Recombinant production of docosahexaenoic acid in a polyketide biosynthesis mode in Escherichia coli

The docosahexaenoic acid (DHA) biosynthesis gene cluster (pDHA3) from the DHA-producing Moritella marina strain MP-1 includes the genes pfaA, pfaB, pfaC, and pfaD, which are similar to the genes of polyketide biosynthesis. When this cluster was co-expressed in Escherichia coli with M. marina MP-1 pfaE, which encodes phosphopantetheinyl transferase, DHA was biosynthesized. The maximum production of DHA (5% of total fatty acids) was observed at 15°C. This is the first report of the recombinant production of DHA in a polyketide biosynthesis mode.     

Culturable diversity and biochemical features of thraustochytrids from coastal waters of Southern China

Thraustochytrids are ubiquitous marine osmo-heterotrophic fungi-like microorganisms with only about 40 identified species till now. In this study, a total of 60 thraustochytrid strains were isolated from marine coastal habitats. Analysis of 18S rRNA gene sequences revealed that they belonged to three genera, i.e., Schizochytrium, Aurantiochytrium, and Thraustochytrium. All of the isolates were found to show considerable cellulolytic and lipolytic activities. Strains of Aurantiochytrium sp. and Thraustochytrium sp. were found to produce the highest levels of extracellular polysaccharides (EPS), which reached 345 μg ml^?1 in the growth media. Fourier transform infrared (FTIR) spectra of the EPS samples derived from two thraustochytrids (PKU#Sed1 and #SW1) displayed peaks for carbohydrates, proteins, lipids, uronic acids, and nucleic acids. Fatty acid profiles of four thraustochytrids comprised of palmitic acid (C16:0) and docosahexaenoic acid (DHA) as their major constituents. Schizochytrium sp. demonstrated the highest DHA production at 44 % of total fatty acids (TFA) with biomass and DHA yield of 7.1 and 1.6 g l^?1, respectively, on the fourth day of growth. All the four isolates exhibited considerable production of palmitic acid (16:0) in their fatty acid profiles ranging from 35 to 50 % TFA. This is the first report on extracellular enzymes, EPS, and DHA production from thraustochytrids isolated from the coastal habitats of China.     

Efficient docosahexaenoic acid production by Schizochytrium sp. via a two-phase pH control strategy using ammonia and citric acid as pH regulators

The cell growth and docosahexaenoic acid (DHA) synthesis of Schizochytrium sp. are closely related to the culture pH. A two-phase pH control strategy based on nitrogen consumption was developed in which pH 7.0 was used for biomass accumulation and pH 5.0 for DHA synthesis. Using this strategy, the cell dry weight and DHA content reached 98.07 and 25.85 g/L, respectively. Furthermore, ammonia and citric acid were used as pH regulators. Application of citric acid further resulted in 7.88 and 4.87% improvements of total lipids and the ratio of DHA to total fatty acids, respectively. Ammonia, as a suitable nitrogen source, promoted non-lipid biomass accumulation. Using this method, a maximum DHA yield of 32.75 g/L was obtained with non-lipid biomass (58.01 g/L) and the ratio of DHA to total fatty acids (52.36%). This study provides an easy strategy for large-scale industrial production of DHA via high-cell-density fermentation of Schizochytrium sp.     

Long-chain n−3 polyunsaturated fatty acid production by members of the marine protistan group the thraustochytrids: screening of isolates and optimisation of docosahexaenoic acid production

Following an isolation programme for thraustochytrids (marine fungoid protists) from three different locations, 57 isolates were screened for biomass, oil and docosahexaenoic acid production (DHA). Although a common fatty acid profile for the thraustochytrid isolates emerged, there was considerable variation in the DHA content of the oil. In some isolates from a cold temperate environment, DHA represented almost 50% of the total fatty acids present. Although isolates from a sub-tropical environment produced higher levels of biomass, with up to 37% (w/w) oil, the DHA fraction of the fatty acids was low. Cool temperate isolates gave intermediate values. Studies to optimise biomass and DHA production by manipulation of growth medium composition were carried out on a tropical strain. Results indicated that medium with a high C:N ratio stimulated DHA production. The use of such media in bioreactor cultivations gave maximum biomass, lipid and DHA content of 14 g l⁻¹, 78 and 25% (w/w), respectively. Optimum DHA production was 2.17 g l⁻¹ after 107 h cultivation.     

Enhancement of polyunsaturated fatty acid production by cerulenin treatment in polyunsaturated fatty acid-producing bacteria

When docosahexaenoic acid (DHA)-producing Moritella marina strain MP-1 was cultured in the medium containing 0.5 μ g cerulenin ml⁻¹, an inhibitor for fatty acid biosynthesis, the cells grew normally, but the␣content of DHA in the total fatty acids increased from 5.9–19.4%. The DHA yield of M. marina strain MP-1 cells also increased from 4 to 13.7 mg l⁻¹ by cerulenin treatment. The same effect of cerulenin was observed in eicosapentaenoic acid (EPA)-producing Shewanella marinintestina strain IK-1 grown in the medium containing 7.5 μg cerulenin ml⁻¹, and the cerulenin treatment increased the EPA yield from 1.6 to 8 mg l⁻¹. The use of cerulenin is, therefore, advantageous to increase the content of intracellular polyunsaturated fatty acids (PUFA) in particular PUFA-containing phospholipids in bacterial cells.An erratum to this article can be found at .     

Batch and fed-batch growth of Pichia pastoris under increased air pressure

Pichia pastoris CBS 2612 behavior under air pressures of 1, 3 and 5 bar in culture media of glycerol (pure and crude) and methanol was studied. Generally, the increase in oxygen transfer rate due to the increase of total pressure improved cellular growth for all carbon sources and for batch and fed-batch processes with different feeding rate strategies. In batch cultures, 1.4-, 1.2-, and 1.5-fold improvement in biomass production was obtained with the increase of air pressure up to 5 bar, using methanol, pure glycerol, and crude glycerol, respectively. The increase of air pressure to 5 bar using exponential feeding rate led to 1.4-fold improvement in biomass yield per glycerol mass consumed, for crude and pure glycerol. The current low cost of crude glycerol from the biodiesel production together with the present results shows the possibility of improving cell mass production of P. pastoris using increased air pressure.     

Optimization of biomass and fatty acid productivity of Scenedesmus obliquus as a promising microalga for biodiesel production

Nowadays, microalgae are discussed as a promising feedstock for biodiesel production. The present study examines the possibility of enhancement of fatty acid productivity of Scenedesmus obliquus by modifications of the culture medium composition. The effect of different concentrations of sodium bicarbonate, salinity, potassium nitrate, glycerol and sugarcane molasses on the enhancement of biomass and esterified fatty acids production was studied. NaHCO₃ caused an increase in the biomass productivity at low concentrations (0.5 g L^?1), while negatively affected fatty acid productivity at all tested concentrations. Increase of salinity enhanced both biomass and fatty acid productivity. The optimum NaCl concentration and sea water ratio were 0.94 g L^?1 and 25 % which resulted in 56 and 39 % increase in fatty acid productivity, respectively. Nitrogen deficiency showed increase in fatty acid content by 54 % over control but fatty acid productivity was decreased as a result of growth inhibition. Nitrogen-free cultures and cultures treated with ?50 % concentrations of KNO₃ showed 96 and 42 % decrease in EFA productivity, respectively, as compared with the control. Addition of 0.05 and 0.1 M of glycerol increased the biomass productivity by 6 and 5 %, respectively but showed no significant effect on fatty acid productivity as a result of decrease in fatty acid content. Finally, usage of sugarcane molasses stimulated both biomass and fatty acid content. The increase in fatty acid productivity was 32, 65 and 73 % above the control level at 1, 3 and 5 g L^?1 of sugarcane molasses, respectively.     

Optimization of docosahexaenoic acid production by Schizochytrium limacinum SR21

Culture conditions of Schizochytrium limacinum SR21 for the purpose of microbial docosahexaenoic acid (DHA) production were investigated. The strain SR21 showed a wide tolerance to salinity; that is, the optimum salinity was between 50% and 200% that of sea water. Monosaccharides (glucose and fructose) and glycerol supported good cell growth and DHA yield. Di- and polysaccharides, oleic acid, and linseed oil gave low DHA yields. A high content of DHA (more than 30% of total fatty acids) was obtained from culture on glucose, fructose, and glycerol, and also the strain had simple polyunsaturated fatty acid profiles. The major polyunsaturated fatty acids other than DHA were n-6 docosapentaenoic acid only, and the contents of icosapentaenoic acid and arachidonic acid were less than 1%. Using corn steep liquor as a nitrogen source, a high total fatty acid content was obtained. The total fatty acid content in the dry cell weight increased as the concentration of the nitrogen source decreased, reached more than 50%. An increase in carbon source concentration led to a high DHA yield. A maximum DHA yield of more than 4 g/l was obtained in both glucose and glycerol media at 9% and 12% respectively. S. limacinum SR21 was thought to be a promising resource for microbial DHA production yielding a good level of productivity as well as a simple polyunsaturated fatty acid profile. Received: 26 June 1997 / Received revision: 29 August 1997  / Accepted: 19 September 1997     

Improvement of Omega-3 Docosahexaenoic Acid Production by Marine Dinoflagellate Crypthecodinium cohnii Using Rapeseed Meal Hydrolysate and Waste Molasses as Feedstock

Rapeseed meal and waste molasses are two important agro-industrial by-products which are produced in large quantities. In this study, solid state fermentation and fungal autolysis were performed to produce rapeseed meal hydrolysate (RMH) using fungal strains of Aspergillus oryzae, Penicillium oxalicum and Neurospora crassa. The hydrolysate was used as fermentation feedstock for heterotrophic growth of microalga Crypthecodinium cohnii that produce docosahexaenoic acid (DHA). The addition of waste molasses as a supplementary carbon source greatly increased the biomass and DHA yield. In the batch fermentations using media composed of diluted RMH (7%) and 1-9% waste molasses, the highest biomass concentration and DHA yield reached 3.43 g/L and 8.72 mg/L, respectively. The algal biomass produced from RMH and molasses medium also had a high percentage of DHA (22-34%) in total fatty acids similar to that of commercial algal biomass. RMH was shown to be rich in nitrogen supply comparable to the commercial nitrogen feedstock like yeast extract. Using RMH as sole nitrogen source, waste molasses excelled other carbon sources and produced the highest concentration of biomass. This study suggests that DHA production of the marine dinoflagellate C. cohnii could be greatly improved by concomitantly using the cheap by-products rapeseed meal hydrolysate and molasses as alternative feedstock.     

Isolation and Characterization of Taiwanese Heterotrophic Microalgae: Screening of Strains for Docosahexaenoic Acid (DHA) Production

Marine heterotrophic microalgal species which are potentially rich in docosahexaenoic acid (DHA, C22:6n−3) have been found in Taiwan; however, there was a lack of detailed analysis and characterization of these indigenous algae which is needed for the development of commercial applications. Hence, the objective of this study was to screen DHA-rich heterotrophic microalgae species indigenous to Taiwan for commercial purposes. Heterotrophic microalgae from a variety of marine habitats were isolated, cultivated, and then identified according to their 18S rRNA gene sequences and morphological characteristics. A comparison was made of their fatty acid profiles, fatty acid content, and amount of biomass. For the strain with highest DHA yield, the optimal growth conditions were determined in order to establish the best fermentation conditions for scale-up. In this study, 25 heterotrophic microalgal strains were successfully isolated from marine habitats around Taiwan. All of the isolated strains showed a close phylogenic relationship with the Thraustochytriaceae family according to their 18S rRNA gene sequences. GC/MS analysis discerned seven distinctive fatty acid profiles of these strains, with the production of eicosapentaenoic acid (C20:5n−3) ranging from 0.02 to 2.61 mg L⁻¹, and DHA ranging from 0.8 to 18.0 mg L⁻¹. An Aurantiochytrium strain BL10 with high DHA production was subsequently chosen for further manipulation. Under optimal growth conditions it could produce up to 59.0 g of dry biomass per liter of culture, with dry biomass containing 73% total fatty acid and 29% DHA, revealing BL10 as an excellent source of microbial DHA.     

Screening and characterization of Isochrysis strains and optimization of culture conditions for docosahexaenoic acid production

Isochrysis is a genus of marine unicellular microalgae that produces docosahexaenoic acid (DHA, C22:6), a very long chain polyunsaturated fatty acid (PUFA) of significant health and nutritional value. Mass cultivation of Isochrysis for DHA production for human consumption has not been established due to disappointing low DHA productivity obtained from commonly used Isochrysis strains. In this study, 19 natural Isochrysis strains were screened for DHA yields and the results showed that the cellular DHA content ranged from 6.8 to 17.0 % of total fatty acids with the highest DHA content occurring in the exponential growth phase. Isochrysis galbana #153180 exhibited the greatest DHA production potential and was selected for further investigation. The effects of different light intensities, forms, and concentrations of nitrogen, phosphorus, and salinity on growth and DHA production of I. galbana #153180 were studied in a bubble column photobioreactor (PBR). Under favorable culture conditions, I. galbana #153180 contained DHA up to 17.5 % of total fatty acids or 1.7 % of cell dry weight. I. galbana #153180 was further tested in outdoor flat-plate PBRs varying in light path length, starting cell density (SCD), and culture mode (batch versus semicontinuous). When optimized, record high biomass and DHA productivity of I. galbana #153180 of 0.72 g L^?1?day^?1 and 13.6 mg?L^?1?day^?1, or 26.4 g?m^?2?day^?1 and 547.7 mg?m^?2?day^?1, respectively, were obtained, suggesting that I. galbana #153180 may be a desirable strain for commercial production of DHA.     

Biomass and oil content of Chlorella sp., Haematococcus sp., Nannochloris sp. and Scenedesmus sp. under mixotrophic growth conditions in the presence of technical glycerol

The growth of algae strains Chlorella sp., Haematococcus sp., Nannochloris sp. and Scenedesmus sp. under mixotrophic conditions in the presence of different concentrations of technical glycerol was investigated with the aim of increasing biomass growth and algae oil content. The highest concentration of lipid obtained in media with 5 g L^?1 glycerol for Chlorella sp., Scenedesmus sp., Nannochloris sp. and Haematococcus sp. was 17.77, 22.34, 27.55 and 34.22 % larger than during the autotrophic growth of these species. Increases in triacylglycerols of up to ten times was observed for Scenedesmus sp. under mixotrophic conditions (using 10 g L^?1 glycerol), whereas an increase of 2.28 times was found for Haematococcus sp. The content of saturated fatty acids of Scenedesmus, Chlorella, Haematococcus and Nannochloris was 67.11, 34.63, 23.39 and 24.23 %, and the amount of unsaturated fatty acids was 32.9, 65.06, 79.61 and 75.78 % of total fatty acids, respectively. Growth on technical glycerol of these strains with light produced higher biomass concentrations and lipid content compared with autotrophic growth. The fatty acid content of oils from these species suggests their potential use as biodiesel feedstock.     

Exploring potential use of Australian thraustochytrids for the bioconversion of glycerol to omega-3 and carotenoids production

Marine microbes have the potential for accumulating large quantities of lipids and are therefore suitable candidate as feedstock in unsaturated fatty acid production. The efficient utilisation of glycerol as an alternative carbon source to glucose was demonstrated in the fermentation of newly isolated thraustochytrid strains from the Queenscliff, Victoria, Australia. The isolates exhibited the presence of omega-3 and omega-6 polyunsaturated fatty acids, with the major fatty acids for all isolates being (as percent total fatty acid), palmitic acid (25.1–40.78%), stearic acid (4.24–13.2%), eicosapentaenoic acid EPA (2.31–8.5%) and docosapentaenoic acid (7.24–10.9%). Glycerol as a carbon source gave promising biomass growth with significant lipid and DHA productivity. An approximate three-fold increase in carotenoid content in all isolates was achieved when glycerol was used as a carbon source in the production medium.     

High cell density cultivation of a novel Aurantiochytrium sp. strain TC 20 in a fed-batch system using glycerol to produce feedstock for biodiesel and omega-3 oils

A recently isolated Australian Aurantiochytrium sp. strain TC 20 was investigated using small-scale (2 L) bioreactors for the potential of co-producing biodiesel and high-value omega-3 long-chain polyunsaturated fatty acids. Higher initial glucose concentration (100 g/L compared to 40 g/L) did not result in markedly different biomass (48 g/L) or fatty acid (12–14 g/L) yields by 69 h. This comparison suggests factors other than carbon source were limiting biomass production. The effect of both glucose and glycerol as carbon sources for Aurantiochytrium sp. strain TC 20 was evaluated in a fed-batch process. Both glucose and glycerol resulted in similar biomass yields (57 and 56 g/L, respectively) by 69 h. The agro-industrial waste from biodiesel production—glycerol—is a suitable carbon source for Aurantiochytrium sp. strain TC 20. Approximately half the fatty acids from Aurantiochytrium sp. strain TC 20 are suitable for development of sustainable, low emission sources of transportation fuels and bioproducts. To further improve biomass and oil production, fortification of the feed with additional nutrients (nitrogen sources, trace metals and vitamins) improved the biomass yield from 56 g/L (34 % total fatty acids) to 71 g/L (52 % total fatty acids, cell dry weight) at 69 h; these yields are to our knowledge around 70 % of the biomass yields achieved, however, in less than half of the time by other researchers using glycerol and markedly greater than achieved using other industrial wastes. The fast growth and suitable fatty acid profile of this newly isolated Aurantiochytrium sp. strain TC 20 highlights the potential of co-producing the drop-in biodiesel and high value omega-3 oils.     

Genome Sequence of the Psychrophilic Deep-Sea Bacterium Moritella marina MP-1 (ATCC 15381)

Moritella marina MP-1 is a bacterial species known for its production of docosahexaenoic acid. We present the draft genome sequence of the type strain Moritella marina MP-1 (ATCC 15381), having 4,636,778 bp with a G+C content of 40.5% and consisting of 83 contigs.     

An EntD-like phosphopantetheinyl transferase gene from Photobacterium profundum SS9 complements pfa genes of Moritella marina strain MP-1 involved in biosynthesis of docosahexaenoic acid

The EntD-like phosphopantetheinyl transferase (PPTase) gene, cloned from the eicosapentaenoic acid-producing bacterium Photobacterium profundum strain SS9, has an ORF of 690 bp encoding a 230-amino acid protein. When this PPTase gene was expressed in Escherichia coli with pfaA, pfaB, pfaC and pfaD derived from Moritella marina MP-1, which were four of five essential genes for biosynthesis of docosahexaenoic acid (DHA), the DHA production of the recombinant was 2% (w/w) of total fatty acids. This is the first report showing that the EntD-like PPTase is involved in producing n-3 polyunsaturated fatty acids. Shinji Sugihara and Yoshitake Orikasa contributed equally to this work.     

Efficient production of triacylglycerols rich in docosahexaenoic acid (DHA) by osmo-heterotrophic marine protists

Thraustochytrids have recently emerged as a promising source for docosahexaenoic acid (DHA) production due to their high growth rate and oil content. In this study, two thraustochytrid isolates, Aurantiochytrium sp. PKU#SW7 and Thraustochytriidae sp. PKU#Mn16 were used for DHA production. Following growth parameters were optimized to maximize DHA production: temperature, pH, salinity, and glucose concentration. Both isolates achieved the highest DHA yield at the cultivation temperature of 28 °C, pH 6, 100 % seawater, and 2 % glucose. A DHA yield of 1.395 g/l and 1.426 g/l was achieved under the optimized culture conditions. Further investigation revealed that both isolates possess simple fatty acids profiles with palmitic acid and DHA as their dominant constituents, accounting for ～79 % of total fatty acids. To date, very few studies have focused on the DHA distribution in various lipid fractions which is an important factor for identifying strains with a potential for industrial DHA production. In the present study, the lipids profiles of each strain both revealed that the majority of DHA was distributed in neutral lipids (NLs), and the DHA distribution in NLs of PKU#SW7 was exclusively in the form of triacylglycerols (TAGs) which suggest that PKU#SW7 could be utilized as an alternative source of DHA for dietary supplements. The fermentation process established for both strains also indicating that Aurantiochytrium sp. PKU#SW7 was more suitable for cultivation in fermenter. In addition, the high percentage of saturated fatty acids produced by the two thraustochytrids indicates their potential application in biodiesel production. Overall, our findings suggest that two thraustochytrid isolates are suitable candidates for biotechnological applications.     

Thraustochytrid Marine Protists: Production of PUFAs and Other Emerging Technologies

Thraustochytrids, the heterotrophic, marine, straminipilan protists, are now established candidates for commercial production of the omega-3 polyunsaturated fatty acid (ω-3 PUFA), docosahexaenoic acid (DHA), that is important in human health and aquaculture. Extensive screening of cultures from a variety of habitats has yielded strains that produce at least 50% of their biomass as lipids, and DHA comprising at least 25% of the total fatty acids, with a yield of at least 5 g L⁻¹. Most of the lipids occur as triacylglycerols and a lesser amount as phospholipids. Numerous studies have been carried out on salinity, pH, temperature, and media optimization for DHA production. Commercial production is based on a fed batch method, using high C/N ratio that favors lipid accumulation. Schizochytrium DHA is now commercially available as nutritional supplements for adults and as feeds to enhance DHA levels in larvae of aquaculture animals. Thraustochytrids are emerging as a potential source of other PUFAs such as arachidonic acid and oils with a suite of PUFA profiles that can have specific uses. They are potential sources of asataxanthin and carotenoid pigments, as well as other lipids. Genes of the conventional fatty acid synthesis and the polyketide-like PUFA synthesis pathways of thraustochytrids are attracting attention for production of recombinant PUFA-containing plant oils. Future studies on the basic biology of these organisms, including biodiversity, environmental adaptations, and genome research are likely to point out directions for biotechnology explorations. Potential areas include enzymes, polysaccharides, and secondary metabolites.     

Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid

Crude glycerol is a major byproduct of the biodiesel industry; previous research has proved the feasibility of producing docosahexaenoic acid (DHA, 22:6 n − 3) through fermentation of the algae Schizochytrium limacinum on crude glycerol. The objective of this work is to investigate the cell growth kinetics, substrate utilization efficiency, and DHA production of the algae through a continuous culture. Steady-state biomass yield, biomass productivity, growth yield on glycerol, specific glycerol consumption rate, and fatty acid composition were investigated within the range of dilution rate (D) from 0.2 to 0.6 day⁻¹, and the range of feed crude glycerol concentration (S₀) from 15 to 120 g/L. The maximum specific growth rate was determined as 0.692 day⁻¹. The cells had a true growth yield of 0.283 g/g but with a relatively high maintenance coefficient (0.2216 day⁻¹). The highest biomass productivity of 3.88 g/L-day was obtained at D = 0.3 day⁻¹ and S₀ = 60 g/L, while the highest DHA productivity (0.52 g/L-day) was obtained at D = 0.3 day⁻¹ and S₀ = 90 g/L due to the higher DHA content at S₀ = 90 g/L. The biomass and DHA productivity of the continuous culture was comparable to those of batch culture, while lower than the fed-batch culture, mainly because of the lower DHA content obtained by the continuous culture. Overall, the results show that continuous culture is a powerful tool to investigate the cell growth kinetics and physiological behaviors of the algae growing on biodiesel-derived crude glycerol.     

Glycerol increases growth,protein production and alters the fatty acids profile of Spirulina (Arthrospira) sp LEB 18

Most of the crude glycerol produced globally is generated by biodiesel production, which makes this byproduct an environmental responsibility of the biofuel industries. Among the forms of this compound in use, microalgae cultivation is a promising alternative that may generate a reduction in crude glycerol treatment costs via using it as an organic, carbon-rich substrate in culture media. In this work, the influence of different concentrations of glycerol in the culture medium, the composition of fatty acids and proteins in Spirulina sp. LEB 18 biomass and their effect on its growth were investigated. The fatty acid profile of the biomass was altered, showing a 20% increase in the unsaturated concentration and a 60% reduction in the saturated concentration in the culture supplemented with 0.05 mol L⁻¹ of glycerol compared to those in the control. The addition of the substrate stimulated an increase in its cellular concentration (3.00 g L⁻¹, 0.05 mol L⁻¹), productivity (0.72 g L⁻¹ d⁻¹, 0.05 mol L⁻¹) and its protein production (69.78% w w⁻¹, 0.05 mol L⁻¹).