Abstract
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.
Highlights
Among all organisms tested, Haematococcus pluvialis can accumulate the highest levels of natural astaxanthin
Growth of H. pluvialis JNU35 under a two‐step culture system To analyze the growth of H. pluvialis JNU35 under a two-stage culture, H. pluvialis JNU35 was first cultured in nitrogen sufficient (NS) [9.0 mM (NH2)2CO] modified BBM medium (mBBM) for 10 days and transferred into a fresh nitrogen free (NF) mBBM for another 10 days
In the later stage of a two-step culture, cell division was blocked due to the lack of nitrogen, so each cell was exposed to relatively higher light intensity than nitrate reductase (NR) stage, and biochemical compounds were rapidly accumulated after the compounds were transferred to the fresh nitrogen-free medium
Summary
Haematococcus pluvialis can accumulate the highest levels of natural astaxanthin. 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. Due to the effects of genetic differences among various strains, many problems arise with the mass cultivation of H. pluvialis These problems include cultures that are contaminated, or display slow growth rates and complex induction conditions. It is vital to optimize the cultivation conditions to increase biomass and promote astaxanthin accumulation of H. pluvialis at large-scale production [4,5,6]. The entire transcriptional regulatory mechanism of H. pluvialis is completely unknown, e.g., how H. pluvialis responds to stress and further accumulates astaxanthin, especially under nitrogen stress
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