Exploring linker's sequence diversity to fuse carotene cyclase and hydroxylase for zeaxanthin biosynthesis

Abstract

Fusion of catalytic domains can accelerate cascade reactions by bringing enzymes in close proximity. However, the design of a protein fusion and the choice of a linker are often challenging and lack of guidance. To determine the impact of linker parameters on fusion proteins, a library of linkers featuring various lengths, secondary structures, extensions and hydrophobicities was designed. Linkers were used to fuse the lycopene cyclase (crtY) and β-carotene hydroxylase (crtZ) from Pantoea ananatis to create fusion proteins to produce zeaxanthin. The fusion efficiency was assessed by comparing the carotenoids content in a carotenoid-producer Escherichia coli strain. It was shown that in addition to the orientation of the enzymes and the size of the linker, the first amino acid of the linker is also a key factor in determining the efficiency of a protein fusion. The wide range of sequence diversity in our linker library enables the fine tuning of protein fusion and this approach can be easily transferred to other enzyme couples.

Keywords: Artificial protein fusion; Linker; Protein engineering; Zeaxanthin.

Fig. 1

Metabolic pathway of zeaxanthin. Each block represents a module of a set of genes clustered on one plasmid (Zhang et al., 2018b). MVAP: phosphomevalonate; DMAPP: dimethylallyl pyrophosphate; GPP: geranyl pyrophosphate.

Fig. 2

A: Carotenoids content in mg/L of the strains harbouring the fusion constructs Y-linker-Z and Z-linker-Y. EAB09 is the control (ctrl) strain expressing independent enzymes. Errors bars represent the standard deviation of two independent experiments. The control strain experiment was repeated four times.

Fig. 3

Classification of the linker properties and their repartition in the library. Heading in dark grey give the category of the linker. Sub-headings in light grey represents a class within the category and numbers in these lines represent the sub total of linkers in each class.

Fig. 4

A: Carotenoid content in mg/L of the strains harbouring the fusion constructs Z-linker-Y with small, medium or large linkers. EAB09 is the control strain with non-fused enzymes. ZY is the strain with enzymes fused directly, without linker. Errors bars represent the standard deviation of two independent experiments. B: Ratio of xanthophylls to carotenes with the strains clustered according to the size of the linker. Data is presented as standard boxplots. Dark bars represent median values, boxes are the range from first to third quartile, whiskers represent minima and maxima. The dashed red line represents the ratio of xanthophylls to carotenes in the strain with independent enzymes (strain EAB09). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

A: Carotenoid content in mg/L of the strains harbouring the fusion constructs crtZ-linker-crtY with small linkers. EAB09 is the control strain with non-fused enzymes. Errors bars represent the standard deviation of two independent experiments. N_I: NON-HELICAL_INFERIOR; H_I: HELICAL_INFERIOR; H_S: HELICAL_SUPERIOR; N_S: NON-HELICAL_SUPERIOR. Non-helical and helical refers to the presumed secondary structure of the linker while inferior and superior refers to the extension of the linker. Among each category, strains are ranged by ascending order of total amount carotenoids. B: Ratio of xanthophylls to carotenes with the strains clustered according to the structure and extension of the linker. Data are presented as standard boxplots. Dark bars represent median values, boxes are the range from first to third quartile, whiskers represent minima and maxima. The dashed red line represents the ratio of xanthophylls to carotenes in the strain with independent enzymes (strain EAB09). C:SDS-PAGE of BL21 strain containing the final plasmid of the pathway with an empty vector, crtY enzyme, fusion enzymes crtZ-SHS01-crtY, crtZ-SNI01-crtY, crtZ-SNS06-crtY. M indicates the line with protein molecular weight marker. Red arrow indicates the proteins of interest. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

A: Carotenoid accumulation profile in mg/L of the strains EAB09, SHS01 and SNI01 over time. Errors bars represent the standard deviation of three independent experiments. B: Flux of carotenoids at selected time expressed in mg/L/hour/OD_600nm. Numbers in green are higher than their control equivalent (EAB09). Numbers in red are lower than their control equivalent (EAB09). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

A: Sequence alignment of small linkers depicted in Fig. 5. The dashed frame surrounds the independent enzymes. The linker sequences are framed by a plain line box. Constructions are ranged based on the ratio of xanthophylls to carotenes accumulated in the strains. Alignment performed with clustalW. B: Amino acid propensity by position in the linker based on the 1280 linkers from the online database. Propensity values are represented as a red to green colour gradient, from smaller to higher values, respectively. Top panel represents position 1 to 10 of linkers from the N-terminal. Bottom panel represents last ten residues of linkers. First raw of table is the one letter code for amino acid. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Ratio of xanthophylls to carotenes produced by crtZ-linker-crtY protein fusion with the five linkers SHI01, SHS01, SNS03, SNS04 and SNS06. The original first amino acid of the linker is indicated at the top of each panel as the Wild-Type (WT) and the ratio of carotenoids in the corresponding strain is represented by the left bar of each panel. Ratio of mutation with amino acids alanine (A), glycine (G), leucine (L), valine (V), glutamic acid (E) and lysine (K) are displayed after the WT left to right. The dashed red line represents the ratio of xanthophylls to carotenes in the strain with independent enzymes. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)