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

Mapping Intimacies ◽

10.21203/rs.3.rs-973743/v1 ◽

2021 ◽

Author(s):

Maryam Sadat Mirbagheri Firoozabad ◽

Hamidreza Akhbariyoon

Keyword(s):

Pichia Pastoris ◽

Animal Feed ◽

Industrial Scale ◽

Recombinant Yeast ◽

Mucor Rouxii ◽

Pastoris Gs115 ◽

Plant Seed ◽

Total Fatty Acids ◽

Delta 6 Desaturase ◽

Gla Production

Abstract 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.

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

10.21203/rs.3.rs-706359/v1 ◽

2021 ◽

Author(s):

Maryam Sadat Mirbagheri Firoozabad ◽

Hamidreza Akhbariyoon

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Pichia Pastoris ◽

Essential Fatty Acids ◽

Industrial Scale ◽

Recombinant Yeast ◽

Recombinant Yeast Strain ◽

Total Fatty Acids ◽

Delta 6 Desaturase ◽

Gla Production

Abstract 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.

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