Construction of energy-conserving sucrose utilization pathways for improving poly-γ-glutamic acid production in Bacillus amyloliquefaciens

Abstract

Background: Sucrose is an naturally abundant and easily fermentable feedstock for various biochemical production processes. By now, several sucrose utilization pathways have been identified and characterized. Among them, the pathway consists of sucrose permease and sucrose phosphorylase is an energy-conserving sucrose utilization pathway because it consumes less ATP when comparing to other known pathways. Bacillus amyloliquefaciens NK-1 strain can use sucrose as the feedstock to produce poly-γ-glutamic acid (γ-PGA), a highly valuable biopolymer. The native sucrose utilization pathway in NK-1 strain consists of phosphoenolpyruvate-dependent phosphotransferase system and sucrose-6-P hydrolase and consumes more ATP than the energy-conserving sucrose utilization pathway.

Results: In this study, the native sucrose utilization pathway in NK-1 was firstly deleted and generated the B. amyloliquefaciens 3Δ strain. Then four combination of heterologous energy-conserving sucrose utilization pathways were constructed and introduced into the 3Δ strain. Results demonstrated that the combination of cscB (encodes sucrose permease) from Escherichia coli and sucP (encodes sucrose phosphorylase) from Bifidobacterium adolescentis showed the highest sucrose metabolic efficiency. The corresponding mutant consumed 49.4% more sucrose and produced 38.5% more γ-PGA than the NK-1 strain under the same fermentation conditions.

Conclusions: To our best knowledge, this is the first report concerning the enhancement of the target product production by introducing the heterologous energy-conserving sucrose utilization pathways. Such a strategy can be easily extended to other microorganism hosts for reinforced biochemical production using sucrose as substrate.

Keywords: Bacillus amyloliquefaciens; Energy-conserving pathway; Phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS); Poly-γ-glutamic acid (γ-PGA); Sucrose permease; Sucrose phosphorylase; Sucrose utilization pathway.

Fig. 1

Possible pathways for sucrose utilization in nature. a Sucrose utilization pathway consists of PTS and sucrose-6-P (β-d-Fru-(2 → 1)-α-d-Glc 6-P) hydrolase; b sucrose utilization pathway consists of a putative PTS system and sucrose-6′-P (β-d-Fru 6-P-(2 → 1)-α-d-Glc) phosphorylase; c non-PTS (non-phosphotransferase system) sucrose utilization pathway consists of sucrose permease and sucrase; d non-PTS sucrose utilization pathway consists of sucrose permease and sucrose phosphorylase

Fig. 2

Schematic for the replacement of the sucrose utilization pathway in Bacillus amyloliquefaciens NK-1 strain. a The native sucrose utilization pathway in NK-1; b the to-be-introduced heterologous energy-conserving sucrose utilization pathway. sacP, PTS sucrose-specific enzyme IIBC component; ptsG, PTS glucose-specific EIIA component; ptsH, phosphocarrier protein HPr; ptsI, PTS enzyme I; sacA, sucrase-6-phosphate hydrolase; RBAM_031820, sucrase-6-phosphate hydrolase; pyk, pyruvate kinase; cscB, sucrose permease; sucP, sucrose phosphorylase; gtfA, sucrose phosphorylase; fruK, fructose kinase; pgcA, phosphoglucomutase; ldh, lactic dehydrogenase; pta, phosphotransacetylase; pfhABC, pyruvate dehydrogenase; ackA, acetate kinase. The purple words indicated the metabolites measured in this work

Fig. 3

Construction of the energy-conserving sucrose utilization pathways. The four combinations of pathways: CEG [cscB (E. coli) + gtfA (S. mutans)], CES [cscB (E. coli) + sucP (B. adolescentis)], CBS [cscB (B. lactis) + sucP (B. adolescentis)] and CBG [cscB (B. lactis) + gtfA (S. mutans)] were put under P₄₃ promoter and expressed by pWH1520 plasmid

Fig. 4

γ-PGA fermentation results of various strains. All the strains were cultured at γ-PGA fermentation medium for 48 h prior to the measurement of the dry cell weight (DCW) and γ-PGA prodcution. Values represent mean ± SD of triplicates

Fig. 5

Measurement of metabolites associated with sucrose utilization in NK-1, 3Δ, 3Δ-CEG, 3Δ-CBG, 3Δ-CES and 3Δ-CBS strains. a Glucose-1-phosphate; b PEP; c Pyruvate; d Acetyl-CoA; e Acetate; f Lactate; g ATP

Fig. 6

Sucrose consumption in NK-1, 3Δ, 3Δ-CEG, 3Δ-CBG, 3Δ-CES and 3Δ-CBS strains. All the strains were cultured in the γ-PGA fermentation medium for 48 h prior to measure their sucrose consumption. The results indicated the sucrose consumption amounts in 1 L medium. Values represent mean ± SD of triplicates