microbial CO 2 -fixation pathways, which have several advantages with respect to
energy prerequisite and competence compared to CBBC (Berg 2011). For example,
engineering Methanococcoides burtonii archaeal RuBisCO to tobacco chloroplasts
could address the kinetic restrictions of key enzymes (Wilson et al. 2016). Recently
Miller et al. (2020) designed a chloroplast imitator by encapsulating and operating
photosynthetic membranes in microdroplets and by using light as an external signal.
They created ‘synthetic chloroplast’ by incorporating the CETCH cycle which could
photosynthesise from inorganic carbon, providing the basis to design an independent, entirely synthetic ‘designer’ carbon metabolism in the artificial organelle.
However, engineering RuBisCOs with superior CO 2 -specificities and/or better
catalytic rates is not easy as it is trapped in an intrinsic exchange of activity with
specificity. Superior specificity for CO 2 usually results in a lower enzyme activity
and vice versa is also factual (Savir et al. 2010). Hence, there is a need to look into
the more appropriate alternate routes, such as cutting down wasteful reactions of
CBBC, to improve photosynthetic efficiency. With the increase in temperature, the
specificity of RuBisCO for CO 2 declines, and unproductive energy-intensive process
of photorespiration (PR) results from RuBisCO catalysed oxygenation of Ribulose
1,5-bisphosphate (RuBP), releasing toxic by-products 2-phosphoglycolate and
glycolate (Long et al. 2006). Moreover in PR, recycling of toxic products of glycine
decarboxylation reaction into nontoxic products comes at the expenditure of energy
and net loss of fixed carbon (Peterhansel et al. 2010; South et al. 2019). Thus
alternative approach is to reduce the rate of photorespiration (PR) by increasing
the concentration of CO 2 at the site of RuBisCO or rerouting PR by opening
synthetic bypasses or de novo pathways that turn PR into a carbon-positive process
(Ort et al. 2015; Erb and Zarzycki 2016; Eisenhut et al. 2019).
Inspired from E. coli glycolate oxidizing pathway, Kebeish et al. (2007)
introduced bacterial glycolate dehydrogenase complex together with glyoxylate
carboxylase and tartronate reductase into Arabidopsis chloroplast stroma. These
plants could fix more carbon than the wild type, as the route adopted by them
prevented the wasteful release of ammonia (NH 3 ) in mitochondria and hence
increased the energy efficiency of the pathway (Peterhansel and Maurino 2011).
Furthermore, instead of mitochondria, CO 2 is released in chloroplasts (avoided
mitochondrial glycine decarboxylation to serine), thereby concentrating more CO 2
in propinquity to RuBisCO and suppressing additional oxygenation. Dalal et al.
(2015) characterised the same bypass in the chloroplasts of Camelina sativa (biofuel
crop) which proved to augment vegetative and reproductive yields by more than
50%. In an in vitro study, Trudeau et al. (2018) engineered an acetyl-CoA synthetase
to convert glycolate into glycolyl-CoA and a propionyl-CoA reductase for higher
glycolyl-CoA selectivity and NADPH specificity, to be reduced to D-glycerate and
re-assimilated into the CBBC. These engineered enzymes were then combined with
downstream condensation and assimilation enzymes (native ones): an aldolase, an
isomerase and the native kinase. Together, this enzymatic sequence converted
glycolate to RuBisCO’s substrate RuBP, thus avoiding loss of carbon via PR. South
et al. (2019) engineered mitochondrial glycolate dehydrogenase from green alga
Chlamydomonas reinhardtii and malate synthase from peroxisomes of Cucurbita
442
P. Singla
energy prerequisite and competence compared to CBBC (Berg 2011). For example,
engineering Methanococcoides burtonii archaeal RuBisCO to tobacco chloroplasts
could address the kinetic restrictions of key enzymes (Wilson et al. 2016). Recently
Miller et al. (2020) designed a chloroplast imitator by encapsulating and operating
photosynthetic membranes in microdroplets and by using light as an external signal.
They created ‘synthetic chloroplast’ by incorporating the CETCH cycle which could
photosynthesise from inorganic carbon, providing the basis to design an independent, entirely synthetic ‘designer’ carbon metabolism in the artificial organelle.
However, engineering RuBisCOs with superior CO 2 -specificities and/or better
catalytic rates is not easy as it is trapped in an intrinsic exchange of activity with
specificity. Superior specificity for CO 2 usually results in a lower enzyme activity
and vice versa is also factual (Savir et al. 2010). Hence, there is a need to look into
the more appropriate alternate routes, such as cutting down wasteful reactions of
CBBC, to improve photosynthetic efficiency. With the increase in temperature, the
specificity of RuBisCO for CO 2 declines, and unproductive energy-intensive process
of photorespiration (PR) results from RuBisCO catalysed oxygenation of Ribulose
1,5-bisphosphate (RuBP), releasing toxic by-products 2-phosphoglycolate and
glycolate (Long et al. 2006). Moreover in PR, recycling of toxic products of glycine
decarboxylation reaction into nontoxic products comes at the expenditure of energy
and net loss of fixed carbon (Peterhansel et al. 2010; South et al. 2019). Thus
alternative approach is to reduce the rate of photorespiration (PR) by increasing
the concentration of CO 2 at the site of RuBisCO or rerouting PR by opening
synthetic bypasses or de novo pathways that turn PR into a carbon-positive process
(Ort et al. 2015; Erb and Zarzycki 2016; Eisenhut et al. 2019).
Inspired from E. coli glycolate oxidizing pathway, Kebeish et al. (2007)
introduced bacterial glycolate dehydrogenase complex together with glyoxylate
carboxylase and tartronate reductase into Arabidopsis chloroplast stroma. These
plants could fix more carbon than the wild type, as the route adopted by them
prevented the wasteful release of ammonia (NH 3 ) in mitochondria and hence
increased the energy efficiency of the pathway (Peterhansel and Maurino 2011).
Furthermore, instead of mitochondria, CO 2 is released in chloroplasts (avoided
mitochondrial glycine decarboxylation to serine), thereby concentrating more CO 2
in propinquity to RuBisCO and suppressing additional oxygenation. Dalal et al.
(2015) characterised the same bypass in the chloroplasts of Camelina sativa (biofuel
crop) which proved to augment vegetative and reproductive yields by more than
50%. In an in vitro study, Trudeau et al. (2018) engineered an acetyl-CoA synthetase
to convert glycolate into glycolyl-CoA and a propionyl-CoA reductase for higher
glycolyl-CoA selectivity and NADPH specificity, to be reduced to D-glycerate and
re-assimilated into the CBBC. These engineered enzymes were then combined with
downstream condensation and assimilation enzymes (native ones): an aldolase, an
isomerase and the native kinase. Together, this enzymatic sequence converted
glycolate to RuBisCO’s substrate RuBP, thus avoiding loss of carbon via PR. South
et al. (2019) engineered mitochondrial glycolate dehydrogenase from green alga
Chlamydomonas reinhardtii and malate synthase from peroxisomes of Cucurbita
442
P. Singla