Cyanobacterial Cell Factories for Improved Carotenoid Biosynthesis through a Synthetic Biology Approach

Sarnaik, Aditya; Sawant, Kaustubh R.; Khadilkar, Jayshri; Pillai, Gayatri; Pandit, Reena; Lali, Arvind

doi:10.1021/bk-2019-1329.ch002

Cited by 8 publications

(6 citation statements)

References 128 publications

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“…The coding sequences in both templates were placed under the control of the anhydrotetracycline-inducible PL22 promoter, the B0034 RBS and the T7 terminator. PL22 was chosen as it has been reported to provide a suitable expression of CRISPRi elements (54, 55). In both templates, the assembled genetic circuits were placed in between homology arms comprising approximately 400 bp for the GGR mutants and approximately 200 bp for the ShMKS mutants, upstream and downstream of the chosen protospacer sequence in slr 0168 (Figure 3a, exact base positions on the genome are provided in Supplementary Table S3 and S4).…”

Section: Resultsmentioning

confidence: 99%

Design of an effective sgRNA for CRISPR/Cas9 knock-ins in polyploidSynechocystis sp. PCC 6803

Nares-Rodriguez,

Karunakaran

2023

Preprint

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Synechocystis sp. PCC 6803 (Synechocystis) is a highly promising organism for the production of diverse recombinant chemicals, including biofuels. However, conventional genetic engineering inSynechocystisis challenging due to its highly polyploid genome which not only leads to low product yields but also makes the recombinant organism less reliable for use in biomanufacturing. Due to its precision, effectiveness and reliability in a vast array of chassis, CRISPR/Cas9 has the potential of overcoming the drawbacks effected by a polyploid genome. Here we identified and developed an effective sgRNA for the knock-in of nucleotide sequences of varying lengths in the neutral siteslr0168 of polyploidSynechocystisusing CRISPR/Cas9. The gene encoding digeranylgeranylglycerophospholipid reductase fromSulfolobus acidocaldariusand the methyl ketone operon fromSolanum habrochaiteswere chosen as the exemplar nucleotide sequences for incorporation into the chromosome ofSynechocystis. It is demonstrated here that our sgRNA design was effective for both knock-ins and that CRISPR/Cas9 achieves complete mutant segregation after a single step of selection and induction.

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Section: Resultsmentioning

confidence: 99%

Design of an effective sgRNA for CRISPR/Cas9 knock-ins in polyploidSynechocystis sp. PCC 6803

Nares-Rodriguez,

Karunakaran

2023

Preprint

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“…Cyanobacteria share a pool of electron acceptors between photosynthesis and respiration, and nitrogen assimilation is tightly coupled at the level of ferredoxin in PSI. , Hence, cultivating them with 1% CO 2 and 200 μmol m –2 s –1 of light would have increased the overall photosynthetic electron transport but reduced the antenna density to avoid photo-oxidation and photobleaching, thereby balancing the photon uptake during carboxysomal carbon fixation. This corroborated with our study where the expression of genes encoding Mn 2+ -ABC transporter ATP-binding protein, ComEC family competence protein, KatG, pilus assembly chaperone, NAD synthetase, cysteine-tRNA ligase, 50S ribosomal protein L7/L12, and UDP-glucose-beta- d -glucan glucosyltransferase was upregulated.…”

Section: Discussionmentioning

confidence: 99%

“…As nitrogen plays a key role during protein biosynthesis, the nitrate uptake rate got influenced. Importantly, as nitrate uptake in cyanobacteria is tightly coupled with photosynthetic electron transport 29 and, in turn, photosynthetic proteins constitute a major protein fraction of the cellular proteome (Supporting Information Table S5), ultimately photosynthetic and respiratory pathways got affected.…”

Section: Global Transcriptome Analysis and Differential Genementioning

confidence: 99%

Global Transcriptome-Guided Identification of Neutral Sites for Engineering Synechococcus elongatus PCC 11801

et al. 2023

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Engineered cyanobacteria are attractive hosts for the phototrophic conversion of CO 2 to chemicals. Synechococcus elongatus PCC11801, a novel, fast-growing, and stress-tolerant cyanobacterium, has the potential to be a platform cell factory, and hence, it necessitates the development of a synthetic biology toolbox. Considering the widely followed cyanobacterial engineering strategy of chromosomal integration of heterologous DNA, it is of interest to discover and validate new chromosomal neutral sites (NSs) in this strain. To that end, global transcriptome analysis was performed using RNA Seq under the conditions of high temperature (HT), carbon (HC), and salt (HS) and ambient growth conditions. We found upregulation of 445, 138, and 87 genes and downregulation of 333, 125, and 132 genes, under HC, HT, and HS, respectively. Following nonhierarchical clustering, gene enrichment, and bioinformatics analysis, 27 putative NSs were predicted. Six of them were experimentally tested, and five showed confirmed neutrality, based on unaltered cell growth. Thus, global transcriptomic analysis was effectively exploited for NS annotation and would be advantageous for multiplexed genome editing.

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“…Artificially or chemically synthesized dyes have been broadly utilized in the pharmaceutical, cosmetic, textile and nutraceutical fields. However, these chemically synthesized dyes constitute serious health risks [ 2 ]. Thus, to avoid the harmful impacts of artificial dyes, the use of natural pigments obtained from organisms like microalgae is greatly encouraged.…”

Section: Introductionmentioning

confidence: 99%

Microalgae: A Promising Source of Bioactive Phycobiliproteins

Tounsi,

Ben Hlima,

Hentati

et al. 2023

Marine Drugs

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Phycobiliproteins are photosynthetic light-harvesting pigments isolated from microalgae with fluorescent, colorimetric and biological properties, making them a potential commodity in the pharmaceutical, cosmetic and food industries. Hence, improving their metabolic yield is of great interest. In this regard, the present review aimed, first, to provide a detailed and thorough overview of the optimization of culture media elements, as well as various physical parameters, to improve the large-scale manufacturing of such bioactive molecules. The second section of the review offers systematic, deep and detailed data about the current main features of phycobiliproteins. In the ultimate section, the health and nutritional claims related to these bioactive pigments, explaining their noticeable potential for biotechnological uses in various fields, are examined.

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Cyanobacterial Cell Factories for Improved Carotenoid Biosynthesis through a Synthetic Biology Approach

Cited by 8 publications

References 128 publications

Design of an effective sgRNA for CRISPR/Cas9 knock-ins in polyploidSynechocystis sp. PCC 6803

Design of an effective sgRNA for CRISPR/Cas9 knock-ins in polyploidSynechocystis sp. PCC 6803

Global Transcriptome-Guided Identification of Neutral Sites for Engineering Synechococcus elongatus PCC 11801

Microalgae: A Promising Source of Bioactive Phycobiliproteins

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