SREBP1 is a transcription factor that influences lipogenesis by regulating key genes associated with lipid biosynthesis, while AMPK, modulates lipid metabolism by regulating acetyl-CoA carboxylase. The exact role of these metabolic regulators in oleaginous microbes remains unclear. This study identified and manipulated the genes encoding SREBP1 (sre1) and alpha 1 subunit of AMPK (ampk-alpha 1) in Mucor circinelloides WJ11. Individual overexpression of sre1 yielded 32.5 % lipids and 21 g/L biomass, while ampk-alpha 1 deletion combined with sre1 overexpression yielded 42.5 % lipids and 25 g/L biomass in mutant strains. This increase correlated with upregulated expression of key lipogenic genes and enzyme activity, enhancing lipid production and biomass. These surges were correlated with the increased mRNA levels of key genes (acl, acc1, acc2, cme1, fas1, g6pdh1, g6pdh2 and 6pgdh2). Enzyme activity analysis further showed that upregulation of ACL, ACC, ME, FAS, G6PDH and 6PGDH might provide more precursors and NADPH for lipid biosynthesis in sre1 overexpressing strains. Conversely, the activities of these genes and enzymes were markedly downregulated in sre1 deleted mutants consistent with lower lipid production and biomass than the control. These findings open new avenues for research by exploring the coordinated role of sre1 and ampk-alpha 1 in lipid metabolism in M. circinelloides.

Cross-contamination on food contact surfaces (FCS) increases the risk of foodborne diseases incidence. Traditional microbial detection methods are time-consuming, prompting the exploration of the rapid method; CytoQuant® mobile flow cytometer which employ perturbation of low-frequency electric fields between intact membrane and cytoplasm. The cross-sectional study used CytoQuant® and total plate count (TPC) involved 30 FCS from six (n = 6) selected restaurants with long operating hours in the Lembah Klang area. Findings showed that the CytoQuant® was able to detect the presence of microbial contamination within 30 seconds without pre-treatment. The results also showed that there were significant differences (p < 0.05) between intact cell and particles readings on FCS. Furthermore, even though there are some differences in individual sample readings from the FCS, the mean values across various restaurants showed no significant difference (p > 0.05) between CytoQuant and TPC, suggesting that both methods provide comparable intact cell measurements on average. Further microbiological and premise rating analysis showed that all six premises exhibited aerobic, coliform counts and patogen exceeding established thresholds on the cutting board, including detection of salmonella in one of the premises, which correlated with the poor premises rating. This study suggest that employing CytoQuant® on-site ensures rapid and reliable results in monitoring of FCS, reducing the risk of foodborne diseases compared to TPC analysis.

Thraustochytrids, including Aurantiochytrium sp., are of great interest to various industries due to their ability to produce docosahexaenoic acid (DHA, C22:6 omega-3) and squalene, highly valued metabolites that play crucial roles in human health and well-being. However, the reliance on costly refined carbon substrates limits their commercial viability. This study investigates the use of pineapple off-cut liquid extract (POCL), a byproduct of the pineapple canning industry, as a cost-effective substrate for producing DHA and squalene from Aurantiochytrium sp. SW1 (SW1). POCL is rich in sugars, amino acids, and trace elements, which are essential to support SW1's growth as well as enhancing its DHA and squalene production. Cultivation with POCL, alone or in combination with other supplementary nutritional components, increased DHA and squalene content by 15-36 % compared to the control medium. Optimization using Response Surface Methodology (RSM) on key medium components (tryptone, monosodium glutamate, yeast extract) identified by the Plackett-Burman design further boosted DHA to 3.25 g/L and squalene to 60 mg/L, achieving improvements of 32-1236 % compared to prior to the optimization, respectively. This study highlights POCL's potential as an economical substrate for sustainable DHA and squalene production.

Thraustokaitrid seperti Aurantiochytrium sp. telah menarik perhatian penyelidik dan industri disebabkan kebolehannya untuk menghasilkan asid lemak politaktepu (PUFA) bernilai tinggi terutamanya asid dokosaheksaenoik (DHA, C22:6 ω-3). Namun begitu, keperluan penggunaan substrat yang mahal khususnya sumber karbon tulen semasa proses pengkulturan telah menyumbang kepada peningkatan kos pengkomersialan DHA daripada thraustokaitrid. Oleh itu, dalam kajian ini, potensi penggunaan sisa kulit nanas (SKN) yang merupakan antara sisa utama industri pertanian di Malaysia sebagai sumber karbon alternatif yang lebih murah untuk penghasilan DHA daripada Aurantiochytrium sp. SW1 telah dikaji. Memandangkan SKN mengandungi lignin, selulosa dan hemiselulosa yang agak kompleks, kaedah perawatan autoklaf, berasid dan beralkali pada kepekatan berbeza telah ditentukan bagi mendapatkan gula jumlah yang optimum daripada SKN. Hasil menunjukkan perawatan autoklaf secara signifikan (p<0.05) menghasilkan hidrolisat dengan kepekatan gula tertinggi (39.6 ± 1.98 g/L) berbanding dengan perawatan berasid dan alkali yang masing-masing dapat mengekstrak dalam julat 31-38 g/L gula jumlah. Kemudian, kebolehan Aurantiochytrium sp. SW1 untuk menggunakan kesemua hidrolisat SKN sebagai sumber karbon alternatif dibandingkan. Didapati, penggunaan hidrolisat SKN dengan rawatan 100 mM H2SO4 bersama nutrien tambahan secara signifikan (p<0.05) menghasilkan biojisim tertinggi (7.74 g/L ± 0.39) manakala kandungan lipid terbaik (60.7% ± 3.04) terhasil apabila SW1 dikulturkan menggunakan hidrolisat SKN dengan rawatan autoklaf tanpa penambahan nutrien. Penghasilan DHA yang maksimum (0.68 ± 0.034 g/L) dicapai apabila Aurantiochytrium sp. SW1 dikultur menggunakan hidrolisat SKN dengan rawatan 100 mM H2SO4 bersama nutrien tambahan yang mana 10-35% lebih tinggi jika dibandingkan dengan penghasilan menggunakan hidrolisat SKN yang lain. Kajian ini dapat menjadi asas ke arah penghasilan DHA yang lebih murah daripada thraustokaitrid dengan menggunakan SKN sebagai sumber karbon alternatif.

Thraustochytrids such as Aurantiochytrium sp. have drawn the attention of researchers and industries due to their ability to produce high-value polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid ( DHA , C22:6 omega-3) from its total fatty acids. However, the requirement for expensive substrates, particularly refined carbon sources during the cultivation process, has contributed to the increased commercialization costs of DHA from thraustochytrids. Therefore, in this study, the potential use of pineapple peel waste (PPW), which is among the major agricultural wastes in Malaysia, as a cheaper alternative carbon source for DHA production from Aurantiochytrium sp. SW1 was investigated. Considering that PPW contains relatively complex lignin, cellulose, and hemicellulose, autoclave, acidic, and alkaline treatment methods at different concentrations were examined to obtain the optimal sugar content from PPW. The results showed that autoclave treatment yielded the highest sugar concentration (39.6 f 0.39 g/L) significantly (p<0.05), compared to acidic and alkaline treatments, each of which could extract sugar within the range of 31-38 g/L. Subsequently, the ability of Aurantiochytrium sp. SW1 to utilize all PPW hydrolysates as a sole carbon source was compared. It was found that the use of PPW hydrolysate with 100 mM H2SO4 treatment along with additional nutrients produced the maximum biomass (7.74 f 0.39 g/L) significantly (p<0.05), and outstanding lipid content (60.7 +/- 3.04%) was obtained when SW1 was cultured using autoclaved PPW hydrolysate without nutrient supplementation. The maximum DHA production (0.68 +/- 0.034 g/L) was achieved when Aurantiochytrium sp. SW1 was cultured using PPW hydrolysate with 100 mM H2SO4 treatment along with additional nutrients, which was 10-35% higher compared to production using other PPW hydrolysates. This study could serve as a foundation towards cheaper DHA production from thraustochytrids using PPW as an alternative carbon source.

Summary This study explores enhancing lipid recovery from the lipid‐rich marine microalgae Aurantiochytrium sp. SW1, known for its robust cell wall posing challenges, which necessitates energy‐intensive cell‐disruption for efficient intracellular lipid extraction. Despite advancements in other microalgae, there is lack of reports on efficient cell disruption for Aurantiochytrium sp. SW1 which is vital to reduce the energy costs and minimising the downstream process while maximising lipid yields. Three process intensification technologies (PIT) – microwave treatment, autoclave treatment and ultrasonication were employed on wet Aurantiochytrium sp. SW1 cells to improve the lipid extraction efficiency. Assessing the disruption effectiveness via lipid content, suspension turbidity and particle size distribution, alongside scanning electron microscopy (SEM) for cellular morphology changes post‐PIT treatments, this study indicates all three methods reduced particle size with prolonged treatment time, implying successful cell wall disruption and intracellular component release. Ultrasonication, especially at 50 W power for 10 min, exhibited the highest efficacy, yielding lipid content of 67.76 ± 0.03%. Microscopic analysis unveiled ultrasonication‐induced cell shrinkage and increased membrane permeability, supporting its effectiveness in enhancing solvent penetration for improved lipid recovery. This research underscores PIT techniques' potential as sustainable, energy‐efficient strategies for lipid extraction from Aurantiochytrium sp. SW1 microalgae.

Astaxanthin is a bioactive natural pigment with antioxidant properties. It has extensive applications within the industrial sector as well as in human and animal health. Mucor circinelloides is a zygomycete fungus that accumulates β-carotene as the main carotenoid compound. M. circinelloides is a well-known model organism among Mucorales for studying carotenogenesis in fungi, which makes it a promising candidate for the biotechnological production of carotenoids. In this study, β-carotene hydroxylase (crtR-B) and ketolase (bkt) genes (codon-optimized) were coexpressed from Haematococcus pluvialis in M. circinelloides using two potent promoters gpd1 and zrt1 respectively to generate an astaxanthin-producing biofactory. Following 72 h of cultivation, the recombinant M. circinelloides Mc-57 obtained in this study produced 135 ± 8 µg/g of astaxanthin. This is the highest reported amount in M. circinelloides to date. The mRNA levels of crtR-B and bkt in Mc-57 were assayed using RT-qPCR. These levels showed a 5.7-fold increase at 72 h and a 5.5-fold increase at 24 h, respectively, compared to the control strain. This demonstrated the successful overexpression of both genes, which correlated with the production of astaxanthin in the Mc-57. Moreover, the addition of glutamate (2 g/L) and mevalonate (15 mM) resulted in an increase in astaxanthin production in the recombinant strain. The results showed that the combined addition of these metabolic precursors resulted in 281 ± 20 µg/g of astaxanthin, which is 2.08-fold higher than the control medium (135 ± 8 µg/g). The addition of metabolic precursors also positively impacted the biomass growth of Mc-57, reaching 11.2 ± 0.57 g/L compared to 9.1 ± 0.23 g/L (control medium). The study successfully addressed the challenge of balancing the accumulation of astaxanthin with biomass growth, which has been regarded as common bottleneck in the metabolic engineering of microbial cells. The development of a recombinant fungal strain of M. circinelloides not only increased astaxanthin content. Additionally, it provided a foundation for further improvement of the biotechnological production of astaxanthin in M. circinelloides.

Sphingolipids are a class of lipids containing the backbone of long-chain amino-alcohol bases in their structure, which are synthesized in the endoplasmic reticulum. Modification of this base gives rise to a variety of such lipids ranging from simple to complex sphingolipids that play a significant structural and functional role in membrane biology as well as regulate various cellular processes. Sphingosine, dihydrosphingosine and phytosphingosine are nature's most frequently occurring bases. Ceramides are the simplest sphingolipids after the backbone. These fatty acids are amide-linked derivatives of sphingoid bases and central intermediates of sphingolipid metabolism. Ceramides perform various biological functions and constitute the hydrophobic backbone of all complex sphingolipids. The best-characterized sphingolipids in fungi and yeast are glycosphingolipids (GSLs), which could be categorized into two groups, neutral GSLs (glucosyl and galactosylceramide) and acidic GSLs, (glycosylinositol-phosphorylceramides). Due to the several important functions of sphingolipids in cell biology, it is crucial to understand the regulation and metabolism of sphingolipids. Despite the diversity of structure and function of sphingolipids, their synthesis and degradation are governed by common synthetic and catabolic pathways. In recent years, significant progress in the field of sphingolipids has been made. Recent developments in sphingolipid biology, including the construction of analytical and genetic tools and the development of computer visualization techniques for sphingolipids analysis, have highlighted the role of sphingolipids in developing anticancer and antifungal therapeutics. Recent advances in sphingolipid biology continue to provoke and inspire vigorous investigations in sphingolipidology

The use of chitosan as a flocculant has become a topic of interest over the years due to its positively charged polymer and biodegradable and non-toxic properties. However, most studies only focus on microalgae and wastewater treatment. This study provides crucial insight into the potential of using chitosan as an organic flocculant to harvest lipids and docosahexaenoic acid (DHA-rich Aurantiochytrium sp. SW1 cells by examining the correlation of flocculation parameters (chitosan concentration, molecular weight, medium pH, culture age, and cell density) toward the flocculation efficiency and zeta potential of the cells. A strong correlation between the pH and harvesting efficiency was observed as the pH increased from 3, with the optimal flocculation efficiency of >95% achieved at a chitosan concentration of 0.5 g/L at pH 6 where the zeta potential was almost zero (3.26 mV). The culture age and chitosan molecular weight have no effect on the flocculation efficiency but increasing the cell density decreases the flocculation efficiency. This is the first study to reveal the potential of chitosan to be used as a harvesting alternative for thraustochytrid cells.

Aurantiochytrium sp., a marine thraustochytrid possesses a remarkable ability to produce lipid rich in polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA). Although gene regulation underlying lipid biosynthesis has been previously reported, proteomic analysis is still limited. In this study, high DHA accumulating strain Aurantiochytrium sp. SW1 has been used as a study model to elucidate the alteration in proteome profile under different cultivation phases i.e. growth, nitrogen-limitation and lipid accumulation. Of the total of 5146 identified proteins, 852 proteins were differentially expressed proteins (DEPs). The largest number of DEPs (488 proteins) was found to be uniquely expressed between lipid accumulating phase and growth phase. Interestingly, there were up-regulated proteins involved in glycolysis, glycerolipid, carotenoid and glutathione metabolism which were preferable metabolic routes towards lipid accumulation and DHA production as well as cellular oxidative defence. Integrated proteomic and transcriptomic data were also conducted to comprehend the gene and protein regulation underlying the lipid and DHA biosynthesis. A significant up-regulation of acetyl-CoA synthetase was observed which suggests alternative route of acetate metabolism for acetyl-CoA producer. This study presents the holistic routes underlying lipid accumulation and DHA production in Aurantiochytrium sp. SW1 and other relevant thraustochytrid.

Fatty acid biosynthesis is a fundamental process that occurs in all living organisms and involves multiple reaction steps. Thus, a systematic transfer of the intermediates between the different catalytic sites is highly required for the efficient regulation of a pathway as well as for sustaining growth. Multienzyme complex, a protein complex that comprises a group of interacting enzymes in a specific metabolic pathway, has been identified to catalyze numerous metabolic pathways, including fatty acid synthesis. The existence of a lipogenic multienzyme complex that involves protein interaction between numerous enzymes that took part in fatty acid biosynthesis plays a key fundamental role in channelling the intermediate substrates. Herein, the growing evidence for the formation of multienzyme complexes in fatty acid synthesis and the properties of the complex will be elucidated in this chapter.

Aurantiochytrium sp. SW1, a marine thraustochytrid, has been regarded as a potential candidate as a docosahexaenoic acid (DHA) producer. Even though the genomics of Aurantiochytrium sp. are available, the metabolic responses at a systems level are largely unknown. Therefore, this study aimed to investigate the global metabolic responses to DHA production in Aurantiochytrium sp. through transcriptome and genome-scale network-driven analysis. Of a total of 13,505 genes, 2527 differentially expressed genes (DEGs) were identified in Aurantiochytrium sp., unravelling the transcriptional regulations behinds lipid and DHA accumulation. The highest number of DEG were found for pairwise comparison between growth phase and lipid accumulating phase where a total of 1435 genes were down-regulated with 869 genes being up-regulated. These uncovered several metabolic pathways that contributing in DHA and lipid accumulation including amino acid and acetate metabolism which involve in the generation of crucial precursors. Upon applying network-driven analysis, hydrogen sulphide was found as potential reporter metabolite that could be associated with the genes related to acetyl-CoA synthesis for DHA production. Our findings suggest that the transcriptional regulation of these pathways is a ubiquitous feature in response to specific cultivation phases during DHA overproduction in Aurantiochytrium sp. SW1.

Lipids are considered a heterogeneous group of organic compounds which contain fats and their derivatives. This chapter achieved the data available on the nature and composition of lipids in filamentous fungi, and their distribution within the cell. The chapter describes some aspects of lipid metabolism, including fatty acid biosynthesis, lipid accumulation mechanisms, and different fermentation strategies. The lipid content of vegetative hyphae varies between 1% and more than 50%, of spores between 1% and 35%, and of yeast cells between 7% and approximately 15% of the tissue dry weights. The amount of lipids produced by a given species of fungus depends on the developmental stage of the growth and on the culture conditions. Culture parameters that influence the growth and the lipid contents of fungi have been found to be temperature, carbon and nitrogen sources, pH, inorganic salts, and others. The qualitative and quantitative nature of the extracellular lipids is influenced by the different growth parameters. The extracellular lipids known in a large number of oleaginous strains include polyol fatty acid esters, glycolipids, hydroxy fatty acids, sugar alcohols, acetylated sphingosines, and acetylated fatty acids. The main purpose of this chapter was to explain the biochemistry behind fungal lipid accumulation in oleaginous filamentous fungi, their distribution and functions, and the current applications of fungal fermentation strategies.

Carotenoids are lipid-soluble compounds that are present in nature, including plants and microorganisms such as fungi, certain bacteria, and algae. In fungi, they are widely present in almost all taxonomic classifications. Fungal carotenoids have gained special attention due to their biochemistry and the genetics of their synthetic pathway. The antioxidant potential of carotenoids may help fungi survive longer in their natural environment. Carotenoids may be produced in greater quantities using biotechnological methods than by chemical synthesis or plant extraction. The initial focus of this review is on industrially important carotenoids in the most advanced fungal and yeast strains, with a brief description of their taxonomic classification. Biotechnology has long been regarded as the most suitable alternative way of producing natural pigment from microbes due to their immense capacity to accumulate these pigments. So, this review mainly presents the recent progress in the genetic modification of native and non-native producers to modify the carotenoid biosynthetic pathway for enhanced carotenoid production, as well as factors affecting carotenoid biosynthesis in fungal strains and yeast, and proposes various extraction methods to obtain high yields of carotenoids in an attempt to find suitable greener extraction methods. Finally, a brief description of the challenges regarding the commercialization of these fungal carotenoids and the solution is also given.

Aliphatic compounds are carbon and hydrogen-containing hydrocarbon complexes and are present in almost every plant, animal and microorganism. In 1929, aliphatic hydrocarbons were first discovered by crude chemical methods. These observations were later confirmed and expanded by more sophisticated instruments, such as gas-liquid chromatography and GLC-mass spectrometry. Aliphatic hydrocarbons were detected in the wax of most studied organisms and mainly contained n-alkanes, but may also include n-alkenes, saturated and unsaturated, cyclic alkanes, and isoprenoid hydrocarbons. Similarly, surfaces of higher plants contain a complex waxy coating that consists of primary and secondary fatty alcohols, long chains of fatty acids, ketones, aldehyde, terpenes, diols, waxy esters, glycerides, etc. The chemical composition of aliphatic hydrocarbons, chain length predominance, branching, the unsaturation of surface wax and their variation among various organisms, such as plants, algae, bacteria, animals and particularly in fungi have been described in the current chapter. The hydrocarbon distribution in animals is reported to be slightly similar to that of higher plants, while in bacteria, a complex mixture of normal, single- double branched, saturated, or unsaturated structural isomers are reported. A brief description of the biotechnological production of various aliphatic compounds using genetic engineering has also been presented in this chapter. The biosynthesis of aliphatic hydrocarbons by two common routes, “elongation decarboxylation” and “head-to-head condensation,” has been studied well in plants and bacteria and are discussed here in detail. Pathways involved in the degradation of hydrocarbons by aerobic and anaerobic microbes and the enzymes involved are also described in this chapter. Aliphatic compounds with different chain lengths have been of biotechnological interest for the past few decades as they perform various biological functions in living organisms apart from their role as the chief component of diesel and jet fuels. The current chapter also highlighted the biological importance of these aliphatic hydrocarbons.

Over the past decades, fungi have been increasingly recognized as the potential source of lipids that can be applied in various sectors, including nutraceutical and biofuel. Thus, many studies have been conducted to understand the structural and functional roles of lipid molecules, particularly in the potential oleaginous strains. Lipids produced by oleaginous fungi comprise different classes, and acylglycerol, which are esters formed from different fatty acids and alcohols such as glycerol, represent the major components of the lipid. The biosynthesis of acylglycerol in fungi involved a series of reactions involving the central carbon and glycerol-3-phosphate (G-3-P), while its catabolism in vivo generally involved the degradation of triacylglycerol (TAG) by intracellular lipase resulting in the release of fatty acids and glycerol. The resulting glycerol will be phosphorylated, oxygenated, and enter glycolysis, whereas the fatty acids will undergo β-oxidation into acetyl-CoA and be used for various physiological functions. On the other hand, several fungi species, particularly from the Mucoralles sp, have been documented to be able to utilize the oil and fat as the alternative substrate for growth and reproduction due to its capability to produce extracellular lipases which hydrolyze the ester bond of the TAG. This chapter will comprehensively discuss the functional role of acylglycerol in fungi, its biosynthesis, as well as in vivo and ex vivo degradation in fungi, which will be a bridge toward the development of the industrial application.

Over the past decades, fungi have been increasingly recognized as the potential source of lipids that can be applied in various sectors, including nutraceutical and biofuel. Thus, many studies have been conducted to understand the structural and functional roles of lipid molecules, particularly in the potential oleaginous strains. Lipids produced by oleaginous fungi comprise different classes, and acylglycerol, which are esters formed from different fatty acids and alcohols such as glycerol, represent the major components of the lipid. The biosynthesis of acylglycerol in fungi involved a series of reactions involving the central carbon and glycerol-3-phosphate (G-3-P), while its catabolism in vivo generally involved the degradation of triacylglycerol (TAG) by intracellular lipase resulting in the release of fatty acids and glycerol. The resulting glycerol will be phosphorylated, oxygenated, and enter glycolysis, whereas the fatty acids will undergo β-oxidation into acetyl-CoA and be used for various physiological functions. On the other hand, several fungi species, particularly from the Mucoralles sp, have been documented to be able to utilize the oil and fat as the alternative substrate for growth and reproduction due to its capability to produce extracellular lipases which hydrolyze the ester bond of the TAG. This chapter will comprehensively discuss the functional role of acylglycerol in fungi, its biosynthesis, as well as in vivo and ex vivo degradation in fungi, which will be a bridge toward the development of the industrial application.

In recent years, the utilisation of endophytes has emerged as a promising biological treatment technology for the degradation of plastic wastes such as biodegradation of synthetic plastics. This study, therefore, aimed to explore and extensively screen endophytic fungi (from selected plants) for efficient in vitro polyvinyl alcohol (PVA) biodegradation. In total, 76 endophytic fungi were isolated and cultivated on a PVA screening agar medium. Among these fungi, 10 isolates showed potential and were subsequently identified based on phenotypical characteristics, ITS ribosomal gene sequences, and phylogenetic analyses. Four strains exhibited a maximum level of PVA-degradation in the liquid medium when cultivated for 10 days at 28 °C and 150 rpm. These strains showed varied PVA removal rates of 81% (Penicillium brevicompactum OVR-5), 67% (Talaromyces verruculosus PRL-2), 52% (P. polonicum BJL-9), and 41% (Aspergillus tubingensis BJR-6) respectively. The most promising PVA biodegradation isolate ‘OVR-5’, with an optimal pH at 7.0 and optimal temperature at 30 °C, produced lipase, manganese peroxidase, and laccase enzymes. Based on analyses of its metabolic intermediates, as identified with GC–MS, we proposed the potential PVA degradation pathway of OVR-5. Biodegradation results were confirmed through scanning electron microscopy and Fourier transform infrared spectroscopy. This study provides the first report on an endophytic P. brevicompactum strain (associated with Orychophragmus violaceus) that has a great ability for PVA degradation providing more insight on potential fungus-based applications in plastic waste degradation.

Thraustochytrids, such as Aurantiochytrium and Schizochytrium, have been shown as a promising sustainable alternative to fish oil due to its ability to accumulate a high level of docosahexaenoic acid (DHA) from its total fatty acids. However, the low DHA volumetric yield by most of the wild type (WT) strain of thraustochytrids which probably be caused by the low oxidative stress tolerance as well as a limited supply of key precursors for DHA biosynthesis has restricted its application for industrial application. Thus, to enhance the DHA production, we aimed to generate Aurantiochytrium SW1 mutant with high tolerance toward oxidative stress and high glucose-6 phosphate dehydrogenase (G6PDH) activities through strategic plasma mutagenesis coupled with chemical screening. The WT strain (Aurantiochytrium sp. SW1) was initially exposed to plasma radiation and was further challenged with zeocin and polydatin, generating a mutant (YHPM1) with a 30, 65, and 80% higher overall biomass, lipid, and DHA production in comparison with the parental strains, respectively. Further analysis showed that the superior growth, lipid, and DHA biosynthesis of the YHMP1 were attributed not only to the higher G6PDH and enzymes involved in the oxidative defense such as superoxide dismutase (SOD) and catalase (CAT) but also to other key metabolic enzymes involved in lipid biosynthesis. This study provides an effective approach in developing the Aurantiochytrium sp. mutant with superior DHA production capacity that has the potential for industrial applications.

Aims: Thraustochytrids have been shown to be excellent lipid producers due to their ability to accumulate over 50% lipid (g/g biomass) containing up to 50% docosahexaenoic acid (DHA). However, efficient and cost-effective cell recovery of lipid-rich biomass has become a significant challenge at the industrial scale. In this study, we attempted to enhance the harvesting efficiency (HE) and the DHA content of Aurantiochytrium sp. through co-cultivation with a gamma-linolenic acid (GLA)-producing oleaginous filamentous fungus, Cunninghamella bainieri 2A1.Methodology and results: A 72 h old C. bainieri 2A1 culture in the form of loose mycelia or pellets of various sizes was added into 72 h old Aurantiochytrium sp. cultures and further incubated for 48 h. The HE of Aurantiochytrium sp. was then determined by comparing the remaining OD values of the supernatant with and without minimal centrifugation at 4000x g. Results showed that 63.23% of HE was achieved without centrifugation from co-cultivation with dispersed mycelia. Higher HE between 96.71-99.55% was achieved when centrifugation was implemented, with the highest value resulting from co-cultivation with dispersed mycelia. These are higher than HE of centrifuged control cultures (80%) consisting of Aurantiochytrium sp. monocultures, suggesting that co-cultivation with C. bainieri 2A1 facilitates the recovery of Aurantiochytrium sp. cells. Moreover, the co-cultivation also resulted in a 28% increase in DHA compared to non-optimized cultures.Conclusion, significance and impact of study: This study provides the first evidence of enhancement in harvesting and DHA content of oleaginous thraustochytrids that could be achieved through co-cultivation with oleaginous fungi.