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Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with special functions. Using Yarrowia lipolytica as an unconventional chassis, we previously showed the performance of the Δ-6 pathway in ARA production. However, a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9 pathway from Isochrysis galbana was constructed via pathway engineering, allowing us to synthesize ARA at 91.5 mg L -1 . To further improve the ARA titer, novel enzyme fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations containing different linkers. Finally, with the integrated pathway engineering and synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/L, was achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS). The results show that the combined pathway and protein engineering can significantly facilitate applications of Y. lipolytica .

To investigate the metabolic regulation against oxygen supply, comparative metabolomics was performed to explore the metabolic responses of Mortierella alpina in the process of arachidonic acid (ARA) production. More than 110 metabolites involved in Embden-Meyerhof-Parnas pathway, pentose phosphate pathway, tricarboxylic acid cycle, inositol phosphate metabolism, fatty acid biosynthesis, and amino acid metabolism were identified by gas chromatography-mass spectrometry. Samples at different aeration rates were clearly distinguished by principal components analysis and partial least squares analysis, indicating that oxygen supply had a profound effect on the metabolism of M. alpina. Eleven major metabolites were identified as potential biomarkers to be primarily responsible for the difference of metabolism. Further study of metabolic changes with the relevant pathways demonstrated that the levels of several intermediate metabolites in relation to central carbon metabolism changed remarkably via both processes and citrate and malate was supposed to play vital roles in polyunsaturated acid (PUFA) synthesis. Increase of myo-inositol and sorbitol were probably for osmo-regulation and redox balance, while enhanced phosphoric acid and pyroglutamic acid were supposed to have function in the activation of signal transduction pathway for stress resistance. The present study provides a novel insight into the metabolic responses of M. alpina to aeration rates and the metabolic characteristics during the ARA fermentation.

The mechanism of arachidonic acid (ARA) content increase during aging of Mortierella alpina was elucidated. Lipid fraction analysis showed that ARA content increased from 46.9% to 66.4% in the triacylglycerol (TAG) molecule, and ARA residue occupation increased in the majority of TAG molecules during the aging process. For the first time, intracellular metabolite analysis was conducted to reveal the pathways closely associated with ARA biosynthesis during aging. The main reason for the increased ARA content was not only at the expense of other fatty acids degradation but also at the expense of further ARA biosynthesis during aging. Furthermore, translocation played a vital role in ARA redistribution among the glycerol moiety, and mycelium did not die immediately with key pathways activated to maintain a relatively stable intracellular environment. This study lays a foundation for further improvement of ARA content in the oil product obtained from M. alpina.

Arachidonic acid (ARA)-rich oil production by Mortierella alpina is a long fermentation period needed process due to the low growth rate of the filamentous fungus used. This causes the low productivity of ARA-rich oil and hinders its industrial mass scale production. In the present study, different fed-batch strategies were conducted to shorten the fermentation period. The result showed that compared with the batch culture, the fermentation period was shortened from 7days to 5days with the productivity of ARA-rich oil increased from 0.9g/(L·d) to 1.3g/(L·d) by using the fed-batch fermentation strategy. Furthermore, repeated fed-batch fermentation strategy was adopted to achieve the purpose of continuous production. By using this strategy, the fermentation period was shortened from 40days to 26days in a four cycle repeated fed-batch fermentation. This strategy proved to be convenient and economical for ARA-rich oil commercial production process., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Fungal arachidonic acid (ARA)-rich oil is an important microbial oil that affects diverse physiological processes that impact normal health and chronic disease. In this article, the historic developments and technological achievements in fungal ARA-rich oil production in the past several years are reviewed. The biochemistry of ARA, ARA-rich oil synthesis and the accumulation mechanism are first introduced. Subsequently, the fermentation and downstream technologies are summarized. Furthermore, progress in the industrial production of ARA-rich oil is discussed. Finally, guidelines for future studies of fungal ARA-rich oil production are proposed in light of the current progress, challenges and trends in the field.

Arachidonic acid (ARA)-rich oil production by Mortierella alpina is a high oxygen demand and shear-sensitive process. In the aerobic fermentation process, oxygen supply is usually a limiting factor owing to the low solubility of oxygen in the fermentation broth. Two kinds of perforated ring gas distributors and a novel microporous ceramic membrane gas distributor were designed and applied to improve oxygen supply. With the decrease of the orifice diameter of perforated ring gas distributors, dry cell weight (DCW), lipids concentration, and ARA content in total fatty acid increased from 17.86 g/L, 7.08 g/L, and 28.08 % to 25.67 g/L, 11.94 g/L, and 36.99 %, respectively. Furthermore, the effect of different dissolved oxygen (DO) on ARA-rich oil production with membrane gas distributor was also studied. The maximum DCW, lipid concentration, and ARA content using membrane gas distributor with DO controlled at 40 % reached 29.67 g/L, 16.74 g/L, and 49.53 %, respectively. The ARA titer increased from 1.99 to 8.29 g/L using the membrane gas distributor to substitute the perforated ring gas distributor. In the further experiment, a novel tubular titanium metal membrane gas distributor was successfully applied in a 7,000 L bioreactor and the results demonstrated that membrane gas distributor was industrially practical.

A three-stage fermentation strategy was designed for efficient arachidonic acid (ARA)-rich oil production by Mortierella alpina. The process at different stages by changing the components of medium was investigated. In the first stage, mycelia were inoculated in a nutrient-rich medium for rapid propagation. In the second stage, mycelia were collected and then cultivated in glucose solution to achieve high cellular lipid contents. In the third stage, mycelia were cultured in a glucose-absent medium to obtain rapid ARA accumulation. Using this fermentation strategy, high dry cell weight, lipid, and ARA concentration reached 41.6, 26.6, and 11.4 g/L, respectively. The results demonstrated that mycelia propagation, lipid biosynthesis, and ARA accumulation process can be significantly spatially separated, allowing further optimization to improve the efficiency of each stage. This was the first report of using a three-stage fermentation strategy for ARA-rich oil production, and it could be applied to other similar oleaginous microorganisms to obtain high related polyunsaturated fatty acids accumulation.

To improve the arachidonic acid (ARA) accumulation in Mortierella alpina, a mixed B-group vitamin addition strategy was developed. The ARA titer reached up to 10.0 g/L, 1.7-fold of the control. At the same time, the highest specific activities of key enzymes involved in ARA biosynthesis, including malic enzyme, glucose-6-phosphate dehydrogenase and ATP: citrate lyase, were 63.3, 38.6 and 53.7% higher than the control, respectively. The possible vitamin triggered improved ARA accumulation mechanism was thus elucidated that B-group vitamins could function as the cofactors of the key enzymes involved in ARA biosynthesis, or precursors for the formation of NADPH and acetyl-CoA which were crucial for ARA synthesis, and strengthened the related metabolic flux.

The fermentation characteristics of Mortierella alpina were investigated in response to various nitrogen sources. Influences on nitrogen source and glucose uptake rate, mycelial morphology of M. alpina, and pH of medium in relation to different nitrogen sources were discussed. Effects of different nitrogen sources on cell growth, fatty acid composition, arachidonic acid (ARA), and total lipid concentration were also evaluated. It revealed that the maximum nitrogen source uptake ratio was obtained when corn steep liquor was used as nitrogen source. When yeast extract was used as the sole nitrogen source, glucose was completely exhausted at the end of fermentation. The maximum dry cell weight obtained from medium with yeast extract as nitrogen source had the highest total lipid concentration. Sodium nitrate was the favorable nitrogen source for ARA accumulation, and the highest ARA percentage in total fatty acids was obtained, 35.9%. Urea was identified as the favorable nitrogen source for ARA production, the highest ARA concentration obtained from urea was 5.8 g/l. Compared with inorganic nitrogen sources, organic nitrogen compounds are favorable for both cell growth and total lipids accumulation.

A novel method for efficient arachidonic acid rich lipids extraction was investigated. Six different enzymes (papain, pectinase, snailase, neutrase, alcalase and cellulase) were used to extract lipids from Mortierella alpina. The effects of enzyme concentration, temperature and hydrolysis time on oil recovery were evaluated using factorial experimental design and polynomial regression for each enzyme. Hydrolysis time is found to be the most important parameter for all enzymes. The ratios of enzyme mixtures were also studied. It showed that the mixtures of pectinase and papain (5:3, v/v), pectinase and alcalase (5:1, v/v) were better combined effects on oil yields. The effects of hydrolysis time and temperature were then analyzed by response surface methodology, and oil recoveries were satisfactory (104.6% for pectinase and papain and 101.3% for pectinase and alcalase). In the whole process, the lipid composition was not affected by the enzyme treatments according to fatty acid profile., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Fungal morphology and aeration play a significant role in the growth process of Mortierella alpina. The production of microbial oil rich in arachidonic acid (ARA) in M. alpina was enhanced by using a multi-stage fermentation strategy which combined fed-batch culture with precise control of aeration and agitation rates at proper times. Results: The fermentation period was divided into four stages according to the cultivation characteristics of M. alpina. The dissolved oxygen concentration was well suited for ARA biosynthesis. Moreover, the ultimate dry cell weight (DCW), lipid, and ARA yields obtained using this strategy reached 41.4, 22.2, 13.5 g/L, respectively. The respective values represent 14.8, 25.8, and 7.8% improvements over traditional fed-batch fermentation processes. Conclusions: This strategy provides promising control insights for the mass production of ARA-rich oil on an industrial scale. Pellet-like fungal morphology was transformed into rice-shaped particles which were beneficial for oxygen transfer and thus highly suitable for biomass accumulation. [ABSTRACT FROM AUTHOR]

[Display omitted] • Alternative strategy for the customized production of ω6-PUFAs was developed. • Optimal biosynthetic pathways for ω6-PUFAs in Y. lipolytica were investigated. • ω6-PUFAs production was improved by relieving the substrate competition bottleneck. • The highest proportion of DGLA at 46.65% of TFAs was achieved in Y. lipolytica. Omega-6 polyunsaturated fatty acids (ω6-PUFAs), such as γ-linolenic acid (GLA), dihomo-γ-linolenic acid (DGLA) and arachidonic acid (ARA), are indispensable nutrients for human health. Harnessing the lipogenesis pathway of Yarrowia lipolytica creates a potential platform for producing customized ω6-PUFAs. This study explored the optimal biosynthetic pathways for customized production of ω6-PUFAs in Y. lipolytica via either the Δ6 pathway from Mortierella alpina or the Δ8 pathway from Isochrysis galbana. Subsequently, the proportion of ω6-PUFAs in total fatty acids (TFAs) was effectively increased by bolstering the provision of precursors for fatty acid biosynthesis and carriers for fatty acid desaturation, as well as preventing fatty acid degradation. Finally, the proportions of GLA, DGLA and ARA synthesized by customized strains accounted for 22.58%, 46.65% and 11.30% of TFAs, and the corresponding titers reached 386.59, 832.00 and 191.76 mg/L in shake-flask fermentation, respectively. This work provides valuable insights into the production of functional ω6-PUFAs. [ABSTRACT FROM AUTHOR]

The accumulation of reactive oxygen species (ROS) can induce oxidative damage which can be detrimental to microbial biomass and product yields. In this study, we used intermittent hypoxic stress (IHS) to eliminate the main source of ROS during the production of arachidonic acid (ARA)-rich lipids by Mortierella alpina . Biomass, lipid- and ARA concentration simultaneously reached their peak values, and the ARA yield increased by about 20% along with a lower level of ROS concentration. Under these conditions, more NADPH, substrate and energy may be available for lipid biosynthesis. The efficacy of this strategy was also confirmed in a bioreactor, with similar results. Interestingly, the total power required for stirring fell by about 4%, which may be of practical value for energy savings. This work offers new insights into the control of ROS generation in M. alpina and might be applicable to other microbial PUFAs producers. [ABSTRACT FROM AUTHOR]

Synthetic multigene pathways enable microbes to synthesize novel products, but generally affect cell growth. In our previous study, an arachidonic acid biosynthesis pathway was constructed in Yarrowia lipolytica , and interestingly, the engineered strain achieved higher biomass production without metabolic burden. In this study, we provide an in-depth analysis of the effects of this multigene pathway on Y. lipolytica . Compared to the original strain, the engineered strain was able to produce arachidonic acid (0.42% of total fatty acids). Moreover, the engineered strain had robust growth and a low titer of extracellular organic acids. qRT-PCR analysis results indicated that the heterologous metabolic pathway influenced the expression of genes related to fatty acid metabolism and the secretion of organic acids. Taken together, it can thus be concluded that the synthetic metabolic pathway is able to redirect the carbon flux towards intracellular fatty acid accumulation at the expense of extracellular organic acid secretion in Y. lipolytica , which is highly desirable and can hopefully be copied in other strains. [ABSTRACT FROM AUTHOR]

Yarrowia lipolytica , a non-conventional oleaginous yeast with special traits, has attracted increasing interest for producing value-added products. Generally, the DNA fragments of these heterologous metabolic pathways are constructed via the classic restriction digestion and ligation method. In contrast, the one-step in vivo pathway assembly method has been only rarely applied to Y. lipolytica . Here, with arachidonic acid biosynthesis as a case study, a one-step in vivo pathway assembly and integration method was used for engineering Y. lipolytica . Using rDNA as integrative locus, this study showed that there was a relation between the assembly efficiency and the length of overlapping region. Especially, with an overlap up to 1 kb, the method was able to rapidly assemble the arachidonic acid biosynthesis pathway (nearly 10 kb) into the chromosome with high efficiency (nearly 23%). Meanwhile, the pathway assembled in Y. lipolytica demonstrated long-term genetic stability and the engineered strain exhibited robust growth. Furthermore, this study demonstrated that the codon-optimized genes from Mortierella alpina can function efficiently in Y. lipolytica : a high level arachidonic acid production (0.4% of total fatty acids) was produced in the engineered strain. To our knowledge, this is the first time that this method is applied to Y. lipolytica for functional polyunsaturated fatty acids production. This method represents a powerful tool with potential for facilitating engineering applications in non-conventional yeasts. [ABSTRACT FROM AUTHOR]