Engineering of a type III rubisco from a hyperthermophilic archaeon in order to enhance catalytic performance in mesophilic host cells

Abstract

The hyperthermophilic archaeon Thermococcus kodakaraensis harbors a type III ribulose 1,5-bisphosphate carboxylase/oxygenase (Rbc(Tk)). It has previously been shown that Rbc(Tk) is capable of supporting photoautotrophic and photoheterotrophic growth in a mesophilic host cell, Rhodopseudomonas palustris Delta3, whose three native Rubisco genes had been disrupted. Here, we have examined the enzymatic properties of Rbc(Tk) at 25 degrees C and have constructed mutant proteins in order to enhance its performance in mesophilic host cells. Initial sites for mutagenesis were selected by focusing on sequence differences in the loop 6 and alpha-helix 6 regions among Rbc(Tk) and the enzymes from spinach (mutant proteins SP1 to SP7), Galdieria partita (GP1 and GP2), and Rhodospirillum rubrum (RR1). Loop 6 of Rbc(Tk) is one residue longer than those found in the spinach and G. partita enzymes, and replacing Rbc(Tk) loop 6 with these regions led to dramatic decreases in activity. Six mutant enzymes retaining significant levels of Rubisco activity were selected, and their genes were introduced into R. palustris Delta3. Cells harboring mutant protein SP6 displayed a 31% increase in the specific growth rate under photoheterotrophic conditions compared to cells harboring wild-type Rbc(Tk). SP6 corresponds to a complete substitution of the original alpha-helix 6 of Rbc(Tk) with that of the spinach enzyme. Compared to wild-type Rbc(Tk), the purified SP6 mutant protein exhibited a 30% increase in turnover number (k(cat)) of the carboxylase activity and a 17% increase in the k(cat)/K(m) value. Based on these results, seven further mutant proteins were designed and examined. The results confirmed the importance of the length of loop 6 in Rbc(Tk) and also led to the identification of specific residue changes that resulted in an increase in the turnover number of Rbc(Tk) at ambient temperatures.

FIG. 1.

Mutant design of Rbc_Tk. Shown is an alignment of the loop 6 and α-helix 6 regions of Rubisco proteins from T. kodakaraensis KOD1 (Tk) (type III; GenBank accession number BAD86479), spinach (Sp) (type I; accession number P00875) (red), G. partita (Gp) (type I; accession number BAA75796) (blue), and R. rubrum (Rr) (type II; accession number P04718) (green). Active-site residues are indicated with asterisks. Residues identical in all four enzymes are shaded. Gaps in the alignment are indicated with hyphens. Secondary structural elements are shown above the alignment. (A and B) Sequences of mutant Rbc_Tk proteins produced in this study. Exchanged residues are indicated with the colors of the sequence on which the mutations were based. The positions where single residues were deleted in ΔT317 and ΔA318 are underlined. (B) Sequences of mutant proteins based on SP4.

FIG. 2.

Activity levels of partially purified wild-type and mutant Rbc_Tk proteins. Carboxylase activities of partially purified wild-type and mutant Rbc_Tk proteins are indicated. Activity measurements were performed at 25°C. Measurements were performed at least in duplicate.

FIG. 3.

Growth of R. palustris Δ3 recombinant strains harboring wild-type and mutant Rbc_Tk proteins. Representative growth curves of Δ3cRbc_Tk (open circles) and strains harboring mutant proteins (closed circles) under photoheterotrophic conditions are shown. Heterotrophically grown cells were washed and inoculated into photoheterotrophic medium. The average specific growth rate of seven independent growth experiments of Δ3cRbc_Tk (μ_w) was 0.30 ± 0.02 day⁻¹. The average specific growth rate of each strain harboring a mutant Rbc_Tk (μ_m) (three to five measurements) was compared with that of μ_w and is indicated in each panel. (A) Growth of strains harboring mutant Rubiscos that displayed relevant levels of activity shown in Fig. 2. (B) Growth of strains harboring mutant Rubisco proteins based on SP4. A representative growth curve for Δ3cSP4 is also indicated with gray circles as a comparison.