Rubisco substitutions predicted to enhance crop performance through carbon uptake modelling.
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ABSTRACT:
SUBMITTER: Iqbal WA
PROVIDER: S-EPMC8411856 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a pivotal enzyme that mediates the fixation of CO2. As the most abundant protein on earth, Rubisco has a significant impact on global carbon, water, and nitrogen cycles. However, the significantly low carboxylation activity and competing oxygenase activity of Rubisco greatly impede high carbon fixation efficiency. This review first summarizes the current efforts in directly or indirectly modifying plant Rubisco, which has been challenging due to its high conservation and limitations in chloroplast transformation techniques. However, recent advancements in understanding Rubisco biogenesis with the assistance of chaperones have enabled successful heterologous expression of all Rubisco forms, including plant Rubisco, in microorganisms. This breakthrough facilitates the acquisition and evaluation of modified proteins, streamlining the measurement of their activity. Moreover, the establishment of a screening system in E. coli opens up possibilities for obtaining high-performance mutant enzymes through directed evolution. Finally, this review emphasizes the utilization of Rubisco in microorganisms, not only expanding their carbon-fixing capabilities but also holding significant potential for enhancing biotransformation processes.
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Project description: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.
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Project description:Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild type in air supplemented with 1% CO2. This study represents a step towards the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.
| S-EPMC9940876 | biostudies-literature