4.5 Article

Hot spot-based engineering of ketopantoate hydroxymethyltransferase for the improvement of D-pantothenic acid production in Escherichia coli

Journal

JOURNAL OF BIOTECHNOLOGY
Volume 364, Issue -, Pages 40-49

Publisher

ELSEVIER
DOI: 10.1016/j.jbiotec.2023.01.010

Keywords

Pantothenic acid; Ketopantoate hydroxymethyltransferase; Protein engineering; Hot spot; Catalytic pocket

Ask authors/readers for more resources

In this study, a hot spot analysis was conducted on the ketopantoate hydroxymethyltransferase from Corynebacterium glutamicum, resulting in an increased production of D-PA. The double-site variant CgKPHMT-K25A/E189S exhibited higher enzyme activity and catalytic efficiency, leading to a significant increase in D-PA yield.
D-Pantothenic acid (D-PA) is an essential vitamin with wide applications. However, the biotechnological pro-duction of D-PA is still not competitive with the chemical synthesis in terms of production cost. Ketopantoate hydroxymethyltransferase is a crucial enzyme in the D-PA synthetic pathway in Escherichia coli encoded by the panB gene. Here a hot spots study was applied to a ketopantoate hydroxymethyltransferase from Corynebacterium glutamicum (CgKPHMT) to relieve the product inhibitory effect and thus improve the D-PA production. Compared with the wild type, the double-site variant CgKPHMT-K25A/E189S showed 1.8 times higher enzyme activity and 2.1 times higher catalytic efficiency, 1.88 and 3.32 times higher inhibitory constant of alpha-ketoisovalerate and D -PA, respectively. The D-PA yield using E. coli W3110 adopted the double-site variant was 41.17 g center dot L-1 within 48 h, a 9.80 g center dot L-1 increase. Structural analysis of K25A/E189S revealed the expansion of the entry channel and the change of the electric charge from negative to uncharged due to the substitution from glutamic acid to serine at site 189. Our study emphasized the positive roles of ketopantoate hydroxymethyltransferase in D-PA production and paved the way by analyzing critical enzymes in the synthetic pathway of E. coli to increase the D-PA yield.

Authors

I am an author on this paper
Click your name to claim this paper and add it to your profile.

Reviews

Primary Rating

4.5
Not enough ratings

Secondary Ratings

Novelty
-
Significance
-
Scientific rigor
-
Rate this paper

Recommended

No Data Available
No Data Available