Activating the d-Tagatose Production Capacity of Escherichia coli with Structural Insights into C4 Epimerase Specificity

Abstract

d-Tagatose, a rare low-calorie sweetener, is ideal for beverages due to its high solubility and low viscosity. Current enzymatic production methods from d-galactose or d-galactitol are limited by reaction reversibility, affecting the yield and purity. This study demonstrates that Escherichia coli harbors a thermodynamically favorable pathway for producing d-tagatose from d-glucose via phosphorylation-epimerization-dephosphorylation steps. GatZ and KbaZ, annotated as aldolase chaperones, exhibit C4 epimerization activity, converting d-fructose-6-phosphate to d-tagatose-6-phosphate. Structural analysis reveals active site differences between these enzymes and class II aldolases, indicating functional divergence. By exploiting the strains' inability to metabolize d-tagatose, carbon starvation was applied to remove sugar byproducts. The engineered strains converted 45 g L^-1 d-glucose to d-tagatose, achieving a titer of 7.3 g L^-1 and a productivity of 0.1 g L^-1 h^-1 under test tube conditions. This approach highlights E. coli as a promising host for efficient d-tagatose production.

Keywords: d-tagatose; metabolic engineering; rare sugars.

Figure 1

Strategies for the biosynthesis of d-tagatose. a) “Izumoring strategies” for d-tagatose production. b) Phosphorylation/dephosphorylation strategy for d-tagatose. c) The proposed biosynthetic production of d-tagatose in E. coli. Deleted steps are in blue. Additionally expressed steps are in red. PTS, the phosphotransferase system; GatZ, putative tagatose-1,6-bisphosphate aldolase 2 chaperone; KbaZ, putative tagatose-1,6-bisphosphate aldolase 1 chaperone; HxpA, hexitol phosphatase A; Zwf, NADP⁺-dependent glucose-6-phosphate dehydrogenase; Pgm, phosphoglucomutase; PfkA, 6-phosphofructokinase 1; PfkB, 6-phosphofructokinase 2; AlsE, d-allulose-6-phosphate 3-epimerase; ManA, mannose-6-phosphate isomerase; GatY, tagatose-1,6-bisphosphate aldolase 2; KbaY, tagatose-1,6-bisphosphate aldolase 1.

Figure 2

d-tagatose production capability ofE. coli. Cells were grown in M9P media with 10 g L^–1 glucose at 37 °C to OD₆₀₀ ∼ 0.4, then grown at 30 °C for 24 h. At OD₆₀₀ ∼ 0.4, 1 mM IPTG was added (c, d). a) d-tagatose production in AL1050 (MG1655 + lacI^qtetR spec^R), AL3755 (AL1050 with ΔpfkA), and AL4240 (AL1050 with ΔpfkA Δzwf) (Table 1). b) Effect of each epimerase gene deletion on d-tagatose production in AL4240. c) d-tagatose production in AL4424 (AL1050 with ΔpfkA Δzwf ΔgatZ) with and without additional expression of gatZ. (Table 1 and 2). d) Effect of additional expression of each phosphatase gene and gatZ on d-tagatose production in AL4424 (AL1050 with ΔpfkA Δzwf ΔgatZ). Error bars indicate s.d. (n = 3 biological replicates).

Figure 3

Modulating expression of genes for d-tagatose production. Cells were grown in M9P media with 10 g L^–1 glucose at 37 °C to OD₆₀₀ ∼ 0.4, then grown at 30 °C for 24 h. At OD₆₀₀ ∼ 0.4, 1 mM IPTG was added when required. a) Each candidate epimerase gene was expressed along with hxpA under P_LlacO1 promoter (Table 2) in AL4424 (AL1050 with ΔpfkA Δzwf ΔgatZ, Table 1). b) Either gatZ or kbaZ and hxpA were expressed under P_LlacO1 or P_gadB. The start codon of hxpA was changed from GTG to ATG (hxpA*). Errors indicate s.d. (n = 3 biological replicates).

Figure 4

Structure analysis of GatZ. a) Predicted substrate-binding mode of the AlphaFold structure of GatZ with F6P. b) Substrate-binding model of FBA (PDB: 3GAY) with d-tagatose-1,6-diphosphate. Zn²⁺ ions are shown as gray spheres in the structural models in (a) and (b). c) Reaction mechanism of FBA catalyzing the conversion of fructose-1,6-bisphosphate to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). d) Reaction mechanism of GatZ catalyzing the conversion of F6P to T6P.

Figure 5

The effect of gene deletions on d-tagatose production. Cells were grown in M9P media with 10 g L^–1 glucose to OD₆₀₀ ∼ 0.4 at 37 °C, then grown at 30 °C for 24 h. Errors indicate s.d. (n = 3 biological replicates).

Figure 6

High cell density d-tagatose production. Cultures were grown in M9P media with 40 g L^–1 (a–c) and 15 g L^–1 (d–f) glucose concentration at 37 °C until an OD₆₀₀ of ∼0.4–0.6. Induced with 1 mM IPTG if necessary and grown for a further 30 min. Cultures were then spun down and resuspended in M9P media with 40 g L^–1 (a–c) and 15 g L^–1 (d–f) glucose, induced with 1 mM IPTG if necessary, to an OD₆₀₀ of ∼10 and grown at 30 °C. Each day M9P media with 40 g L^–1 (a–c) and 15 g L^–1 (d–f) glucose was added to the production media. (a and d) d-tagatose production, (b and e) d-fructose production, and (c and f) d-mannitol production. Error bars indicate s.d. (n = 3 biological replicates).