Systemic properties of autotrophic growth1cw henning knoop, yvonne zilliges, wolfgang lockau, and ralf steuer. Codon optimization was performed using the optimizer application and the codon usage table of the cyanobase website. Extracellular erythritol is not consumed by synechocystis and exhibits low toxicity. Genomic structure of the cyanobacterium synechocystis sp. Improving heterologous protein expression in synechocystis. Next, tm1254 was cloned in an operon together with cmer or gcy1p, which was expressed with a trc1 promoter, after integration into the synechocystis genome together with a kanamycin resistance cassette, at the neutral. Experimental analysis of recently transposed insertion. Sll1951 is the surface layer slayer protein of the cyanobacterium synechocystis sp. Pcc 6803 jones pr 2012 ethylene synthesis and regulated expression of recombinant protein in synechocystis sp. An improved plasmid vector system for genetic engineering of synechocystis sp pcc 6803 arul varman washington university in st. Vermaasa a department of botany and center for the study of early events in photosynthesis, arizona state university, tempe. Evidence of multifaceted functions of codon usage in translation. The freeliving cynobacterium synechocystis pcc 6803 is an interesting model organism to study oxygenic photosynthesis as well as other metabolic processes.
Our current model is that psb28 and psb282 compete for the same binding. Ethylene synthesis and regulated expression of recombinant. The organization of the different genes in the operons have been found to resemble that of the cyanobacteria synechococcus sp. Highlights in the genome of the cyanobacterium synechocystis sp. Genomic and transcriptomic comparisons between closely related bacteria are powerful approaches to infer insights into their metabolic potentials and regulatory networks. Systemic properties of autotrophic growth1cw henning knoop, yvonne zilliges, wolfgang lockau, and ralf steuer institute for theoretical biology h. We have searched the genome for small hypothetical chlorophyll binding proteins. The ratio of codon usage frequency is one of the indicators to measure codon usage bias of different species. Nucleotide sequence of the second psb g gene in synechocystis.
Here, we report for the first time that the cyanobacterium synechocystis pcc 6803 is able to produce caffeic acid from pcoumaric acid upon heterologous expression of c3h. In this study, continuous and stable ethylene prodn. Exploring native genetic elements as plugin tools for. Insights into isoprene production using the cyanobacterium. To evaluate the effects of the addition of extracellular erythritol on the growth of synechocystis, cells were inoculated in the absence and presence of 1, 10, 20, 30, 50 and 95 gl erythritol in bg11 medium and with illumination optimized for rapid growth see methods section. For a brief explanation how to use this program, go here. The ethylene production rate of the engineered synechocystis 6803 can reach to 171 mgl per day after the optimization of efe expression levels, light intensity, and nutrient status 112. Kf444508, encoding the diterpene synthases that convert geranylgeranyl diphosphate ggpp to rmanoyl oxide, were codon optimized using the optimumgene optimization tool for synechocystis, available from genscript and inserted into the pdftrc shuttle vector. Metabolic engineering of the pentose phosphate pathway for. The sll1951 gene encodes the surface layer protein of. Isoprene was produced in synechocystis 6803 by the expression of a codon optimized isoprene synthase from pueraria montana.
Yy is funded by the national basic research program of china 973 project, grant. Synechocystis sp pcc 6803 strains lacking photosystem i. A portal site for the genome databases related to the plants whose genome sequences were determined or genome analyses were conducted mainly at kazusa dna research institute. Engineering of genetic control tools in synechocystis sp. Pcc 6803 metabolism that spans a 12hr light12hr dark cycle by segmenting it into 12. Pcc 6803 has a bidirectional nifehydrogenase hox hydrogenase which reversibly reduces protons to h2. Synechocystis 6803 can use its oxygen sensitive and bidirectional.
Toxinantitoxin systems on the large defense plasmid psysa of synechocystis sp. Experimental analysis of recently transposed insertion sequences in the cyanobacterium synechocystis sp. Ethylene has been biosynthesized in synechocystis 6803 by the expression of codon optimized ethyleneformingenzyme encoding gene efe from pseudomonas syringae 111,112. Aug, 20 moreover, ethylene and isoprene are important precursors for the production of synthetic rubbers and polymer. In synechocystis 6803 a unicellular cyanobacterium the presence of homologous genes to glga sll0945. Pcc 6803 using a commercial labscale high density cultivation hdc platform in the. Insights into isoprene production using the cyanobacterium synechocystis sp. Improving a synechocystisbased photoautotrophic chassis through. Evidence of multifaceted functions of codon usage in translation within the model beetle tribolium castaneum. For conversion of the genetic code, the codon usage of synechocystis sp.
The genome of the unicellular cyanobacterium synechocystis sp. Ethylene production with engineered synechocystis sp pcc 6803. Synechocystis sp pcc 6803 strains lacking photosystem i and. The sequence determination of the entire genome of the synechocystis sp. Resultsthis study focuses on synechocystis sp pcc 6803 and shows stable ethylene production through the integration of a codon optimized version of the efe gene under control of the ptrc promoter. The functions of nearly half of the genes has been deduced using similarity searches.
Comparative genome analysis of the closely related. Bioinformatic tool to adapt codon usage to sequenced prokaryotes. Pcc 6803 with enhanced transforming capacity pcc 6803t was obtained from s. An expression system was established for the comparison of selected rbs sequences in synechocystis, using several alternative reporter proteins syfp2, codon optimized gfpmut3 and ethylene forming enzyme as quantitative indicators of the. Identification of the start codon for sphs encoding the. Functional expression of an arabidopsis p450 enzyme, p. A single plasmid based crispr interference in synechocystis 6803. Photosystem ii component lifetimes in the cyanobacterium.
This is especially true for species such as synechocystis, which do not retain. Pcc 6803 homolog of the circadian clock output regulator rpaa in day. The genomic sequence has revealed the structure of the genome and its gene constituents 3167 genes, as well as the relative map positions of each gene. All these genes were codonoptimized for expression in synechocystis and obtained through chemical synthesis see methods section. We first validate the biological fidelity of cyclesyn predictions by comparing them to experimental observations for a diurnally cultured synechocystis sp. Cultures were supplemented with 20 mm teskoh, ph 8. A glucosetolerant 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. In our study we found that the two glgp genes in synechocystis 6803 have different functions. Toxinantitoxin systems on the large defense plasmid psysa of. This is achieved by exploiting the large difference in timescales between metabolic reactions and cell growth.
Structural analysis of four large plasmids harboring in a. Ethylene synthesis and regulated expression of recombinant protein in synechocystis sp. Resultsthis study focuses on synechocystis sp pcc 6803 and shows stable ethylene production through the integration of a codonoptimized version of the efe gene under control of the ptrc promoter and the core shinedalgarno sequence 5. Two open reading frames, located antisense to known genes, fulfilled the criteria.
High density cultivation for efficient sesquiterpenoid. The plasmids were introduced into escherichia coli bl21 codon. We have adopted a 3fold approach to aid purification of unassembled cp47 and cp43 complexes. Synechocystis is a genus of unicellular, freshwater cyanobacteria in the family merismopediaceae. Here we present cyclesyn, a periodic model of synechocystis sp. This database was constructed based on the codon usage of genes provided from ncbi genbank. Pcc 6803 play a central role in photosynthesis, respiration and other important metabolic pathways. Vermaas department of botany and the centerfor the study of early events in photosynthesis, arizona state university, tempe, az 852871601, usa received 8. Spacer1 and spacer2 were designed to target sequences starting at 5 bp upstream and 60 bp downstream of the start codon, respectively. Synechocystis 6803 was able to grow at 42c, but a sll56 mutant was unable to grow at 40c. Structural analysis of four large plasmids harboring in a unicellular cyanobacterium, synechocystis sp. The software, automated mass spectral deconvolution and.
Pcc 6803 is the most popular cyanobacterial model for prokaryotic photosynthesis and for metabolic engineering to produce biofuels. Pcc 6803 is a widelyused cyanobacterial model strain that is used to study many biological processes and is also applied for the production of biopolymers. For the production of erythritol by synechocystis pcc 6803, reductase genes from yeast and fungal catabolic pathways were chosen for expression in the cyanobacterium. Genomescale modeling of synechocystis sp pcc 6803 and prediction of pathway insertion. At least three ribosome binding sites most were designed using.
Genetic engineering of synechocystis pcc6803 for the. The search for new chlorophyllbinding proteins in the. The two operons atp1 and atp2, encoding the subunits of the f o f 1 atpsynthase, have been cloned and sequenced from the cyanobacterium synechocystis sp. The low abundance of psii assembly intermediates in wt synechocystis 6803 is a major barrier to their isolation. Limonene production by engineered synechocystis 6803 was tested by incubating cultures for 7 days. Pcc6803 models shastri aa, morgan ja 2005 flux balance analysis of photoautotrophic metabolism. Up to date, approximately 900 known or predicted membrane proteins, consisting 24. Molecular microbiology volume 0, issue ja research article the role of the synechocystis sp.
This study focuses on synechocystis sp pcc 6803 and shows stable ethylene production through the integration of a codonoptimized version of the efe gene under control of the ptrc promoter and the core shinedalgarno sequence 5. Because of the volatility of limonene, a dodecane overlay was applied on cultures to collect limonene in the organic layer. Subunit composition of cp43less photosystem ii complexes of. This study confirms those findings and extends them to 4 additional prokaryotic organisms. The photosynthetic bacteria rhodobacter capsulatus and. Low carbon technology research and development program alca, japan. Bisabolene synthase codon usage variants were synthesized by genscript. The ethyleneforming enzyme efe from pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. Expression of the ggps gene, involved in osmolyte synthesis. Here we analyze a gene expression profiling methodology, rcbs the scores of relative codon usage bias to elucidate expression patterns of genes in the synechocystis genome. Improving heterologous protein expression in synechocystis sp.
Analysis of codon usageq correspondence analysis of. Isolation of cp47his and cp43his from synechocystis 6803. Chlamydia trachomatis, chlamydophila pneumoniae ar39, synechocystis sp. This thesis is brought to you for free and open access by washington university open scholarship. An aug codon, about 50 bases upstream from the end of psbdi had. To broaden the range of microbial systems for the functional heterologous biosynthesis of plant triterpenes, we chose to employ the physiologically versatile photosynthetic bacteria rhodobacter capsulatus sb1003 and synechocystis sp. Both are wellestablished model organisms for anaerobic and. Pcc 6803, foreseeing the use of this cyanobacterium as a photoautotrophic chassis. Volume 260, number 1, 57 feb 08030 january 1990 the psbc start codon in synechocystis sp. Pcc 6803 by introducing a codon optimized ethyleneforming enzyme efe from pseudomonas syringae pv. Data in table 3 reveals there are 17 codons showing distinct usage differences between the. Investigating the early stages of photosystem ii assembly in. Dna sequence was optimized for the preferred codon usage in synechocystis. Department of genetics thesis submitted to the university of nottingham for the degree of doctor.
F5a and f3a, f5a and f3b, f5b and f3a, and f5b and f3b. Utr sequence before the translational start codon of each gene was amplified by pcr as an intact promoter, which was then directly placed before the gene coding an enhanced yellow fluorescence protein eyfp as a reporter. This study focuses on synechocystis sp pcc 6803 and shows stable. Synechocystis sp pcc 6803 and phycobilisome function strains lacking photosystem i gaozhong shen, sammy boussiba,aib and wim f. Transformation in the cyanobacterium synechocystis sp. Photosystem ii component lifetimes in the cyanobacterium synechocystis sp. Pdf ethylene production with engineered synechocystis sp. Seawater cultivation of freshwater cyanobacterium synechocystis sp. To ensure efficient expression of the plant cdna in the cyanobacterial host, the codon usage was adopted to that of synechocystis. A comparison of constitutive and inducible nonendogenous. A synthetic codon optimized gene encoding nterminal histagged efe efeh was expressed in synechocystis sp. Pcc 6803 consists of a single chromosome and several plasmids of different sizes, and the nucleotide sequences of. Gene expression profile of the cynobacterium synechocystis.
Codon usage table with amino acids a style like codonfrequency output in gcg wisconsin package tm. Adherence to codon usage biases for each of these 6 organisms is inversely correlated with a coding regions average amino acid biosynthetic. Identification of alcohol stress tolerance genes of. Exploiting day and nighttime metabolism of synechocystis sp. Pcc 6803 open reading frames below 50 codons were not annotated. Sequence analysis of the genome of the unicellular.
The role of psb28 is currently unknown, but on the basis of phenotype of a null mutant of synechocystis 6803, it might regulate chlorophyll availability during the biogenesis of psi and psii. Ethylene production with engineered synechocystis sp pcc. An improved plasmid vector system for genetic engineering of. However, because stress against the produced alcohol is a major barrier for industrial applications, it is highly desirable to engineer organisms with strong alcohol tolerance. An sll1951 deletion mutation was introduced into synechocystis and was easily segregated to homozygosity under laboratory conditions. A dna fragment from the cyanobacterium synechocystis sp. Recently, stable and continuous ethylene production was achieved in synechocystis sp. Translation efficiency of heterologous proteins is. Pcc 6803 hereafter referred to as synechocystis in a study in which a codon optimized efe gene with mutational hotspots eliminated was expressed from a constitutive, pea plant chloroplast psba promoter, and integrated at the slr0168 neutralsite. Transcription and differential regulation of these antisense genes. Identification and bioinformatic analysis of the membrane. Atcc stands ready to support our customers needs during the coronavirus pandemic.
Pcc 6803 is the most popular cyanobacterial strain, serving as a standard in the research fields of photosynthesis, stress response, metabolism and so on. Comprehensive identification of the membrane proteins is of importance for a better understanding of the diverse functions of its unique membrane structures. Accordingly, genome modification in synechocystis pcc6803 requires a. Comparisons of amino acid composition and codon usage data not shown do not reveal dramatic dif ferences which might suggest why synechocystis 6803 harbours two psbg copies. We show here that a second psb28 homologue, psb282, also associates with rc47. The arabidopsis thaliana ref8 gene, which encodes a c3h, was synthesized and codon optimized for enhanced expression in synechocystis. However, there are not many studies on the use of seawater for cultivating. A synthetic codonoptimized gene encoding nterminal histagged efe efeh was expressed in synechocystis sp.
This enzyme is composed of a hydrogenase domain and a. Journal of chemical technology and biotechnology 844. Sequence analysis of the cdna encoding human liver glycogen phosphorylase reveals tissuespecific codon usage. Pcc 6803 was the first phototrophic organism to be fully sequenced. Pcc 6803 shinobu okamoto masahik, ikeuchio an, d masayuk ohmorii departrment of life sciences, graduate school of art and sciences, university of tokyo, komaba, meguro, tokyo 1538902, japan received 27 april 1999. Amino acid cost and codonusage biases in 6 prokaryotic. A novel tool to adapt codon usage of a target gene to its. In order to compare the strengths of native promoters in synechocystis 6803, 5.
Pcc 6803 extracellular phosphate levels are relayed to the pho regulon via the sphs histidine kinase. In this cyanobacterium, the start codon of sphs has been assigned as a gug, thereby predicting sphs to be a cytosolic protein lacking a putative nterminal region found in the phor orthologue from escherichia coli. Pdf ethylene synthesis and regulated expression of. This large, hemolysinlike protein was found in the supernatant of a strain that was deficient in slayer attachment. A single plasmid based crispr interference in synechocystis 6803 a proof of concept. Sequence features in the 1 mb region from map positions 64% to 92% of the genome.
Sequence analysis of the genome of the unicellular cyanobacterium synechocystis sp. Vermaas department of botany and the center for the study of early events in photosynthesis, arizona state university, tempe, az 852871601, usa received 8 november 1989 the translation start codon for psbc, the gene encoding. Six histidine residues histag were introduced in the protein nterminal right after the first methionine codon. Vermaasa a department of botany and center for the study of early events in photosynthesis, arizona state university, tempe, arizona 852871601. Hydropathicity plots for psbg1 and psbg2 are also very similar fig. Pcc 6803 is an attractive organism for the production of alcohols, such as isobutanol and ethanol. Pdf improving heterologous protein expression in synechocystis. Supplementing the wildtype gene to the mutant restored its growth at high temperatures, and inactivation of the downstream genes showed no effect.
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