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. 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.

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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).
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