Rapid Production of High-Titer d-Mannitol and Gluconate Catalyzed by a Combination of Whole-Cell and an Enzyme at High Temperatures

Abstract

d-Mannitol and d-gluconate are value-added biobased chemicals with diverse applications in food, medical, and chemical industries. d-Mannitol can be hydrogenated from hexoses (e.g., d-fructose) catalyzed by microbial fermentation, whole-cell biocatalysis, and purified-enzyme cascade biocatalysis. Here we designed a cell-enzyme system comprised of the whole cells co-expressing both hyperthermophilic mannitol dehydrogenase (MDH) and glucose dehydrogenase (GDH) as well as a hyperthermophilic xylose isomerase (XI). The whole cells have its inherent NAD enabled to implement NAD-self sufficient coupled redox reactions without externally-added NAD and aeration. Four cases of whole cells co-expressed MDH and GDH in E. coli BL21(DE3) were compared and optimized by expressing two genes separately or in tandem and changing gene alignment. Also, two-step biotransformation was developed to convert 300 g/L glucose to 129 g/L mannitol and 161 g/L gluconate in a pH-controlled bioreactor at 70 °C. This cell-enzyme system had a high volumetric productivity (10.7 g/L/h mannitol and 13.4 g/L/h gluconate) and a high product yield (91.7%). This study implied that using hyperthermophilic enzymes and cell-enzyme system could open great opportunities for industrial biomanufacturing.

Keywords

d-Mannitol / Gluconate / Whole cell catalysis / Thermophilic enzyme / NAD self-sufficient

Supplementary Materials

The following supporting information can be found at: https://www.sciepublish.com/article/pii/611. Figure S1. SDS-PAGE analysis of XI. Lane M, protein marker; Lane T, total proteins of E. coli/ pET20b-XI; Lane S, supernatant of E. coli/ pET20b-XI; Lane H, heat-treated supernatant of E. coli/ pET20b-XI.

Author Contributions

Y.-H.P.J.Z. conceived the synthetic pathway. X.L. and Y.-H.P.J.Z. designed the experiments, analyzed the data, and wrote the manuscript. P.H. conducted experiments, X.L. performed the experiments. All authors read and approved the manuscript.

Ethical Statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding

This research was funded by the National Key Research and Development Program of China, grant number 2022YFA0912000.

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

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