40% 3HP) copolymers produced by the engineered H. bluephagenesis TD27 overexpressing pcs. TMS tetramethylsilane (internal standard).