Development of a CRISPR/Cas9-mediated gene-editing method to isolate a mutant of the unicellular green alga Parachlorella kessleri strain NIES-2152 with improved lipid productivity.

BACKGROUND: Previously, we isolated a mutant of Parachlorella kessleri named strain PK4 that accumulated higher concentrations of lipids than the wild-type strain. Resequencing of the PK4 genome identified mutations in three genes which may be associated with the high-lipid phenotype. The first gene, named CDMT1, encodes a protein with a calcium-dependent membrane association domain; the second gene, named DMAN1, encodes endo-1,4-ß-mannanase, while the third gene, named AATPL1, encodes a plastidic ATP/ADP antiporter-like protein. RESULTS: To determine which of these mutant genes are directly responsible for the phenotype of strain PK4, we delivered Cas9-gRNA ribonucleoproteins targeting each of the three genes into the wild-type cells by electroporation and successfully disrupted these three genes separately. The lipid productivity in the disruptants of CDMT1 and DMAN1 was similar to and lower than that in the wild-type strain, while the disruptants of AATPL1 exhibited > 30% higher lipid productivity than the wild-type strain under diurnal conditions. CONCLUSIONS: We succeeded in improving the lipid productivity of P. kessleri by CRISPR/Cas9-mediated gene disruption of AATPL1. The effective gene-editing method established in this study will be useful to improve Parachlorella strains for industrial applications.

Seasonal and geographical differences in the ruminal microbial and chloroplast composition of sika deer (Cervus nippon) in Japan.

To understand the nutritional status of culled wild sika deer (Cervus nippon), we compared the ruminal microbes of deer living in habitats differing in food composition (Nagano winter, Nagano spring, and Hokkaido winter) using next-generation sequencing. Twenty-nine sika deer were sampled. Alpha and beta diversity metrics determined via 16S and 18S rRNA amplicon-seq analysis showed compositional differences. Prevotella, Entodinium, and Piromyces were the dominant genera of bacteria, fungi and protozoa, respectively. Moreover, 66 bacterial taxa, 44 eukaryotic taxa, and 46 chloroplastic taxa were shown to differ significantly among the groups by the linear discriminant analysis effect size (LEfSe) technique. Total RNA-seq analysis yielded 397 significantly differentially expressed transcripts (q < 0.05), of which 48 (q < 0.01) were correlated with the bacterial amplicon-seq results (Pearson correlation coefficient > 0.7). The ruminal microbial composition corresponded with the presence of different plants because the amplicon-seq results indicated that chloroplast from broadleaf trees and Stramenopiles-Alveolates-Rhizaria (SAR) were enriched in Nagano, whereas chloroplast from graminoids, Firmicutes and the dominant phylum of fungi were enriched in Hokkaido. These results could be related to the severe snow conditions in Hokkaido in winter and the richness of plants with leaves and acorns in Nagano in winter and spring. The findings are useful for understanding the nutritional status of wild sika deer.

Supra-Optimal Temperature: An Efficient Approach for Overaccumulation of Starch in the Green Alga Parachlorella kessleri.

Green algae are fast-growing microorganisms that are considered promising for the production of starch and neutral lipids, and the chlorococcal green alga Parachlorella kessleri is a favorable model, as it can produce both starch and neutral lipids. P. kessleri commonly divides into more than two daughter cells by a specific mechanism-multiple fission. Here, we used synchronized cultures of the alga to study the effects of supra-optimal temperature. Synchronized cultures were grown at optimal (30 °C) and supra-optimal (40 °C) temperatures and incident light intensities of 110 and 500 µmol photons m-2 s-1. The time course of cell reproduction (DNA replication, cellular division), growth (total RNA, protein, cell dry matter, cell size), and synthesis of energy reserves (net starch, neutral lipid) was studied. At 40 °C, cell reproduction was arrested, but growth and accumulation of energy reserves continued; this led to the production of giant cells enriched in protein, starch, and neutral lipids. Furthermore, we examined whether the increased temperature could alleviate the effects of deuterated water on Parachlorella kessleri growth and division; results show that supra-optimal temperature can be used in algal biotechnology for the production of protein, (deuterated) starch, and neutral lipids.

Characterization of Growth and Cell Cycle Events Affected by Light Intensity in the Green Alga Parachlorella kessleri: A New Model for Cell Cycle Research.

Multiple fission is a cell cycle variation leading to the production of more than two daughter cells. Here, we used synchronized cultures of the chlorococcal green alga Parachlorella kessleri to study its growth and pattern of cell division under varying light intensities. The time courses of DNA replication, nuclear and cellular division, cell size, total RNA, protein content, dry matter and accumulation of starch were observed at incident light intensities of 110, 250 and 500 µmol photons m-2s-1. Furthermore, we studied the effect of deuterated water on Parachlorella kessleri growth and division, to mimic the effect of stress. We describe a novel multiple fission cell cycle pattern characterized by multiple rounds of DNA replication leading to cell polyploidization. Once completed, multiple nuclear divisions were performed with each of them, immediately followed by protoplast fission, terminated by the formation of daughter cells. The multiple fission cell cycle was represented by several consecutive doublings of growth parameters, each leading to the start of a reproductive sequence. The number of growth doublings increased with increasing light intensity and led to division into more daughter cells. This study establishes the baseline for cell cycle research at the molecular level as well as for potential biotechnological applications, particularly directed synthesis of (deuterated) starch and/or neutral lipids as carbon and energy reserves.

Ultrafast Simultaneous Raman-Fluorescence Spectroscopy.

Raman and fluorescence spectroscopies offer complementary approaches in bioanalytical chemistry, particularly in microbiological assays. The former method is used to detect lipids, metabolites, and nonspecific proteins and nucleic acids in a label-free manner, while the latter is used to investigate targeted proteins, nucleic acids, and their interactions via labeling or transfection. Despite their complementarity, these regimes are seldom used in conjunction due to fluorescent signals overwhelming inherently weak Raman signals by more than several orders of magnitude. Here we report a multimodal spectrometer that simultaneously performs Raman and fluorescence spectroscopies at high speed. It is made possible by Fourier-transform-coherent anti-Stokes Raman scattering (FT-CARS) and Fourier-transform-two-photon excitation (FT-TPE) measurements powered by a femtosecond pulse laser coupled to a homemade rapid-scan Michelson interferometer, operating at 24 000 spectra per second. As a proof-of-principle demonstration, we validate the ultrafast fluoRaman spectrometer by measuring coumarin dyes in organic solvents. To show its potential for applications that require rapid fluoRaman spectroscopy, we also demonstrate fluoRaman flow cytometry of Haematococcus pluvialis cells under varying culture conditions with a high throughput of ∼10 events per second to perform large-scale single-cell analysis of their metabolic stress response.

Deciphering the relationship among phosphate dynamics, electron-dense body and lipid accumulation in the green alga Parachlorella kessleri.

Phosphorus is an essential element for life on earth and is also important for modern agriculture, which is dependent on inorganic fertilizers from phosphate rock. Polyphosphate is a biological polymer of phosphate residues, which is accumulated in organisms during the biological wastewater treatment process to enhance biological phosphorus removal. Here, we investigated the relationship between polyphosphate accumulation and electron-dense bodies in the green alga Parachlorella kessleri. Under sulfur-depleted conditions, in which some symporter genes were upregulated, while others were downregulated, total phosphate accumulation increased in the early stage of culture compared to that under sulfur-replete conditions. The P signal was detected only in dense bodies by energy dispersive X-ray analysis. Transmission electron microscopy revealed marked ultrastructural variations in dense bodies with and without polyphosphate. Our findings suggest that the dense body is a site of polyphosphate accumulation, and P. kessleri has potential as a phosphate-accumulating organism.

Highly efficient lipid production in the green alga Parachlorella kessleri: draft genome and transcriptome endorsed by whole-cell 3D ultrastructure.

BACKGROUND: Algae have attracted attention as sustainable producers of lipid-containing biomass for food, animal feed, and for biofuels. Parachlorella kessleri, a unicellular green alga belonging to the class Trebouxiophyceae, achieves very high biomass, lipid, and starch productivity levels. However, further biotechnological exploitation has been hampered by a lack of genomic information. RESULTS: Here, we sequenced the whole genome and transcriptome, and analyzed the behavior of P. kessleri NIES-2152 under lipid production-inducing conditions. The assembly includes 13,057 protein-coding genes in a 62.5-Mbp nuclear genome. Under conditions of sulfur deprivation, lipid accumulation was correlated with the transcriptomic induction of enzymes involved in sulfur metabolism, triacylglycerol (TAG) synthesis, autophagy, and remodeling of light-harvesting complexes. CONCLUSIONS: Three-dimensional transmission electron microscopy (3D-TEM) revealed extensive alterations in cellular anatomy accompanying lipid hyperaccumulation. The present 3D-TEM results, together with transcriptomic data support the finding that upregulation of TAG synthesis and autophagy are potential key mediators of the hyperaccumulation of lipids under conditions of nutrient stress.

Naturally grown rucola, Eruca sativa, contains more α-linolenic acid than conventionally grown rucola.

The ratio of dietary n-6/n-3 polyunsaturated fatty acids is higher in the modern Western meal when compared with that of the Paleolithic era. Evidences have been accumulating that the extremely high ratio of dietary n-6/n-3 polyunsaturated fatty acids increases the risk of disease and the deterioration of physical conditions among humans. In this study, the ratio of linoleic acid (C18:2, n-6) and α-linolenic acid (C18:3, n-3) of rucola, Eruca sativa, was compared between naturally grown samples and conventional farming products (n=3 for each). We found that the naturally grown rucola contained significantly higher amount of α-linolenic acid (p=0.026), resulting in the lower ratio of linoleic acid and α-linolenic acid than the conventional (p=0.016). This finding suggests that vegetables cultured in conventional farming method could decrease the health promotion effects that the vegetables originally possess in natural environment.

Starch and lipid accumulation in eight strains of six Chlorella species under comparatively high light intensity and aeration culture conditions.

The microalgae family Chlorella species are known to accumulate starch and lipids. Although nitrogen or phosphorous deficiencies promote starch and lipids formation in many microalgae, these deficiencies also limit their growth and productivity. Therefore, the Chlorellaceae strains were attempted to increase starch and lipids productivity under high-light-intensity conditions (600-µmol photons m(-2)s(-1)). The 12:12-h light-dark (LD) cycle conditions elicited more stable growth than the continuous light (LL) conditions, whereas the starch and lipids yields increased in LL conditions. The amount of starch and lipids per cell increased in Chlorella viscosa and Chlorella vulgaris in sulfur-deficient medium, and long-chain fatty acids with 20 or more carbon atoms accumulated in cells grown in sulfur-deficient medium. Accumulation of starch and lipids was investigated in eight strains. The accumulation was strain-dependent, and varied according to the medium and light conditions. Five of the eight Chlorella strains exhibited similar accumulation patterns.

Phenotypic spectrum of Parachlorella kessleri (Chlorophyta) mutants produced by heavy-ion irradiation.

Heavy-ion mutagenesis is a technology used for effective production of genetic mutants. This study demonstrates that algal breeding using a unicellular alga, Parachlorella kessleri, by heavy-ion mutagenesis can improve lipid yield in laboratory experiments. The primary screening yielded 23 mutants among which a secondary screening yielded 7 strains, which were subjected to phenotypic assays. P. kessleri strains produced by heavy-ion radiation spanned a broad spectrum of phenotypes that differed in lipid content and fatty acid profiles. Starch grain morphology was distinctively altered in one of the mutants. The growth of strain PK4 was comparable to that of the wild type under stress-free culture conditions, and the mutant also produced large quantities of lipids, a combination of traits that may be of commercial interest. Thus, heavy-ion irradiation is an effective mutagenic agent for microalgae and may have potential in the production of strains with gain-of-function phenotypes.

Sequential accumulation of starch and lipid induced by sulfur deficiency in Chlorella and Parachlorella species.

The influence of sulfur deficiency on biomass production was analyzed in the four Chlorellaceae species, Chlorella vulgaris, Chlorella sorokiniana, Chlorella lobophora, and Parachlorella kessleri. Culturing under sulfur-deficient conditions promoted transient accumulation of starch followed by a steady increase in lipid storage. Transmission electron microscopy indicated an increase and decrease in starch granules and subsequent enlargement of lipid droplets under sulfur-deficient conditions. Chlorellaceae spp. accumulated 1.5-2.7-fold higher amounts of starch and 1.5-2.4-fold higher amounts of lipid under sulfur-deficient conditions than under sulfur-sufficient conditions. More than 75% of the fatty acids that accumulated in Chlorellaceae spp. under the sulfur-sufficient condition were unsaturated and culturing under sulfur-deficient conditions increased the saturated fatty acid content from 24.3% to 59.7% only in P. kessleri. These results indicate that the sequential accumulation of starch and lipid is a response to the sulfur depletion that commonly occurs in Chlorellaceae spp.

Sex determination and individual identification of American minks (Neovison vison) on Hokkaido, northern Japan, by fecal DNA analysis.

To determine the sex and identity of individual American minks (Neovison vsion), a species Introduced into Japan, molecular genetic methods were employed on fecal samples collected from the Kushiro Wetland, eastern Hokkaido. We examined the sex chromosome-linked genes ZFX and ZFY and 11 microsatellite loci to identify individuals. From microsatellite genotypes, the probability of identity was calculated to distinguish between individuals with 99% certainty. To evaluate the accuracy of the genotyping results, we used two approaches for several randomly selected samples. In the first approach, we genotyped all samples from the results of a maximum of three independent polymerase chain reactions (PCRs). In the second approach, we genotyped 10% of the samples from the results of five independent PCRs. Samples subsequent genotypings disagreed with the first genotype were counted as one of three categories of error. The results indicated that genotyping more than 10 microsatellite loci was required to reduce the probability of error in identity to less than 0.01. Twenty of 72 fecal samples were genotyped at 10 or 11 microsatellite loci and sex-determined by ZFX/ZFY genes, resulting in identification of five males and nine females. In assessing the accuracy of the results, genotyping errors were found to have occurred in 20% of the first genotypes. The main type of error was 'missing data', which can be prevented by increasing the number of replicate PCRs.

Genetic identification of mammalian carnivore species in the Kushiro Wetland, eastern Hokkaido, Japan, by analysis of fecal DNA.

To identify mammalian carnivore species distributed in the Kushiro Wetland, eastern Hokkaido, Japan, we developed molecular-genetic methods for identification of the species from fecal samples collected from the field. Species-specific primers and PCR programs were established for five native and six alien species of carnivores: Martes zibellina, Mustela nivalis, Mustela erminea, Vulpes vulpes, and Nyctereutes procyonoides as native species, and Neovison vison, Martes melampus, Mustela itatsi, Canis familiaris, Felis catus, and Procyon lotor as alien species in Hokkaido. Touchdown PCR, in which the annealing temperature is decreased 1 degrees C every cycle, was more effective for some species from which fecal DNA was not amplified species-specifically with standard PCR programs. Of 405 fecal samples collected from the Kushiro Wetland, the species of origin of 246 samples were successfully identified: 88 samples for N. vison, 140 for M. zibellina, 13 for V. vulpes, four for C. familiaris and one for F. catus. The results show the particular applicability of this method to monitoring M. zibellina and N. vison. In addition, methods to PCR-amplify DNA from two crayfish species (Pacifastacus leniusculus and Cambaroides japonicus) were developed to determine whether the carnivore fecal samples contained detectable DNA from the prey crayfishes. DNA from P. leniusculus was amplified from feces of N. vison identified in the present study, but no DNA from C. japonicus was detected. This indicates that N. vison preys on the alien species P. leniusculus.