Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Jan 26;5(1):e8860.
doi: 10.1371/journal.pone.0008860.

A novel whole-cell biocatalyst with NAD+ regeneration for production of chiral chemicals

Zijun Xiao  1 , Chuanjuan Lv, Chao Gao, Jiayang Qin, Cuiqing Ma, Zhen Liu, Peihai Liu, Lixiang Li, Ping Xu
Affiliations

A novel whole-cell biocatalyst with NAD+ regeneration for production of chiral chemicals

Zijun Xiao et al. PLoS One. .

Abstract

Background: The high costs of pyridine nucleotide cofactors have limited the applications of NAD(P)-dependent oxidoreductases on an industrial scale. Although NAD(P)H regeneration systems have been widely studied, NAD(P)(+) regeneration, which is required in reactions where the oxidized form of the cofactor is used, has been less well explored, particularly in whole-cell biocatalytic processes.

Methodology/principal findings: Simultaneous overexpression of an NAD(+) dependent enzyme and an NAD(+) regenerating enzyme (H(2)O producing NADH oxidase from Lactobacillus brevis) in a whole-cell biocatalyst was studied for application in the NAD(+)-dependent oxidation system. The whole-cell biocatalyst with (2R,3R)-2,3-butanediol dehydrogenase as the catalyzing enzyme was used to produce (3R)-acetoin, (3S)-acetoin and (2S,3S)-2,3-butanediol.

Conclusions/significance: A recombinant strain, in which an NAD(+) regeneration enzyme was coexpressed, displayed significantly higher biocatalytic efficiency in terms of the production of chiral acetoin and (2S,3S)-2,3-butanediol. The application of this coexpression system to the production of other chiral chemicals could be extended by using different NAD(P)-dependent dehydrogenases that require NAD(P)(+) for catalysis.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. SDS-PAGE analysis of recombinant E. coli.
Lane M, marker proteins; lane 1, E. coli BL21(DE3); lane 2, E. coli BL21(DE3) (pETDuet); lane 3, E. coli BL21(DE3) (pETDuet-ydjL); lane 4, E. coli BL21(DE3) (pETDuet-nox); and lane 5, E. coli BL21(DE3) (pETDuet-ydjLnox).
Figure 2
Figure 2. A recombinant E. coli whole-cell biocatalyst with BDH and the enzymatic NAD regeneration system for the production of chiral AC and (2S,3S)-2,3-BD.
Figure 3
Figure 3. Effects of biocatalysis conditions on the reaction rate.
A. pH; B. substrate concentration. E. coli BL21(DE3) (pETDuet-ydjLnox) was incubated with meso-2,3-BD in the reaction.
Figure 4
Figure 4. Time course of the recombinant E. coli whole-cell biocatalyst-mediated (3S)-AC production from meso-2,3-BD by the recombined E. coli.
The concentration of (3S)-AC (□) and meso-2,3-BD (▪) in E. coli BL21(DE3) (pETDuet-ydjLnox) ; concentrations of (3S)-AC (○) and meso-2,3-BD (•) in E. coli BL21(DE3) (pETDuet-ydjL); and concentrations of (3S)-AC (▵) and meso-2,3-BD (▴) in E. coli BL21(DE3).
Figure 5
Figure 5. GC analyses of substrates and products of the catalytic reaction (* Isoamyl alcohol was used as the internal standard).
A. Conversion of meso-2,3-BD to (3S)-AC: A1-before the reaction; A2-after the reaction; B. Conversion of (2R,3R)-2,3-BD to (3R)-AC: B1-before the reaction; B2-after the reaction; C. The starting material and products of kinetic resolution from a mixture of 2,3-BD: C1-before the reaction; C2-after the reaction.
See this image and copyright information in PMC

References

    1. Agranat I, Caner H, Caldwell J. Putting chirality to work: the strategy of chiral switches. Nat Rev Drug Discov. 2002;1:753–768. - PubMed
    1. Schoemaker HE, Mink D, Wubbolts MG. Dispelling the myths—biocatalysis in industrial synthesis. Science. 2003;299:1694–1697. - PubMed
    1. van der Donk WA, Zhao H. Recent developments in pyridine nucleotide regeneration. Curr Opin Biotechnol. 2003;14:421–426. - PubMed
    1. Kim MJ, Whitesides GM. L-Lactate dehydrogenase: substrate specificity and use as a catalyst in the synthesis of homochiral 2-hydroxy acids. J Am Chem Soc. 1988;110:2959–2964. - PubMed
    1. Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts M, et al. Industrial biocatalysis today and tomorrow. Nature. 2001;409:258–268. - PubMed

Publication types