Harvesting Aurantiochytrium sp. SW1 via Flocculation Using Chitosan: Effects of Flocculation Parameters on Flocculation Efficiency and Zeta Potential.

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.

Mucor circinelloides: a model organism for oleaginous fungi and its potential applications in bioactive lipid production.

Microbial oils have gained massive attention because of their significant role in industrial applications. Currently plants and animals are the chief sources of medically and nutritionally important fatty acids. However, the ever-increasing global demand for polyunsaturated fatty acids (PUFAs) cannot be met by the existing sources. Therefore microbes, especially fungi, represent an important alternative source of microbial oils being investigated. Mucor circinelloides-an oleaginous filamentous fungus, came to the forefront because of its high efficiency in synthesizing and accumulating lipids, like γ-linolenic acid (GLA) in high quantity. Recently, mycelium of M. circinelloides has acquired substantial attraction towards it as it has been suggested as a convenient raw material source for the generation of biodiesel via lipid transformation. Although M. circinelloides accumulates lipids naturally, metabolic engineering is found to be important for substantial increase in their yields. Both modifications of existing pathways and re-formation of biosynthetic pathways in M. circinelloides have shown the potential to improve lipid levels. In this review, recent advances in various important metabolic aspects of M. circinelloides have been discussed. Furthermore, the potential applications of M. circinelloides in the fields of antioxidants, nutraceuticals, bioremediation, ethanol production, and carotenoids like beta carotene and astaxanthin having significant nutritional value are also deliberated.

Role of Snf-ß in lipid accumulation in the high lipid-producing fungus Mucor circinelloides WJ11.

BACKGROUND: Mucor circinelloides WJ11 is a high-lipid producing strain and an excellent producer of γ-linolenic acid (GLA) which is crucial for human health. We have previously identified genes that encode for AMP-activated protein kinase (AMPK) complex in M. circinelloides which is an important regulator for lipid accumulation. Comparative transcriptional analysis between the high and low lipid-producing strains of M. circinelloides showed a direct correlation in the transcriptional level of AMPK genes with lipid metabolism. Thus, the role of Snf-ß, which encodes for ß subunit of AMPK complex, in lipid accumulation of the WJ11 strain was evaluated in the present study. RESULTS: The results showed that lipid content of cell dry weight in Snf-ß knockout strain was increased by 32 % (from 19 to 25 %). However, in Snf-ß overexpressing strain, lipid content of cell dry weight was decreased about 25 % (from 19 to 14.2 %) compared to the control strain. Total fatty acid analysis revealed that the expression of the Snf-ß gene did not significantly affect the fatty acid composition of the strains. However, GLA content in biomass was increased from 2.5 % in control strain to 3.3 % in Snf-ß knockout strain due to increased lipid accumulation and decreased to 1.83 % in Snf-ß overexpressing strain. AMPK is known to inactivate acetyl-CoA carboxylase (ACC) which catalyzes the rate-limiting step in lipid synthesis. Snf-ß manipulation also altered the expression level of the ACC1 gene which may indicate that Snf-ß control lipid metabolism by regulating ACC1 gene. CONCLUSIONS: Our results suggested that Snf-ß gene plays an important role in regulating lipid accumulation in M. circinelloides WJ11. Moreover, it will be interesting to evaluate the potential of other key subunits of AMPK related to lipid metabolism. Better insight can show us the way to manipulate these subunits effectively for upscaling the lipid production. Up to our knowledge, it is the first study to investigate the role of Snf-ß in lipid accumulation in M. circinelloides.

Annotation of AMP-activated protein kinase genes and its comparative transcriptional analysis between high and low lipid producing strains of Mucor circinelloides.

BACKGROUND: AMP-activated protein kinase (AMPK) is an important regulator for lipid accumulation, potentially known to have an inhibitory role in lipid synthesis. It inactivates acetyl-CoA carboxylase (ACC), an important regulatory enzyme required for lipid synthesis. However, in Mucor circinelloides, AMPK and its association with lipid accumulation has not been studied yet. OBJECTIVES: To identify AMPK genes in M. circinelloides and to compare their expression levels in high and low lipid-producing strains of M. circinelloides to predict the possible roles of AMPK in lipid metabolism and to select candidate genes for further studies to enhance lipid accumulation. RESULTS: Two genes for α-subunit, one for ß-subunit and six for γ-subunit were identified and annotated. Bioinformatic analysis confirmed the presence of typical conserved domains in these genes. Furthermore, transcriptional profiling displayed marked differences in expression kinetics of subunits among the selected strains. The expression of AMPK genes decreased rapidly in WJ11, high lipid producer strain during the lipid accumulation phase while contrasting profile of expression was observed in CBS 277.49, low lipid producer strain. CONCLUSION: The present study has shown the association of AMPK genes with lipid metabolism at the transcriptional level. The involvement of Snf-α1, Snf-α2, Snf-ß, Snf-γ1, Snf-γ4, Snf-γ5 subunits were shown to be more pronounced and could potentially be further explored in future studies.

Biodegradation of poly (vinyl alcohol) by an orychophragmus rhizosphere-associated fungus Penicillium brevicompactum OVR-5, and its proposed PVA biodegradation pathway.

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.

Industrially Important Fungal Carotenoids: Advancements in Biotechnological Production and Extraction.

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.

Strategic Development of Aurantiochytrium sp. Mutants With Superior Oxidative Stress Tolerance and Glucose-6-Phosphate Dehydrogenase Activity for Enhanced DHA Production Through Plasma Mutagenesis Coupled With Chemical Screening.

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.

Whole genome analysis and elucidation of docosahexaenoic acid (DHA) biosynthetic pathway in Aurantiochytrium sp. SW1.

Aurantiochytrium sp., a fungoid marine protist that belongs to Stramenophila has proven its potential in the production of polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acids (DHA). In this study, genomic characterisation of a potential producer for commercial production of DHA, Aurantiochytrium sp. SW1 has been carried out via whole genome sequencing analysis. The genome size of this strain is 60.89 Mb, with a total of 11,588 protein-coding genes. Among these, 9,127 genes could be functionally annotated into a total of 7,248 (62.5 %) from UniProt, 6,554 (56.6 %) from KEGG and 8,643 (74.6 %) genes from eggNOG protein database. The highest proportion of genes belongs to the protein family of metabolism were further assigned into 11 metabolic categories. The highest number of genes belonging to lipid metabolism (321 genes) followed by carbohydrate metabolism (290 genes), metabolism of cofactors and vitamins (197 genes) and amino acid metabolism (188 genes). Further analysis into the biosynthetic pathway for DHA showed evidence of all genes involved in PKS (polyketide synthase)-like PUFA synthase pathway and incomplete fatty acid synthase-elongase/desaturase pathway. Analysis of PUFA synthase showed the presence of up to ten tandem acyl carrier protein (ACP) domains which might have contributed to high DHA production in this organism. In addition, a hybrid system incorporating elements of FAS, Type I PKS and Type II PKS systems were found to be involved in the biosynthetic pathways of fatty acids in Aurantiochytrium sp. SW1. This study delivers an important reference for future research to enhance the lipid, especially DHA production in Aurantiochytrium sp, SW1 and establishment of this strain as an oleaginous thraustochytrid model.

Coculturing of Mucor plumbeus and Bacillus subtilis bacterium as an efficient fermentation strategy to enhance fungal lipid and gamma-linolenic acid (GLA) production.

This study aimed to improve lipid and gamma-linolenic acid (GLA) production of an oleaginous fungus, Mucor plumbeus, through coculturing with Bacillus subtilis bacteria, optimising the environmental and nutritional culture conditions, and scaling them for batch fermentation. The maximum levels of biomass, lipid, fatty acid, and GLA in a 5 L bioreactor containing cellobiose and ammonium sulfate as the optimal carbon and nitrogen sources, respectively, achieved during the coculturing processes were 14.5 ± 0.4 g/L, 41.5 ± 1.3, 24 ± 0.8, and 20 ± 0.5%, respectively. This strategy uses cellobiose in place of glucose, decreasing production costs. The nutritional and abiotic factor results suggest that the highest production efficiency is achieved at 6.5 pH, 30 °C temperature, 10% (v/v) inoculum composition, 200 rpm agitation speed, and a 5-day incubation period. Interestingly, the GLA concentration of cocultures (20.0 ± 0.5%) was twofold higher than that of monocultures (8.27 ± 0.11%). More importantly, the GC chromatograms of cocultures indicated the presence of one additional peak corresponding to decanoic acid (5.32 ± 0.20%) that is absent in monocultures, indicating activation of silent gene clusters via cocultivation with bacteria. This study is the first to show that coculturing of Mucor plumbeus with Bacillus subtilis is a promising strategy with industrialisation potential for the production of GLA-rich microbial lipids and prospective biosynthesis of new products.

Evaluation of Different Standard Amino Acids to Enhance the Biomass, Lipid, Fatty Acid, and γ-Linolenic Acid Production in Rhizomucor pusillus and Mucor circinelloides.

In this study, 18 standard amino acids were tested as a single nitrogen source on biomass, total lipid, total fatty acid (TFA) production, and yield of γ-linolenic acid (GLA) in Rhizomucor pusillus AUMC 11616.A and Mucor circinelloides AUMC 6696.A isolated from unusual habitats. Grown for 4 days at 28°C, shaking at 150 rpm, the maximum fungal biomass for AUMC 6696.A was 14.6 ± 0.2 g/L with arginine and 13.68 ± 0.1 g/L with asparagine, when these amino acids were used as single nitrogen sources, while AUMC 11616.A maximum biomass was 10.73 ± 0.8 g/L with glycine and 9.44 ± 0.6 g/L with valine. These were significantly higher than the ammonium nitrate control (p < 0.05). The highest levels of TFA were achieved with glycine for AUMC 11616.A, 26.2 ± 0.8% w/w of cell dry weight, and glutamic acid for AUMC 6696.A, 23.1 ± 1.3%. The highest GLA yield was seen with proline for AUMC 11616.A, 13.4 ± 0.6% w/w of TFA, and tryptophan for AUMC 6696.A, 12.8 ± 0.3%, which were 38% and 25% higher than the ammonium tartrate control. The effects of environmental factors such as temperature, pH, fermentation time, and agitation speed on biomass, total lipids, TFA, and GLA concentration of the target strains have also been investigated. Our results demonstrated that nitrogen assimilation through amino acid metabolism, as well as the use of glucose as a carbon source and abiotic factors, are integral to increasing the oleaginicity of tested strains. Few studies have addressed the role of amino acids in fermentation media, and this study sheds light on R. pusillus and M. circinelloides as promising candidates for the potential applications of amino acids as nitrogen sources in the production of lipids.

Two-level factorial analysis of the effect of fructose on DHA biosynthetic capacity of Aurantiochytrium sp. SW1.

Thraustochytrids are getting increasingly popular due to their high potential role as alternative producers of the high-valued ω-3 polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA). While most thraustochytrids prefer glucose as the major carbon source, few strains have been reported to prefer fructose. One such strain is Aurantiochytrium sp. SW1. In this study, the effect of fructose on DHA accumulation by SW1 was investigated using a two-level full factorial design. Besides, biomass, lipid and DHA accumulation profiles of SW1 cultivated in fructose and glucose media were compared. Results revealed that fructose has a very significant positive effect on the volumetric DHA content. Meanwhile, its involvement in affecting DHA biosynthetic capacity, though significant, is not very profound. It was also found that when cultivated in fructose medium, SW1 had a less steep log phase compared to that of glucose medium. However, after 48h of cultivation, biomass and lipid accumulation in fructose medium outweighed the other. Volumetric DHA content in fructose medium at 96h was 11% higher than that of glucose medium. Overall, fructose was found to be a more suitable substrate for biomass, lipid and DHA accumulation in SW1 compared to the conventional source, glucose.

Recent Molecular Tools for the Genetic Manipulation of Highly Industrially Important Mucoromycota Fungi.

Mucorales is the largest and most well-studied order of the phylum Mucormycota and is known for its rapid growth rate and various industrial applications. The Mucorales fungi are a fascinating group of filamentous organisms with many uses in research and the industrial and medical fields. They are widely used biotechnological producers of various secondary metabolites and other value-added products. Certain members of Mucorales are extensively used as model organisms for genetic and molecular investigation and have extended our understanding of the metabolisms of other members of this order as well. Compared with other fungal species, our understanding of Mucoralean fungi is still in its infancy, which could be linked to their lack of effective genetic tools. However, recent advancements in molecular tools and approaches, such as the construction of recyclable markers, silencing vectors, and the CRISPR-Cas9-based gene-editing system, have helped us to modify the genomes of these model organisms. Multiple genetic modifications have been shown to generate valuable products on a large scale and helped us to understand the morphogenesis, basic biology, pathogenesis, and host-pathogen interactions of Mucoralean fungi. In this review, we discuss various conventional and modern genetic tools and approaches used for efficient gene modification in industrially important members of Mucorales.

Molecular Mechanism of Citrate Efflux by the Mitochondrial Citrate Transporter CT in Filamentous Fungus Mucor circinelloides WJ11.

The mitochondrial citrate transporter (MCT) plays an important role in citrate efflux from the mitochondria in eukaryotes, and hence provides a direct correlation between carbohydrate metabolism and lipid synthesis. Our previous studies on transporters confirmed the presence of two MCTs (TCT and CT) in oleaginous Mucor circinelloides WJ11 associated with high lipid accumulation. However, the molecular mechanism of citrate efflux from the mitochondria by MCT in M. circinelloides is still unclear. To study the citrate transport mechanism of CT, the citrate transporter gene was expressed in Escherichia coli, and its product was purified. The citrate transport activity of the protein was studied in CT reconstituted liposomes. Our results showed high efficiency of CT for [14C] citrate/citrate exchange with K m 0.01 mM at 25°C. Besides citrate, other molecules such as oxaloacetate, malate, fumarate, succinate aconitate, oxoadipate, isocitrate, and glutamate also promote citrate transport. In addition, the ct overexpression and knockout plasmids were constructed and transferred into M. circinelloides WJ11, and the mitochondria were isolated, and the transport activity was studied. Our findings showed that in the presence of 10 mM malate, the mitochondria of ct-overexpressing transformant showed 51% increase in the efflux rate of [14C] citrate, whereas the mitochondria of the ct-knockout transformant showed 18% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. This study provided the first mechanistic evidence of citrate efflux from the mitochondria by citrate transporter in oleaginous filamentous fungus M. circinelloides, which is associated with high lipid accumulation.

Genetic Modification of Mucor circinelloides for Canthaxanthin Production by Heterologous Expression of ß-carotene Ketolase Gene.

Canthaxanthin is a reddish-orange xanthophyll with strong antioxidant activity and higher bioavailability than carotenes, primarily used in food, cosmetics, aquaculture, and pharmaceutical industries. The spiking market for natural canthaxanthin promoted researchers toward genetic engineering of heterologous hosts for canthaxanthin production. Mucor circinelloides is a dimorphic fungus that produces ß-carotene as the major carotenoid and is considered as a model organism for carotenogenic studies. In this study, canthaxanthin-producing M. circinelloides strain was developed by integrating the codon-optimized ß-carotene ketolase gene (bkt) of the Haematococcus pluvialis into the genome of the fungus under the control of strong promoter zrt1. First, a basic plasmid was constructed to disrupt crgA gene, a negative regulator of carotene biosynthesis resulted in substantial ß-carotene production, which served as the building block for canthaxanthin by further enzymatic reaction of the ketolase enzyme. The genetically engineered strain produced a significant amount (576 ± 28 µg/g) of canthaxanthin, which is the highest amount reported in Mucor to date. Moreover, the cell dry weight of the recombinant strain was also determined, producing up to more than 9.0 g/L, after 96 h. The mRNA expression level of bkt in the overexpressing strain was analyzed by RT-qPCR, which increased by 5.3-, 4.1-, and 3-folds at 24, 48, and 72 h, respectively, compared with the control strain. The canthaxanthin-producing M. circinelloides strain obtained in this study provided a basis for further improving the biotechnological production of canthaxanthin and suggested a useful approach for the construction of more valuable carotenoids, such as astaxanthin.

Deletion of Plasma Membrane Malate Transporters Increased Lipid Accumulation in the Oleaginous Fungus Mucor circinelloides WJ11.

Malate as an important intermediate metabolite, its subcellular location, and concentration have a significant impact on fungal lipid metabolism. Previous studies showed that the mitochondrial malate transporter plays an important role in lipid accumulation in Mucor circinelloides by manipulating intracellular malate concentration. However, the role of plasma membrane malate transporters in oleaginous fungi remains unexplored. Therefore, in this work, two plasma membrane malate transporters "2-oxoglutarate:malate antiporters" (named SoDIT-a and SoDIT-b) of M. circinelloides WJ11 were deleted, and the consequences in growth capacity, lipid accumulation, and metabolism were analyzed. The results showed that deletion of sodit-a or/and sodit-b reduced the extracellular malate, confirming that the products of both genes participate in malate transportation. In parallel, the lipid contents in mutants increased approximately 10-40% higher than that in the control strain, suggesting that the defect in plasma membrane malate transport results in an increase of malate available for lipid biosynthesis. Furthermore, transcriptional analysis showed that the expression levels of multiple key genes involved in the lipid biosynthesis were also increased in the knockout mutants. To the best of our knowledge, this is the first report that demonstrated the association between plasma membrane malate transporters and lipid accumulation in M. circinelloides.

Comparative Analysis of Different Isolated Oleaginous Mucoromycota Fungi for Their γ-Linolenic Acid and Carotenoid Production.

γ-Linolenic acid (GLA) and carotenoids have attracted much interest due to their nutraceutical and pharmaceutical importance. Mucoromycota, typical oleaginous filamentous fungi, are known for their production of valuable essential fatty acids and carotenoids. In the present study, 81 fungal strains were isolated from different Egyptian localities, out of which 11 Mucoromycota were selected for further GLA and carotenoid investigation. Comparative analysis of total lipids by GC of selected isolates showed that GLA content was the highest in Rhizomucor pusillus AUMC 11616.A, Mucor circinelloides AUMC 6696.A, and M. hiemalis AUMC 6031 that represented 0.213, 0.211, and 0.20% of CDW, respectively. Carotenoid analysis of selected isolates by spectrophotometer demonstrated that the highest yield of total carotenoids (640 µg/g) was exhibited by M. hiemalis AUMC 6031 and M. hiemalis AUMC 6695, and these isolates were found to have a similar carotenoid profile with, ß-carotene (65%), zeaxanthin (34%), astaxanthin, and canthaxanthin (5%) of total carotenoids. The total fatty acids of all tested isolates showed moderate antimicrobial activity against Staphylococcus aureus and Salmonella Typhi, and Penicillium chrysogenum. To the best of our knowledge, this is the first report on the highest yield of total lipid accumulation (51.74% CDW) by a new oleaginous fungal isolate R. pusillus AUMC 11616.A. A new scope for a further study on this strain will be established to optimize and improve its total lipids with high GLA production. So, R. pusillus AUMC 11616.A might be a potential candidate for industrial application.

Comparative Analysis of ß-Carotene Production by Mucor circinelloides Strains CBS 277.49 and WJ11 under Light and Dark Conditions.

Carotenoids are natural potent antioxidants and free radical scavengers which are able to modulate the pathogenesis of some cancers and heart diseases in human, indicating their importance in being provided through the diet. Mucor circinelloides accumulates ß-carotene as the main carotenoid compound and has been used as a model organism in carotenogenic studies. In the present study, the potential of two M. circinelloides strains to accumulate ß-carotene was investigated under light and dark conditions. The results, which were quantitated by HPLC, showed that CBS 277.49 accumulated higher pigment in comparison to WJ11 under both conditions. Continuous illumination triggered the pigment accumulation up to 2.7-fold in strain CBS 277.49 and 2.2-fold in strain WJ11 in comparison to dark. The mRNA analysis of the four key genes involved in isoprenoid pathway by RT-qPCR showed higher transcriptional levels in CBS 277.49 as compared to WJ11, indicating that the pigment production metabolic machinery is more active in CBS 277.49 strain. A new scope for further research was established by this work for improved ß-carotene production in the high producing strain CBS 277.49.

Redirecting Metabolic Flux towards the Mevalonate Pathway for Enhanced ß-Carotene Production in M. circinelloides CBS 277.49.

Carotenoids produced by microbial sources are of industrial and medicinal importance due to their antioxidant and anticancer properties. In the current study, optimization of ß-carotene production in M. circinelloides strain 277.49 was achieved using response surface methodology (RSM). Cerulenin and ketoconazole were used to inhibit fatty acids and the sterol biosynthesis pathway, respectively, in order to enhance ß-carotene production by diverting metabolic pool towards the mevalonate pathway. All three variables used in screening experiments were found to be significant for the production of ß-carotene. The synergistic effect of the C/N ratio, cerulenin, and ketoconazole was further evaluated and optimized for superior ß-carotene production using central composite design of RSM. Our results found that the synergistic combination of C/N ratios, cerulenin, and ketoconazole at different concentrations affected the ß-carotene productions significantly. The optimal production medium (std. order 11) composed of C/N 25, 10 µg/mL cerulenin, and 150 mg/L ketoconazole, producing maximum ß-carotene of 4.26 mg/L (0.43 mg/g) which was 157% greater in comparison to unoptimized medium (1.68 mg/L, 0.17 mg/g). So, it was concluded that metabolic flux had been successfully redirected towards the mevalonate pathway for enhanced ß-carotene production in CBS 277.49.

Different Classes of Phytohormones Act Synergistically to Enhance the Growth, Lipid and DHA Biosynthetic Capacity of Aurantiochytrium sp. SW1.

In the present study, the impact of eight phytohormones from six different classes on the growth, lipid and docosahexaenoic acid (DHA) biosynthetic capacity of Aurantiochytrium sp. SW1 (SW1) was evaluated. Kinetin (KIN), jasmonic acid (JA) and gibberellic acid (GA) significantly enhanced the growth and DHA production of SW1 by 16%-28% and 66%-84% in comparison to the control, respectively. The synergistic effect of these three phytohormones, evaluated by the response surface methodology (RSM), showed that a combination of 3.6 mg/L GA, 2.0 mg/L KIN and 20.0 mg/L JA further increased the growth and DHA production of SW1 by 16% to 28% and 22% to 36%, respectively, in comparison to the individual supplementation. The synergistic effect of these phytohormones was also shown to be time-dependent, where feeding at 24 h of cultivation led to 15%, 26% and 35% further increments in the biomass, lipid and DHA production in comparison to that of 0 h, respectively. The determination of stress markers, antioxidant enzymes and key enzymes involved in fatty acid biosynthesis aided to elucidate the potential mechanism underlying the improvement of growth and DHA production by SW1 at various times of feeding. Supplementation with the phytohormones at 24 h exhibited the maximum impact on reducing the level of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as augmented the antioxidants (superoxide dismutase and catalase) and key metabolic enzymes involved in lipogenesis (malic, glucose-6-phosphate dehydrogenase and ATP-citrate lyase) in comparison to the control and other time points. This study signifies the potential application of phytohormones for improving the growth, lipid and DHA production in Aurantiochytrium spp.

Optimization of Culture Conditions for Enhanced Growth, Lipid and Docosahexaenoic Acid (DHA) Production of Aurantiochytrium SW1 by Response Surface Methodology.

In this study, optimization of growth, lipid and DHA production of Aurantiochytrium SW1 was carried out using response surface methodology (RSM) in optimizing initial fructose concentration, agitation speed and monosodium glutamate (MSG) concentration. Central composite design was applied as the experimental design and analysis of variance (ANOVA) was used to analyze the data. ANOVA analysis revealed that the process which adequately represented by quadratic model was significant (p < 0.0001) for all the response. All the three factors were significant (p < 0.005) in influencing the biomass and lipid data while only two factors (agitation speed and MSG) gave significant effect on DHA production (p < 0.005). The estimated optimal conditions for enhanced growth, lipid and DHA production were 70 g/L fructose, 250 rpm agitation speed and 10 g/L MSG. Consequently, the quadratic model was validated by applying the estimated optimum conditions, which confirmed the model validity where 19.0 g/L biomass, 9.13 g/L lipid and 4.75 g/L of DHA were produced. The growth, lipid and DHA were 28, 36 and 35% respectively higher than that produced in the original medium prior to optimization.