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. 2019 Sep 18;10(1):4248.
doi: 10.1038/s41467-019-12247-w.

Modular enzyme assembly for enhanced cascade biocatalysis and metabolic flux

Wei Kang  1 , Tian Ma  2 , Min Liu  1 , Jiale Qu  1 , Zhenjun Liu  1 , Huawei Zhang  3 , Bin Shi  2 , Shuai Fu  4 , Juncai Ma  3 , Louis Tung Faat Lai  3 , Sicong He  5 , Jianan Qu  5 , Shannon Wing-Ngor Au  3 , Byung Ho Kang  3 , Wilson Chun Yu Lau  3 , Zixin Deng  2   6 , Jiang Xia  7 , Tiangang Liu  8
Affiliations

Modular enzyme assembly for enhanced cascade biocatalysis and metabolic flux

Wei Kang et al. Nat Commun. .

Abstract

Enzymatic reactions in living cells are highly dynamic but simultaneously tightly regulated. Enzyme engineers seek to construct multienzyme complexes to prevent intermediate diffusion, to improve product yield, and to control the flux of metabolites. Here we choose a pair of short peptide tags (RIAD and RIDD) to create scaffold-free enzyme assemblies to achieve these goals. In vitro, assembling enzymes in the menaquinone biosynthetic pathway through RIAD-RIDD interaction yields protein nanoparticles with varying stoichiometries, sizes, geometries, and catalytic efficiency. In Escherichia coli, assembling the last enzyme of the upstream mevalonate pathway with the first enzyme of the downstream carotenoid pathway leads to the formation of a pathway node, which increases carotenoid production by 5.7 folds. The same strategy results in a 58% increase in lycopene production in engineered Saccharomyces cerevisiae. This work presents a simple strategy to impose metabolic control in biosynthetic microbe factories.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Hierarchical MenD-MenH assemblies mediated by the RIAD–RIDD peptide interaction for biocatalysis. a The assembly of tri-enzyme units. E1, E2: enzymes; green and blue structure: RIDD dimer; black line: linker; pink structure: RIAD; one orange circle: cysteine; two orange circles: disulfide bond. b Disulfide-stabilized MenD-MenH tri-enzyme units resolved by SDS-PAGE. Blue filled circle: MenD; orange filled circle: MenH; black semilunar line: RIDD-RIAD trimer. c Hierarchical enzymes assemblies A, B, and C having different stoichiometries and sizes. Black line: assembly structure; red line: protomers of MenD; blue line: protomers of MenH. d Tetrameric structures of the assemblies on TEM. Scale bars: 100 nm (the first row) and 20 nm (the second and third row). e MenD and MenH catalyzed conversion of isochorismate to SEPHCHC and then SHCHC. f Measurement of the cascade biocatalyst by product generation in three enzyme assembly systems. Red column: Free enzyme control; purple column: Assembly A; blue column: Assembly B; dark blue column: Assembly C. g Schematic diagram of Assembly A, B, and C. Error bars indicate the standard deviations of three biological replicates. Source data are provided as a Source Data file
Fig. 2
Fig. 2
Assembly of the limiting enzymes in carotenoid biosynthesis in the bacterium cells. a The Idi and CrtE enzymes represent the limiting step between the two pathways. b Physical association of Idi and CrtE changes the transfer of C5 precursors. Red filled circle: CrtE; blue filled circle: Idi; green circle: E. coli cells. c The RIAD–RIDD peptide interaction induces redistribution of Idi-specific immunogold particles from the cytosol to the plasma membrane. Red arrows: Idi on the plasma membrane; black arrows: Idi in the cytosol. Scale bars: 500 nm. d FRET between Idi-CFP and CrtE-YFP measured by fluorescence lifetime imaging microscopy (FLIM). The cells were excited at 810 nm in a two-photon microscope and the fluorescent signals were collected at 481 nm. Error bars indicate the standard deviations of ten replicates. e Co-localization of assembled Idi-CFP and CrtE-YFP under the fluorescent microscopy. Scale bars: 2 μm. Source data are provided as a Source Data file
Fig. 3
Fig. 3
Idi-CrtE assembly increases the metabolic flux of carotenoid biosynthesis in shake-flask fermentation. a A scheme showing that the Idi-CrtE assembly installs a metabolic node that guide the flux towards carotenoid synthesis. Green filled circle: substrate; green filled rhombus: intermediate; green drop: product. Seven horns star, hexagon, octagon: enzymes of the upstream. Notched circle, blue filled rhombus: enzymes of the downstream. Black arrow: substrate intake; purple arrow: upstream pathway; blue arrow: downstream pathway; blue dotted arrow: non-target pathway. b Enzyme assembly increases carotenoid production and the cell mass. Black line: Car1; red line: Car2. c Global changes of the metabolic intermediates. Black column: Car1; red column: Car2. Error bars indicate the standard deviations of three biological replicates. Source data are provided as a Source Data file
Fig. 4
Fig. 4
Enzyme assembly gearing metabolic flux towards carotenoid biosynthesis. a Comparison of the growth curve of Car2 and Car1 in fed-batch fermentation. b Comparison of the yield of overall carotenoids of Car2 and Car1 in fed-batch fermentation. Black line: Car1; red line: Car2. c Comparison of the product of main carotenoids. d Changes of the metabolic intermediates in responsive to enzyme assembly. Black column: Car1; red column: Car2. Error bars indicate the standard deviations of three replicates. Source data are provided as a Source Data file
Fig. 5
Fig. 5
Idi-CrtE assembly increases lycopene production in yeast in fed-batch fermentation. a A scheme showing that the Idi-CrtE assembly of lycopene biosynthesis in S. cerevisiea. b Enzyme assembly increases lycopene production and the cell mass. Black line: TM606; red line: TM624. Error bars indicate the standard deviations of two replicates. Source data are provided as a Source Data file
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