CRISPRi as a Tool to Repress Multiple Copies of Extracellular Polymeric Substances (EPS)-Related Genes in the Cyanobacterium Synechocystis sp. PCC 6803.
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ABSTRACT:
SUBMITTER: Santos M
PROVIDER: S-EPMC8620461 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cyanobacteria are model organisms for photosynthesis and are attractive for biotechnology applications. To aid investigation of genotype-phenotype relationships in cyanobacteria, we develop an inducible CRISPRi gene repression library in Synechocystis sp. PCC 6803, where we aim to target all genes for repression. We track the growth of all library members in multiple conditions and estimate gene fitness. The library reveals several clones with increased growth rates, and these have a common upregulation of genes related to cyclic electron flow. We challenge the library with 0.1 M L-lactate and find that repression of peroxiredoxin bcp2 increases growth rate by 49%. Transforming the library into an L-lactate-secreting Synechocystis strain and sorting top lactate producers enriches clones with sgRNAs targeting nutrient assimilation, central carbon metabolism, and cyclic electron flow. In many examples, productivity can be enhanced by repression of essential genes, which are difficult to access by transposon insertion.
| S-EPMC7125299 | biostudies-literature
Project description:Purpose: To obtain an overview of the metabolic changes associated to the absence of KpsM in Synechocystis, in particular on carbon-related metabolic pathways Methods: The trancriptomes of Synechocystis wild type and kpsM mutant were analyzed by RNA sequencing, using three biological replicates. Cultures were grown until reaching an OD730nm of 1.5. Sequencing libraries were generated using NEBNext® Ultra TM RNA Library Prep Kit for Illumina®. PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina). Raw data (raw reads) of FASTQ format were firstly processed through in-house scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter and poly-N sequences and reads with low quality from raw data. Paired-end clean reads were mapped to the reference genome using HISAT2 software.HTSeq was used to count the read numbers mapped of each gene, including known and novel genes. And then RPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. RPKM, Reads Per Kilobase of exon model per Million mapped reads, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels. Differential expression analysis between two conditions/groups (three biological replicates per condition) was performed using DESeq2 R package. DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using the Benjamini and Hochberg’s approach for controlling the False Discovery Rate (FDR). Genes with an adjusted P value < 0.05 found by DESeq2 were assigned as differentially expressed. Results: Approximately 700 genes (out of the 3636 identified and 215 quantified, representing a coverage of 85,12%) were significantly differentially expressed (P 216 value < 0.05) in the kpsM mutant compared to the wild type. Of those, 406 were down217 regulated, while 297 were up-regulated. Conclusions: Overall, our transcriptomic data indicates alterations in the mechanisms of energy production and conversion in the kpsM mutant compared to the wild type. The approach resulted in the identification of altered levels of transcripts belonging to a variety of functional categories and highlighting a number of key metabolic processes affected by the disruption of kpsM, namely photosynthesis, oxidative phosphorylation and carbon metabolism.
2021-01-20 | GSE165073 | GEO
Project description:Many cyanobacteria produce extracellular polymeric substances (EPS), composed mainly of heteropolysaccharides, that play a variety of physiological roles, being crucial for cell protection, motility, and biofilm formation. However, due to their complexity, the EPS biosynthetic pathways as well as their assembly and export mechanisms are still far from being fully understood. Here, we show that the absence of a putative EPS-related protein, KpsM (Slr0977), has a pleiotropic effect on Synechocystis sp. strain PCC 6803 physiology, with a strong impact on the export of EPS and carbon fluxes. The kpsM mutant exhibits a significant reduction of released polysaccharides and a smaller decrease of capsular polysaccharides, but it accumulates more polyhydroxybutyrate (PHB) than the wild type. In addition, this strain shows a light/cell density-dependent clumping phenotype and exhibits an altered protein secretion capacity. Furthermore, the most important structural component of pili, the protein PilA, was found to have a modified glycosylation pattern in the mutant compared to the wild type. Proteomic and transcriptomic analyses revealed significant changes in the mechanisms of energy production and conversion, namely, photosynthesis, oxidative phosphorylation, and carbon metabolism, in response to the inactivation of slr0977 Overall, this work shows for the first time that cells with impaired EPS secretion undergo transcriptomic and proteomic adjustments, highlighting the importance of EPS as a major carbon sink in cyanobacteria. The accumulation of PHB in cells of the mutant, without affecting significantly its fitness/growth rate, points to its possible use as a chassis for the production of compounds of interest.IMPORTANCE Most cyanobacteria produce extracellular polymeric substances (EPS) that fulfill different biological roles depending on the strain/environmental conditions. The interest in the cyanobacterial EPS synthesis/export pathways has been increasing, not only to optimize EPS production but also to efficiently redirect carbon flux toward the production of other compounds, allowing the implementation of industrial systems based on cyanobacterial cell factories. Here, we show that a Synechocystis kpsM (slr0977) mutant secretes less EPS than the wild type, accumulating more carbon intracellularly, as polyhydroxybutyrate. Further characterization showed a light/cell density-dependent clumping phenotype, altered protein secretion, and modified glycosylation of PilA. The proteome and transcriptome of the mutant revealed significant changes, namely, in photosynthesis and carbon metabolism. Altogether, this work provides a comprehensive overview of the impact of kpsM disruption on Synechocystis physiology, highlighting the importance of EPS as a carbon sink and showing how cells adapt when their secretion is impaired, and the redirection of the carbon fluxes.
| S-EPMC7885315 | biostudies-literature
Project description:Many cyanobacteria produce extracellular polymeric substances (EPS) mainly composed of heteropolysaccharides with unique characteristics that make them suitable for biotechnological applications. However, manipulation/optimization of EPS biosynthesis/characteristics is hindered by a poor understanding of the production pathways and the differences between bacterial species. In this work, genes putatively related to different pathways of cyanobacterial EPS polymerization, assembly, and export were targeted for deletion or truncation in the unicellular Synechocystis sp. PCC 6803. No evident phenotypic changes were observed for some mutants in genes occurring in multiple copies in Synechocystis genome, namely ∆wzy (∆sll0737), ∆wzx (∆sll5049), ∆kpsM (∆slr2107), and ∆kpsM∆wzy (∆slr2107∆sll0737), strongly suggesting functional redundancy. In contrast, Δwzc (Δsll0923) and Δwzb (Δslr0328) influenced both the amount and composition of the EPS, establishing that Wzc participates in the production of capsular (CPS) and released (RPS) polysaccharides, and Wzb affects RPS production. The structure of Wzb was solved (2.28 Å), revealing structural differences relative to other phosphatases involved in EPS production and suggesting a different substrate recognition mechanism. In addition, Wzc showed the ATPase and autokinase activities typical of bacterial tyrosine kinases. Most importantly, Wzb was able to dephosphorylate Wzc in vitro, suggesting that tyrosine phosphorylation/dephosphorylation plays a role in cyanobacterial EPS production.
| S-EPMC6562117 | biostudies-literature
Project description:Arsenic is a ubiquitous contaminant and a toxic metalloid which presents two main redox states in nature: arsenite [As(III)] and arsenate [As(V)]. Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by the arsBHC operon and two additional arsenate reductases encoded by the arsI1 and arsI2 genes. Here we describe the genome-wide responses to the presence of arsenate and arsenite in wild type and mutants in the arsenic resistance system. Both forms of arsenic produced similar responses in the wild type strain, including induction of several stress related genes and repression of energy generation processes. These responses were transient in the wild type strain but maintained in time in an arsB mutant strain, which lacks the arsenite transporter. In contrast, the responses observed in a strain lacking all arsenate reductases were somewhat different and included lower induction of genes involved in metal homeostasis and Fe-S cluster biogenesis, suggesting that these two processes are targeted by arsenite in the wild type strain. Finally, analysis of the arsR mutant strain revealed that ArsR seems to only control 5 genes in the genome. Furthermore, the arsR mutant strain exhibited hypersentivity to nickel, copper and cadmium and this phenotype was suppressed by mutation in arsB but not in arsC gene suggesting that overexpression of arsB is detrimental in the presence of these metals in the media.
| S-EPMC4010505 | biostudies-literature
Project description:BackgroundCyanobacteria are phototrophic prokaryotes that convert inorganic carbon as CO2 into organic compounds at the expense of light energy. They need only inorganic nutrients and can be cultivated to high densities using non-arable land and seawater. This has made cyanobacteria attractive organisms for the production of biofuels and chemical feedstock. Synechocystis sp. PCC 6803 is one of the most widely used cyanobacterial model strains. Based on its available genome sequence and genetic tools, Synechocystis has been genetically modified to produce different biotechnological products. Efficient isoprene production is an attractive goal because this compound is widely used as chemical feedstock.ResultsHere, we report on our attempts to generate isoprene-producing strains of Synechocystis using a plasmid-based strategy. As previously reported, a codon-optimized plant isoprene synthase (IspS) was expressed under the control of different Synechocystis promoters that ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR and Western blotting, while the amount of isoprene was quantified using GC-MS. In addition to isoprene measurements in the headspace of closed culture vessels, single photon ionization time-of-flight mass spectrometry (SPI-MS) was applied, which allowed online measurements of isoprene production in open-cultivation systems under various conditions. Under standard conditions, a good correlation existed between ispS expression and isoprene production rate. The cultivation of isoprene production strains under NaCl-supplemented conditions decreased isoprene production despite enhanced ispS mRNA levels. The characterization of the metabolome of isoprene-producing strains indicated that isoprene production might be limited by insufficient precursor levels. Transcriptomic analysis revealed the upregulation of mRNA and regulatory RNAs characteristic of acclimation to metabolic stress.ConclusionsOur best production strains produced twofold higher isoprene amounts in the presence of low NaCl concentrations than previously reported strains. These results will guide future attempts to establish isoprene production in cyanobacterial hosts.
| S-EPMC4836186 | biostudies-literature
Project description:Synechocystis sp. PCC 6803 is the most popular cyanobacterial strain, serving as a standard in the research fields of photosynthesis, stress response, metabolism and so on. A glucose-tolerant (GT) derivative of this strain was used for genome sequencing at Kazusa DNA Research Institute in 1996, which established a hallmark in the study of cyanobacteria. However, apparent differences in sequences deviating from the database have been noticed among different strain stocks. For this reason, we analysed the genomic sequence of another GT strain (GT-S) by 454 and partial Sanger sequencing. We found 22 putative single nucleotide polymorphisms (SNPs) in comparison to the published sequence of the Kazusa strain. However, Sanger sequencing of 36 direct PCR products of the Kazusa strains stored in small aliquots resulted in their identity with the GT-S sequence at 21 of the 22 sites, excluding the possibility of their being SNPs. In addition, we were able to combine five split open reading frames present in the database sequence, and to remove the C-terminus of an ORF. Aside from these, two of the Insertion Sequence elements were not present in the GT-S strain. We have thus become able to provide an accurate genomic sequence of Synechocystis sp. PCC 6803 for future studies on this important cyanobacterial strain.
| S-EPMC3190959 | biostudies-literature
Project description:The naturally transformable cyanobacterium Synechocystis sp. PCC 6803 is a widely used chassis strain for the photosynthetic production of chemicals. However, Synechocystis possesses multiple genome copies per cell which means that segregating mutations across all genome copies can be time-consuming. Here we use flow cytometry in combination with DNA staining to investigate the effect of phosphate deprivation on the genome copy number of the glucose-tolerant GT-P sub-strain of Synechocystis 6803. Like the PCC 6803 wild type strain, the ploidy of GT-P cells grown in BG-11 medium is growth phase dependent with an average genome copy number of 6.05 ± 0.27 in early growth (OD740 = 0.1) decreasing to 2.49 ± 0.11 in late stationary phase (OD740 = 7). We show that a 10-fold reduction in the initial phosphate concentration of the BG-11 growth medium reduces the average genome copy number of GT-P cells from 4.51 ± 0.20 to 2.94 ± 0.13 and increases the proportion of monoploid cells from 0 to 6% after 7 days of growth. In addition, we also show that the DnaA protein, which unusually for bacteria is not required for DNA replication in Synechocystis, plays a role in restoring polyploidy upon subsequent phosphate supplementation. Based on these observations, we have developed an alternative natural transformation protocol involving phosphate depletion that decreases the time required to obtain fully segregated mutants.
| S-EPMC7174562 | biostudies-literature
Project description:Copper is supplied to plastocyanin for photosynthesis and cytochrome c oxidase for respiration in the thylakoids of Synechocystis PCC 6803 by the membrane-bound P-type ATPases CtaA and PacS, and the metallochaperone Atx1. We have determined the Cu(I) affinities of all of the soluble proteins and domains in this pathway. The Cu(I) affinities of the trafficking proteins range from 5 × 10(16) to 5 × 10(17) M(-1) at pH 7.0, consistent with values for homologues. Unusually, Atx1 binds Cu(I) significantly tighter than the metal-binding domains (MBDs) of CtaA and PacS (CtaA(N) and PacS(N)), and equilibrium copper exchange constants of approximately 0.2 are obtained for transfer to the MBDs. Dimerization of Atx1 increases the affinity for Cu(I), but the loop 5 His61 residue has little influence. The MBD of the zinc exporter ZiaA (ZiaA(N)) exhibits an almost identical Cu(I) affinity, and Cu(I) exchange with Atx1, as CtaA(N) and PacS(N), and the relative stabilities of the complexes must enable the metallochaperone to distinguish between the MBDs. The binding of potentially competing zinc to the trafficking proteins has been studied. ZiaA(N) has the highest Zn(II) affinity and thermodynamics could be important for zinc removal from the cell. Plastocyanin has a Cu(I) affinity of 2.6 × 10(17) M(-1), 15-fold tighter than that of the Cu(A) site of cytochrome c oxidase, highlighting the need for specific mechanisms to ensure copper delivery to both of these targets. The narrow range of Cu(I) affinities for the cytoplasmic copper proteins in Synechocystis will facilitate relocation when copper is limiting.
| S-EPMC3156197 | biostudies-literature
Project description:Understanding energy and redox homeostasis and carbon partitioning is crucial for systems metabolic engineering of cell factories. Carbon metabolism alone cannot achieve maximal accumulation of metabolites in production hosts, since an efficient production of target molecules requires energy and redox balance, in addition to carbon flow. The interplay between cofactor regeneration and heterologous production in photosynthetic microorganisms is not fully explored. To investigate the optimality of energy and redox metabolism, while overproducing alkenes-isobutene, isoprene, ethylene and 1-undecene, in the cyanobacterium Synechocystis sp. PCC 6803, we applied stoichiometric metabolic modelling. Our network-wide analysis indicates that the rate of NAD(P)H regeneration, rather than of ATP, controls ATP/NADPH ratio, and thereby bioproduction. The simulation also implies that energy and redox balance is interconnected with carbon and nitrogen metabolism. Furthermore, we show that an auxiliary pathway, composed of serine, one-carbon and glycine metabolism, supports cellular redox homeostasis and ATP cycling. The study revealed non-intuitive metabolic pathways required to enhance alkene production, which are mainly driven by a few key reactions carrying a high flux. We envision that the presented comparative in-silico metabolic analysis will guide the rational design of Synechocystis as a photobiological production platform of target chemicals.
| S-EPMC10516873 | biostudies-literature