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Chapter 20 Engineering Photosynthetic Enzymes Involved in CO2–Assimilation by Gene Shuffling

  • Genhai Zhu
  • Itzhak Kurek
  • Lu Liu
Part of the Advances in Photosynthesis and Respiration book series (AIPH, volume 31)

Summary

Enhancing photosynthesis is a promising approach for increasing plant productivity. Advances in plant transformation technology make it possible to manipulate photosynthesis by overexpressing particular genes for alleviating bottleneck steps, diverting the flux of Calvin cycle intermediates and photoassimilates, or introducing new enzymes and pathways that can positively influence photosynthesis. Furthermore, directed molecular evolution makes it possible to target selected key enzymes in photosynthetic pathways for modifying their specific catalytic or protein properties and tailoring them to best function under specified growth conditions. In this chapter, advances in directed molecular evolution technology and the use of gene shuffling methodology to modify Rubisco and Rubisco activase to enhance plant photosynthesis and growth are described. By shuffling the Chlamydomonas reinhardtii Rubisco large subunit and utilizing competitive growth selection, several mutated Rubisco enzymes with increased carboxylase activity or CO2/O2 specificity were identified. The mutations identified in the modified Chlamydomonas Rubisco variants were then introduced into the tobacco enzyme by site-directed mutagenesis. Enzyme kinetic assays indicated that the modified tobacco Rubisco enzymes displayed increased CO2/O2 specificity, carboxylase activity and reduced Km for CO2. Similarly, gene shuffling technology was used to generate several Arabidopsis thaliana Rubisco activase variants exhibiting improved thermostability in order to alleviate the inhibition of plant photosynthesis by elevated temperatures. The thermostable activase variants were then expressed in an Arabidopsis Rubisco activase deletion line created by fast-neutron mutagenesis. The positive effects of the shuffled thermostable Rubisco activase variants on Rubisco activation state, rates of photosynthesis, and growth under moderate heat stress were demonstrated.

Keywords

Directed Evolution Glycine Betaine Plant Photosynthesis Rubisco Activase Competitive Growth 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

aadA

aminoglycoside 3” adenyltransferase gene

ble

bleomycin resistant gene

GFP

green fluorescent protein

HTP

high-throughput

ictB

A gene involved in HCO 3 accumulation within the cyanobacterium Synechococcus sp. PCC 7942

kccat

Rubisco kcat for carboxylation

Km

the substrate concentration at which an enzyme yields one half maximum velocity

kocat

Rubisco kcat for oxygenation

LAI

the ratio of total leaf surface area of a crop to the surface area of the land on which the crop grows

LSU

Rubisco large subunit

otsA

trehalose synthase gene

PGA

3-phosphoglyceric acid

rbcL

Rubisco large subunit gene

RbcS

Rubisco small subunit gene

RCA

Rubisco activase

RuBP

ribulose-1,5-bisphosphate

SBPase

sedoheptulose-1,7-bisphosphatase

SSU

Rubisco small subunit

Vc

maximum velocity of Rubisco carboxylation reaction

Ω

Rubisco CO2/O2 specificity

Notes

Acknowledgements

This work was supported in part by grant from the National Institute of Standards and Technology-Advanced Technology Program. The authors would like to thank Thom True, Xin Zhang, Tiger Hu, Monica Majumdar, Eva Lin, Lik Hsueh and Alfred Madrigal for technical assistance; Shelly A. Straight for isolating the Arabidopsis activase deletion mutant; Dr. Cornelia Stettner of Icon Genetics AG for tobacco transformation; Dr. John A. Kiser for statistic analysis; Dr. Daniel Siehl for scientific discussions; Hoa M. Giang and Ingrid Udranszky for help in preparing the manuscript; Dr. Michael Lassner for scientific guidance; and Dr. Michael E. Salvucci from the USDA-ARS, Western Cotton Research Laboratory, Phoenix, AZ and Dr. Robert J. Spreitzer from the University of Nebraska for consultation to the project.

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.Pioneer Hi-Bred International Inc.HaywardUSA
  2. 2.Pioneer Hi-Bred International Inc.Redwood CityUSA

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