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. 2024 Mar 20;9(2):388-398.
doi: 10.1016/j.synbio.2024.03.005. eCollection 2024 Jun.

Engineering and finetuning expression of SerC for balanced metabolic flux in vitamin B6 production

Kai Chen  1   2   3   4 , Linxia Liu  2   3   4 , Jinlong Li  2   4   5 , Zhizhong Tian  2 , Hongxing Jin  1 , Dawei Zhang  2   3   4   5
Affiliations

Engineering and finetuning expression of SerC for balanced metabolic flux in vitamin B6 production

Kai Chen et al. Synth Syst Biotechnol. .

Abstract

Vitamin B6 plays a crucial role in cellular metabolism and stress response, making it an essential component for growth in all known organisms. However, achieving efficient biosynthesis of vitamin B6 faces the challenge of maintaining a balanced distribution of metabolic flux between growth and production. In this study, our focus is on addressing this challenge through the engineering of phosphoserine aminotransferase (SerC) to resolve its redundancy and promiscuity. The enzyme SerC was semi-designed and screened based on sequences and predicted kcat values, respectively. Mutants and heterologous proteins showing potential were then fine-tuned to optimize the production of vitamin B6. The resulting strain enhances the production of vitamin B6, indicating that different fluxes are distributed to the biosynthesis pathway of serine and vitamin B6. This study presents a promising strategy to address the challenge posed by multifunctional enzymes, with significant implications for enhancing biochemical production through engineering processes.

Keywords: Metabolic flux distribution; Multifunctional enzymes; Phosphoserine aminotransferase SerC; Protein engineering; Vitamin B6.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for [Journal name] and was not involved in the editorial review or the decision to publish this article.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests.

Figures

Fig. 1
Fig. 1
Mutation sites of SerC based on sequence. (a) The active sites of SerC protein from E. coli (PDB: 1BJO). (b) Phylogenetic tree constructed on the basis of SerC homologs chosen from γ-proteobacteria (orange) and the α-proteobacteria (purple). (c) The PN titer of SerC overexpression mutants. Data are presented as mean values ± SD from two independent biological replicates (n = 2).
Fig. 2
Fig. 2
Interactions networks of different substrates with the WT and T153G enzymes. (a), (b), and (c) represent the interaction networks of WT with substrates PLP_GLU, PLP_OHPB, and PLP_3PHP, respectively. (d), (e), and (f) depict the interaction networks of T153G with substrates PLP_GLU, PLP_OHPB, and PLP_3PHP, respectively. Ligands are shown in cyan and protein residues in magenta. Hydrogen bonds (solid blue line), hydrophobic contacts (dashed gray line), and salt bridges (dashed yellow line with yellow balls) are detected between ligands and targets.
Fig. 3
Fig. 3
Screening of heterologous SerC genes. (a) The heterologous genes mined from the GotEnzymes database based on kcat values. Magq, Magnetospira sp. QH-2; Palw, Pseudooceanicola algae; NOE, Nostoc sp. CENA543; Ava, Trichormus variabilis; Asz, Acetobacter senegalensis. (b) Multiple sequence alignment by the program ESPript (Easy Sequencing in PostScript). The red asterisk represented the position of 153 and 176 of native SerC from E. coli. (c)The PN titer and cell growth of heterologous SerC mutants. Data are presented as mean values ± SD from three independent biological replicates (n = 3), the circles or squares represent individual data points.
Fig. 4
Fig. 4
Interactions networks of different substrates with NOE_SerC. (a), (b), and (c) show the interaction networks of NOE_SerC with substrates PLP_GLU, PLP_OHPB, and PLP_3PHP, respectively. Ligands are shown in cyan and protein residues in magenta. Hydrogen bonds (solid blue line), hydrophobic contacts (dashed gray line), and salt bridges (dashed yellow line with yellow balls) are detected between ligands and targets.
Fig. 5
Fig. 5
Regulation of the expression of SerC based on the high-yield plasmid form. (a), (d), (g) Schematic diagram of the strategies for SerC regulation. (b), (e), (h) The PN titer of diferent mutants in 24 h and 48 h. (c), (f), (i) The cell growth (OD600) and residual glycerol. Eco_SerC, the native SerC of E. coli. Data are presented as mean values ± SD from three independent biological replicates (n = 3), the circles or squares represent individual data points.
Fig. 6
Fig. 6
PN and serine biosynthetic pathways and the concentrations of specific amino acids. (a). The simplified metabolic pathways of serine and PN biosynthesis by engineered E. coli strain. Enzymes: Epd erythrose 4-phosphate dehydrogenase, PdxB 4-phosphoerythronate dehydrogenase, SerC 3-phosphserine aminotransferase, PdxA 4-phosphohydroxy-l-threonine dehydrogenase, PdxJ PNP synthase, Dxs 1-deoxyxylulose 5-phosphate synthase, PdxP PNP phosphatase, SerA D-3-phosphoglycerate dehydrogenase, SerB phosphoserine phosphatase, GlyA glycine hydroxymethyltransferase. Metabolites: E4P erythrose 4-phosphate, 4 PE 4-phosphoerythronate, OHPB 2-oxo-3-hydroxy-4-phosphobutanoate, 4HTP 4-phosphohydroxy-l-threonine, PHA 3-phosphohydroxy-1-aminoacetone, DXP 1-deoxy-d-xylulose 5-phosphate, G3P glyceraldehyde 3-phosphate, 3 PG 3-phosphoglycerate, PYR pyruvate, 3PHP 3-phosphohydroxylpyruvate, Glu glutamate, α-KG α-ketoglutarate. (b) and (c). The intracellular concentrations of glutamate (Glu), serine (Ser) and glycine (Gly). Eco_SerC was the control strain (LL388 N), and the Ava_SerC was the SerC engineered strain (CK034).
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