Improved recombinant expression and purification of functional plant Rubisco

Wilson, Robert H.; Thieulin-Pardo, Gabriel; Hartl, F. Ulrich; Hayer‐Hartl, Manajit

doi:10.1002/1873-3468.13352

Cited by 32 publications

(29 citation statements)

References 45 publications

(71 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An alternative to evolving Rs Rubisco might be to evolve plant Rubisco itself using an RDE screen incorporating the chloroplast protein folding machinery [53,56]. As the components of the plant Rubisco expression systems are primarily T7-promoter regulated [37,57], our JM109(DE3)-based RDE3 screen appears to be a suitable selection host for such a directed evolution endeavor.…”

Section: Discussionmentioning

confidence: 99%

Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction

Zhou

Whitney

2019

IJMS

View full text Add to dashboard Cite

Inaccuracies in biochemically characterizing the amount and CO2-fixing properties of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase continue to hamper an accurate evaluation of Rubisco mutants selected by directed evolution. Here, we outline an analytical pipeline for accurately quantifying Rubisco content and kinetics that averts the misinterpretation of directed evolution outcomes. Our study utilizes a new T7-promoter regulated Rubisco Dependent Escherichia coli (RDE3) screen to successfully select for the first Rhodobacter sphaeroides Rubisco (RsRubisco) mutant with improved CO2-fixing properties. The RsRubisco contains four amino acid substitutions in the large subunit (RbcL) and an improved carboxylation rate (kcatC, up 27%), carboxylation efficiency (kcatC/Km for CO2, increased 17%), unchanged CO2/O2 specificity and a 40% lower holoenzyme biogenesis capacity. Biochemical analysis of RsRubisco chimers coding one to three of the altered amino acids showed Lys-83-Gln and Arg-252-Leu substitutions (plant RbcL numbering) together, but not independently, impaired holoenzyme (L8S8) assembly. An N-terminal Val-11-Ile substitution did not affect RsRubisco catalysis or assembly, while a Tyr-345-Phe mutation alone conferred the improved kinetics without an effect on RsRubisco production. This study confirms the feasibility of improving Rubisco by directed evolution using an analytical pipeline that can identify false positives and reliably discriminate carboxylation enhancing amino acids changes from those influencing Rubisco biogenesis (solubility).

show abstract

Section: Discussionmentioning

confidence: 99%

Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction

Zhou

Whitney

2019

IJMS

View full text Add to dashboard Cite

show abstract

“…NosL8S8ΔN exhibited wild-type carboxylation activity, but could no longer be reactivated by NosRca upon inhibition with CABP ( Figure 3D). To determine whether this mechanism is conserved in plants, we produced the analogous N-terminal RbcL truncation in Rubisco of N. tabacum (NtL8S8ΔN) by recombinant expression in the presence of chloroplast chaperonins and assembly factors (Aigner et al, 2017;Lin et al, 2019;Wilson et al, 2019) ( Figure S1A…”

Section: Nosrca Binds the N-terminus Of Rbcl In The Central Porementioning

confidence: 99%

Dual Role of a Rubisco Activase in Metabolic Repair and Carboxysome Organization

Hayer‐Hartl

Flecken

Wang

et al. 2020

Preprint

View full text Add to dashboard Cite

Rubisco, the key enzyme of CO2 fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone Rubisco activase (Rca) in a process that is not well understood. Here we performed a structural and mechanistic analysis of cyanobacterial Rca, a close homolog of plant Rca. In the Rca:Rubisco complex, Rca is positioned over the Rubisco catalytic site under repair and pulls the N-terminal tail of the large Rubisco subunit (RbcL) into the hexamer pore. Simultaneous displacement of the C-terminus of the adjacent RbcL opens the catalytic site for inhibitor release.An alternative interaction of Rca with Rubisco is mediated by C-terminal domains that resemble the small Rubisco subunit. These domains, together with the N-terminal AAA+ hexamer, ensure that Rca is packaged with Rubisco into carboxysomes. Cyanobacterial Rca is a dual-purpose protein with functions in Rubisco repair and carboxysome organization.

show abstract

“…Previously, the development of a tobacco master line, where the rbc L gene had been replaced with a homolog from Rhodospirillum rubrum encoding a form II Rubisco, allowed modification of the rbc L gene and rapid characterization of the subsequent mutant Rubisco enzymes within 6–9 weeks of transformation, although this master line required a high‐CO 2 environment to grow in soil (Whitney and Sharwood, 2008). Recently, functional Rubisco enzymes from Arabidopsis and tobacco were successfully assembled in E. coli with the co‐expression of at least five chaperones (Aigner et al ., 2017; Wilson et al ., 2019; Lin et al ., 2020). Thus, modified Rubisco enzymes from plants can now be readily produced in E. coli , and once modifications that lead to superior carboxylation kinetics are identified they can be introduced into host plants for further characterization of their effects on photosynthesis and plant growth.…”

Section: Discussionmentioning

confidence: 99%

A procedure to introduce point mutations into the Rubisco large subunit gene in wild‐type plants

et al. 2021

View full text Add to dashboard Cite

Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast-encoded Rubisco large subunit rbcL is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation of rbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid's efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbcL within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild-type N. tabacum with stable point mutations within rbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modification of rbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast-encoded genes.

show abstract

Improved recombinant expression and purification of functional plant Rubisco

Cited by 32 publications

References 45 publications

Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction

Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction

Dual Role of a Rubisco Activase in Metabolic Repair and Carboxysome Organization

A procedure to introduce point mutations into the Rubisco large subunit gene in wild‐type plants

Contact Info

Product

Resources

About