Comparison of alternative integration sites in the chromosome and the native plasmids of the cyanobacterium Synechocystis sp. PCC 6803 in respect to expression efficiency and copy number

Abstract

Background Synechocystis sp. PCC 6803 provides a well-established reference point to cyanobacterial metabolic engineering as part of basic photosynthesis research, as well as in the development of next-generation biotechnological production systems. This study focused on expanding the current knowledge on genomic integration of expression constructs in Synechocystis, targeting a range of novel sites in the chromosome and in the native plasmids, together with established loci used in literature. The key objective was to obtain quantitative information on site-specific expression in reference to replicon copy numbers, which has been speculated but never compared side by side in this host. Results An optimized sYFP2 expression cassette was successfully integrated in two novel sites in Synechocystis chromosome (slr0944; sll0058) and in all four endogenous megaplasmids (pSYSM/slr5037-slr5038; pSYSX/slr6037; pSYSA/slr7023; pSYSG/slr8030) that have not been previously evaluated for the purpose. Fluorescent analysis of the segregated strains revealed that the expression levels between the megaplasmids and chromosomal constructs were very similar, and reinforced the view that highest expression in Synechocystis can be obtained using RSF1010-derived replicative vectors or the native small plasmid pCA2.4 evaluated in comparison. Parallel replicon copy number analysis by RT-qPCR showed that the expression from the alternative loci is largely determined by the gene dosage in Synechocystis, thereby confirming the dependence formerly proposed based on literature. Conclusions This study brings together nine different integrative loci in the genome of Synechocystis to demonstrate quantitative differences between target sites in the chromosome, the native plasmids, and a RSF1010-based replicative expression vector. To date, this is the most comprehensive comparison of alternative integrative sites in Synechocystis, and provides the first direct reference between expression efficiency and replicon gene dosage in the context. In the light of existing literature, the findings support the view that the small native plasmids can be notably more difficult to target than the chromosome or the megaplasmids, and that the RSF1010-derived vectors may be surprisingly well maintained under non-selective culture conditions in this cyanobacterial host. Altogether, the work broadens our views on genomic integration and the rational use of different integrative loci versus replicative plasmids, when aiming at expressing heterologous genes in Synechocystis.

Pilot scale production, extraction and purification of a thermostable phycocyanin from Synechocystis sp. PCC 6803

“…On day 18, the presence of the CT.353 TeBACD vector was confirmed in all carboys by PCR (see supplementary materials ), which demonstrated that the cultures retained the self-replicating vector for producing Te-PC in the absence of a selective pressure for the duration of the cultivation period. This is consistent with recent work that has demonstrated that self-replicating RSF1010-based plasmids appear stable in cultures without antibiotic selection for at least six weeks ( Nagy et al, 2021 ). Furthermore, the results suggested that the absence of kanamycin reduced cell stress and/or metabolic burden, which allowed for a small, but significant increase in Te-PC productivity.…”

“…The yields of Te-PC in the Synechocystis mutant strains were comparable to that of native PC in wild-type Synechocystis , while the thermostability properties matched those from T. elongatus . Recent work has demonstrated that the level of eYFP expression from an RSF1010-based vector was 3-fold higher than eYFP expression from chromosomal integrations by natural transformation ( Nagy et al, 2021 ). Importantly, that study also showed that the self-replicating vector was retained for 42 days without antibiotic selection at lab scale, thereby suggesting that scale-up batch cultivation without antibiotic selection is feasible.…”

Low-Carbon-Footprint Production of High-End 5-Aminolevulinic Acid via Integrative Strain Engineering and RuBisCo-Equipped Escherichia coli

“…Besides, more biomass and ALA with lower CO 2 emission were observed by using PL instead of PLP in BY strains, since PLP may be an inhibitor to RuBisCo and Prk. 45 The biomass was significantly improved in RSSA-BY by 176% and CO 2 was reduced by 53.8%. In conclusion, this study achieved a robust strain A-RcYI with outstanding ALAS activity for efficient ALA production in E. coli.…”

“…The results of Figure B and Table supplement that pRARE enhanced ALA production while CO 2 release was reduced, which may be attributed to the improvement on the cell behavior and protein quality with sufficient tRNA as aforementioned. Besides, more biomass and ALA with lower CO 2 emission were observed by using PL instead of PLP in BY strains, since PLP may be an inhibitor to RuBisCo and Prk . The biomass was significantly improved in RSSA-BY by 176% and CO 2 was reduced by 53.8%.…”

Inducible CRISPR/Cas9 allows for multiplexed and rapidly segregated single target genome editing in Synechocystis sp. PCC 6803

“…This method would likely be inefficient for the CRISPR/Cas9 vectors developed in this study, as RSF1010-replicon vectors are stably maintained in S6803 for prolonged periods under non-selective conditions. 46 Another option is to add a counter-selection marker to the vector, such as sacB from Bacillus subtilis that causes sucrose-sensitivity. 47 However, the sacB -method is only functional for glucose-tolerant wild type S6803 strains.…”