Engineering and Fermenter Optimization of Fungi GLA Productions in Pichia Pastoris GS115 Using Oil Waste

γ-Linolenic acid (GLA) is an important n-6 polyunsaturated fatty acid (PUFA) that has received considerable attention in both levels 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 main 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, which is one of the important enzymes in the GLA production pathway was isolated from Mucor rouxii DSM1194 and expressed in Pichia pastoris GS115 by pPICZC vector. The recombinant yeast expressed the GLA up to 19.2% (72 mg/g) of total fatty acids. GLA production of recombinant yeast was studied in fermenter by oil waste along 5 days and results detected 6.3 g / l lipid and 103 mg/g GLA was produced in 72 hours. The present study may provide an opportunity for the development of an alternative host for manufacturing GLA on an industrial scale.

An Integrated Approach to Produce a Recombinant Yeast Pichia Pastoris For GLA Production

γ-Linolenic acid (GLA) is an important n-6 polyunsaturated fatty acid (PUFA) used in many nutritional and medicinal applications such as the treatment of cancer, inflammatory disorders, and diabetes. However, GLA level of the total fatty acids in plant seeds and nuts as prominent sources of GLA is not enough to utilize on an industrial scale. The study aimed to improve the expression of delta-6 desaturase, which is one of the important enzymes in GLA production pathway. The expression vector pPICZC was selected for clone M.rouxii delta-6 desaturase. The engineered vector was first cloned into E. coli DH5α and after plasmid extraction and confirmation of sequencing was transformed by electroporation into Pichia pastoris GS115. The results indicated that the recombinant yeast strain expressed the gene in the presence of methanol 0.5%. The lipids and essential fatty acids especially GLA were extracted to confirm the expression. The results of studies of lipid and fatty acid production by Sudan black and Nile red staining, GC, and flow cytometry revealed that recombinant strain can produce GLA levels up to 19.2% of total fatty acids. The present study may provide an opportunity for the development of an alternative host for manufacturing GLA on an industrial scale.

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.

Accumulation of the Mycotoxin Patulin in the Presence of Gluconic Acid Contributes to Pathogenicity of Penicillium expansum

Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest fruit maceration through secretion of D-gluconic acid (GLA) and secondary metabolites such as the mycotoxin patulin in colonized tissue. GLA involvement in pathogenicity has been suggested but the mechanism of patulin accumulation and its contribution to P. expansum pathogenicity remain unclear. The roles of GLA and patulin accumulation in P. expansum pathogenicity were studied using i) glucose oxidase GOX2-RNAi mutants exhibiting decreased GOX2 expression, GLA accumulation, and reduced pathogenicity; ii) IDH-RNAi mutants exhibiting downregulation of IDH (the last gene in patulin biosynthesis), reduced patulin accumulation, and no effect on GLA level; and iii) PACC-RNAi mutants exhibiting downregulation of both GOX2 and IDH that reduced GLA and patulin production. Present results indicate that conditions enhancing the decrease in GLA accumulation by GOX2-RNAi and PACC-RNAi mutants, and not low pH, affected patulin accumulation, suggesting GLA production as the driving force for further patulin accumulation. Thus, it is suggested that GLA accumulation may modulate patulin synthesis as a direct precursor under dynamic pH conditions modulating the activation of the transcription factor PACC and the consequent pathogenicity factors, which contribute to host-tissue colonization by P. expansum.

A Penicillium expansum Glucose Oxidase–Encoding Gene, GOX2, Is Essential for Gluconic Acid Production and Acidification During Colonization of Deciduous Fruit

Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest maceration of fruit through secretion of total, d-gluconic acid (GLA). Two P. expansum glucose oxidase (GOX)-encoding genes, GOX1 and GOX2, were analyzed. GOX activity and GLA accumulation were strongly related to GOX2 expression, which increased with pH to a maximum at pH 7.0, whereas GOX1 was expressed at pH 4.0, where no GOX activity or extracellular GLA were detected. This differential expression was also observed at the leading edge of the decaying tissue, where GOX2 expression was dominant. The roles of the GOX genes in pathogenicity were further studied through i) development of P. expansum goxRNAi mutants exhibiting differential downregulation of GOX2, ii) heterologous expression of the P. expansum GOX2 gene in the nondeciduous fruit-pathogen P. chrysogenum, and iii) modulation of GLA production by FeSO4 chelation. Interestingly, in P. expansum, pH and GLA production elicited opposite effects on germination and biomass accumulation: 26% of spores germinated at pH 7.0 when GOX activity and GLA were highest whereas, in P. chrysogenum at the same pH, when GLA did not accumulate, 72% of spores germinated. Moreover, heterologous expression of P. expansum GOX2 in P. chrysogenum resulted in enhanced GLA production and reduced germination, suggesting negative regulation of spore germination and GLA production. These results demonstrate that pH modulation, mediated by GLA accumulation, is an important factor in generating the initial signal or signals for fungal development leading to host-tissue colonization by P. expansum.

Enhancing the production of gamma-linolenic acid in Hansenula polymorpha by fed-batch fermentation using response surface methodology

Gamma-linolenic acid (GLA, C18:3Δ6,9,12) is an n-6 polyunsaturated fatty acid (PUFA) that has been used for the alleviation and treatment of a number of symptoms and diseases. Increasing GLA demand has led to a search for alternative producers and potential strategies for GLA production. Based on the successful performance of Hansenula polymorpha, a methylotrophic yeast, as a “cell factory” for the production of valuable bioproducts, a bioprocess development approach was implemented for GLA production in the recombinant yeast carrying the mutated Δ6-desaturase gene of Mucor rouxii. Using a substrate-feeding strategy under glycerol-limited conditions, the physical-chemical variables during the fed-batch fermentation of the recombinant H. polymorpha were optimised for GLA production through response surface methodology using a Box-Behnken design. The medium composition, including yeast extract and trace elements, and dissolved oxygen tension (DOT) were targeted. We found that DOT was the most effective variable for enhancing GLA yield. These results also suggest that the optimum conditions for GLA production are 28 % saturation of DOT, 1 g L−1 of yeast extract and 3.6 mL L−1 of the Pichia trace metals 1 (PTM1).

Media Optimization for the Production of ?- Linolenic Acid by Cunninghamella echinulata var.elegans MTCC 552 Using Response Surface Methodology

?- Linolenic acid (GLA) is an intracellular primary metabolite produced by some plants and fungal species via several distinct biosynthetic pathways. It is an essential fatty acid, and is mainly used in treatment of various diseases such as rheumatoid arthritis, multiple sclerosis, schizophrenia, atopic eczema and premenstrual syndrome. It is also a precursor of a number of biologically active compounds. In present work, strains of Cunninghamella echinulata var.elegans MTCC 552, Mucor rouxii MTCC 386, Mucor hiemallis MTCC 1278 and Rhizopus stolnifer MTCC 2591 were screened for maximum production of GLA. Cunninghamella echinulata var. elegans MTCC 552 produced maximum GLA (13.7 ± 0.31 mg/g of DCW), and hence selected for further work. Many crucial factors that affect GLA production were optimized in two steps. In the first step, one factor at a time method was used to investigate the effects of media constituents. Subsequently, in the second step, concentration of media components was optimized using Response Surface Methodology (RSM). These optimizations increased the GLA production to 19.8 ± 0.35 mg/g of DCW.