The effects of low temperature on docosahexaenoic acid biosynthesis in Schizochytrium sp TIO01 and the underlying mechanism

Schizochytrium are known for their abundant production of docosahexaenoic acid (DHA). Low temperatures can promote the biosynthesis of polyunsaturated fatty acids in many species. In this study, the effects of low temperature on the biosynthesis of DHA in Schizochytrium sp TIO01 and the underlying mechanism was investigated. Based on the de novo genome assembly (contig N50=2.86 Mb) and iTRAQ-based protein identification, we first reconstructed the detailed Schizochytrium fatty acid biosynthesis pathway. Our findings revealed that desaturases, involved in DHA synthesis via the fatty acid synthase (FAS) pathway, were completely absent. The polyketide synthase (PKS) pathway and the FAS pathway are separately responsible for DHA and saturated fatty acid synthesis in Schizochytrium . Analysis of fatty acid composition profiles indicates that low temperature has a significant impact on the production of DHA in Schizochytrium , increasing the DHA content and overall total fatty acids from 43% to 65%. The increased DHA content, however, was not a result of the expression of the PKS pathway genes. Further gene expression analysis indicated that low temperatures may promote DHA accumulation by the up-regulation of both the pentose phosphate pathway and the branched-chain amino acid degradation pathway (increasing the production of the substrates for polyunsaturated fatty acid synthesis: acetyl-CoA and NADPH). In addition, low temerpatures result in a down-regulation of the FAS pathway (reducing the consumption of the substrates for saturated fatty acid synthesis) and malic enzyme, leading to a decreased saturated fatty acid content. These detailed , metabolism, global and overview maps, signal transduction, the endocrine system, and so on. The top20 KEGG pathways of differentially expressed genes

revealing an underlying mechanism by which low temperatures promote the accumulation of DHA in Schizochytrium. The high-quality and nearly complete genome sequence of Schizochytrium provides a valuable reference for further investigation of the regulation of polyunsaturated fatty acids biosynthesisand the evolutionary characteristics in Thraustochytriidae species.

Background
Docosahexaenoic acid (DHA, C22:6), a major ω-3 polyunsaturated fatty acid (PUFA), is widely distributed among phospholipids in the human brain and retina, playing a vital role in human health [1]. A variety of marine-microorganisms are rich in DHA [2,3]. As one of the most promising DHA-producing species, Schizochytrium have the ability to accumulate DHA to more than 40% of the total fatty acid (TFA) concentration and ~ 40% of the dry cell weight [4]. On account of this characteristic, the PUFA biosynthesis pathway in Schizochytrium has attracted increasing attention and is currently being exploited by several companies [5].
The entire PUFA synthesis pathway was conventionally considered to occur via two routes: that knock-outs of the PKS gene led to PUFA auxotrophic behavior [8]. Although these biochemical and genetic studies on Schizochytrium have suggested that the PKS pathway is responsible for DHA biosynthesis [7][8][9], recent studies revealed that enhancement of the FAS pathway contributes significantly to increased DHA productivity in Schizochytrium 4 [10,11]. Following these results, questions arise as to which route is employed by Schizochytrium for DHA synthesis. Genome sequencing is an ideal approach for investigating this issue, however, information for high-quality whole-genomes for Schizochytrium available in public databases are currently limited [12], preventing the identification of the Schizochytrium PUFA biosynthesis pathway.
Low temperatures have been shown to impact polyunsaturated fatty acid productivity in many PUFA-producing organisms [13][14][15][16][17]. However, few studies have investigated the underlying mechanism of these effects. Ma et al pioneered in-depth studies in Aurantiochytrium, showing that the upregulation of the PKS pathway and downregulation of the FAS pathway play important roles in promoting DHA accumulation [13,18]. Min et al reported that significant upregulation of the fatty acid desaturases associated with the FAS pathway, leads to PUFA accumulation in Bangia fuscopurpurea [ 19]. In Shewanella piezotlerans and Photobacterium profundum, upregulation of the PKS pathway genes was not detected despite the increase in PUFA accumulation at a low temperature. [20,21]. In Schizochytrium, Zeng et al have reported that low temperatures have a significant impact on DHA production, increasing DHA levels of up to 50% of the TFA level [22]. The mechanism underlying this low-temperature-induced DHA accumulation remains unknown in Schizochytrium.
Previously, we isolated a Schizochytrium strain from sea water (designated Schizochytrium sp TIO01) [23]. In this study, the effects of low temperature on fatty acid synthesis in Schizochytrium sp TIO01 was analysis and the underlying mechanism was investigated.
Based on the de novo genome assembly and iTRAQ-based LC-MS-MS, we reconstructed the fatty acid biosynthesis pathway of Schizochytrium sp TIO01. Next, the effects of low temperature on fatty acid synthesis was investigated by analysis of differential expression of genes related to fatty acid biosynthesis.

Schizochytrium genome assembly, assessment and annotation
To reveal the fatty acid biosynthesis pathways and further improve our understanding of the underlying mechanism by which low temperatures promote DHA accumulation in Schizochytrium, we performed de novo whole-genome assembly and annotation. Using 13.9 Gb of PacBio RS II subreads (217 × genomic data) and 31 Gb of Illumina PE250 clean data (480 × genomic data), a genome size of 64 Mb with a 45% GC base ratio, containing 34 scaffolds and 3 circular contigs (one of the circular contigs is mtDNA genome which was previously reported [24]) was obtained. The N50 scaffold and N50 contig were 5.83 Mb and 2.86 Mb, respectively. Table 1 shows the detailed information regarding the Schizochytrium assembled genome and the genome for the thraustochytriaceae species.
To inspect the de novo assembly accuracy, multiple independent sources of reads were 6 used to assess the assembled genome. Among the 62,457,386 paired Illumina PE250 reads (approximately 480 × coverage of the genome), over 99% could be aligned to the genome.
The overall alignment rate of the transcriptomic reads from 14 independent culture conditions (approximately 900 × coverage of the genome) ranged from 95-97%, as shown in Table S1). These results suggested that the Schizochytrium assembled genome was of high-quality and nearly complete. We also evaluated the completeness of the genome assembly using BUSCO-v3.0 (database: eukaryota_odb9) [  To identify Schizochytrium proteins, six samples of Schizochytrium cells were grown at both normal temperatures as well as low temperatures and subjected to iTRAQ-based proteomic analysis. Tandem mass spectra were searched against the Schizochytrium protein database containing the genomics-predicted proteins and transcriptomicspredicted novel proteins. A total of 4,008 proteins were quantified (Detailed information regarding the iTRAQ identified proteins are shown in Table S2), of which 3,196 were annotated by the reference genome and 812 were predicted by transcriptomic analysis.
Using KEGG pathway analysis, 2,939 proteins among the 4,008 were annotated in 45 pathways, these identified proteins are involved in global and overview maps, signal transduction, carbohydrate metabolism, lipid metabolism, amino acid metabolism, energy metabolism among others. The top20 KEGG pathways of iTRAQ identified proteins were 8 shown in Figure 2a.

Fatty acid biosynthesis pathway
Based on genome annotations and protein identification, the fatty acid biosynthesis pathways of Schizochytrium, including the saturated fatty acids synthesis pathway and the PUFA synthesis pathway were reconstructed (Figure 3). Annotations regarding these proteins and their detailed LC-MS-MS information are listed in Table S3 & Table S2, respectively.

9
The entire pathway of polyunsaturated fatty acid (PUFAs) synthesis was conventionally thought to occur via two routes. The first is the fatty acid synthase (FAS) pathway which involves serial desaturation and elongation of saturated fatty acids (C16:0 or C18:0); the second is the polyketide synthase (PKS) pathway [6]. According to Schizochytrium genome annotation, we found that the desaturase involved in the FAS pathway for PUFA synthesis was completely absent (the intermediates of PUFA compounds involved in the FAS pathway for PUFA synthesis, such as C16:1, C18:1, C18:2, C18:3, and C20:3, were not detected by GC). This indicates that the FAS pathway for PUFA synthesis in Schizochytrium is incomplete and that the PKS pathway is responsible for PUFA synthesis. Similar to other Thraustochytriidae species [32], Schizochytrium contains three large multifunctional PKS pathway genes, namely, PfaA, PfaB and PfaC. These three genes were highly homologous (>99%) to proteins from another PUFA-producing species Aurantiochytrium sp. L-BL10.

Transcriptomic profiling under cold temperatures
To improve the understanding of the mechanism by which low temperatures promote DHA accumulation, transcriptomic analysis was performed on six samples at both normal and lowertemperatures as described previously. The obtained reads represented an average of 207.11 times the Schizochytrium genome length. Of these,11,215 expressed genes were detected, including 9,660 genome predicted genes and 1,555 novel predicted genes. A total of 1,546 genes among 11,215 expressed genes were significantly associated with the low-temperature response (|Fold change| >= 2 and adjusted p-value <= 0.001), including

Conclusions
In the present study, multi-omicss approaches were used to investigate the effects of low  Transcriptomics sample preparation for gene prediction: fourteen samples were cultured in 7 different media at 28 °C or 16 °C (the medium components are shown in Table S4).
Harvesting was performed separately when the OD 650 reached 0.7. Total RNA was extracted and sequencing on each sample was performed on the BGISEQ-500 (PE100) or Illumina platform (PE150) at the BGI, Shenzhen, China.
Genome estimation and assembly: The genome size was estimated based on the 17-mer spectrum using gce-v1.0.0 as a heterozygosity model [40]; Falcon [41] (fc_env_180425) as used to assemble the PacBio long reads multiple times to produce the longest contig N50.

Ethics approval and consent to participate
Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The raw sequencing reads used for genome assembly and annotation have been

Supplementary Materials
Table S1 Summary of data and reads alignments used in genome assembly and assessment      Diagrammatic representation for significantly differentially expressed genes associated with the fatty acid biosynthesis pathways, glycolysis, pentose phosphate and TCA cycle when Schizochytrium was cultured under cold conditons.
Genes that were differentially regulated (|Fold change| >=2 and adjusted p-value