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Sufficient NADPH Supply and pknG Deletion Improve 4-hydroxyisoleucine Production in Recombinant Corynebacterium Glutamicum

Sufficient NADPH Supply and pknG Deletion Improve 4-hydroxyisoleucine Production in Recombinant Corynebacterium Glutamicum

Feng Shi, Meiling Zhang, Yongfu Li, Huimin Fang

Enzyme Microb Technol. 2018 Aug;115:1-8.

PMID: 29859597

Abstract:

Cofactor engineering is a common strategy to improve amino acid production. 4-hydroxyisoleucine (4-HIL), a nonproteinogenic amino acid, exhibits unique insulinotropic and insulin-sensitizing activities, therefore has potential medical value in treating diabetes. In our previous study, l-isoleucine (Ile) dioxygenase gene ido was overexpressed in an Ile-producing Corynebacterium glutamicum strain, and 4-HIL was de novo synthesized from glucose. In this study, to increase the NADPH supply, the endogenous NAD+ kinase gene ppnK and glucose-6-phosphate dehydrogenase gene zwf were co-expressed with ido. The resulting strain SL01 produced 81.12 ± 5.96 mM 4-HIL, 62% higher than the ido-mere expressing strain SN02. However, the strain SL02 co-expressing exogenous NADH kinase gene POS5 with ido grew slowly and its 4-HIL production decreased by 12%, perhaps due to the lower 2-oxoglutarate (OG) level and slightly weaker membrane permeability. To increase OG availability for 4-HIL conversion, the serine/threonine protein kinase G gene pknG was deleted and replaced by ido gene in SL02. The growth of the resulting strain SL04 was restored and 4-HIL production was improved to 84.14 ± 6.38 mM; meanwhile, the conversion ratio of Ile to 4-HIL reached up to 0.98 ± 0.01 mol/mol. Therefore, sufficient NADPH supply and OG availability may be benefit to 4-HIL de novo biosynthesis in recombinant C. glutamicum.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP781658239	4-Hydroxyisoleucine		781658-23-9	Price

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