Isolation and Characterization of the Diatom Phaeodactylum Δ5-Elongase Gene for Transgenic LC-PUFA Production in Pichia pastoris

The diatom Phaeodactylum tricornutum can accumulate eicosapentaenoic acid (EPA) up to 30% of the total fatty acids. This species has been targeted for isolating gene encoding desaturases and elongases for long-chain polyunsaturated fatty acid (LC-PUFA) metabolic engineering. Here we first report the cloning and characterization of Δ5-elongase gene in P. tricornutum. A full-length cDNA sequence, designated PhtELO5, was shown to contain a 1110 bp open reading frame encoding a 369 amino acid polypeptide. The putative protein contains seven transmembrane regions and two elongase characteristic motifs of FLHXYHH and MYSYY, the latter being typical for microalgal Δ5-elongases. Phylogenetic analysis indicated that PhtELO5 belongs to the ELO5 group, tightly clustered with the counterpart of Thalassiosira pseudonana. Heterologous expression of PhtELO5 in Pichia pastoris confirmed that it encodes a specific Δ5-elongase capable of elongating arachidonic acid and eicosapentaenoic acid. Co-expression of PhtELO5 and IsFAD4 (a ∆4-desaturase from Isochrysis sphaerica) demonstrated that the high-efficiency biosynthetic pathway of docosahexaenoic acid was assembled in the transgenic yeast. Substrate competition revealed that PhtELO5 exhibited higher activity towards n-3 PUFA than n-6 PUFA. It is hypothesized that Phaeodactylum ELO5 may preferentially participate in biosynthesis of transgenic LC-PUFA via a n-3 pathway in the yeast host

Direct bio-utilization of untreated rapeseed meal for effective iturin A production by Bacillus subtilis in submerged fermentation.

The feasibility of using untreated rapeseed meal as a nitrogen source for iturin A production by Bacillus subtilis 3-10 in submerged fermentation was first evaluated by comparison with two different commercial nitrogen sources of peptone and ammonium nitrate. A significant promoting effect of rapeseed meal on iturin A production was observed and the maximum iturin A concentration of 0.60 g/L was reached at 70 h, which was 20% and 8.0 fold higher than that produced from peptone and ammonium nitrate media, respectively. It was shown that rapeseed meal had a positive induction effect on protease secretion, contributing to the release of soluble protein from low water solubility solid rapeseed meal for an effective supply of available nitrogen during fermentation. Moreover, compared to raw rapeseed meal, the remaining residue following fermentation could be used as a more suitable supplementary protein source for animal feed because of the great decrease of major anti-nutritional components including sinapine, glucosinolate and its degradation products of isothiocyanate and oxazolidine thione. The results obtained from this study demonstrate the potential of direct utilization of low cost rapeseed meal as a nitrogen source for commercial production of iturin A and other secondary metabolites by Bacillus subtilis

Transmission electron micrograph of <i>C</i>. <i>cohnii</i> cells.

<p>Microalga cells were grown on seawater based GYE containing glucose and yeast extract (<b>A</b>, <b>B</b>) or RMH-molasses medium composed of the meal hydrolysate and waste molasses (<b>C</b>, <b>D</b>) for 7 days.</p

The contents of free amino nitrogen and inorganic phosphorus in <i>N</i>. <i>crassa</i> derived hydrolysate.

<p>The rapeseed meal was fermented with <i>N</i>. <i>crassa</i> J2 at 30°C and sampled at various time points for analytical experiments; Data are means and standard deviations from triplicate fermentations.</p

Congo red staining of fungal strains with cellulolytic activity.

<p>The fungal strains were grown on agar plate containing 1% (wt/vol) sodium carboxymethyl cellulose (CMC-Na) and 1.4% (wt/vol) agar. CMC hydrolysis was detected by the Congo red staining after being incubated for 3 d.</p

Effect of supplemented yeast extract on lipid production by <i>C</i>. <i>cohnii</i> on individual RMH medium.

<p>^a TFA, total fatty acids;</p><p>^b UFA, unsaturated fatty acids</p><p>Effect of supplemented yeast extract on lipid production by <i>C</i>. <i>cohnii</i> on individual RMH medium.</p

Effect of supplemented waste molasses on lipid production by <i>C. cohnii</i> on individual RMH medium.

<p>^a TFA, total fatty acids;</p><p>^b UFA, unsaturated fatty acids</p><p>Effect of supplemented waste molasses on lipid production by <i>C. cohnii</i> on individual RMH medium.</p

Biomass concentration of <i>C</i>. <i>cohnii</i> grown on RMH supplemented with yeast extract.

<p>The meal hydrolysates produced by individual fungal stains were supplemented with different levels of yeast extract. Data of biomass concentration are means of three replicates, and error bars show standard deviation.</p

Schematic diagram of the novel DHA production process using by-products for microalgal fermentation.

<p>The rapeseed meal hydrolysate was obtained through the treatment of solid state fermentation followed by fungal autolysis. It was used as organic nitrogen source, and the hydrolyzed waste molasses was used as organic carbon source.</p