Towards the Industrial Production of Omega-3 Long Chain Polyunsaturated Fatty Acids from a Genetically Modified Diatom Phaeodactylum tricornutum.
Towards the Industrial Production of Omega-3 Long Chain Polyunsaturated Fatty Acids from a Genetically Modified Diatom Phaeodactylum tricornutum.
复制标题
DOI：
10.1371/journal.pone.0144054
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Sayanova O
中科院分区：
文献类型：
--
作者：
Hamilton ML;Warwick J;Terry A;Allen MJ;Napier JA;Sayanova O
海洋硅藻褐藻（Phaeodactylum tricornutum）可积累高达30%的omega-3长链多不饱和脂肪酸（LC-PUFA）二十碳五烯酸（EPA），因此被认为是EPA工业生产的良好来源。然而，三角棘豆不会自然积累大量更有价值的omega-3 LC-PUFA二十二碳六烯酸（DHA）。在此之前，我们通过过度表达编码LC-PUFA生物合成途径酶活性的外源基因，设计了三角角藻，使其积累了较高水平的DHA和二十二碳五烯酸（DPA）。本文研究了转基因菌株Pt_Elo5在EPA和DHA的规模化生产中的应用。在实验室规模上对培养物进行了研究，培养物生长在高达1l的烧瓶、3.5 L的气泡柱、550 L的封闭光生物反应器和1250 L的人工照明的环形池中。研究了不同培养基、碳源和光照对转基因菌株Pt_Elo5和野生型三角霉在3.5 L气泡柱中产生omega-3 LC-PUFAs的影响。在连续光照条件下，添加F/2营养物质（F2N）的海水盐（Instant Ocean， IO）中培养的转基因菌株DHA含量最高，占总脂肪酸（TFA）的7.5%。在小规模实验中确定了omega-3 LC-PUFA积累的最佳条件后，我们比较了在较大的栅栏式管状光生物反应器和跑道池中生长的转基因菌株的EPA和DHA水平。我们观察到DHA的显著产量超过EPA，在光生物反应器中生长的细胞中产生的EPA/DPA/DHA分别为TFA的8.7%/4.5%/12.3%，相当于中等指数生长的藻类培养中6.4 μg/mg干重DHA。在自然海水和F/2营养物中，在常温下添加人工照明（110 μmol光子m-2s-1）、16:8h明暗循环条件下，Omega-3 LC-PUFAs产量为24.8% EPA和10.3% DHA。在RP中生长的转基因菌株产生最高水平的EPA（12.8%）纳入中性脂。然而，在PBR中培养的DHA在中性脂中的分配最高（7.1%）。我们的研究结果清楚地证明了转基因Pt_Elo5作为EPA和DHA商业化生产平台的发展潜力。
The marine diatom Phaeodactylum tricornutum can accumulate up to 30% of the omega-3 long chain polyunsaturated fatty acid (LC-PUFA) eicosapentaenoic acid (EPA) and, as such, is considered a good source for the industrial production of EPA. However, P. tricornutum does not naturally accumulate significant levels of the more valuable omega-3 LC-PUFA docosahexaenoic acid (DHA). Previously, we have engineered P. tricornutum to accumulate elevated levels of DHA and docosapentaenoic acid (DPA) by overexpressing heterologous genes encoding enzyme activities of the LC-PUFA biosynthetic pathway. Here, the transgenic strain Pt_Elo5 has been investigated for the scalable production of EPA and DHA. Studies have been performed at the laboratory scale on the cultures growing in up to 1 L flasks a 3.5 L bubble column, a 550 L closed photobioreactor and a 1250 L raceway pond with artificial illumination. Detailed studies were carried out on the effect of different media, carbon sources and illumination on omega-3 LC-PUFAs production by transgenic strain Pt_Elo5 and wild type P. tricornutum grown in 3.5 L bubble columns. The highest content of DHA (7.5% of total fatty acids, TFA) in transgenic strain was achieved in cultures grown in seawater salts, Instant Ocean (IO), supplemented with F/2 nutrients (F2N) under continuous light. After identifying the optimal conditions for omega-3 LC-PUFA accumulation in the small-scale experiments we compared EPA and DHA levels of the transgenic strain grown in a larger fence-style tubular photobioreactor and a raceway pond. We observed a significant production of DHA over EPA, generating an EPA/DPA/DHA profile of 8.7%/4.5%/12.3% of TFA in cells grown in a photobioreactor, equivalent to 6.4 μg/mg dry weight DHA in a mid-exponentially growing algal culture. Omega-3 LC-PUFAs production in a raceway pond at ambient temperature but supplemented with artificial illumination (110 μmol photons m-2s-1 ) on a 16:8h light:dark cycle, in natural seawater and F/2 nutrients was 24.8% EPA and 10.3% DHA. Transgenic strain grown in RP produced the highest levels of EPA (12.8%) incorporated in neutral lipids. However, the highest partitioning of DHA in neutral lipids was observed in cultures grown in PBR (7.1%). Our results clearly demonstrate the potential for the development of the transgenic Pt_Elo5 as a platform for the commercial production of EPA and DHA.