Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine

BACKGROUND: Biosynthesis of L-tert-leucine (L-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of L-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully.

RESULTS: In this work, a novel fusion enzyme (GDH-R3-LeuDH) for the efficient biosynthesis of L-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH-R3-LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of L-tle by GDH-R3-LeuDH was all enhanced by twofold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose).

CONCLUSIONS: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize L-tle and reach the highest space-time yield up to now. These results demonstrated the great potential of the GDH-R3-LeuDH fusion enzyme for the efficient biosynthesis of L-tle.

Medienart:

E-Artikel

Erscheinungsjahr:

2021

Erschienen:

2021

Enthalten in:

Zur Gesamtaufnahme - volume:20

Enthalten in:

Microbial cell factories - 20(2021), 1 vom: 06. Jan., Seite 3

Sprache:

Englisch

Beteiligte Personen:

Liao, Langxing [VerfasserIn]
Zhang, Yonghui [VerfasserIn]
Wang, Yali [VerfasserIn]
Fu, Yousi [VerfasserIn]
Zhang, Aihui [VerfasserIn]
Qiu, Ruodian [VerfasserIn]
Yang, Shuhao [VerfasserIn]
Fang, Baishan [VerfasserIn]

Links:

Volltext

Themen:

EC 1.1.1.47
EC 1.4.1.9
Fusion enzyme
GMW67QNF9C
Glucose 1-Dehydrogenase
Glucose dehydrogenase
Journal Article
L-tert-leucine
Leucine
Leucine Dehydrogenase
Leucine dehydrogenase
Recombinant Fusion Proteins
Whole cells

Anmerkungen:

Date Completed 06.09.2021

Date Revised 06.09.2021

published: Electronic

Citation Status MEDLINE

doi:

10.1186/s12934-020-01501-2

funding:

Förderinstitution / Projekttitel:

PPN (Katalog-ID):

NLM319712230