Engineering Carboxysomes into C3 Plants

Photosynthetic efficiency of C3 plants suffers from the slow catalytic rate and the reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) with O2 instead of CO2, leading to the costly process of photorespiration. Cyanobacteria are able to utilize a form of Rubisco exhibiting a higher catalytic rate and a lower specificity for CO2 by encapsulating the enzyme within a microcompartment known as a carboxysome. Engineering the cyanobacterial CO2 concentrating mechanism (CCM) into chloroplasts is an approach to enhance photosynthesis or to compartmentalize other biochemical reactions to confer new capabilities on transgenic plants. We have explored the expression of cyanobacterial Rubisco in the model plant tobacco as a first step to enhance carbon fixation through installation of the CCM from Synechococcus elongatus PCC7942, a freshwater cyanobacterium. Using the agroinfiltration technique, we have transiently expressed multiple β-carboxysomal proteins (CcmK2, CcmM, CcmL, CcmO and CcmN) in Nicotiana benthamiana with fusions that target these proteins into chloroplasts and that provide fluorescent labels for visualizing the resultant structures. By confocal and electron microscopic analysis, we have observed that the shell proteins of the β-carboxysome are able to assemble in plant chloroplasts into highly organized structures resembling empty microcompartments. Using chloroplast transformation, we have shown that the tobacco Rubisco enzyme can be replaced with the kinetically faster cyanobacterial enzyme, which assembles and confers autotrophic growth. Our initial chloroplast transformants exhibited much slower growth than wild-type, even when provided with exogenous CO2. We have altered the chloroplast operon containing the cyanobacterial rbcL and rbcS genes and have obtained morphologically normal chloroplast transformants that grow almost as rapidly as wild-type tobacco in elevated CO2. Current transformation experiments are in progress to incorporate additional elements of the CCM into chloroplasts.