maxima into chloroplasts of tobacco plants. In these transgenic tobacco lines,
glycolate is oxidized to glyoxylate in chloroplast by glycolate dehydrogenase and
glyoxylate reacts with acetyl-CoA to form malate by the catalytic activity of malate
synthase. Indigenous chloroplast enzymes malic enzyme and pyruvate dehydrogenase convert malate to acetyl-CoA and consequently to two molecules of CO 2 . In
addition, they maximized flux by introducing transcriptional downregulation by
RNA interference of the plastidic glycerate/glycolate transporter 1 (PLGG1) that
inhibited glycolate export from the chloroplast. Computational modelling of these
alternative pathways highlighted the importance of optimized expression of
non-native genes in preventing photoinhibition and subsequently attaining significantly improved harvestable photosynthetic quantum yield. Shen et al. (2019)
designed the bypass for PR in rice by redirecting the native enzymes of peroxisomes
(glycolate oxidase, oxalate oxidase and catalase) to chloroplast stroma and thus
enabled superior carbon fixation in these plants over wild ones. Beyond photosynthesis, PR is associated with other essential functions such as C1
À
, N and S
metabolism, thus demanding a holistic approach while using advanced engineering
strategies that completely substitute endogenous PR with synthetic ones (Eisenhut
et al. 2019).
Cyanobacterial CO 2 and bicarbonate transporters add to inorganic carbon concentration in the cytoplasm, which then diffuses into carboxysomes and gets
dehydrated to generate CO 2 . Interestingly, low permeability of carboxysomes
toward CO 2 and O 2 further guarantees high CO 2 /O 2 ratio at the vicinity of RuBisCO,
thereby dropping the oxygenation reaction. Thus, constructing self-assembling
proteinaceous microcompartments like carboxysomes, that co-localize the RuBisCO
and carbonic anhydrase (CA), can increase RuBisCO turnover by reducing
PR. Using a diffusion-reaction model, McGrath and Long (2014) suggested that
the execution of a carboxysome-type CCM in plant chloroplasts can potentially
boost photosynthetic competence. Lin et al. (2014) used the agroinfiltration technique, to transfer multiple β-carboxysomal proteins (CcmK2, CcmM, CcmL, CcmO
and CcmN) from Synechococcus elongatus PCC7942 to the chloroplast of Nicotiana
benthamiana with sensors that perceive and provide fluorescent labels for
visualizing the resultant structures. The shell proteins assemble in plant chloroplasts
into highly organized microcompartments, thus establishing the likelihood of
introducing carboxysomes into chloroplasts for probable compartmentalization of
RuBisCO. Giessen and Silver (2017) proposed a synthetic minimal organelle,
utilizing encapsulin nanocompartments with engineered catalytic RuBisCO and
CA components to engender a CO 2 -fixing arrangement. Further, encapsulin pores
need to be engineered to imitate the selective permeability of natural carboxysomes.
The intended route also includes implantation of bicarbonate transporters targeted to
the chloroplast inner membrane, encapsulin nanocompartments into chloroplast
stroma replacing endogenous lower turnover RuBisCO and minimizing stromal
carbonic anhydrase (CA) activity to attain the best possible carbon flux to an
engineered CO 2 -fixing organelle. Long et al. (2018) characterised simplified
carboxysomes, isometric with those in Cyanobium, within tobacco chloroplasts
where they replaced the endogenous RuBisCO large subunit gene with
15 Plant Synthetic Biology: A Paradigm Shift Targeting Stress Mitigation,. . .
443
glycolate is oxidized to glyoxylate in chloroplast by glycolate dehydrogenase and
glyoxylate reacts with acetyl-CoA to form malate by the catalytic activity of malate
synthase. Indigenous chloroplast enzymes malic enzyme and pyruvate dehydrogenase convert malate to acetyl-CoA and consequently to two molecules of CO 2 . In
addition, they maximized flux by introducing transcriptional downregulation by
RNA interference of the plastidic glycerate/glycolate transporter 1 (PLGG1) that
inhibited glycolate export from the chloroplast. Computational modelling of these
alternative pathways highlighted the importance of optimized expression of
non-native genes in preventing photoinhibition and subsequently attaining significantly improved harvestable photosynthetic quantum yield. Shen et al. (2019)
designed the bypass for PR in rice by redirecting the native enzymes of peroxisomes
(glycolate oxidase, oxalate oxidase and catalase) to chloroplast stroma and thus
enabled superior carbon fixation in these plants over wild ones. Beyond photosynthesis, PR is associated with other essential functions such as C1
À
, N and S
metabolism, thus demanding a holistic approach while using advanced engineering
strategies that completely substitute endogenous PR with synthetic ones (Eisenhut
et al. 2019).
Cyanobacterial CO 2 and bicarbonate transporters add to inorganic carbon concentration in the cytoplasm, which then diffuses into carboxysomes and gets
dehydrated to generate CO 2 . Interestingly, low permeability of carboxysomes
toward CO 2 and O 2 further guarantees high CO 2 /O 2 ratio at the vicinity of RuBisCO,
thereby dropping the oxygenation reaction. Thus, constructing self-assembling
proteinaceous microcompartments like carboxysomes, that co-localize the RuBisCO
and carbonic anhydrase (CA), can increase RuBisCO turnover by reducing
PR. Using a diffusion-reaction model, McGrath and Long (2014) suggested that
the execution of a carboxysome-type CCM in plant chloroplasts can potentially
boost photosynthetic competence. Lin et al. (2014) used the agroinfiltration technique, to transfer multiple β-carboxysomal proteins (CcmK2, CcmM, CcmL, CcmO
and CcmN) from Synechococcus elongatus PCC7942 to the chloroplast of Nicotiana
benthamiana with sensors that perceive and provide fluorescent labels for
visualizing the resultant structures. The shell proteins assemble in plant chloroplasts
into highly organized microcompartments, thus establishing the likelihood of
introducing carboxysomes into chloroplasts for probable compartmentalization of
RuBisCO. Giessen and Silver (2017) proposed a synthetic minimal organelle,
utilizing encapsulin nanocompartments with engineered catalytic RuBisCO and
CA components to engender a CO 2 -fixing arrangement. Further, encapsulin pores
need to be engineered to imitate the selective permeability of natural carboxysomes.
The intended route also includes implantation of bicarbonate transporters targeted to
the chloroplast inner membrane, encapsulin nanocompartments into chloroplast
stroma replacing endogenous lower turnover RuBisCO and minimizing stromal
carbonic anhydrase (CA) activity to attain the best possible carbon flux to an
engineered CO 2 -fixing organelle. Long et al. (2018) characterised simplified
carboxysomes, isometric with those in Cyanobium, within tobacco chloroplasts
where they replaced the endogenous RuBisCO large subunit gene with
15 Plant Synthetic Biology: A Paradigm Shift Targeting Stress Mitigation,. . .
443