A parametric logistic equation with light flux and medium concentration for cultivation planning of microalgae.

Microalgae are considered to be promising producers of bioactive chemicals, feeds and fuels from carbon dioxide by photosynthesis. Thus, the prediction of microalgal growth profiles is important for the planning of cost-effective and sustainable cultivation-harvest cycles. This paper proposes a mathematical model capable of predicting the effect of light flux into culture and medium concentration on the growth profiles of microalgae by incorporating these growth-limiting factors into a logistic equation. The specific form of the equation is derived based on the experimentally measured growth profiles of Monoraphidium sp., a microalgal strain isolated by the authors, under 16 conditions consisting of combinations of incident light fluxes into culture and initial medium concentrations. Using a cross-validation method, it is shown that the proposed model has the ability to predict necessary incident light flux into culture and initial medium concentration for harvesting target biomass at a target time. Finally, model-guided cultivation planning is performed and is evaluated by comparing the result with experimental data.

Screening, gene cloning, and characterization of orsellinic acid decarboxylase from Arthrobacter sp. K8 for regio-selective carboxylation of resorcinol derivatives.

Toward a sustainable synthesis of value-added chemicals, the method of CO2 utilization attracts great interest in chemical process engineering. Biotechnological CO2 fixation is a promising technology; however, efficient methods that can fix carbon dioxide are still limited. Instead, some parts of microbial decarboxylases allow the introduction of carboxy group into phenolic compounds using bicarbonate ion as a C1 building block. Here, we identified a unique decarboxylase from Arthrobacter sp. K8 that acts on resorcinol derivatives. A high-throughput colorimetric decarboxylase assay facilitated gene cloning of orsellinic acid decarboxylase from genomic DNA library of strain K8. Sequence analysis revealed that the orsellinic acid decarboxylase belonged to amidohydrolase 2 family, but shared low amino acid sequence identity with those of related decarboxylases. Enzymatic characterization unveiled that the decarboxylase introduces a carboxy group in a highly regio-selective manner. We applied the decarboxylase to enzymatic carboxylation of resorcinol derivatives. Using Escherichia coli expressing the decarboxylase gene as a whole cell biocatalyst, orsellinic acid, 2,4-dihydroxybenzoic acid, and 4-methoxysalicylic acid were produced in the presence of saturated bicarbonate. These findings could provide new insights into the production of useful phenolic acids from resorcinol derivatives.

Enhancement of acetyl-CoA flux for photosynthetic chemical production by pyruvate dehydrogenase complex overexpression in Synechococcus elongatus PCC 7942.

Genetic manipulation in cyanobacteria enables the direct production of valuable chemicals from carbon dioxide. However, there are still very few reports of the production of highly effective photosynthetic chemicals. Several synthetic metabolic pathways (e.g., isopropanol, acetone, isoprene, and fatty acids) have been constructed by branching from acetyl-CoA and malonyl-CoA, which are key intermediates for photosynthetic chemical production downstream of pyruvate decarboxylation. Recent reports of the absolute determination of cellular metabolites in Synechococcus elongatus PCC 7942 have shown that its acetyl-CoA levels corresponded to about one hundredth of the pyruvate levels. In short, one of the reasons for lower photosynthetic chemical production from acetyl-CoA and malonyl-CoA was the smaller flux to acetyl-CoA. Pyruvate decarboxylation is a primary pathway for acetyl-CoA synthesis from pyruvate and is mainly catalyzed by the pyruvate dehydrogenase complex (PDHc). In this study, we tried to enhance the flux toward acetyl-CoA from pyruvate by overexpressing PDH genes and, thus, catalyzing the conversion of pyruvate to acetyl-CoA via NADH generation. The overexpression of PDH genes cloned from S. elongatus PCC 7942 significantly increased PDHc enzymatic activity and intracellular acetyl-CoA levels in the crude cell extract. Although growth defects were observed in overexpressing strains of PDH genes, the combinational overexpression of PDH genes with the synthetic metabolic pathway for acetate or isopropanol resulted in about 7-fold to 9-fold improvement in its production titer, respectively (9.9 mM, 594.5 mg/L acetate, 4.9 mM, 294.5 mg/L isopropanol). PDH genes overexpression would, therefore, be useful not only for the production of these model chemicals, but also for the production of other chemicals that require acetyl-CoA as a key precursor.

Modification of carbon metabolism in Synechococcus elongatus PCC 7942 by cyanophage-derived sigma factors for bioproduction improvement.

Many cyanophages, which infect cyanobacteria, most of possess putative sigma factors that have high amino acid sequence homology with the σ70-type sigma factor present in cyanobacteria, allowing them to obtain energy and metabolites for their own propagation. In this study, we aimed to modify the carbon metabolism of Synechococcus elongatus PCC 7942 by expressing putative sigma factors from Synechococcus phages to improve bioproduction. Four cyanophage-derived putative sigma factors-putative RpsD4 from Synechococcus phage S-CBS1, putative RpoD and putative RpoS from S-CBS2, and putative RpsD4 from S-CBS3-were selected for this purpose. These were introduced into S. elongatus PCC 7942, and their expression was controlled with a theophylline-dependent riboswitch. The expression of the putative RpoD from S-CBS2 and putative RpsD4 from S-CBS3 resulted in a significant decrease in the growth rate of S. elongatus PCC 7942. In addition, metabolome analysis showed a 3.2-fold increase in acetyl-CoA concentration with the expression of the putative RpoD from S-CBS2 and a 1.9-fold increase with the putative RpsD4 from S-CBS3. The results of RT-qPCR showed that several sugar metabolism genes were repressed by the putative RpoD and activated by the putative RpsD4. In particular, the engineered strain overexpressing the putative RpsD4 and expressing phosphate acetyltransferase succeeded in improving the productivity of the model target product acetate to 217% of its previous value. To the best of our knowledge, this study is the first to modify the metabolism of S. elongatus PCC 7942 by expressing their putative sigma factors from cyanophages.

Mutations responsible for alcohol tolerance in the mutant of Synechococcus elongatus PCC 7942 (SY1043) obtained by single-cell screening system.

The production of alcohols directly from carbon dioxide by engineered cyanobacteria is an attractive technology for a sustainable future. Enhanced tolerance to the produced alcohols would be a desirable feature of the engineered cyanobacterial strains with higher alcohol productivity. We have recently obtained the mutant strains of Synechococcus elongatus PCC 7942 with higher tolerance to isopropanol using a single-cell screening system (Arai et al., Biotechnol. Bioeng., 114, 1771-1778, 2017). Among the mutant strains, SY1043 showed the highest isopropanol tolerance. Interestingly, SY1043 also showed higher tolerance to other alcohols such as ethanol and 1-butanol, however, the mechanisms involved in enhancing this alcohol tolerance were unclear. To reveal the alcohol tolerance mechanism of SY1043, we investigated the relationship between alcohol tolerance and four mutations found in SY1043 by genome resequencing analysis. Isopropanol tolerance was enhanced by amino acid substitution (Leu285Pro) in a hypothetical protein encoded by Synpcc7942_0180 of the wild type strain TA1297. TA4135, into which this mutation was introduced, showed a same tendency of tolerance to other alcohols (ethanol and 1-butanol).

Metabolic engineering of Synechococcus elongatus PCC 7942 for improvement of 1,3-propanediol and glycerol production based on in silico simulation of metabolic flux distribution.

BACKGROUND: Production directly from carbon dioxide by engineered cyanobacteria is one of the promising technologies for sustainable future. Previously, we have successfully achieved 1,3-propanediol (1,3-PDO) production using Synechococcus elongatus PCC 7942 with a synthetic metabolic pathway. The strain into which the synthetic metabolic pathway was introduced produced 3.48 mM (0.265 g/L) 1,3-PDO and 14.3 mM (1.32 g/L) glycerol during 20 days of incubation. In this study, the productivities of 1,3-PDO were improved by gene disruption selected by screening with in silico simulation. METHODS: First, a stoichiometric metabolic model was applied to prediction of cellular metabolic flux distribution in a 1,3-PDO-producing strain of S. elongatus PCC 7942. A genome-scale model of S. elongatus PCC 7942 constructed by Knoop was modified by the addition of a synthetic metabolic pathway for 1,3-PDO production. Next, the metabolic flux distribution predicted by metabolic flux balance analysis (FBA) was used for in silico simulation of gene disruption. As a result of gene disruption simulation, NADPH dehydrogenase 1 (NDH-1) complexes were found by screening to be the most promising candidates for disruption to improve 1,3-PDO production. The effect of disruption of the gene encoding a subunit of the NDH-1 complex was evaluated in the 1,3-PDO-producing strain. RESULTS AND CONCLUSIONS: During 20 days of incubation, the ndhF1-null 1,3-PDO-producing strain showed the highest titers: 4.44 mM (0.338 g/L) 1,3-PDO and 30.3 mM (2.79 g/L) glycerol. In this study, we successfully improved 1,3-PDO productivity on the basis of in silico simulation of gene disruption.

Alcohol-tolerant mutants of cyanobacterium Synechococcus elongatus PCC 7942 obtained by single-cell mutant screening system.

Enhancement of alcohol tolerance in microorganisms is an important strategy for improving bioalcohol productivity. Although cyanobacteria can be used as a promising biocatalyst to produce various alcohols directly from CO2 , low productivity, and low tolerance against alcohols are the main issues to be resolved. Nevertheless, to date, a mutant with increasing alcohol tolerance has rarely been reported. In this study, we attempted to select isopropanol (IPA)-tolerant mutants of Synechococcus elongatus PCC 7942 using UV-C-induced random mutagenesis, followed by enrichment of the tolerant candidates in medium containing 10 g/L IPA and screening of the cells with a high growth rate in the single cell culture system in liquid medium containing 10 g/L IPA. We successfully acquired the most tolerant strain, SY1043, which maintains the ability to grow in medium containing 30 g/L IPA. The photosynthetic oxygen-evolving activities of SY1043 were almost same in cells after 72 h incubation under light with or without 10 g/L IPA, while the activity of the wild-type was remarkably decreased after the incubation with IPA. SY1043 also showed higher tolerance to ethanol, 1-butanol, isobutanol, and 1-pentanol than the wild type. These results suggest that SY1043 would be a promising candidate to improve alcohol production using cyanobacteria. Biotechnol. Bioeng. 2017;114: 1771-1778. © 2017 Wiley Periodicals, Inc.

Construction of a novel d-lactate producing pathway from dihydroxyacetone phosphate of the Calvin cycle in cyanobacterium, Synechococcus elongatus PCC 7942.

Using engineered cyanobacteria to produce various chemicals from carbon dioxide is a promising technology for a sustainable future. Lactate is a valuable commodity that can be used for the biodegradable plastic, polylactic acid. Typically, lactate production using engineered cyanobacteria was via the conversion of pyruvate in glycolysis by lactate dehydrogenase. In cyanobacteria, the metabolic flux in the Calvin cycle is higher than that in glycolysis under photoautotrophic conditions. The construction of a novel lactate producing pathway that uses metabolites from the Calvin cycle could potentially increase lactate productivity in cyanobacteria. In order to develop such a novel lactate production pathway, we engineered a cyanobacterium Synechococcus elongatus PCC 7942 strain that produced lactate directly from carbon dioxide using dihydroxyacetone phosphate (DHAP) via methylglyoxal. We confirmed that wild-type strain of S. elongatus PCC 7942 could produce lactate using exogenous methylglyoxal. A methylglyoxal synthase gene, mgsA, from Escherichia coli was introduced into Synechococcus elongates PCC 7942 for conversion of DHAP to methylglyoxal. This engineered strain produced lactate directly from carbon dioxide. Genes encoding intrinsic putative glyoxalase I, II (Synpcc7942_0638, 1403) and the lactate/H+ symporter from E. coli (lldP) were additionally introduced to enhance the production. For higher lactate production, it was important to maintain elevated extracellular pH due to the characteristics of lactate exporting system. In this study, the highest lactate titer of 13.7 mM (1.23 g/l) was achieved during a 24-day incubation with the engineered S. elongatus PCC 7942 strain possessing the novel lactate producing pathway.

Improvement of 1,3-propanediol production using an engineered cyanobacterium, Synechococcus elongatus by optimization of the gene expression level of a synthetic metabolic pathway and production conditions.

The introduction of a synthetic metabolic pathway consisting of multiple genes derived from various organisms enables cyanobacteria to directly produce valuable chemicals from carbon dioxide. We previously constructed a synthetic metabolic pathway composed of genes from Escherichia coli, Saccharomyces cerevisiae, and Klebsiella pneumoniae. This pathway enabled 1,3-propanediol (1,3-PDO) production from cellular DHAP via glycerol in the cyanobacterium, Synechococcus elongatus PCC 7942. The production of 1,3-PDO (3.79mM, 0.29g/l) directly from carbon dioxide by engineered S. elongatus PCC 7942 was successfully accomplished. However, the constructed strain accumulated a remarkable amount of glycerol (12.6mM, 1.16g/l), an intermediate metabolite in 1,3-PDO production. Notably, enhancement of latter reactions of synthetic metabolic pathway for conversion of glycerol to 1,3-PDO increases 1,3-PDO production. In this study, we aimed to increase the observed 1,3-PDO production titer. First, the weaker S. elongatus PCC 7942 promoter, PLlacO1, was replaced with a stronger promoter (Ptrc) to regulate genes involved in the conversion of glycerol to 1,3-PDO. Second, the induction timing for gene expression and medium composition were optimized. Promoter replacement resulted in higher 1,3-PDO production than glycerol accumulation, and the amount of products (1,3-PDO and glycerol) generated via the synthetic metabolic pathway increased with optimization of medium composition. Accordingly, we achieved the highest titer of 1,3-PDO (16.1mM, 1.22g/l) and this was higher than glycerol accumulation (9.46mM, 0.87g/l). The improved titer was over 4-fold higher than that of our previous study.

Metabolic engineering for isopropanol production by an engineered cyanobacterium, Synechococcus elongatus PCC 7942, under photosynthetic conditions.

Cyanobacteria engineered for production of biofuels and biochemicals from carbon dioxide represent a promising area of research in relation to a sustainable economy. Previously, we have succeeded in producing isopropanol from cellular acetyl-CoA by means of Synechococcus elongatus PCC 7942 into which a synthetic metabolic pathway was introduced. The isopropanol production by this synthetic metabolic pathway requires acetate; therefore, the cells grown under photosynthetic conditions have to be transferred to a dark and anaerobic conditions to produce acetate. In this study, we achieved acetate production under photosynthetic conditions by S. elongatus PCC 7942 into which we introduced the pta gene encoding phosphate acetyltransferase from Escherichia coli. The metabolic modification (via pta introduction) of the isopropanol-producing strain enabled production of isopropanol under photosynthetic conditions. During 14 days of production, the titer of isopropanol reached 0.55 mM (33.1 mg/l) with an intermediate product, acetone, at 0.21 mM (12.2 mg/l).

Pleurochrysome: A Web Database of Pleurochrysis Transcripts and Orthologs Among Heterogeneous Algae.

Pleurochrysis is a coccolithophorid genus, which belongs to the Coccolithales in the Haptophyta. The genus has been used extensively for biological research, together with Emiliania in the Isochrysidales, to understand distinctive features between the two coccolithophorid-including orders. However, molecular biological research on Pleurochrysis such as elucidation of the molecular mechanism behind coccolith formation has not made great progress at least in part because of lack of comprehensive gene information. To provide such information to the research community, we built an open web database, the Pleurochrysome (http://bioinf.mind.meiji.ac.jp/phapt/), which currently stores 9,023 unique gene sequences (designated as UNIGENEs) assembled from expressed sequence tag sequences of P. haptonemofera as core information. The UNIGENEs were annotated with gene sequences sharing significant homology, conserved domains, Gene Ontology, KEGG Orthology, predicted subcellular localization, open reading frames and orthologous relationship with genes of 10 other algal species, a cyanobacterium and the yeast Saccharomyces cerevisiae. This sequence and annotation information can be easily accessed via several search functions. Besides fundamental functions such as BLAST and keyword searches, this database also offers search functions to explore orthologous genes in the 12 organisms and to seek novel genes. The Pleurochrysome will promote molecular biological and phylogenetic research on coccolithophorids and other haptophytes by helping scientists mine data from the primary transcriptome of P. haptonemofera.

Cyanobacterial production of 1,3-propanediol directly from carbon dioxide using a synthetic metabolic pathway.

Production of chemicals directly from carbon dioxide using light energy is an attractive option for a sustainable future. The 1,3-propanediol (1,3-PDO) production directly from carbon dioxide was achieved by engineered Synechococcus elongatus PCC 7942 with a synthetic metabolic pathway. Glycerol dehydratase catalyzing the conversion of glycerol to 3-hydroxypropionaldehyde in a coenzyme B12-dependent manner worked in S. elongatus PCC 7942 without addition of vitamin B12, suggesting that the intrinsic pseudovitamin B12 served as a substitute of coenzyme B12. The highest titers of 1,3-PDO (3.79±0.23 mM; 288±17.7 mg/L) and glycerol (12.62±1.55 mM; 1.16±0.14 g/L), precursor of 1,3-PDO, were reached after 14 days of culture under optimized conditions in this study.

Optimization of isopropanol production by engineered cyanobacteria with a synthetic metabolic pathway.

Cyanobacterium is an attractive host for the production of various chemicals and alternative fuels using solar energy and carbon dioxide. In previous study, we succeeded to produce isopropanol using engineered Synechococcus elongatus PCC 7942 under dark and anaerobic conditions (0.43 mM, 26.5 mg/l). In the present study, we report the further optimization of this isopropanol producing condition. We then optimized growth conditions for production of isopropanol by the engineered cyanobacteria, including the use of cells in early stationary phase and buffering of the production medium to neutral pH. We observed that shifting of cultures from dark and anaerobic to light and aerobic conditions during the production phase dramatically increased isopropanol production by conversion to isopropanol from acetate, byproduct under dark and anaerobic condition. Under the optimized production conditions, the titer of isopropanol was elevated 6-fold, to 2.42 mM (146 mg/l).

Engineering a synthetic pathway in cyanobacteria for isopropanol production directly from carbon dioxide and light.

Production of alternate fuels or chemicals directly from solar energy and carbon dioxide using engineered cyanobacteria is an attractive method to reduce petroleum dependency and minimize carbon emissions. Here, we constructed a synthetic pathway composed of acetyl-CoA acetyl transferase (encoded by thl), acetoacetyl-CoA transferase (encoded by atoAD), acetoacetate decarboxylase (encoded by adc) and secondary alcohol dehydrogenase (encoded by adh) in Synechococcus elongatus strain PCC 7942 to produce isopropanol. The enzyme-coding genes, heterogeneously originating from Clostridium acetobutylicum ATCC 824 (thl and adc), Escherichia coli K-12 MG1655 (atoAD) and Clostridium beijerinckii (adh), were integrated into the S. elongatus genome. Under the optimized production conditions, the engineered cyanobacteria produced 26.5 mg/L of isopropanol after 9 days.

Genetic manipulations restored the growth fitness of reduced-genome Escherichia coli.

Microbes with smaller genomes would be better chassis for analysis, design, and improvement in the fields of metabolic engineering, synthetic biology, and molecular breeding. To create an Escherichia coli strain with a smaller genome, we used a stepwise genome reduction approach. Beginning with strain MGF-01, which has a genome of 3.62 megabase pairs (Mbp), we generated two E. coli K-12 strains without any insertion sequence (IS), DGF-327 and DGF-298, with reduced genome sizes of 3.27 and 2.98 Mbp, respectively. During the strain construction, intrinsic mutations of ilvG and rph were functionally restored to accelerate initial growth after inoculation. The genomes of the two strains were sequenced, and their structures were confirmed. Both strains showed no auxotrophy, and had better growth fitness, especially in the initial phase, and better cell yield in a rich medium than the wild type K-12 strain. Transcriptome analysis revealed that ibpAB and lon, which encode a heat-shock chaperone and a protease for abnormal proteins, respectively, are down-regulated in DGF strains, compared to the ancestral strains with larger genomes. We concluded that down-regulation of the genes encoding chaperones and proteases is one of the factors that improve the fitness of DGF strains. The DGF strains with fewer genes and better cell yield will be good hosts for applications.

Glutathione production by efficient ATP-regenerating Escherichia coli mutants.

There is an ongoing demand to improve the ATP-regenerating system for industrial ATP-driven bioprocesses because of the low efficiency of ATP regeneration. To address this issue, we investigated the efficiency of ATP regeneration in Escherichia coli using the Permeable Cell Assay. This assay identified 40 single-gene deletion strains that had over 150% higher total cellular ATP synthetic activity relative to the parental strain. Most of them also showed higher ATP-driven glutathione synthesis. The deleted genes of the identified strains that showed increased efficiency of ATP regeneration for glutathione production could be divided into the following four groups: (1) glycolytic pathway-related genes, (2) genes related to degradation of ATP or adenosine, (3) global regulatory genes, and (4) genes whose contribution to the ATP regeneration is unknown. Furthermore, the high glutathione productivity of DeltanlpD, the highest glutathione-producing mutant strain, was due to its reduced sensitivity to the externally added ATP for ATP regeneration. This study showed that the Permeable Cell Assay was useful for improving the ATP-regenerating activity of E. coli for practical applications in various ATP-driven bioprocesses, much as that of glutathione production.

Long-term survival of Escherichia coli lacking the HipBA toxin-antitoxin system during prolonged cultivation.

We designed and constructed six major toxin-antitoxin disruptants (DeltachpBIK, DeltadinJ-yafQ, DeltahipBA, DeltamazEF, DeltarelBE, and DeltayefM-yoeB) of Escherichia coli K-12 W3110. On prolonged cultivation of these disruptants with minimal M9 medium, the DeltahipBA cells exhibited a significantly longer life span than that of the other disruptants and of wild-type cells, as analyzed with a LIVE/DEAD BacLight kit (Invitrogen, Carlsbad, CA) in combination with flow cytometry analysis. The gene expression level of hipA in the wild-type cells was highest at the stationary phase of 40 h. The DeltahipBA cells showed higher macromolecular synthesis activity than the wild-type cells at the stationary phase. Stationary phase cells of DeltahipBA and the wild-type strain showed a significantly extended life span under anaerobic conditions. Furthermore, the DeltahipBA cells showed higher resistance to H(2)O(2) than the wild type. These results suggest that HipBA induces cell death with oxidative stress during prolonged cultivation. This is the first report that an E. coli toxin-antitoxin (TA) system affects frequency of survival during the long-term stationary phase.

Structural and physiological studies on the storage beta-polyglucan of haptophyte Pleurochrysis haptonemofera.

The storage beta-polyglucan and catabolic enzyme activities of the haptophyte Pleurochrysis haptonemofera were characterized. The storage beta-polyglucan was prepared by the dimethylsulfoxide-extraction method. (13)C- and (1)H-NMR spectroscopy revealed that the polyglucan consists of beta-(1-->3)- and beta-(1-->6)-linked glucose polymers, with a beta-(1-->6)- to beta-(1-->3)-linkage ratio of 1.5. Gel permeation chromatography showed that the molecular weight of the polyglucan is 1.1-8.4 x 10(4) Da, with a peak at 3.4 x 10(4) Da. The degree of polymerization, which was estimated from the amounts of total carbohydrate and reduced ends, was 203, corresponding to 3.3 x 10(4) Da. A method for measurement of the beta-polyglucan in a small amount of liquid culture involving a mixture of beta-glucanases, Westase, was established. The beta-polyglucan was localized in the soluble fraction of cells. The amount of beta-polyglucan per cell increased at the stationary phase under continuous illumination and decreased in the dark, like those of storage alpha-polyglucans, starch of green algae and glycogen of cyanobacteria. The activities of beta-1,3- and beta-1,6-glucanases involved in the degradation of the storage beta-polyglucan were assayed in vitro, both being optimal at pH 5.0. The beta-1,3-glucanase activity, which was detected on active staining after native polyacrylamide gel electrophoresis, was partially purified by ammonium sulfate precipitation and anion exchange chromatography.

Gene expression profiling of coccolith-bearing cells and naked cells in haptophyte Pleurochrysis haptonemofera with a cDNA macroarray system.

Pleurochrysis haptonemofera is a unicellular marine coccolithophorid that has calcified scales, coccoliths, on the cell surface. Some coccolithophorids including P. haptonemofera have a coccolith-bearing stage and a naked stage in their life cycles. To characterize genes involved in the coccolithogenesis, we generated a total of 9550 expressed sequence tags (EST) from a normalized cDNA library that was prepared using both coccolith-bearing cells (C-cells) and naked cells (N-cells), constructed a cDNA macroarray using the EST clones, and then analyzed the gene expression specificity in C-cells and N-cells. When cDNA clones whose expression ratio exceeded 3-fold were selected, as many as 180 clones were identified as C-cell-specific ones, while only 12 were found to be N-cell-specific ones. These clones were sequenced, assembled, and homology-searched against a public nonredundant protein database. As a result, they were grouped into 54 C-cell-specific and 6 N-cell-specific genes, and 59% and 50% of these genes exhibited significant similarity to those of other known proteins, respectively. To assess mRNA expression further, Northern hybridization was performed for 12 of the C-cell-specific genes and one of the N-cell-specific ones. These clones, together with the new cDNA macroarray, will provide a powerful tool for the future genome-wide functional analysis of uncharacterized genes related to the regulation of the calcification and life cycle of coccolithophorids.

Protoplast formation of the coccolithophorid Pleurochrysis haptonemofera in hypoosmotic K+ solution: shedding of the coccosphere and regrowth of the protoplast in normal medium.

Both coccolith-bearing cells (C-cells) and naked cells (N-cells) of the coccolithophorid Pleurochrysis haptonemofera can grow in salinities of more than 7 per thousand (about 20% of a "normal" sea water salinity [35 per thousand]), with the highest growth rates in salinities of more than 14 per thousand. Microscopic observations of cells suspended in 100 mM NaCl (7 per thousand) showed that, while N-cells were swelling uniformly all over the cell surface, C-cells were bulging the plasma membrane from the hole of the coccosphere at the apical (flagellar) pole of the cell. Effects of several cations and anions on the morphological change of C-cells under hypoosmotic pressure were investigated. When 100 mM K(+) was used, protoplasts were released from the coccosphere completely in almost all the cells. This phenomenon was shown with K(+) most effectively. The protoplasts could grow in the fresh medium and form the first coccolith within 9 h.