Reconstruction of a Cofactor Self-Sufficient Whole-Cell Biocatalyst System for Efficient Biosynthesis of Allitol from d-Glucose

Abstract

The combined catalysis of glucose isomerase (GI), d-psicose 3-epimerase (DPEase), ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH) provides a convenient route for the biosynthesis of allitol from d-glucose; however, the low catalytic efficiency restricts its industrial applications. Here, the supplementation of 0.32 g/L NAD+ significantly promoted the cell catalytic activity by 1.18-fold, suggesting that the insufficient intracellular NAD­(H) content was a limiting factor in allitol production. Glucose dehydrogenase (GDH) with 18.13-fold higher activity than FDH was used for reconstructing a cofactor self-sufficient system, which was combined with the overexpression of the rate-limiting genes involved in NAD+ salvage metabolic flow to expand the available intracellular NAD­(H) pool. Then, the multienzyme self-assembly system with SpyTag and SpyCatcher effectively channeled intermediates, leading to an 81.1% increase in allitol titer to 15.03 g/L from 25 g/L d-glucose. This study provided a facilitated strategy for large-scale and efficient biosynthesis of allitol from a low-cost substrate.

Phosphorylation-Driven Production of d-Allulose from d-Glucose by Coupling with an ATP Regeneration System

“…The biomass of strains was evaluated at OD 600 using an ultraviolet–visible spectrophotometer (Mapada, Shanghai, China). The concentration of glucose, fructose, sucrose, and d -allulose of samples was determined by high-performance liquid chromatography (HPLC; G1362A, Agilent Technologies Inc., China) containing a 1260 refractive index detector (RID) and a Carbomix Pb-NP column (7.8 × 300 mm, 10 μm, 78 °C, Sepax Technologies, Suzhou, China) . In addition, intracellular ATP concentrations were detected using an ATP assay kit (Jiancheng, Nanjing, China) .…”

“…and the DAEase-encoding gene, dpe, of Clostridium bolteae were from a previous report. 25 The RhaB-encoding gene, rhaB, the PPK1 gene, ppk1, the adenylate kinase (ADK) gene, adk, and the acid phosphatase (AphA) gene, aphA, were amplified from the genome of E. coli BL21 (DE3). The gene fragments of gi, dpe, rhaB, ppk1, adk, and aphA were ligated into pRSFDuet-1/ pETDuet-1/pACYCDuet-1/pET-32a(+) plasmids using BamH I and Hind III restriction sites or Bgl II and Xho I.…”

“…The concentration of glucose, fructose, sucrose, and D-allulose of samples was determined by high-performance liquid chromatography (HPLC; G1362A, Agilent Technologies Inc., China) containing a 1260 refractive index detector (RID) and a Carbomix Pb-NP column (7.8 × 300 mm, 10 μm, 78 °C, Sepax Technologies, Suzhou, China). 25 In addition, intracellular ATP concentrations were detected using an ATP assay kit (Jiancheng, Nanjing, China). 27 The substrate conversion rate was the consumption of D-glucose/the total D-glucose supplemented, and the yield (g/g) was the D-allulose synthesized/the total D-glucose consumed.…”

“…To improve cane molasses utilization, molasses was pretreated by the microwave-assisted sulfuric acid (MASA) method described in a previous work. 25 In detail, the molasses was diluted twice with water, and dilute sulfuric acid (2.5 mol/L) was used to adjust the pH to 2.0. Then, the molasses was heated for 5 min using a microwave oven (Galanz, China) to accelerate the hydrolysis of sucrose, and the pH was adjusted to pH 7.0 with Ca(OH) 2 (2.5 mol/L) after cooling molasses to room temperature.…”

“…The GI-encoding gene, gi , of Bacillus sp. and the DAEase-encoding gene, dpe , of Clostridium bolteae were from a previous report . The RhaB-encoding gene, rhaB , the PPK1 gene, ppk1 , the adenylate kinase (ADK) gene, adk , and the acid phosphatase (AphA) gene, aphA , were amplified from the genome of E.…”