The phylogeny and systematics of cordycipitoid fungi have been extensively studied in the last two decades. However, systematic positions of some taxa in the family Cordycipitaceae have not yet been thoroughly resolved. In this study, a new phylogenetic framework of Cordycipitaceae is reconstructed using multigene (nrSSU, nrLSU, tef-1α, rpb1 and rpb2) sequence data with large-scale taxon sampling. In addition, ITS sequence data of species belonging to the Lecanicillium lineage in the family Cordycipitaceae are used to further determine their phylogenetic placements. Based on molecular phylogenetic data together with morphological evidence, two new genera (Flavocillium and Liangia), 16 new species and four new combinations are introduced. In the new genus Flavocillium, one new species F. bifurcatum and three new combinations previously described as Lecanicillium, namely F. acerosium, F. primulinium and F. subprimulinium, are proposed. The genus Liangia is built by the new species Lia. sinensis with Lecanicillium-like asexual morph, isolated from an entomopathogenic fungus Beauveria yunnanensis. Due to the absence of Paecilomyces hepiali, an economically and medically significant fungus, in the earlier phylogenetic analyses, its systematic position has been puzzling in both business and academic communities for a long time. Here, P. hepiali is recharacterized using the holotype material along with seven additional samples. It is assigned to the genus Samsoniella (Cordycipitaceae, Hypocreales) possessing Cordyceps-like sexual morph and Isaria-like asexual morph, and thus a new combination, namely S. hepiali is proposed. An additional nine new species in Samsoniella are described: S. alpina, S. antleroides, S. cardinalis, S. cristata, S. lanmaoa, S. kunmingensis, S. ramosa, S. tortricidae and S. yunnanensis. Four new species in Cordyceps are described: C. chaetoclavata, C. cocoonihabita, C. shuifuensis and C. subtenuipes. Simplicillium yunnanense, isolated from synnemata of Akanthomyces waltergamsii, is described as a new species.

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BackgroundAmong all organisms tested, Haematococcus pluvialis can accumulate the highest levels of natural astaxanthin. Nitrogen starvation and high irradiance promote the accumulation of starch, lipid, and astaxanthin in H. pluvialis, yet their cell division is significantly retarded. Accordingly, adaptive regulatory mechanisms are very important and necessary to optimize the cultivation conditions enabling an increase in biomass; as well as promoting astaxanthin accumulation by H. pluvialis. To clarify the intrinsic mechanism of high-level astaxanthin and biomass accumulation in the newly isolated strain, H. pluvialis JNU35, nitrogen-sufficiency and nitrogen-depletion conditions were employed. Time-resolved comparative transcriptome analysis was also conducted by crossing the two-step culture process.ResultsIn the present study, we report the overall growth and physiological, biochemical, and transcriptomic characteristics of H. pluvialis JNU35 in response to nitrogen variation. From eight sampling time-points (2 days, 4 days, 8 days, 10 days, 12 days, 14 days, 16 days, and 20 days), 25,480 differentially expressed genes were found. These genes included the significantly responsive unigenes associated with photosynthesis, astaxanthin biosynthesis, and nitrogen metabolic pathways. The expressions of all key and rate-limiting genes involved in astaxanthin synthesis were significantly upregulated. The photosynthetic pathway was found to be attenuated, whereas the ferredoxin gene was upregulated, which might activate the cyclic electron-transport chain as compensation. Moreover, the expressions of genes related to nitrogen transport and assimilation were upregulated. The expressions of genes in the proteasome pathway were also upregulated. In contrast, the chloroplasts and nonessential proteins were gradually degraded, activating the specific ornithine–urea cycle pathway. These changes may promote the sustained accumulation of astaxanthin and biomass.ConclusionsTo the best of our knowledge, this paper is the first to investigate the long-term differences of gene expression from two-step culture process in the astaxanthin producer, H. pluvialis JNU35. According to our results, β-carotene ketolase (bkt1 and bkt2) serves as the key enzyme regulating astaxanthin accumulation in H. pluvialis JNU35. The cyclic electron-transport chain and novel nitrogen metabolic process were used adaptively as the regulatory mechanism compensating for different levels of stress. The in-depth study of these metabolic pathways and related key genes can reveal the underlying relationship between cell growth and astaxanthin accumulation in H. pluvialis JNU35.Electronic supplementary materialThe online version of this article (10.1186/s13068-019-1355-5) contains supplementary material, which is available to authorized users.

Violaxanthin is a major xanthophyll pigment in the microalga Eustigmatos cf. polyphem, but the amount produced after propagation can vary depending upon culture conditions. In this study, the effects of cultivation time, nitrogen concentration, light intensity, and culture mode on violaxanthin production were investigated. The results showed that this microalga vigorously grew and maintained a high level of violaxanthin in the fed-batch culture, and the highest violaxanthin productivity of 1.10 ± 0.03 mg L−1 d−1 was obtained under low light illumination with 18 mM of initial nitrogen supply for ten days. Additionally, violaxanthin was purified from E. cf. polyphem by silica gel chromatography and preparative high-performance liquid chromatography (PHPLC), and identified with high-resolution mass spectrometry (HRMS). The antioxidant activity of the purified violaxanthin was evaluated by three tests in vitro: reducing power assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2-azobis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical assays. The strongest inhibition of purified violaxanthin occurred during the scavenging of ABTS+ radicals, with EC50 of 15.25 μg mL−1. In conclusion, this is the first report to investigate the effects of different culture conditions on violaxanthin accumulation in E. cf. polyphem and provide a novel source for the production of violaxanthin that can be used for food and pharmaceutical applications.

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Background Heterotrophic cultivation of microalgae has been proposed as a viable alternative method for novel high-value biomolecules, enriched biomass, and biofuel production because of their allowance of high cell density levels, as well as simple production technology. Tetradesmus bernardii, a newly isolated high-yielding oleaginous microalga under photoautotrophic conditions, is able to grow heterotrophically, meaning that it can consume organic carbon sources in dark condition. We investigated the effect of different carbon/nitrogen (C/N) ratios on the growth and lipid accumulation of T. bernardii in heterotrophic batch culture under two nitrogen sources (NaNO3 and CO(NH2)2). In addition, we conducted time-resolved transcriptome analysis to reveal the metabolic mechanism of T. bernardii in heterotrophic culture. Results T. bernardii can accumulate high biomass concentrations in heterotrophic batch culture where the highest biomass of 46.09 g/L was achieved at 100 g/L glucose concentration. The rate of glucose to biomass exceeded 55% when the glucose concentration was less than 80 g/L, and the C/N ratio was 44 at urea treatment. The culture was beneficial to lipid accumulation at a C/N ratio between 110 and 130. NaNO3 used as a nitrogen source enhanced the lipid content more than urea, and the highest lipid content was 45% of dry weight. We performed RNA-seq to analyze the time-resolved transcriptome of T. bernardii. As the nitrogen was consumed in the medium, nitrogen metabolism-related genes were significantly up-regulated to speed up the N metabolic cycle. As chloroplasts were destroyed in the dark, the metabolism of cells was transferred from chloroplasts to cytoplasm. However, storage of carbohydrate in chloroplast remained active, mainly the synthesis of starch, and the precursor of starch synthesis in heterotrophic culture may largely come from the absorption of organic carbon source (glucose). With regard to lipid metabolism, the related genes of fatty acid synthesis in low nitrogen concentration increased gradually with the extension of cultivation time. Conclusion T. bernardii exhibited rapid growth and high lipid accumulation in heterotrophic culture. It may be a potential candidate for biomass and biofuel production. Transcriptome analysis showed that multilevel regulation ensured the conversion from carbon to the synthesis of carbohydrate and lipid.

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