The locally isolated filamentous fungus Cunninghamella bainieri 2A1 was cultivated in a 5 L bioreactor to produce lipid and gamma-linolenic acid (GLA). The optimization was carried out using response surface methodology based on a central composite design. A statistical model, second-order polynomial model, was adjusted to the experimental data to evaluate the effect of key operating variables, including aeration rate and agitation speed on lipid production. Process analysis showed that linear and quadratic effect of agitation intensity significantly influenced lipid production process (P < 0.01). The quadratic model also indicated that the interaction between aeration rate and agitation speed had a highly significant effect on lipid production (P < 0.01). Experimental results showed that a lipid content of 38.71% was produced in optimum conditions using an airflow rate and agitation speed of 0.32 vvm and 599 rpm, respectively. Similar results revealed that 0.058(g/g) gamma-linolenic acid was produced in optimum conditions where 1.0 vvm aeration rate and 441.45 rpm agitation rate were used. The regression model confirmed that aeration and agitation were of prime importance for optimum production of lipid in the bioreactor.
Delta 6-asid lemak desaturase dan delta 12-asid lemak desaturase merupakan enzim yang diperlukan bagi langkah desaturasi semasa proses biosintesis asid gamma-linolenik (GLA) oleh kulat oleaginus. Objektif kajian ini ialah untuk menganalisis profil pengekspresan gen mengekod enzim delta 6-asid lemak desaturase (des6) dan delta 12-asid lemak desaturase (des12) kulat oleaginus Cunninghamella bainieri semasa penghasilan GLA. Jujukan gen separa bersaiz 1372 pb bagi des6 dan 1008 pb bagi des12 telah dipencil daripada C. bainieri. Analisis pengekspresan gen menggunakan kaedah tindak balas berantai polimerase kuantitatif masa sebenar (RT-qPCR) menunjukkan perubahan kadar pengekspresan des6 adalah lebih tinggi berbanding kadar pengekspresan des12 semasa penghasilan GLA. Pengekspresan des6 adalah tertinggi selepas 24 jam dikultur dalam medium penghasilan GLA. Namun, kadar pengekspresannya menurun hingga jam ke-96 pertumbuhan tetapi meningkat semula pada jam ke-120. Bagi des12, kadar pengekspresannya adalah lebih sekata dengan pengekspresan tertinggi dikesan pada jam ke-120. Analisis penghasilan GLA menunjukkan jumlah GLA dalam sel berkolerasi dengan kadar pengekspresan des6. Hasil kajian mencadangkan bahawa aras pengekspresan des6 adalah penting dalam menentukan aras GLA dalam C. bainieri.
Strategic feeding of ammonium and metal ions (Mg(2+), Mn(2+), Fe(3+), Cu(2+), Ca(2+), Co(2+), and Zn(2+)) for enhanced GLA-rich lipid accumulation in C. bainieri 2A1 was established. When cultivated in nitrogen-limited medium, the fungus produced up to 30% lipid (g/g biomass) with 12.9% (g/g lipid) GLA. However, the accumulation of lipid stopped at 48 hours of cultivation although glucose was abundant. This event occurred in parallel to the diminishing activity of malic enzyme (ME), fatty acid synthase (FAS), and ATP citrate lyase (ACL) as well as the depletion of metal ions in the medium. Reinstatement of the enzymes activities was achieved by feeding of ammonium tartrate, but no increment in the lipid content was observed. However, increment in lipid content from 32% to 50% (g/g biomass) with 13.2% GLA was achieved when simultaneous feeding of ammonium, glucose, and metal ions was carried out. This showed that the cessation of lipid accumulation was caused by diminishing activities of the enzymes as well as depletion of the metal ions in the medium. Therefore, strategic feeding of ammonium and metal ions successfully reinstated enzymes activities and enhanced GLA-rich lipid accumulation in C. bainieri 2A1.
Aquilaria malaccensis produces agarwood in response to wounding and fungal attack. However, information is limited regarding Aquilaria's interaction with its diverse fungal community. In this study, time-related changes of three natural fungal colonizers in two wounded wild A. malaccensis were tracked, beginning a few hours after wounding up to 12 months. Using species-specific primers derived from their nrITS sequences in quantitative real-time PCR (qPCR), we quantified the amount of Cunninghamella bainieri, Fusarium solani and Lasiodiplodia theobromae. Because time is a major factor affecting agarwood quantity and quality, 14 wood samples were collected at different time points, i.e., 0-18 h, 2-13 days, 2-18 weeks, and 6-12 months after wounding. qPCR data revealed that the abundance of the three species decreased over time. The fungi were detected in high numbers during the first few hours and days after wounding (40- to 25,000-fold higher levels compared with initial counts) and in low numbers (<1- to 3,200-fold higher than initially) many months later. Consistent with its role in defense response, the accumulation of secondary metabolites at the wounding site could have caused the decline in fungal abundance. Succession patterns of the two trees were not identical, indicating that fungal populations may have been affected by tree environment and wound microclimate. Our results are important for understanding the diversity of microbial community in wild Aquilaria species and their association to wound-induced agarwood formation. Fungi could be secondary triggers to agarwood production in situations where trees are wounded in attempt to induce agarwood.
This research was performed based on a comparative study on fungal lipid production by a locally isolated strain Cunninghamella bainieri 2A1 in batch culture and repeated-batch culture using a nitrogen-limited medium. Lipid production in the batch culture was conducted to study the effect of different agitation rates on the simultaneous consumption of ammonium tartrate and glucose sources. Lipid production in the repeated-batch culture was studied by considering the effect of harvesting time and harvesting volume of the culture broth on the lipid accumulation. The batch cultivation was carried out in a 500 ml Erlenmeyer flask containing 200 ml of the fresh nitrogen-limited medium. Microbial culture was incubated at 30 °C under different agitation rates of 120, 180 and 250 rpm for 120 h. The repeated-batch culture was performed at three harvesting times of 12, 24 and 48 h using four harvesting cultures of 60%, 70%, 80% and 90%. Experimental results revealed that nitrogen source (ammonium tartrate) was fully utilized by C. bainieri 2A1 within 24 h in all agitation rates tested. It was also observed that a high amount of glucose in culture medium was consumed by C. bainieri 2A1 at 250 rpm agitation speed during the batch fermentation. Similar results showed that the highest lipid concentration of 2.96 g/L was obtained at an agitation rate of 250 rpm at 120 h cultivation time with the maximum lipid productivity of 7.0 × 10(-2) mg/ml/h. On the other hand, experimental results showed that the highest lipid concentration produced in the repeated-batch culture was 3.30 g/L at the first cycle of 48 h harvesting time using 70% harvesting volume, while 0.23 g/L gamma-linolenic acid (GLA) was produced at the last cycle of 48 h harvesting time using 80% harvesting volume.
The biosynthesis of biomedical products including lipid and gamma-linolenic acid (GLA) by Cunninghamella bainieri 2A1 was studied in repeated batch fermentation. Three key process variables, namely, glucose concentration, ammonium tartrate concentration, and harvesting time, were optimized using response surface methodology. Repeated batch fermentation was carried out by the cultivation of Cunninghamella bainieri 2A1 in nitrogen-limited medium with various nitrogen concentration (1-4 g/L) and glucose concentration (20-40 g/L) at three time intervals (12 h, 24 h, and 48 h). Experimental results showed that the highest lipid concentration of 6.2 g/L and the highest GLA concentration of 0.4 g/L were obtained in optimum conditions, where 20.2 g/L glucose, 2.12 g/L ammonium tartrate, and 48 h harvesting time were utilized. Statistical results showed that the interaction between glucose and ammonium tartrate concentration had highly significant effects on lipid and GLA biosynthesis (P < 0.01). Moreover, harvesting time had a significant interaction effect with glucose and ammonium tartrate concentration on lipid production (P < 0.05).
Lipid biosynthesis and fatty acids composition of oleaginous zygomycetes, namely Cunninghamella bainieri 2A1, cultured in media with excess or limited nitrogen were quantitatively determined at different times of culture growth. Accumulation of lipids occurred even when the activity of NAD(+)-ICDH (β-Nicotinamide adenine dinucleotide-isocitrate dehydrogenase) was still detectable in both media. In C. bainieri 2A1, under nitrogen limitation, the ratio of lipids was around 35%, whereas in nitrogen excess medium (feeding media supplemented with ammonium tartarate), the lipid ratio decreased. The amount of this decrease depended on the level of ammonium tartarate in the media. The main findings in this paper were that C. bainieri 2A1 has the ability to accumulate lipid although nitrogen concentration detected inside the media and that NAD-ICDH was active in all culture periods. These results proved that the strain C. bainieri 2A1 has an alternative behavior in lipid biosynthesis that differs from yeast. According to the old hypotheses, yeasts could not accumulate lipid more than 10% when nitrogen was detected inside the media. Nitrogen-limited and excess media both contained the same fatty acids (palmitic acid, stearic acid, olic acid, linoleic acid and γ-linolenic acid), but at different concentrations. The C:N ratio was also studied and showed no effects on total lipid accumulation, but a significant effect on γ-linolenic acid concentration.
The fungal transformations of medroxyrogesterone (1) were investigated for the first time using Cunninghamella elegans, Trichothecium roseum, and Mucor plumbeus. The metabolites obtained are as following: 6β, 20-dihydroxymedroxyprogesterone (2), 12β-hydroxymedroxyprogesterone (3), 6β, 11β-dihydroxymedroxyprogesterone (4), 16β-hydroxymedroxyprogesterone (5), 11α, 17-dihydroxy-6α-methylpregn-4-ene-3, 20-dione (6), 11-oxo-medroxyprogesterone (7), 6α-methyl-17α-hydroxypregn-1,4-diene-3,20-dione (8), and 6β-hydroxymedroxyprogesterone (9), 15β-hydroxymedroxyprogesterone (10), 6α-methyl-17α, 11β-dihydroxy-5α-pregnan-3, 20-dione (11), 11β-hydroxymedroxyprogesterone (12), and 11α, 20-dihydroxymedroxyprogesterone (13). Among all the microbial transformed products, the newly isolated biotransformed product 13 showed the most potent activity against proliferation of SH-SY5Y cells. Compounds 12, 5, 6, 9, 11, and 3 (in descending order of activity) also showed some extent of activity against SH-SY5Y tumour cell line. The never been reported biotransformed product, 2, showed the most potent inhibitory activity against acetylcholinesterase. Molecular modelling studies were carried out to understand the observed experimental activities, and also to obtain more information on the binding mode and the interactions between the biotransformed products, and enzyme.
Malic enzyme (ME) plays a vital role in determining the extent of lipid accumulation in oleaginous fungi being the major provider of NADPH for the activity of fatty acid synthase (FAS). We report here the first direct evidence of the existence of a lipogenic multienzyme complex (the lipid metabolon) involving ME, FAS, ATP: citrate lyase (ACL), acetyl-CoA carboxylase (ACC), pyruvate carboxylase (PC) and malate dehydrogenase (MDH) in Cunninghamella bainieri 2A1. Cell-free extracts prepared from cells taken in both growth and lipid accumulation phases were prepared by protoplasting and subjected to Blue Native (BN)-PAGE coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). A high molecular mass complex (approx. 3.2 MDa) consisting of the above enzymes was detected during lipid accumulation phase indicating positive evidence of multienzyme complex formation. The complex was not detected in cells during the balanced phase of growth or when lipid accumulation ceased, suggesting that it was transiently formed only during lipogenesis.