Recent advances in synthetic biology of cyanobacteria for improved chemicals production

Wang, Fen; Gao, Yuanyuan; Yang, Guang

doi:10.1080/21655979.2020.1837458

Cited by 33 publications

(16 citation statements)

References 129 publications

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“…The most extensively studied cyanobacterium is the unicellular freshwater species Synechocystis , for which many distinct wild type substrains have been actively used [ 23 – 25 ]. Despite the increasing interest in expanding towards alternative strains with specific physiological properties such as faster growth rate or tolerance to high light intensities and salinity [ 26 , 27 ], Synechocystis continues to have an important role in many leading laboratories due to the wealth of existing biological information, synthetic biology tools and technical know-how [ 28 – 30 ]. From the perspective of the current work, considerations that relate to the rational engineering of Synechocystis genome include: (i) polyploidy of the chromosome (Table 1 ) which complicates the preparative steps due to time-consuming strain segregation, (ii) differences between putative integration sites that may affect system stability due to site-specific functions, (iii) expression efficiency that is related to the copy number of the target replicon, and (iv) the choice between available replicative plasmids offering alternatives to genomic integration.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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“…2 ). Whereas most of the genetic tools for cyanobacteria reported are frequently focused on Synechococcus or Synechocystis [ 18 ], the SEVA vectors described in this work may broaden the potential applications for other strains, such as Anabaena and Chroococcidiopsis tested here and, by extension, to other cyanobacteria .…”

Section: Resultsmentioning

confidence: 99%

SEVA-Cpf1, a CRISPR-Cas12a vector for genome editing in cyanobacteria

2022

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Background Cyanobacteria are photosynthetic autotrophs that have tremendous potential for fundamental research and industrial applications due to their high metabolic plasticity and ability to grow using CO2 and sunlight. CRISPR technology using Cas9 and Cpf1 has been applied to different cyanobacteria for genome manipulations and metabolic engineering. Despite significant advances with genome editing in several cyanobacteria strains, the lack of proper genetic toolboxes is still a limiting factor compared to other model laboratory species. Among the limitations, it is essential to have versatile plasmids that could ease the benchwork when using CRISPR technology. Results In the present study, several CRISPR-Cpf1 vectors were developed for genetic manipulations in cyanobacteria using SEVA plasmids. SEVA collection is based on modular vectors that enable the exchangeability of diverse elements (e.g. origins of replication and antibiotic selection markers) and the combination with many cargo sequences for varied end-applications. Firstly, using SEVA vectors containing the broad host range RSF1010 origin we demonstrated that these vectors are replicative not only in model cyanobacteria but also in a new cyanobacterium specie, Chroococcidiopsis sp., which is different from those previously published. Then, we constructed SEVA vectors by harbouring CRISPR elements and showed that they can be easily assimilated not only by conjugation, but also by natural transformation. Finally, we used our SEVA-Cpf1 tools to delete the nblA gene in Synechocystis sp. PCC 6803, demonstrating that our plasmids can be applied for CRISPR-based genome editing technology. Conclusions The results of this study provide new CRISPR-based vectors based on the SEVA (Standard European Vector Architecture) collection that can improve editing processes using the Cpf1 nuclease in cyanobacteria.

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“…Cyanobacteria are having a peculiar thylakoid membrane that harbours a complete set of respiratory enzymes as well as photosynthetic apparatus (Cooley & Vermaas, 2001 ). Alkane synthesis occurs primarily in the thylakoid membrane (Wang et al, 2020 ). In freshwater cyanobacteria, predominantly detected hydrocarbons are heptadecane, while marine cyanobacteria produce pentadecane (Shakeel et al, 2015 ).…”

Section: Introductionmentioning

confidence: 99%

Insights into cyanobacterial alkane biosynthesis

Parveen

Yazdani

2021

Journal of Industrial Microbiology and Biotechnology

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Alkanes are high-energy molecules that are compatible with enduring liquid fuel infrastructures, which make them highly suitable for being next-generation biofuels. Though biological production of alkanes has been reported in various microorganisms, the reports citing photosynthetic cyanobacteria as natural producers have been the most consistent for the long-chain alkanes and alkenes (C15–C19). However, the production of alkane in cyanobacteria is low, leading to its extraction being uneconomical for commercial purposes. In order to make alkane production economically feasible from cyanobacteria, the titre and yield need to be increased by several orders of magnitude. In the recent past, efforts have been made to enhance alkane production, although with a little gain in yield, leaving space for much improvement. Genetic manipulation in cyanobacteria is considered challenging, but recent advancements in genetic engineering tools may assist in manipulating the genome in order to enhance alkane production. Further, advancement in a basic understanding of metabolic pathways and gene functioning will guide future research for harvesting the potential of these tiny photosynthetically efficient factories. In this review, our focus would be to highlight the current knowledge available on cyanobacterial alkane production, and the potential aspects of developing cyanobacterium as an economical source of biofuel. Further insights into different metabolic pathways and hosts explored so far, and possible challenges in scaling up the production of alkanes will also be discussed.

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Recent advances in synthetic biology of cyanobacteria for improved chemicals production

Cited by 33 publications

References 129 publications

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

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

SEVA-Cpf1, a CRISPR-Cas12a vector for genome editing in cyanobacteria

Insights into cyanobacterial alkane biosynthesis

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