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
. 2021 Dec 17:9:795885.
doi: 10.3389/fbioe.2021.795885. eCollection 2021.

Using Unnatural Protein Fusions to Engineer a Coenzyme Self-Sufficiency System for D-Phenyllactic Acid Biosynthesis in Escherichia coli

Zhao Qin  1 , Dan Wang  1 , Ruoshi Luo  1 , Tinglan Li  1 , Xiaochao Xiong  2 , Peng Chen  1
Affiliations

Using Unnatural Protein Fusions to Engineer a Coenzyme Self-Sufficiency System for D-Phenyllactic Acid Biosynthesis in Escherichia coli

Zhao Qin et al. Front Bioeng Biotechnol. .

Abstract

The biosynthetic production of D-penyllactic acid (D-PLA) is often affected by insufficient supply and regeneration of cofactors, leading to high production cost, and difficulty in industrialization. In this study, a D-lactate dehydrogenase (D-LDH) and glycerol dehydrogenase (GlyDH) co-expression system was constructed to achieve coenzyme NADH self-sufficiency and sustainable production of D-PLA. Using glycerol and sodium phenylpyruvate (PPA) as co-substrate, the E. coli BL21 (DE3) harboring a plasmid to co-express LfD-LDH and BmGlyDH produced 3.95 g/L D-PLA with a yield of 0.78 g/g PPA, similar to previous studies. Then, flexible linkers were used to construct fusion proteins composing of D-LDH and GlyDH. Under the optimal conditions, 5.87 g/L D-PLA was produced by expressing LfD-LDH-l3-BmGlyDH with a yield of 0.97 g/g PPA, which was 59.3% increased compared to expression of LfD-LDH. In a scaled-up reaction, a productivity of 5.83 g/L/h was reached. In this study, improving the bio-catalytic efficiency by artificial redox self-equilibrium system with a bifunctional fusion protein could reduce the bio-production cost of D-PLA, making this bio-production of D-PLA a more promising industrial technology.

Keywords: D-PLA; coenzyme self-sufficiency; d-Lactate dehydrogenase; fusion protein; glycerol dehydrogenase; phenyllactic acid.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The program of redox self-balanced coenzyme regeneration and whole-cell synthesis of D-PLA.
FIGURE 2
FIGURE 2
(A) Time course of the consumption of PPA (by CP101, CP102), and glycerol (by CP103, CP104); (B) Time course of the production of PLA by CP100, CP101, CP102, CP103, and CP104; (C) The effect of D-LDH and NADH concentration on D-PLA titer; (D) Time course of the production of D-PLA by enzyme catalysis. Data are means ± SD (n = 3).
FIGURE 3
FIGURE 3
(A) Time course of the production of D-PLA by D-LDH and GlyDH co-expressed strains CP201, CP202, CP203, and CP204; (B) Time course of intracellular NADH concentration. Data are means ± SD (n = 3).
FIGURE 4
FIGURE 4
Time course of the production of D-PLA by fusion protein strains CP301, CP302, CP303, and CP304. Data are means ± SD (n = 3).
FIGURE 5
FIGURE 5
(A) Effect of IPTG concentration on D-PLA titer; (B) Effect of pH on D-PLA titer; (C) Effect of temperature on D-PLA titer; (D) Effect of substrate (glycerol, relative to PPA) on D-PLA titer. Data are means ± SD (n = 3).
FIGURE 6
FIGURE 6
D-PLA production by whole-cell bioconversion using the CP303 strain in a 5 L bioreactor. Data are means ± SD (n = 3).
See this image and copyright information in PMC

References

    1. Aalbers F. S., Fraaije M. W. (2017). Coupled Reactions by Coupled Enzymes: Alcohol to Lactone cascade with Alcohol Dehydrogenase-Cyclohexanone Monooxygenase Fusions. Appl. Microbiol. Biotechnol. 101 (20), 7557–7565. 10.1007/s00253-017-8501-4 - DOI - PMC - PubMed
    1. Aalbers F. S., Fraaije M. W. (2019). Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes. Chembiochem 20 (1), 20–28. 10.1002/cbic.201800394 - DOI - PMC - PubMed
    1. Abadli M., Dewasme L., Tebbani S., Dumur D., Vande Wouwer A. (2021). An Experimental Assessment of Robust Control and Estimation of Acetate Concentration in Escherichia coli BL21(DE3) Fed-Batch Cultures. Biochem. Eng. J. 174, 108103. 10.1016/j.bej.2021.108103 - DOI
    1. Anami Y., Yamazaki C. M., Xiong W., Gui X., Zhang N., An Z., et al. (2018). Glutamic Acid-Valine-Citrulline Linkers Ensure Stability and Efficacy of Antibody-Drug Conjugates in Mice. Nat. Commun. 9 (1), 2512. 10.1038/s41467-018-04982-3 - DOI - PMC - PubMed
    1. Béguin P. (1999). Hybrid Enzymes. Curr. Opin. Biotechnol. 10 (4), 336–340. 10.1016/s0958-1669(99)80061-5 - DOI - PubMed

LinkOut - more resources