Forizymes - functionalised artificial forisomes as a platform for the production and immobilisation of single enzymes and multi-enzyme complexes

Abstract

The immobilisation of enzymes plays an important role in many applications, including biosensors that require enzyme activity, stability and recyclability in order to function efficiently. Here we show that forisomes (plant-derived mechanoproteins) can be functionalised with enzymes by translational fusion, leading to the assembly of structures designated as forizymes. When forizymes are expressed in the yeast Saccharomyces cerevisiae, the enzymes are immobilised by the self-assembly of forisome subunits to form well-structured protein bodies. We used glucose-6-phosphate dehydrogenase (G6PDH) and hexokinase 2 (HXK2) as model enzymes for the one-step production and purification of catalytically active forizymes. These structures retain the typical stimulus-response reaction of the forisome and the enzyme remains active even after multiple assay cycles, which we demonstrated using G6PDH forizymes as an example. We also achieved the co-incorporation of both HXK2 and G6PDH in a single forizyme, facilitating a two-step reaction cascade that was 30% faster than the coupled reaction using the corresponding enzymes on different forizymes or in solution. Our novel forizyme immobilisation technique therefore not only combines the sensory properties of forisome proteins with the catalytic properties of enzymes but also allows the development of multi-enzyme complexes for incorporation into technical devices.

Figure 1. Production of eYFP-tagged artificial forisomes in Saccharomyces cerevisiae.

Representative confocal microscopy images of control cells expressing eYFP alone (a) or in combination with the untagged MtSEO-F1 (b) or MtSEO-F4 (c) subunits show cytosolic fluorescence as well as non-fluorescent artificial forisomes (indicated by red asterisks) in (b,c). Yeast cells expressing N-terminal eYFP-MtSEO-F fusion proteins (d–i) or C-terminal MtSEO-F-eYFP fusion proteins (j–o) either alone or in combination with each untagged MtSEO-F protein reveal the most suitable combinations for functionalisation by forming fluorescent forisome structures. (p–s) Yeast cells expressing candidate MtSEO-F combinations as eYFP-glucose-6-phosphate dehydrogenase (G6PDH)-cIL-MtSEO-F fusion proteins for the confirmation of functional forisome assembly when larger enzyme tags are present.

Figure 2. Production and catalytic activity of G6PDH and HXK2 forizymes.

The forizymes were composed of G6PDH-cIL-MtSEO-F1 and MtSEO-F1 subunits (G6PDH forizymes) or HXK2-HAL-MtSEO-F1 and MtSEO-F1 subunits (HXK2 forizymes) and were produced in S. cerevisiae cells. (a) SDS-PAGE analysis for the verification of G6PDH forizyme production. Control forisomes composed solely of MtSEO-F1 were produced and purified simultaneously. Symbols: *untagged MtSEO-F1, •G6PDH-cIL-MtSEO-F1 fusion. (b) Western blot for the detection of the G6PDH-cIL-MtSEO-F1 fusion protein (•) in purified G6PDH forizymes using a Myc epitope-specific antibody (the Myc epitope is part of the synthetic linker cIL). The antibody did not detect the purified control forisomes composed solely of MtSEO-F1. (c) Activity assay for the purified G6PDH forizymes and MtSEO-F1 control forisomes based on measuring the formation of NADPH by monitoring the absorbance at 340 nm in a G6PDH enzyme assay. (d) SDS-PAGE analysis for the verification of HXK2 forizyme production. Control forisomes composed solely of MtSEO-F1 were produced and purified simultaneously. Symbols: *untagged MtSEO-F1, ▼HXK2-HAL-MtSEO-F1 fusion. (e) Western blot for the detection of the HXK2-HIL-MtSEO-F1 fusion protein (▼) in purified HXK2 forizymes using an HA-specific antibody. The antibody did not detect the purified control forisomes composed solely of MtSEO-F1. (f) Activity assay for the purified HXK2 forizymes and MtSEO-F1 control forisomes based on a coupled reaction with soluble G6PDH leading to the formation of NADPH. Each value was corrected for the blank measurement. Graphs show means ± SD of technical triplicates representing one of three independent forizyme purifications.

Figure 3. Stimulus-response characteristics and stability of forizymes.

(a) Microscopic images of G6PDH forizyme conformational changes caused by the addition of Ca²⁺ and subsequent removal. (b) Thermal stability of forizymes and soluble G6PDH. Both enzyme preparations were incubated at 40 °C and aliquots were withdrawn at different time points to determine residual enzyme activity. (c) To demonstrate recyclability, forizyme and agarose-immobilised G6PDH activity was monitored during multiple reaction cycles. Forizymes were centrifuged to the bottom of the reaction well prior to the reaction (small picture). Measurements are shown as means ± SD of three independent experiments for each purification. Relative enzyme activity is shown as a percentage of initial activity.

Figure 4. Assembly and incorporation efficiency of multi-enzyme forizymes that display fluorescence-tagged G6PDH and HXK2.

(a) Vector constructs for the expression of forizymes with two fluorophore-tagged enzymes, and confocal microscopy images of a corresponding yeast cell expressing a Cerulean-HXK2-cIL-MtSEO-F1/eYFP-G6PDH-cIL-MtSEO-F1/MtSEO-F1 forizyme. GPD: gluceraldehyde-3-phosphate dehydrogenase promoter; white arrowhead indicates start codon; black asterisk indicates stop codon; T indicates terminator. (b) Confocal microscopy images of purified forizymes composed of Cerulean-HXK2-cIL-MtSEO-F1/eYFP-G6PDH-cIL-MtSEO-F1/MtSEO-F1. Forizymes contain either both (white in overlay) or only one of the fluorophore-tagged enzyme-MtSEO-F1 fusions (blue or yellow, respectively). (c) Percentage of forizymes composed of Cerulean-HXK2-cIL-MtSEO-F1/MtSEO-F1, eYFP-G6PDH-cIL-MtSEO-F1/MtSEO-F1, or both fluorophore-enzyme fusions. Data are shown as means ± SD of two independent purifications with n = 183 fluorescent forizymes counted.

Figure 5. Production and analysis of HXK2-G6PDH forizymes for a two-step reaction cascade.

(a) SDS-PAGE analysis and immunoblot detection for the verification of HXK2-G6PDH forizyme production. HXK2-G6PDH forizymes are composed of MtSEO-F1 subunits and two enzyme-MtSEO-F1 fusion proteins, namely HXK2-HIL-MtSEO-F1 (HA epitope) and G6PDH-cIL-MtSEO-F1 (Myc epitope). Artificial forisomes composed of only MtSEO-F1, and single enzyme forizymes (HXK2 and G6PDH forizymes) were produced and purified simultaneously as controls. (b) Enzyme assay to detect the coupled activity of HXK2-G6PDH forizymes and controls. Coupled enzyme activity was detected using glucose (the HXK2 substrate) in the reaction mixture and no soluble G6PDH was added. Values are shown as means ± SD of technical triplicates from one of three independent forizyme purifications. The initial absorption of each forizyme preparation was set to zero. (c) Comparison of the coupled HXK2-G6PDH reaction in HXK2-G6PDH forizymes, mixed monofunctional forizymes and soluble enzymes. The activities of the HXK2 forizymes and soluble HXK were adjusted to be equivalent to the HXK2 activity of the HXK2-G6PDH forizymes, and the same applied to G6PDH activities. The graph shows one representative measurement from three experiments with independent forizyme purifications. Absorption values are means ± SD of triplicate measurements. The initial absorption of each sample was set to zero. (d) Relative reaction rate of the coupled HXK2-G6PDH reaction calculated from maximum initial rate velocities obtained in (c). One-way ANOVA and a post hoc Holm-Sidak multiple comparison test were carried out to detect significant differences between the coupled reactions of the HXK2-G6PDH forizymes, monofunctional forizymes and soluble enzymes. ***Indicates significant difference (p ≤ 0.001) compared to HXK2-G6PDH forizymes. Boxplots show the 25–75% range (box), median (horizontal line), mean (small rectangle within the box), SD (error bars) and minimum and maximum values (asterisks) from three experiments with independent forizyme purifications.