Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts.

The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO2-concentrating mechanism. BCT1 could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex.

A carboxysome-based CO concentrating mechanism for C crop chloroplasts: advances and the road ahead.

The introduction of the carboxysome-based CO concentrating mechanism (CCM) into crop plants has been modelled to significantly increase crop yields. This projection serves as motivation for pursuing this strategy to contribute to global food security. The successful implementation of this engineering challenge is reliant upon the transfer of a microcompartment that encapsulates cyanobacterial Rubisco, known as the carboxysome, alongside active bicarbonate transporters.

The Chlamydomonas reinhardtii chloroplast envelope protein LCIA transports bicarbonate in planta.

LCIA (low CO2-inducible protein A) is a chloroplast envelope protein associated with the CO2-concentrating mechanism of the green alga Chlamydomonas reinhardtii. LCIA is postulated to be a HCO3- channel, but previous studies were unable to show that LCIA was actively transporting bicarbonate in planta. Therefore, LCIA activity was investigated more directly in two heterologous systems: an Escherichia coli mutant (DCAKO) lacking both native carbonic anhydrases and an Arabidopsis mutant (βca5) missing the plastid carbonic anhydrase βCA5.

Towards engineering a hybrid carboxysome.

Carboxysomes are bacterial microcompartments, whose structural features enable the encapsulated Rubisco holoenzyme to operate in a high-CO environment. Consequently, Rubiscos housed within these compartments possess higher catalytic turnover rates relative to their plant counterparts. This particular enzymatic property has made the carboxysome, along with associated transporters, an attractive prospect to incorporate into plant chloroplasts to increase future crop yields.

Engineered Accumulation of Bicarbonate in Plant Chloroplasts: Known Knowns and Known Unknowns.

Heterologous synthesis of a biophysical CO-concentrating mechanism (CCM) in plant chloroplasts offers significant potential to improve the photosynthetic efficiency of C plants and could translate into substantial increases in crop yield. In organisms utilizing a biophysical CCM, this mechanism efficiently surrounds a high turnover rate Rubisco with elevated CO concentrations to maximize carboxylation rates. A critical feature of both native biophysical CCMs and one engineered into a C plant chloroplast is functional bicarbonate (HCO ) transporters and vectorial CO-to-HCO converters.

Structural Basis for the Allosteric Regulation of the SbtA Bicarbonate Transporter by the P-like Protein, SbtB, from sp. PCC7001.

Cyanobacteria have evolved a suite of enzymes and inorganic carbon (C) transporters that improve photosynthetic performance by increasing the localized concentration of CO around the primary CO-fixating enzyme, Rubisco. This CO-concentrating mechanism (CCM) is highly regulated, responds to illumination/darkness cycles, and allows cyanobacteria to thrive under limiting C conditions. While the transcriptional control of CCM activity is well understood, less is known about how regulatory proteins might allosterically regulate C transporters in response to changing conditions.

Factors Altering Pyruvate Excretion in a Glycogen Storage Mutant of the Cyanobacterium, Synechococcus PCC7942.

Interest in the production of carbon commodities from photosynthetically fixed CO2 has focused attention on cyanobacteria as a target for metabolic engineering and pathway investigation. We investigated the redirection of carbon flux in the model cyanobacterial species, Synechococcus elongatus PCC 7942, under nitrogen deprivation, for optimized production of the industrially desirable compound, pyruvate. Under nitrogen limited conditions, excess carbon is naturally stored as the multi-branched polysaccharide, glycogen, but a block in glycogen synthesis, via knockout mutation in the gene encoding ADP-glucose pyrophosphorylase (glgC), results in the accumulation of the organic acids, pyruvate and 2-oxoglutarate, as overflow excretions into the extracellular media.

Characterisation of cyanobacterial bicarbonate transporters in E. coli shows that SbtA homologs are functional in this heterologous expression system.

Cyanobacterial HCO3(-) transporters BCT1, SbtA and BicA are important components of cyanobacterial CO2-concentration mechanisms. They also show potential in applications aimed at improving photosynthetic rates and yield when expressed in the chloroplasts of C3 crop species. The present study investigated the feasibility of using Escherichia coli to assess function of a range of SbtA and BicA transporters in a heterologous expression system, ultimately for selection of transporters suitable for chloroplast expression.