Squalene Production Research Articles

Towards a Sustainable Route for the Production of Squalene Using Cyanobacteria

Treatment of slaughterhouse wastewater is a huge industrial problem. The use of an algae biorefinery platform could be a sustainable technological alternative that produces value-added compounds instead of dumping the wastewater. For this reason, this research aimed to evaluate squalene production from the microalgae Phormidium autumnale cultivated using agroindustrial wastewater. A derivatization method was performed to determine the squalene and fatty acids content, evaluated by gas chromatography with flame ionization and mass spectrometry detectors. A total of 0.18 g/kg of squalene were found in the biomass, with a high content of unsaturated fatty acids (52%). Sensitivity analysis estimated production of 727–72,750 kg/year in industries with different capacities. In this sense, P. autumnale in agroindustrial wastewater could offer a potential alternative method of squalene production.

English

2-C-Methyl-D-erythritol-4-phosphate (MEP) pathway has been extensively employed for terpenoids biosynthesis in Escherichia coli. In this study, to obtain key-enzymes of MEP pathway for squalene production, overexpression of different combination of MEP pathway genes were compared. Squalene production in strain YSS12 with overexpressed dxs, idi and ispA of MEP pathway from E. coli was improved by 71-fold when compared with strain YSS3 which only contained double copy SQS. Analysis of transcriptional levels of MEP pathway genes in engineering strains showed that different squalene production can be attributed to changed transcriptional levels of co-overexpressed genes dxs, idi, ispG and ispA in engineering strains. Furthermore, different E. coli expression hosts were compared for squalene production, among which BL21(DE3) was the best squalene producer. These results illustrate that dxs, idi and ispA of the MEP pathway from E. coli were key-enzymes for squalene production in E. coli. These key-enzymes of MEP pathway could also be applied to other terpenoids production in E. coli.   Key words: Squalene, key-enzyme, 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, Escherichia coli.

Optimization of fermentation conditions for squalene production by heterotrophic marine microalgae Schizochytrium mangrovei PQ6

Squalene is currently known as chemopreventive agents in reducing the incident of coronary heart disease and cancer. It is also a strong antioxidant used extensively in the food and cosmetic industries. Microbial sources of squalene are being explored in recent years. The objective of this study was to investigate fermentation conditions of Schizochytrium mangrovei PQ6 for high squalene production in 30L bioreactor. Squalene production was influenced by various nutritional factors such as glucose, nitrogen, vitamins etc. and physical factors such as batch and fed-batch fermentation. Our study established that fed-batch fermentation with nitrogen source of 1.2% yeast extract and 6.4% monosodium glutamate and 0.4% mixture of vitamin B1, 6 and 12 as optimal culture condition for maximal production of squalene by S. mangrovei PQ6. In fed-batch culture, the glucose concentration feeding was added up to 22% into the culture at 36 h of fermentation. The maximum squalene production was achieved 6.9 ± 0.1 g/L at 48 h of fed-batch fermentation, respectively. The values were 2-3 times more than that in fed-batch cultivation without adding mixture of vitamins and 4-6 times more than that in batch cultivation. Our obtained results are a breakthrough for the development of alternative squalene source from heterotrophic marine microalgae of Schizochytrium mangrovei PQ6 species for health-promoting functional foods. Citation: Nguyen Cam Ha, Hoang Thi Minh Hien, Le Thi Thom, Hoang Thi Huong Quynh, Dang Diem Hong, 2017. Optimization of fermentation conditions for squalene production by heterotrophic marine microalgae Schizochytrium mangrovei PQ6. Tap chi Sinh hoc, 39(3): 349-358. DOI: 10.15625/0866-7160/v39n3.9130. *Corresponding author: hoangminhhien@yahoo.com; ddhong60vn@yahoo.com Received 6 January 2017, accepted 20 August 2017

Tetraterpene Synthase Substrate and Product Specificity in the Green Microalga Botryococcus braunii Race L.

Recently, the biosynthetic pathway for lycopadiene, a C40 tetraterpenoid hydrocarbon, was deciphered from the L race of Botryococcus braunii, an alga that produces hydrocarbon oils capable of being converted into combustible fuels. The lycopadiene pathway is initiated by the squalene synthase (SS)-like enzyme lycopaoctaene synthase (LOS), which catalyzes the head-to-head condensation of two C20 geranylgeranyl diphosphate (GGPP) molecules to produce C40 lycopaoctaene. LOS shows unusual substrate promiscuity for SS or SS-like enzymes by utilizing C15 farnesyl diphosphate (FPP) and C20 phytyl diphosphate in addition to GGPP as substrates. These three substrates can be combined by LOS individually or in combinations to produce six different hydrocarbons of C30, C35, and C40 chain lengths. To understand LOS substrate and product specificity, rational mutagenesis experiments were conducted based on sequence alignment with several SS proteins as well as a structural comparison with the human SS (HSS) crystal structure. Characterization of the LOS mutants in vitro identified Ser276 and Ala288 in the LOS active site as key amino acids responsible for controlling substrate binding, and thus the promiscuity of this enzyme. Mutating these residues to those found in HSS largely converted LOS from lycopaoctaene production to C30 squalene production. Furthermore, these studies were confirmed in vivo by expressing LOS in E. coli cells metabolically engineered to produce high FPP and GGPP levels. These studies also offer insights into tetraterpene hydrocarbon metabolism in B. braunii and provide a foundation for engineering LOS for robust production of specific hydrocarbons of a desired chain length.

Iterative integration of multiple-copy pathway genes in Yarrowia lipolytica for heterologous β-carotene production

β-Carotene is a terpenoid molecule with high hydrophobicity that is often used as an additive in foods and feed. Previous work has demonstrated the heterologous biosynthesis of β-carotene from an intrinsic high flux of acetyl-CoA in 12 steps through 11 genes in Yarrowia lipolytica. Here, an efficient biosynthetic pathway capable of producing 100-fold more β-carotene than the baseline construct was generated using strong promoters and multiple gene copies for each of the 12 steps. Using fed-batch fermentation with an optimized medium, the engineered pathway could produce 4g/L β-carotene, which was stored in lipid droplets within engineered Y. lipolytica cells. Expansion of these cells for squalene production also demonstrated that Y. lipolytica could be an industrially relevant platform for hydrophobic terpenoid production.

Improvement of Squalene Production from CO2 in Synechococcus elongatus PCC 7942 by Metabolic Engineering and Scalable Production in a Photobioreactor.

The push-and-pull strategy for metabolic engineering was successfully demonstrated in Synechococcus elongatus PCC 7942, a model photosynthetic bacterium, to produce squalene from CO2. Squalene synthase (SQS) was fused to either a key enzyme (farnesyl diphosphate synthase) of the methylerythritol phosphate pathway or the β-subunit of phycocyanin (CpcB1). Engineered cyanobacteria with expression of a fusion CpcB1-SQS protein showed a squalene production level (7.16 ± 0.05 mg/L/OD730) that was increased by 1.8-fold compared to that of the control strain expressing SQS alone. To increase squalene production further, the gene dosage for CpcB1·SQS protein expression was increased and the fusion protein was expressed under a strong promoter, yielding 11.98 ± 0.49 mg/L/OD730 of squalene, representing a 3.1-fold increase compared to the control. Subsequently, the best squalene producer was cultivated in a scalable photobioreactor (6 L) with light optimization, which produced 7.08 ± 0.5 mg/L/OD730 squalene (equivalent to 79.2 mg per g dry cell weight). Further optimization for photobioprocessing and strain development will promote the construction of a solar-to-chemical platform.

Production of squalene by microbes: an update

Squalene, a naturally occurring linear triterpene formed via MVA or MEP biosynthetic pathway, is widely distributed in microorganisms, plants and animals. At present, squalene is used extensively in the food, cosmetic and medicine industries because of its antioxidant, antistatic and anti-carcinogenic properties. Increased consumer demand has led to the development of microbial bioprocesses for the commercial production of squalene, in addition to the traditional methods of isolating squalene from the liver oils of deep-sea sharks and plant seed oils. As knowledge of the biosynthetic enzymes and of regulatory mechanisms modulating squalene production increases, opportunities arise for the genetic engineering of squalene production in hosts. In this review, we present the various strategies used up to date to improve and/or engineer squalene production in microbes and analyze yields.

Biosynthetic Approaches to Squalene Production: The Case of Yeast.

Squalene is a precursor in the eukaryotic sterol biosynthesis. It is a valuable compound with several human health-related applications. Since the traditional natural resources of squalene are limited, alternatives for the production of squalene on industrial scale have been intensively explored during past years. The yeast Saccharomyces cerevisiae represents an attractive option due to elaborated techniques of genetic and metabolic engineering that can be applied to improve squalene yields. We discuss in this chapter some theoretical aspects of genetic manipulations of the ergosterol biosynthesis pathway aimed at increased squalene production and describe analytical methods for squalene purification and determination of its content in yeast cells.

Photosynthetic conversion of CO2 to farnesyl diphosphate-derived phytochemicals (amorpha-4,11-diene and squalene) by engineered cyanobacteria.

BackgroundMetabolic engineering of cyanobacteria has enabled photosynthetic conversion of CO2 to value-added chemicals as bio-solar cell factories. However, the production levels of isoprenoids in engineered cyanobacteria were quite low, compared to other microbial hosts. Therefore, modular optimization of multiple gene expressions for metabolic engineering of cyanobacteria is required for the production of farnesyl diphosphate-derived isoprenoids from CO2.ResultsHere, we engineered Synechococcus elongatus PCC 7942 with modular metabolic pathways consisting of the methylerythritol phosphate pathway enzymes and the amorphadiene synthase for production of amorpha-4,11-diene, resulting in significantly increased levels (23-fold) of amorpha-4,11-diene (19.8 mg/L) in the best strain relative to a parental strain. Replacing amorphadiene synthase with squalene synthase led to the synthesis of a high amount of squalene (4.98 mg/L/OD730). Overexpression of farnesyl diphosphate synthase is the most critical factor for the significant production, whereas overexpression of 1-deoxy-d-xylulose 5-phosphate reductase is detrimental to the cell growth and the production. Additionally, the cyanobacterial growth inhibition was alleviated by expressing a terpene synthase in S. elongatus PCC 7942 strain with the optimized MEP pathway only (SeHL33).ConclusionsThis is the first demonstration of photosynthetic production of amorpha-4,11-diene from CO2 in cyanobacteria and production of squalene in S. elongatus PCC 7942. Our optimized modular OverMEP strain (SeHL33) with either co-expression of ADS or SQS demonstrated the highest production levels of amorpha-4,11-diene and squalene, which could expand the list of farnesyl diphosphate-derived isoprenoids from CO2 as bio-solar cell factories.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0617-8) contains supplementary material, which is available to authorized users.

Engineering of the terpenoid pathway in Saccharomyces cerevisiae co-overproduces squalene and the non-terpenoid compound oleic acid

The balanced and optimal expression of genes in an engineered pathway is a prerequisite for the overproduction of a natural compound in a customized strain. Hence, 13 new constitutive promoters were characterized and optimized for overexpressing genes in the squalene biosynthetic pathway (SB) and squalene overproduction in Saccharomyces cerevisiae. Subsequently, these newly characterized promoters were employed for overexpressing genes in the mevalonate pathway; as a result, 29.41-fold (100mg/L) more squalene was produced in the engineered strain (FOH-0) than in the wild-type strain (WS). Afterward, the entire squalene biosynthetic pathway was upregulated with the newly characterized promoters to engineer the FOH-2 strain, and it resulted in the production of 304.16mg/L of squalene after inhibition of squalene monooxygenase with terbinafine. The FOH-2 strain also overproduced 154.94mg/L oleic acid, and this was perhaps because of an indirect upregulation of the oleic acid biosynthetic pathway by an unknown regulatory mechanism. The production of ergosterol and biomass in the FOH-2 strain was also increased by up to 34mg/L and 13.5g DW/L, respectively, compared to the WS strain. Consequently, the overexpression of genes in the SB pathway increased the production of squalene, oleic acid, ergosterol and biomass by 89.46-, 4.66-, 3.37- and 2.25-fold, respectively, in the FOH-2 strain compared to the WS strain. The growth rate of the FOH-2 strain was also 1.3-fold higher than the WS strain.Finally, our study reports the enhanced coproduction of squalene, ergosterol, oleic acid and biomass in yeast due to the overexpression of genes in the SB pathway by newly characterized constitutive promoters. It also shows that ergosterol overproduction indirectly upregulates its complementary pathway.

Overproduction of squalene synergistically downregulates ethanol production in Saccharomyces cerevisiae

Metabolic engineering strategies are often devised to redirect precursor flux in an engineered pathway. To develop a new strategy, the effect of overexpression of the squalene biosynthesis (SB) pathway and squalene overproduction on the ethanol production (EP) and post-squalene biosynthesis (PB) pathways was determined in Saccharomyces cerevisiae. Through overexpression of the HMG1, IDI1, ERG20 and ERG9 genes of the SB pathway, production of squalene increased 10-fold in the M1EG strain compared to the wild-type strain (WT) [(34mg/L), without terbinafine, an inhibitor of squalene monooxygenase, and 35.02-fold (119.08mg/L) with terbinafine]. However, due to overexpression of the SB pathway and squalene overproduction, production of ethanol and functionality of the EP and PB pathways were synergistically downregulated by 51.61%, 95.86% and 81.79%, respectively, in the M1EG strain compared to the WT strain. Overexpression of the entire SB pathway also enhanced production of squalene by 76.12-fold (304.49mg/L) and synergistically downregulated production of ethanol and functionality of the EP and PB pathways by 66.13%, 97.02% and 87.56% in the FOH-2 strain compared to the WT strain, respectively. The EP and PB pathways were strongly downregulated in the FOH-2 strain compared to the M1EG strain because the FOH-2 strain overexpresses the entire SB pathway and produces more squalene than the M1EG strain.These data suggest that overexpression of the SB pathway and squalene overproduction downregulate the EP and PB pathways in engineered strains. Therefore, we speculate that a cryptic regulation mechanism may downregulate these pathways, and characterization of such a mechanism may enable us to divert the precursor flux from the EP and PB pathways to the squalene biosynthesis pathway.

Metabolic engineering of Rhodopseudomonas palustris for squalene production.

Squalene is a strong antioxidant used extensively in the food, cosmetic and medicine industries. Rhodopseudomonas palustris TIE-1 was used as the host because of its ability to grow photosynthetically using solar energy and carbon dioxide from atmosphere. The deletion of the shc gene resulted in a squalene production of 3.8 mg/g DCW, which was 27-times higher than that in the wild type strain. For constructing a substrate channel to elevate the conversion efficiency, we tried to fuse crtE gene with hpnD gene. By fusing the two genes, squalene content was increased to 12.6 mg/g DCW, which was 27.4 % higher than that resulted from the co-expression method. At last, the titer of squalene reached 15.8 mg/g DCW by co-expressing the dxs gene, corresponding to 112-fold increase relative to that for wild-type strain. This study provided novel strategies for improving squalene yield and demonstrated the potential of producing squalene by Rhodopseudomonas palustris.

Thraustochytrids as production organisms for docosahexaenoic acid (DHA), squalene, and carotenoids.

Thraustochytrids have been applied for industrial production of the omega-3 fatty acid docosahexaenoic (DHA) since the 1990s. During more than 20years of research on this group of marine, heterotrophic microorganisms, considerable increases in DHA productivities have been obtained by process and medium optimization. Strains of thraustochytrids also produce high levels of squalene and carotenoids, two other commercially interesting compounds with a rapidly growing market potential, but where yet few studies on process optimization have been reported. Thraustochytrids use two pathways for fatty acid synthesis. The saturated fatty acids are produced by the standard fatty acid synthesis, while DHA is synthesized by a polyketide synthase. However, fundamental knowledge about the relationship between the two pathways is still lacking. In the present review, we extract main findings from the high number of reports on process optimization for DHA production and interpret these in the light of the current knowledge of DHA synthesis in thraustochytrids and lipid accumulation in oleaginous microorganisms in general. We also summarize published reports on squalene and carotenoid production and review the current status on strain improvement, which has been hampered by the yet very few published genome sequences and the lack of tools for gene transfer to the organisms. As more sequences now are becoming available, targets for strain improvement can be identified and open for a system-level metabolic engineering for improved productivities.

Botrydial and botcinins produced by Botrytis cinerea regulate the expression of Trichoderma arundinaceum genes involved in trichothecene biosynthesis

Trichoderma arundinaceum IBT 40837 (Ta37) and Botrytis cinerea produce the sesquiterpenes harzianum A (HA) and botrydial (BOT), respectively, and also the polyketides aspinolides and botcinins (Botcs), respectively. We analysed the role of BOT and Botcs in the Ta37-B. cinerea interaction, including the transcriptomic changes in the genes involved in HA (tri) and ergosterol biosynthesis, as well as changes in the level of HA and squalene-ergosterol. We found that, when confronted with B. cinerea, the tri biosynthetic genes were up-regulated in all dual cultures analysed, but at higher levels when Ta37 was confronted with the BOT non-producer mutant bcbot2Δ. The production of HA was also higher in the interaction area with this mutant. In Ta37-bcbot2Δ confrontation experiments, the expression of the hmgR gene, encoding the 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is the first enzyme of the terpene biosynthetic pathway, was also up-regulated, resulting in an increase in squalene production compared with the confrontation with B. cinerea B05.10. Botcs had an up-regulatory effect on the tri biosynthetic genes, with BotcA having a stronger effect than BotcB. The results indicate that the interaction between Ta37 and B. cinerea exerts a stimulatory effect on the expression of the tri biosynthetic genes, which, in the interaction zone, can be attenuated by BOT produced by B. cinerea B05.10. The present work provides evidence for a metabolic dialogue between T. arundinaceum and B. cinerea that is mediated by sesquiterpenes and polyketides, and that affects the outcome of the interaction of these fungi with each other and their environment.

Cloning and Characterization of a Squalene Synthase Gene from the Chaga Medicinal Mushroom, Inonotus obliquus (Agaricomycetes).

Squalene synthase catalyzes the condensation of 2 molecules of farnesyl diphosphate to produce squalene, the first committed precursor for sterol, brassinosteroid, and triterpene biosynthesis. A squalene synthase gene, designated IoSQS, was isolated from Inonotus obliquus, a medicinal mushroom that produces a plethora of bioactive triterpenes. IoSQS complementary DNA was found to contain an open reading frame of 1476 bp, encoding a protein of 491 amino acids with a calculated molecular mass of 55.85 kDa. The IoSQS genomic DNA sequence consisted of 1813 bp and contained 4 exons and 3 introns. The restriction fragment polymorphisms revealed by Southern blot analysis suggested that IoSQS was a single-copy gene. Promoter analysis indicated that the 5' upstream region of IoSQS possessed various potential elements associated with physiological and environmental factors. The expression pattern of IoSQS in different stages and under methyl jasmonate treatment correlated with the accumulation of total triterpenoids and was consistent with the predicted results of the IoSQS promoter region. The N-terminal 466 residues of the hydrophilic sequence were expressed as a His-tagged protein in Escherichia coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. Squalene was detected in vitro in reaction mixture by high-performance liquid chromatography analysis. These results suggest that the IoSQS enzyme is involved in squalene production in I. obliquus.

Advances in Biochemistry and Microbial Production of Squalene and Its Derivatives.

Squalene is a linear triterpene formed via the MVA or MEP biosynthetic pathway and is widely distributed in bacteria, fungi, algae, plants, and animals. Metabolically, squalene is used not only as a precursor in the synthesis of complex secondary metabolites such as sterols, hormones, and vitamins, but also as a carbon source in aerobic and anaerobic fermentation in microorganisms. Owing to the increasing roles of squalene as an antioxidant, anticancer, and anti-inflammatory agent, the demand for this chemical is highly urgent. As a result, with the exception of traditional methods of the isolation of squalene from animals (shark liver oil) and plants, biotechnological methods using microorganisms as producers have afforded increased yield and productivity, but a reduction in progress. In this paper, we first review the biosynthetic routes of squalene and its typical derivatives, particularly the squalene synthase route. Second, typical biotechnological methods for the enhanced production of squalene using microbial cell factories are summarized and classified. Finally, the outline and discussion of the novel trend in the production of squalene with several updated events to 2015 are presented.

Inhibitory effect of imperatorin on insulin-like growth factor-1-induced sebum production in human sebocytes cultured in vitro.

Acne is a common skin disease that originates in the sebaceous gland. The pathogenesis of acne is very complex, involving the increase of sebum production and perifollicular inflammation. In this study, we screened the anti-lipogenic material and demonstrated its effect using cultured human sebocytes. Normal human sebocytes were cultured by explanting the sebaceous glands. To evaluate the anti-lipogenic effect, sebocytes were treated with test materials and (14)C-acetate incorporation assay was performed. To screen the anti-lipogenic materials, we tested the effect of many herbal plant extracts. We found that Angelica dahurica extract inhibited the insulin-like growth factor-1 (IGF-1)-induced sebum production in terms of squalene synthesis in sebocytes. Furthermore, imperatorin isolated from A. dahurica showed remarkable inhibitory effect on squalene production as well as squalene synthase promoter activity. To investigate the putative action mechanism, we tested the effect of imperatorin on intracellular signaling. The results showed that imperatorin inhibited IGF-1-induced phosphorylation of Akt. In addition, imperatorin significantly down-regulated PPAR-γ and SREBP-1, the important transcription factors for lipid synthesis. These results suggest that imperatorin has a potential for reducing sebum production in sebocytes, and can be applicable for acne treatment.

Influence of Rhizoctonia solani and Trichoderma spp. in growth of bean (Phaseolus vulgaris L.) and in the induction of plant defense-related genes

Many Trichoderma species are well-known for their ability to promote plant growth and defense. We study how the interaction of bean plants with R. solani and/or Trichoderma affect the plants growth and the level of expression of defense-related genes. Trichoderma isolates were evaluated in vitro for their potential to antagonize R. solani. Bioassays were performed in climatic chambers and development of the plants was evaluated. The effect of Trichoderma treatment and/or R. solani infection on the expression of bean defense-related genes was analyzed by real-time PCR and the production of ergosterol and squalene was quantified. In vitro growth inhibition of R. solani was between 86 and 58%. In in vivo assays, the bean plants treated with Trichoderma harzianum T019 always had an increased size respect to control and the plants treated with this isolate did not decrease their size in presence of R. solani. The interaction of plants with R. solani and/or Trichoderma affects the level of expression of seven defense-related genes. Squalene and ergosterol production differences were found among the Trichoderma isolates, T019 showing the highest values for both compounds. T. harzianum T019 shows a positive effect on the level of resistance of bean plants to R. solani. This strain induces the expression of plant defense-related genes and produces a higher level of ergosterol, indicating its ability to grow at a higher rate in the soil, which would explain its positive effects on plant growth and defense in the presence of the pathogen.

High Production of Squalene Using a Newly Isolated Yeast-like Strain Pseudozyma sp. SD301.

A yeast-like fungus, termed strain SD301, with the ability to produce a high concentration of squalene, was isolated from Shuidong Bay, China. The nucleotide sequence analysis of the internal transcribed spacer (ITS) region of SD301 indicated the strain belonged to Pseudozyma species. The highest biomass and squalene production of SD301 were obtained when glucose and yeast extracts were used as the carbon and nitrogen sources, respectively, with a C/N ratio of 3. The optimal pH and temperature were 6 and 25 °C, with 15 g L(-1) of supplemented sea salt. The maximum squalene productivity reached 0.039 g L(-1) h(-1) in batch fermentation, while the maximum squalene yield of 2.445 g L(-1) was obtained in fed-batch fermentation. According to our knowledge, this is the highest squalene yield produced thus far using fermentation technology, and the newly isolated strain Pseudozyma sp. SD301 is a promising candidate for commercial squalene production.

Karyotype analysis of Schizochytrium mangrovei pq6 using dapi (4, 6-diamidino-2-phenylidole) counterstaining and pulsed-field gel electrophosis (pfge) techniques

Schizochytrium mangrovei PQ6 is a heterotrophic marine microalga which was isolated from Phu Quoc Island, Kien Giang province. It has many unique characteristics, including ability to be adapted to varying culture environmental conditions, such as temperature, salinity, pH as well as producing high biomass (30-40 g of dry cell weight - DCW per liter), lipid content (up to 70% DCW), and especially rich in polyunsaturated fatty acids, such as docosahexaenoic acid-DHA, C22: 6n-3; eicosapentaenoic acid-EPA, C20: 5n-3, docosapentaenoic acid-DPA, C22: 5n-6. The biomass of this strain was studied to use for aquaculture feed, functional food, biodiesel and some bioactive compounds (polyunsaturated fatty acid, squalene) production. However, until now no scientific reports published in the world pertaining karyotype analysis of this species as well as others species belonging to the genus Schizochytrium. In this paper, for karyotype analysis experiment, we used colchicine to arrest the cell cycle at the metaphase. To obtain clear images, the cells of PQ6 strain were treated with maceration enzymes and chromosomes were stained with DAPI (4’, 6-diamidino-2-phenylindole) and observed under fluorescence microscopy. The experimental results showed that PQ6 strain has three chromosomes. On the other hand, four bands (equal four chromosomes) were separated by pulsed-field gel electrophoresis (PFGE). The different of obtained number of chromosome allowed us to suggest that maybe, this microalga has a plasmid. And thus, this question needs to be clarified in the near future. However, the result of our research provides first evidence about karyotype and served as an initial basic for genomic assembly and annotation of this microalga.