Studies On The Substrate Specificity Of Delta 6 Desaturase And Its Application In Mortierella Alpina

Polyunsaturated fatty acids(PUFAs) in creatures are synthesized via fatty acid pathway, during which delta 6 fatty acid desaturase(FADS6) converting linoleic acid(LA, 18:2Δ9,12) to γ-linolenic acid(GLA, 18:3Δ6,9,12) or α-linolenic acid(ALA, 18:3Δ9,12,15) to stearidonic acid(SDA, 18:4Δ6,9,12,15) by introducing a double bond between the carbon 6 and 7 from carboxyl end of the substrate, is a critical enzyme in the ω6/ω3 polyunsaturated fatty acids(PUFAs) metabolic flux. The substrate specificity of FADS6 directly determines the fatty acid metabolic flux in vivo. The substrate specificity of FADS6 for LA and ALA is likely to be different in species which produce greatly divergent levels of ω6 and ω3 PUFAs, or even different. Mortierella alpina has been extensively studied for its production of AA, which are synthesized mainly via the ω6 pathway, and AA level accounts for 40% of its total fatty acid, but its ω3 PUFAs level is low(producing only in low temperature, about 1-3%). However, Micromonas pusilla has a higher eicosapentaenoic acid(EPA, 20:5Δ5,8,11,14,17) up to yield 26% of total fatty acid, whereas its ω6 PUFAs level is low(about 0.1%). This study began from analyzing the substrate specificity of two FADS6 enzymes, and revealed key domain and amino acid sites affecting on their substrate specificity in FADS6 primary structure. Finally, FADS6 from Micromonas pusilla was applied on EPA production in Mortierella alpina. The main results are described as follows:1. Study on substrate specificity for FADS6: The heterologous expression vectors for MaFADS6-I, MaFADS6-II and MpFADS6 were constructed under the skeleton of plasmid pYES2/NT C and expressed in Saccharomyces cerevisiae. By adding exogenous substrate, the specificity of all FADS6 s were determined. The results showed that MaFADS6-I had specificity for LA(ω6), while MaFADS6-II had no specificity and MpFADS6 had specificity for ALA(ω3).2. Analysis of the domain that influencing FADS6 substrate specificity: To figure out the reason that MaFADS6-I and MpFADS6 had different substrate specificity, both amino acid sequences were divided into five regions. Ten kinds of fusion genes were constructed by overlap extension PCR and their activities were determined in Saccharomyces cerevisiae. The results revealed that sequences between the His boxes I and II played a pivotal role in substrate specificity, and the sequences between cytochrome b5 region and His boxes I also of vital importance to substrate specificity, and we also made a conclusion that varying substrate concentration did not alter FADS6 specificity.3. Analysis of the key sites that influencing FADS6 activities: Based on the result of the key domains affecting FADS6 substrate specificity, nine amino acid residues between the His boxes I and II domain were targeted for further study by site-directed mutagenesis. The result indicated that G194 L, E222 S, M227 K and V399I/I400 E substitutions interfered with substrate recognition, suggesting that the corresponding amino acid residues were crucial during this process. The amino acid residues between cytochrome b5 region and His boxes I also had effect on substrate specificity, between which five sites(single and double site) downstream MaFADS6-I cytochrome b5 region were targeted by site-directed mutagenesis. The results revealed that K61 and D68 sites played a key role in the catalysis of LA. Therefore, key sites in key domain of FADS6 affecting on its substrate specificity were located.4. Heterogenous expression of MpFADS6 gene preferring to ALA(ω3) in Mortierella alpina: First, a plasmid harboring the MpFADS6 gene was constructed under the skeleton of plasmid pBIG2-ura5 s and overexpressed in a uracil-auxotrophic strain of M. alpina using the Agrobacterium tumefaciens-mediated transformation(ATMT) method; after screening and characterization of transformants, Ma-MpFADS6 recombinant strains were obtained.5. Application of Ma-MpFADS6 recombinant strain on EPA production in Mortierella alpina: the EPA production reached 80.0 mg/L and 90.4 mg/L in Ma-MpFADS6 transformants grown at 28°C and at 12°C, respectively. To raise the level of ALA, free form fatty acid was used as exogenous substrate, which increased the EPA production up to 114.5 mg/L. Considering the cost of EPA production in M. alpina and the stability of free form substrate, peony seed oil(PSO) and peony seed meal(PSM) were used as source of ALA, and EPA production was improved to 149.3 mg/L and 515.29 mg/L. The EPA yield was further increased to 588.5 mg/L in a 5-L bioreactor by a 16-day fermentation under 50 g/L PSM, accounting for 7.8% of its total fatty acid and resulting in a 9.8-fold increase compared to EPA percentage in wild-type M. alpina(0.8%). EPA yield was reached 638.5 mg/L by fed-batch fermentation in 16 days which resulted in a 28.4-fold increase compared to EPA production in wild-type M. alpina, indicating that we succeeded in EPA production in M. alpina through ω3 PUFAs synthesis pathway.