GLA Production Research Articles

Construction of Eicosatetraenoic Acid Producing Cell Factory by Genetic Engineering of Mucor circinelloides

Eicosatetraenoic acid (ETA, 20:4, ω-3) is the desaturation product of dihomo-gamma linolenic acid (DGLA, 20:3, ω-6) catalyzed by delta-17 desaturase, which is considered as a healthy product that helps to lower risk of heart diseases. The oleaginous filamentous fungus, Mucor circinelloides, has been used for a long time as a model micro-organism for GLA production at industrial scales. However, M. circinelloides lacks the key enzymes to synthesize C20 polyunsaturated fatty acids (PUFAs). M. circinelloides could produce DGLA by overexpressing the D6E(GLELO) gene, which could be a useful tool to produce ETA due to the availability of established genetic manipulation tools. Therefore, in this study, delta-17 desaturase (PpD17 and PaD17) genes from Phytophthora parasitica and Pythium aphanidermatum, respectively, were introduced into M. circinelloides to construct an ETA-producing cell factory. Our results showed that the PaD17 and PpD17 overexpression strains’ biomass increased by 25.98 and 23.34 g/L (39.98 and 25.75%), respectively, compared with the control strain. Meanwhile, the lipid contents of the recombinant strains also increased and reached up to 28.88% in Mc-PaD17 and 30.95% in Mc-PpD17, respectively, compared with the control strain (23.38% in Mc-2076). The RT-qPCR results showed that overexpression of delta-17 desaturase genes promoted the expression of cme2, fas2, and D6E, thereby contributing to lipid biosynthesis in M. circinelloides. Meanwhile, the content of ETA reached up to 1.95%, and the yield of ETA was up to 114.69 mg/L in PpD17 overexpression mutants at 96 h. This study provided the first report on the construction of an ETA-producing cell factory by heterologous overexpression of the PpD17 gene in M. circinelloides, which established a new scope for further research in the production of ETA in oleaginous fungi.

Simultaneous overexpression of ∆6-, ∆12- and ∆9-desaturases enhanced the production of γ-linolenic acid in Mucor circinelloides WJ11.

Mucor circinelloides WJ11, an oleaginous filamentous fungus, produces 36% lipid of its cell dry weight when cultured in a high C/N ratio medium, however, the yield of γ-linolenic acid (GLA) is insufficient to make it competitive with other plant sources. To increase the GLA content in M. circinelloides WJ11, this fungus was engineered by overexpression of its key genes such as Δ6-, Δ12-, and Δ9-desaturases involved in GLA production. Firstly, we tried to overexpress two Δ6-desaturase isozymes to determine which one played important role in GLA synthesis. Secondly, Δ6-and Δ12-desaturase were co-overexpressed to check whether linoleic acid (LA), the precursor for GLA synthesis, is a limiting factor or not. Moreover, we tried to explore the effects of simultaneous overexpression of Δ6-, Δ12-, and Δ9-desaturases on GLA production. Our results showed that overexpression (1 gene) of DES61 promoted higher GLA content (21% of total fatty acids) while co-overexpressing (2 genes) DES61 and DES12 and simultaneous overexpressing (3 genes) DES61, DES12, and DES91 increased the GLA production of engineered strains by 1.5 folds and 1.9 folds compared to the control strain, respectively. This study provided more insights into GLA biosynthesis in oleaginous fungi and laid a foundation for further increase in GLA production into fungus such as M. circinelloides.

Engineering and fermenter production of fungi GLA in Pichia pastoris GS115 using oil waste.

γ-Linolenic acid (GLA) is an essential n-6 polyunsaturated fatty acid (PUFA) that has received considerable attention in human and animal feed. GLA is used in many nutritional and medicinal applications, such as the treatment of cancer, inflammatory disorders, and diabetes. Currently, plant seed is the primary dietary source of GLA that is not enough to utilize on an industrial scale. To generate a sustainable novel source of GLA, the gene of delta-6 desaturase, one of the essential enzymes in the GLA production pathway, was isolated from Mucor rouxii DSM1194 and expressed in P. pastoris GS115 by pPICZC vector. The recombinant yeast expressed the GLA up to 19.2% (72mg/g) of total fatty acids. GLA production of recombinant yeast was studied in a fermenter by oil waste for 5 days, and results detected 6.3g/l lipid, and 103mg/g GLA was produced in 72h. The present study may provide an opportunity to develop an alternative host for manufacturing GLA on an industrial scale.

Dysregulation of Immune Response Mediators and Pain-Related Ion Channels Is Associated with Pain-like Behavior in the GLA KO Mouse Model of Fabry Disease.

Fabry disease (FD) is a rare life-threatening disorder caused by deficiency of the alpha-galactosidase A (GLA) enzyme with a characteristic pain phenotype. Impaired GLA production or function leads to the accumulation of the cell membrane compound globotriaosylceramide (Gb3) in the neurons of the dorsal root ganglia (DRG) of FD patients. Applying immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT PCR) analysis on DRG tissue of the GLA knockout (KO) mouse model of FD, we address the question of how Gb3 accumulation may contribute to FD pain and focus on the immune system and pain-associated ion channel gene expression. We show a higher Gb3 load in the DRG of young (<6 months) (p < 0.01) and old (≥12 months) (p < 0.001) GLA KO mice compared to old wildtype (WT) littermates, and an overall suppressed immune response in the DRG of old GLA KO mice, represented by a reduced number of CD206+ macrophages (p < 0.01) and lower gene expression levels of the inflammation-associated targets interleukin(IL)1b (p < 0.05), IL10 (p < 0.001), glial fibrillary acidic protein (GFAP) (p < 0.05), and leucine rich alpha-2-glycoprotein 1 (LRG1) (p < 0.01) in the DRG of old GLA KO mice compared to old WT. Dysregulation of immune-related genes may be linked to lower gene expression levels of the pain-associated ion channels calcium-activated potassium channel 3.1 (KCa3.1) and transient receptor potential ankyrin 1 channel (TRPA1). Ion channel expression might further be disturbed by impaired sphingolipid recruitment mediated via the lipid raft marker flotillin-1 (FLOT1). This impairment is represented by an increased number of FLOT1+ DRG neurons with a membranous expression pattern in old GLA KO mice compared to young GLA KO, young WT, and old WT mice (p < 0.001 each). Further, we provide evidence for aberrant behavior of GLA KO mice, which might be linked to dysregulated ion channel gene expression levels and disturbed FLOT1 distribution patterns. Behavioral testing revealed mechanical hypersensitivity in young (p < 0.01) and old (p < 0.001) GLA KO mice compared to WT, heat hypersensitivity in young GLA KO mice (p < 0.001) compared to WT, age-dependent heat hyposensitivity in old GLA KO mice (p < 0.001) compared to young GLA KO mice, and cold hyposensitivity in young (p < 0.001) and old (p < 0.001) GLA KO mice compared to WT, which well reflects the clinical phenotype observed in FD patients.

Improved γ-Linolenic Acid Production from Cellulose in Mucor circinelloides via Coexpression of Cellobiohydrolase and Delta-6 Desaturase.

The present study was aimed at facilitating the production of γ-linolenic acid (GLA) from the cellulosic substrate with the engineered oleaginous fungus Mucor circinelloides WJ11. Here, the homologous recombination technology was used to overexpress the cellobiohydrolase (CBH2) derived from Trichoderma longibrachiatum and the original delta-6 fatty acid desaturase (D6) in M. circinelloides to construct genetically engineered strains capable of effectively using cellulose to enhance GLA synthesis. When cultivated in modified K&R medium supplemented with microcrystalline cellulose, the CBH2 and D6 coexpressing strains led to increases in the biomass (up to 12.8 g/L) and lipid yield (up to 3.7 g/L) of 87% and 2.4-fold, respectively, compared to that of the control strain. Notably, when CBH2 and D6 were coexpressed in M. circinelloides, the yield of GLA reached 608 mg/L, which was a dramatic increase of 3.9-fold compared to that of the control strain. This is the first report on promoting the GLA production from the cellulosic substrate via coexpression of CBH2 and delta-6 desaturase. This work provides a theoretical basis for efficient transformation from the cellulosic substrate to functional GLA by CBH2 and D6 coexpressing strains, which might play a positive role in promoting the sustainable development of biological industry.

Iron and methyl jasmonate increase high-value PUFA production by elevating the expression of desaturase genes in marine microalga Isochrysis sp.

This study investigated the effect of several metabolic enhancers on the expression of fatty acid biosynthetic genes and their influence on the production of high-value PUFA in the marine microalgae Isochrysis sp., CASA CC 101. The effect of the presence of iron (Fe), nicotinic acid (NIC), methyl jasmonate (MJ) and thidiazuron (TDZ) on the expression of the fatty acid desaturase genes Δ6Des, Δ5Des and Δ4Des was studied in cultures of the marine microalga Isochrysis sp., CASA CC 101.The production of high-value PUFA like γ-linolenic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was correlated with these gene expressions. The results showed that MJ, Fe and TDZ significantly increased the lipid content than the control. MJ specifically up-regulated ∆6Des gene expression and thereby increased GLA production. Whereas Fe specifically increased ∆5Des gene expression and thereby increased EPA production. However, Fe and TDZ-treated cells effectively upregulated the expression of ∆4Des and increased the production of DHA when compared with control cells. Our findings suggest that addition of Fe and MJ in the culture medium triggers the expression of PUFA biosynthetic genes, especially ∆6Des and ∆4Des, in marine microalga Isochrysis sp., CASA CC 101 their presence resulted in increased production of the PUFAs GLA, EPA and DHA. This study shows that the addition of Fe and MJ to the culture media ofIsochrysis sp., CASA CC 101results in up-regulation of its genes Δ4Des, Δ6Des and Δ5Des, and improves the production of PUFA. Therefore, the addition of Fe and MJ to the culture medium is useful to increase the production of high-value PUFA in Isochrysis sp., CASA CC 101 and also to the other micro algal species.

An Efficient Method for Extraction and Enrichment of γ-Linolenic Acid (GLA) from Spirulina

γ-linolenic acid (GLA) is useful for improving and treating various diseases, including immune system-related diseases. In the world of commerce, GLA production has problems that compel scientists to search for a simpler, safer, and less costly way. Some microalgae have been introduced as a rich source of fatty acids; Spirulina platensis has attracted scientists due to the presence of 31% GLA in its fatty acid profile. In the present study, the Iranian strain of spirulina was investigated. Cell culture was performed under optimized conditions and after dry biomass production; the powder was used to continue the study. Finally, the extraction and enrichment steps were performed, and the IR spectra and gas chromatography methods were used for identification and final measurements, respectively. After enrichment by one-step urea crystallization, the percentage of GLA enriched in the total lipid was increased to 61.17%. These results suggest that employing simple, relatively inexpensive, and modified methods for the GLA isolation from the Iranian strain of spirulina may have a potential for industrialization.

Isolation, characterization and fatty acid analysis of Gilbertella persicaria DSR1: A potential new source of high value single-cell oil

Oleaginous fungi accumulate a high amount of lipids in their dry biomass and have attracted more attention, due to their broad potential in biotechnological applications. The present study aimed to isolate and evaluate efficient oleaginous endophytic fungi from selected plants, followed by optimizing the potential strain's growth parameters. A total of 51 morphologically distinct fungal isolates were isolated and examined for their oleaginous character. From the preliminary screening and fermentation assay, 13 fungal isolates were identified based on their high lipid with biomass or polyunsaturated fatty acids in their fatty acid profile. During our screening process, the fungal isolate identified as Gilbertella persicaria DSR1 was found to have desirable features, such as the highest cell dry biomass (12.7 g L−1) and lipid content (34.5% of biomass) with 7% γ-linolenic acid (GLA) of total fatty acids. The least investigated isolated oleaginous strain DSR1 was subjected to optimization of various growth parameters for exploiting its full lipid accumulation potential. Under optimized conditions in the fermentor, the strain DSR1 produced 14 ± 0.3 g L−1 dry cell biomass with 42 ± 1.4% total lipid content. The major fatty acid composition of lipid confirmed by GC analysis was palmitic acid (14.7%), stearic acid (10.6%), oleic acid (37.4%), linoleic acid (20.6%), γ-linolenic acid (13.4%). This is the first report showing the oleaginous nature of G. persicaria and this isolate may be further exploited as a potential candidate for biodiesel and GLA production in larger-scale applications.

Single-cell oil production by Mortierella isabellina DSM 1414 using different sugars as carbon source.

Pure carbon sources, especially carbohydrates which are raw materials deriving from agro-industrial processes, are often used for small-scale single-cell oil production by fermentation. The aim of this study was to investigate the effects of different pure carbon sources on cell growth, lipid accumulation, and γ-linolenic acid (GLA) production by the filamentous fungus Mortierella isabellina DSM 1414 (Deutsche Sammlung von Mikroorganismen). The sugars utilized in this study are found extensively and abundantly in nature, especially in food raw materials and, in consequence, in agro-food industry wastes or surpluses. Thus, the potential of many waste materials containing these sugars to be used in the production of single-cell oil by fermentation could also be evaluated. The effects of the sugars utilized on cell growth, biomass production, and lipid production were investigated. Fatty acids were also analysed in the lipids produced at the end of the fermentations. Results showed that the maximum biomass production was 10.80 g/L in lactose-based media, while the maximum oil production was 5.44 g/L in maltose-based media. Oleic (20.42%-42.94%), palmitic (14.96%-22.19%), and stearic (9.00%-26.92%) acids were the major fatty acids along with linoleic acid (11.35%-18.67%) and GLA (3.56%-8.04%). The production of GLA as the target fatty acid was remarkable. This study indicates that agro-industrial waste including most of the sugars utilized (except for arabinose and sucrose with lipid production of 0.81 and 0.28 g/L, respectively) can be employed for production of single-cell oil by M. isabellina DSM 1414 which contains a high amount of GLA.

Effects of the electrical conductivity of a soilless culture system on gamma linolenic acid levels in borage seed oil.

Borage is a well-known plant of great importance in human nutrition and health. Expanding knowledge of particular plants that have anti-cancer products is a global concern. There is substantial information regarding the benefits, presence and extraction of gamma linolenic acid (GLA; 18:3n6) in different plants around the world, especially in borage seeds. However, there is little information concerning the effects of the salinity of the nutrient solution on the growth and presence of GLA in borage seeds. The objective of this work was to determine the optimal salinity of the nutrient solution for obtaining GLA in soilless cultivation systems. Borage plants were grown in coconut fibre and provided three treatments of nutrient solution of 2.20, 3.35 and 4.50 dS m-1, increasing solution salinity with the standard nutrient solution of concentrated macronutrients as a reference. Vegetative growth, seed production and GLA ratio were measured. The results of vegetative development and GLA production doubled and tripled with the increase in salinity of the nutrient solution, respectively.

Production of High-Value Polyunsaturated Fatty Acids Using Microbial Cultures.

Microbes can produce not only commodity fatty acids, such as palmitic acid (16:0) and stearic acid (18:0), but also high-value fatty acids (essential fatty acids). Most high value fatty acids belong to long chain polyunsaturated fatty acids (PUFA), such as omega-3 fatty acids (e.g., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) and omega-6 fatty acids (e.g., arachidonic acid (ARA) and γ-linolenic acid (GLA)). EPA (20:5n-3) is a 20-carbon fatty acid with five double bonds, and the first double bond is in the n-3 position. DHA (22:6n-3) is a 22-carbon fatty acid with 6 double bonds and the first double bond is also in the n-3 position. Both EPA and DHA play an essential role in cardiovascular health including prevention of atherosclerotic disease development (Zehr and Walker, Prostaglandins Other Lipid Mediat 134:131-140, 2018). ARA (20:4n-6) is a 20-carbon fatty acid with four double bonds, and the first double bond is in the n-6 position. GLA (18:3n-6) is an 18-carbon fatty acid with three double bonds, and the first double bond is in the n-6 position. ARA and GLA have multiple biological effects, such as lowering blood cholesterol, and lowering cardiovascular mortality (Poli and Visioli, Eur J Lipid Sci Technol 117(11):1847-1852, 2015). This chapter provides details on microbial production of EAP, DHA, ARA, and GLA.

Optimization of Process Parameters for the Production of γ-Linolenic Acid by Cunninghamella elegans CFR C07 in Submerged Fermentation.

The production of γ-linolenic acid (GLA) by the fungus Cunninghamella elegans CFR C07 in submerged fermentation was studied. Culture parameters such as carbon source and incubation time were optimized. Four different extraction methods using solvents with acid washed sand, glass beads, lyophilization and Soxhlet extraction were evaluated for improved extraction of lipids from the fungal biomass after fermentation. The GLA production was initially optimized in 250-mL flask and the process was demonstrated in a 3-litre fermentor. The maximum GLA production was 882 mg/L in shake flask culture and 733 mg/L in the fermentor. The study shows that Cunninghamella elegans CFR C07 is a potent organism for the production of GLA under submerged conditions.

Productivity of γ-Linoleic acid by oleaginous fungus Cunninghamella echinulata using a pulsed high magnetic field

Abstract γ-Linolenic acid (GLA) plays a crucial role in brain function and normal growth and development. High percentage of the population cannot effectively produce GLA, resulting in the use of GLA dietary supplements. Several oleaginous fungi can produce low level of GLA. The present study aimed to improve the production of GLA using physical mutagenesis. Spore suspensions of Cunninghamella echinulata were treated with pulsed high magnetic field. A total of 64 strains were isolated and screened using Nile red staining and fluorescence detection. Three mutants were selected after fermentation screening. GLA yields of Mut-29, Mut-15, and Mut-64 increased by 46.2%, 23.1%, and 19.2%, respectively, compared with that of the parent strain. Biomass, lipid content and yield, GLA content, and GLA yield of Mut-29 increased significantly (P

Improved \u03b3-linolenic acid production in Mucor circinelloides by homologous overexpressing of delta-12 and delta-6 desaturases

Backgroundγ-Linolenic acid (GLA) is important because of its nutritional value and medicinal applications. Although the biosynthetic pathways of some plant and microbial GLA have been deciphered, current understanding of the correlation between desaturases and GLA synthesis in oleaginous fungi is incomplete. In previous work, we found that a large amount of oleic acid (OA) had not been converted to linoleic acid (LA) or GLA in Mucor circinelloides CBS 277.49, which may be due to inadequate activities of the delta-12 or delta-6 desaturases, and thus leading to the accumulation of OA and LA. Thus, it is necessary to explore the main contributing factor during the process of GLA biosynthesis in M. circinelloides.ResultsTo enhance GLA production in M. circinelloides, homologous overexpression of delta-12 and two delta-6 desaturases (named delta-6-1 and delta-6-2, respectively) were analyzed. When delta-6 desaturase were overexpressed in M. circinelloides, up to 43% GLA was produced in the total fatty acids, and the yield of GLA reached 180 mg/l, which were, respectively, 38 and 33% higher than the control strain.ConclusionThese findings revealed that delta-6 desaturase (especially for delta-6-1 desaturase) plays an important role in GLA synthesis by M. circinelloides. The strain overexpressing delta-6-1 desaturase may have potential application in microbial GLA production.

Evening primrose (Oenothera biennis) Δ6 fatty acid desaturase gene family: cloning, characterization, and engineered GLA and SDA production in a staple oil crop

γ-Linolenic acid (GLA), stearidonic acid (SDA), and other polyunsaturated fatty acids (PUFAs) play important roles in human health, and Δ6 fatty acid desaturase (D6D) is a key enzyme for PUFA biosynthesis. Oenothera biennis is a traditional GLA-producing oil crop. Here we report the cloning, characterization, and metabolic engineering of its D6D gene family. The intronless 1922-bp ObD6D1 and 1752-bp ObD6D2 both have alternative transcription start sites, alternative polyadenylation sites, and repeated stop codons. Phylogenetic study revealed five large groups of front-end desaturases which are not simply correlated with desaturase types or organism origins. Any type of front-end enzyme has independently originated for multiple times. “Higher plants,” “higher animals,” and cyanobacteria each has front-end enzymes of single ancient origin, but other taxa are contrary. ObD6D1 and ObD6D2 are most abundantly expressed in bud and root, respectively. In yeast expression, ObD6D1 and ObD6D2 both catalyzed linoleic acid into GLA, and α-linolenic acid into SDA. Based on a newly constructed double-selection vector system, four constructs of ObD6D1 and ObD6D2 both individually driven by double CaMV35S promoter PD35S and seed-specific promoter PNAP were transformed into Brassica napus via Agrobacterium. T1 plants, T2 elite lines, and homozygous T3 elite lines accumulated average GLA + SDA levels of 6.04, 15.77, and 21.56% of seed fatty acids, respectively. ObD6D2 showed higher effect than ObD6D1. PD35S-D6D engineering has little side effect but is weaker than PNAP-D6D engineering in terms of GLA + SDA productivity.

Engineering Yarrowia lipolytica for efficient γ-linolenic acid production

γ-Linolenic acid (GLA) has various well-documented beneficial physiological effects and high biological significance. Because the natural supply of GLA is insufficient, microbial GLA production is a promising method for pharmaceutical and nutraceutical purposes. To establish and develop a biotechnological process for GLA production by Yarrowia lipolytica, the codon-optimized △6-desaturase from Mortierella alpina was introduced into this yeast under the control of the strong hp4d promoter. A recombinant Y. lipolytica strain was constructed, which produced 4.6% GLA in total fatty acids. By using a temperature-shift strategy of cultivation, consisting in preliminary growth at 28°C followed by 6-day culture at 20°C, optimal levels of dry cell weight (DCW), lipid content and GLA concentration were obtained from the recombinant strain: 18.55g/L, 1.16g/L and 71.6mg/L, respectively. These DCW, lipid and GLA values were respectively 25.7%, 19.6% and 60.9% higher than those obtained in the control cultivation experiment at the standard constant temperature of 28°C. This work demonstrates the excellent capacity of Y. lipolytica for GLA production, by combining metabolic engineering with a temperature-shift strategy.

PUFA-induced cell death is mediated by Yca1p-dependent and -independent pathways, and is reduced by vitamin C in yeast.

Polyunsaturated fatty acids (PUFA) such as linoleic acid (LA, n-6, C18:2) and γ-linolenic acid (GLA, n-6, C18:3) are essential and must be obtained from the diet. There has been a growing interest in establishing a bio-sustainable production of PUFA in several microorganisms, e.g. in yeast Saccharomyces cerevisiae. However, PUFAs can also be toxic to cells because of their susceptibility to peroxidation. Here we investigated the negative effects of LA and GLA production on S. cerevisiae by characterizing a strain expressing active Δ6 and Δ12 desaturases from the fungus Mucor rouxii. Previously, we showed that the PUFA-producing strain has low viability, down-regulated genes for oxidative stress response, and decreased proteasome activity. Here we show that the PUFA strain accumulates high levels of reactive oxygen species (ROS) and lipid peroxides, and accumulates damaged proteins. The PUFA strain also showed great increase in metacaspase Yca1p activity, suggesting cells could die by caspase-mediated cell death. When treated with antioxidant vitamin C, ROS, lipid peroxidation and protein carbonylation were greatly reduced, and the activity of the metacaspase was significantly decreased too, ultimately doubling the lifespan of the PUFA strain. When deleting YCA1, the caspase-like activity and the oxidative stress decreased and although the lifespan was slightly prolonged, the phenotype could not be fully reversed, pointing that Yca1p was not the main executor of cell death.

Determination of sex-based differences in serum γ-linolenic [corrected] acid and dihomo-γ-linolenic [corrected] acid using gas chromatography-mass spectrometry.

Because serum unsaturated fatty acids can provide useful information on disease diagnosis, the simultaneous determination of several fatty acids in small volumes of human serum would be beneficial for clinical applications. In the present study, serum fatty acids were extracted with n-heptane/chloroform from 10μL of serum collected from 26 healthy Japanese subjects (11 men, ages 23-37 years; 15 women, ages 18-37 years) after deproteinization with perchloric acid, derivatization to their methyl ester using p-toluenesulfonic acid as an acid catalyst, and subsequent separation and measurement by gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode. Nine types of fatty acids (palmitoleic acid [PLA], oleic acid [OA], linoleic [corrected] acid [LA], γ-linolenic acid [GLA], α-linolenic acid [ALA], dihomo-GLA [DGLA], arachidonic acid [AA], eicosapentaenoic acid [EPA], and docosahexaenoic acid [DHA]) were analyzed in the serum within 35 min by GC-MS. The concentrations of these fatty acids in serum ranged from 3.64±0.38μM (GLA) to 413±26.3 μM (LA). Among these nine fatty acids, GLA and DGLA levels were significantly lower in women than in men (p=0.0034 and 0.0012, respectively), suggesting that there may be sex-based differences in the biosynthetic production or metabolic processes of GLA and DGLA in humans.

Absence of α-galactosidase cross-correction in Fabry heterozygote cultured skin fibroblasts

Fabry disease (FD) is an X-linked lysosomal storage disorder resulting from deficiency of α-galactosidase A (GLA). Traditionally, heterozygotes were considered asymptomatic carriers of FD, but it is now apparent that the asymptomatic female carrier is the exception and most heterozygotes suffer significant multisystemic disease. To determine why the process of cross-correction does not occur effectively in FD heterozygotes, we investigated GLA production and secretion in cultured skin fibroblasts as well as GLA levels in plasma. The maturation of GLA was similar in FD heterozygotes and control fibroblasts, confirming that both produce the 46kDa mature form; the same as that present in control plasma. However, the proportion of GLA secreted into the culture media was substantially less than eight other lysosomal proteins. Artificial generation of FD heterozygotes in cellulo, along with another lysosomal storage disorder, mucopolysaccharidosis type II, revealed no cross-correction in the FD system, whereas MPS II fibroblasts were able to cross-correct. In plasma, GLA was present as the 46kDa mature form, which lacks the mannose 6-phosphorylated moiety and is not able to be efficiently endocytosed by affected cells. Our evidence shows that fibroblasts secrete minimal amounts of GLA and consequently normal fibroblasts are unable to cross-correct FD fibroblasts. We suggest that symptomatic FD heterozygotes arise due to the secretion of primarily the mature form, with only small amounts of the mannose 6-phosphorylated form of GLA from unaffected cells. This limits capacity for enzyme cross correction of affected cells, despite uptake of exogenous recombinant GLA.

Comparison of intracellular and secretion-based strategies for production of human α-galactosidase A in the filamentous fungus Trichoderma reesei

BackgroundTrichoderma reesei is known as a good producer of industrial proteins but has hitherto been less successful in the production of therapeutic proteins. In order to elucidate the bottlenecks of heterologous protein production, human α-galactosidase A (GLA) was chosen as a model therapeutic protein. Fusion partners were designed to compare the effects of secretion using a cellobiohydrolase I (CBHI) carrier and intracellular production using a gamma zein peptide from maize (ZERA) which accumulates inside the endoplasmic reticulum (ER). The two strategies were compared on the basis of expression levels, purification performance, enzymatic activity, bioreactor cultivations, and transcriptional profiling.ResultsConstructs were cloned into the cbh1 locus of the T. reesei strain Rut-C30. The secretion and intracellular strains produced 20 mg/l and 636 mg/l of GLA respectively. Purifications of secreted product were accomplished using Step-Tactin affinity columns and for intracellular product, a method was developed for gravity-based density separation and protein body solubilisation. The secreted protein had similar specific activity to that of the commercially available mammalian form. The intracellular version had 5-10-fold lower activity due to the enzymes incompatibility with alkaline pH. The secretion strain achieved 10% lower total biomass than either the parental or the intracellular strain. The patterns of gene induction for intracellular and parental strains were similar, whereas the secretion strain had a broader spectrum of gene expression level changes. Identification of the genes involved indicated strong secretion stress in the secretion strain and to a lesser extent also in intracellular production. Genes involved in the unfolded protein response (UPR) and ER-associated degradation were induced by GLA production, including; hac1, pdi1, prp1, cnx1, der1, and bap31.ConclusionsActive human α-galactosidase could most effectively be produced intracellularly in Trichoderma reesei at >0.5 g/l by avoidance of the extracellular environment, although purification was challenging due to specific activity losses. Strain analysis revealed that in addition to the issues with secreted proteases, the processes of secretion stress including UPR and ER degradation remain as bottlenecks for heterologous protein production. Genetic engineering to eliminate these bottlenecks is the logical path towards establishing a strain capable of producing sensitive heterologous proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-014-0091-y) contains supplementary material, which is available to authorized users.