Dipartimento di Biotecnologie, Università Degli Studi di Verona, Strada le Grazie 15, 37134, Verona, Italy.
Bioprocess Engineering Chair Group, Wageningen University and Research, Wageningen, 6700 AA, The Netherlands.
Microb Cell Fact. 2024 Nov 29;23(1):322. doi: 10.1186/s12934-024-02599-4.
Canthaxanthin is a ketocarotenoid with high antioxidant activity, and it is primarily produced by microalgae, among which Nannochloropsis oceanica, a marine alga widely used for aquaculture. In the last decade, N. oceanica has become a model organism for oleaginous microalgae to develop sustainable processes to produce biomolecules of interest by exploiting its photosynthetic activity and carbon assimilation properties. N. oceanica can accumulate lipids up to 70% of total dry weight and contains the omega-3 fatty acid eicosapentaenoic acid (EPA) required for both food and feed applications. The genome sequence, other omics data, and synthetic biology tools are available for this species, including an engineered strain called LP-tdTomato, which allows homologous recombination to insert the heterologous genes in a highly transcribed locus in the nucleolus region. Here, N. oceanica was engineered to induce high ketocarotenoid and canthaxanthin production.
We used N. oceanica LP-tdTomato strain as a background to express the key enzyme for ketocarotenoid production, a β-carotene ketolase (CrBKT) from Chlamydomonas reinhardtii. Through the LP-tdTomato strain, the transgene insertion by homologous recombination in a highly transcribed genomic locus can be screened by negative fluorescence. The overexpression of CrBKT in bkt transformants increased the content of carotenoids and ketocarotenoids per cell, respectively, 1.5 and 10-fold, inducing an orange/red color in the bkt cell cultures. Background (LP) and bkt lines productivity were compared at different light intensities from 150 to 1200 µmol m s: at lower irradiances, the growth kinetics of bkt lines were slower compared to LP, while higher productivity was measured for bkt lines at 1200 µmol m s. Despite these results, the highest canthaxanthin and ketocarotenoids productivity were obtained upon cultivation at 150 µmol m s.
Through targeted gene redesign and heterologous transformation, ketocarotenoids and canthaxanthin content were significantly increased, achieving 0.3% and 0.2% dry weight. Canthaxanthin could be produced using CO as the only carbon source at 1.5 mg/L titer. These bkt-engineered lines hold potential for industrial applications in fish or poultry feed sectors, where canthaxanthin and ketocarotenoids are required as pigmentation agents.
角黄素是一种具有高抗氧化活性的酮类类胡萝卜素，主要由微藻产生，其中海洋微藻球等角藻被广泛用于水产养殖。在过去的十年中，球等角藻已成为一种模式生物，用于开发可持续的工艺，通过利用其光合作用和碳同化特性来生产有兴趣的生物分子。球等角藻可以积累高达总干重 70%的油脂，并且含有食品和饲料应用所需的ω-3 脂肪酸二十碳五烯酸（EPA）。该物种拥有基因组序列、其他组学数据和合成生物学工具，包括一种名为 LP-tdTomato 的工程菌株，该菌株允许通过同源重组将异源基因插入核仁区域的高转录基因座。在这里，球等角藻被工程化以诱导高产酮类类胡萝卜素和角黄素。
我们使用球等角藻 LP-tdTomato 菌株作为背景，表达来自莱茵衣藻的酮类类胡萝卜素生产的关键酶，β-胡萝卜素酮化酶（CrBKT）。通过 LP-tdTomato 菌株，可以通过负荧光筛选同源重组在高转录基因组座中的转基因插入。bkt 转化体中 CrBKT 的过表达分别使细胞内类胡萝卜素和酮类类胡萝卜素的含量增加了 1.5 倍和 10 倍，使 bkt 细胞培养物呈现橙色/红色。在 150 至 1200 µmol m s 的不同光照强度下比较了背景（LP）和 bkt 系的生产力：在较低的辐照度下，bkt 系的生长动力学比 LP 系慢，而在 1200 µmol m s 时，bkt 系的生产力更高。尽管有这些结果，但在 150 µmol m s 的培养条件下，获得了最高的角黄素和酮类类胡萝卜素的生产力。
通过靶向基因重新设计和异源转化，角黄素和角黄素的含量显著增加，达到干重的 0.3%和 0.2%。角黄素可以使用 CO 作为唯一的碳源，在 1.5 mg/L 的浓度下生产。这些经过 bkt 工程改造的系具有在鱼类或家禽饲料行业中应用的潜力，角黄素和酮类类胡萝卜素是这些行业中作为着色剂所必需的。
