Engineering of chimeric eukaryotic/bacterial Rubisco large subunits in <i>Escherichia coli</i>

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a rate-limiting photosynthetic enzyme that catalyzes carbon fixation in the Calvin cycle. Much interest has been devoted to engineering this ubiquitous enzyme with the goal of increasing plant growth. However, experiments that have successfully produced improved Rubisco variants, via directed evolution in Escherichia coli, are limited to bacterial Rubisco because the eukaryotic holoenzyme cannot be produced in E. coli. The present study attempts to determine the specific differences between bacterial and eukaryotic Rubisco large subunit primary structure that are responsible for preventing heterologous eukaryotic holoenzyme formation in E. coli. A series of chimeric Synechococcus Rubiscos were created in which different sections of the large subunit were swapped with those of the homologous Chlamydomonas Rubisco. Chimeric holoenzymes that can form in vivo would indicate that differences within the swapped sections do not disrupt holoenzyme formation. Large subunit residues 1-97, 198-247 and 448-472 were successfully swapped without inhibiting holoenzyme formation. In all ten chimeras, protein expression was observed for the separate subunits at a detectable level. As a first approximation, the regions that can tolerate swapping may be targets for future engineering.

Dissection of Synechococcus Rubisco Large Subunit Sections Involved in Holoenzyme Formation in Escherichia coli by Combinatorial Section Swapping and Sequence Analyses

“…Chimeric Rubisco plasmids (Figure 1(B)) were mainly created by using primers and existing plasmids from Koay et al (2016) to amplify selected fragments, which were then digested with restriction enzymes as indicated in the primer names and then ligated into the pTrcHisB vector backbone of NcoI/PstI-digested pTrcSynLS (Mueller-Cajar & Whitney 2008). pL 1+10 S was constructed by ligating fragments from pTrcSynL(Chl1-50)S (Koay et al 2016) and pTrcSynL(Chl451-475)S (Koay et al 2016), which were amplified with primer pairs ChlN-NcoI/Syn150-rev-BsmBI (Koay et al 2016) and Syn150-fwd-BsmBI/SynSS-C-PstI (Koay et al 2016), respectively.…”

“…Chimeric Rubisco plasmids (Figure 1(B)) were mainly created by using primers and existing plasmids from Koay et al (2016) to amplify selected fragments, which were then digested with restriction enzymes as indicated in the primer names and then ligated into the pTrcHisB vector backbone of NcoI/PstI-digested pTrcSynLS (Mueller-Cajar & Whitney 2008). pL 1+10 S was constructed by ligating fragments from pTrcSynL(Chl1-50)S (Koay et al 2016) and pTrcSynL(Chl451-475)S (Koay et al 2016), which were amplified with primer pairs ChlN-NcoI/Syn150-rev-BsmBI (Koay et al 2016) and Syn150-fwd-BsmBI/SynSS-C-PstI (Koay et al 2016), respectively. pL 1+2+5+10 S had fragments from pTrcChlLS-SynSS (a plasmid with Synechococcus rbcL replaced by Chlamydomonas, unpublished), pTrcSynL(Chl200-250) S (Koay et al 2016) and pTrcSynL(Chl451-475)S (Koay et al 2016), which were amplified with primer pairs ChlN-NcoI/Chl100-rev-BsmBI (Koay et al 2016), Syn100-fwd-BsmBI/Syn400-rev-BsmBI (Koay et al 2016) and Syn400-fwd-BsmBI/SynSS-C-PstI (Koay et al 2016), respectively.…”

“…pL 1+10 S was constructed by ligating fragments from pTrcSynL(Chl1-50)S (Koay et al 2016) and pTrcSynL(Chl451-475)S (Koay et al 2016), which were amplified with primer pairs ChlN-NcoI/Syn150-rev-BsmBI (Koay et al 2016) and Syn150-fwd-BsmBI/SynSS-C-PstI (Koay et al 2016), respectively. pL 1+2+5+10 S had fragments from pTrcChlLS-SynSS (a plasmid with Synechococcus rbcL replaced by Chlamydomonas, unpublished), pTrcSynL(Chl200-250) S (Koay et al 2016) and pTrcSynL(Chl451-475)S (Koay et al 2016), which were amplified with primer pairs ChlN-NcoI/Chl100-rev-BsmBI (Koay et al 2016), Syn100-fwd-BsmBI/Syn400-rev-BsmBI (Koay et al 2016) and Syn400-fwd-BsmBI/SynSS-C-PstI (Koay et al 2016), respectively. pL 1+2 S had fragments from pL 1+2+5+10 S and pTrcSynLS (Mueller-Cajar & Whitney 2008), which were amplified with primer pairs ChlN-NcoI/Syn150-rev-BsmBI (Koay et al 2016) and Syn150-fwd-BsmBI/SynSS-C-PstI (Koay et al 2016), respectively.…”

“…In a previous study, an effort was made to determine the RbcL domains of cyanobacteria Rubisco that are essential for successful holoenzyme formation in Escherichia coli (Koay et al 2016). Sections spanning the whole coding sequence of cyanobacterial rbcL from Synechococcus PCC6301 were swapped sequentially with homologous sections of the eukaryotic rbcL gene from Chlamydomonas reinhardtii to generate ten chimeric mutants.…”

“…Sections spanning the whole coding sequence of cyanobacterial rbcL from Synechococcus PCC6301 were swapped sequentially with homologous sections of the eukaryotic rbcL gene from Chlamydomonas reinhardtii to generate ten chimeric mutants. In the end, it was established that four Synechococcus RbcL sections with residues 1-47, 48-97, 198-247 and 448-472 could be separately swapped without nullifying holoenzyme formation in E. coli (Koay et al 2016).…”