海岸带生物学与生物资源保护实验室知识库

虾青素(astaxanthin，3，3'-二羟基-4，4'-二酮基-β，β'-胡萝卜素，C₄₀H₅₂O₄)是一种次生类胡萝卜素，具有抗氧化、增强机体免疫力、抗癌、着色等作用，在食品及保健品、药品、饲料工业、化妆品等方面有着广泛的应用。虾青素主要是在藻源(雨生红球藻)、红发夫酵母及人工合成中通过各种方法进行合成。不同来源的虾青素结构不同。含虾青素的藻源主要为雨生红球藻。来自雨生红球藻的虾青素的结构是3S,3'S，属于100％的左旋结构，结构稳定，人或动物摄入体内后才能发挥一些重要的保护功能，最大程度上发挥其应有的多种生物功效。红发夫酵母中虾青素为3R,3'R，属于右旋，而人工合成虾青素含3S,3'S、3S,3'R、3R,3'R。在这3种虾青素结构中，左旋的抗氧化性最强，只有雨生红球藻能够通过生物合成途径合成。

雨生红球藻在多种逆境胁迫条件下能够迅速合成并大量积累天然虾青素，其含量最高可达细胞干重的5%。因此，它是目前公认的合成天然虾青素的首选工具，但利用雨生红球藻大规模生产虾青素仍然是一个挑战，具有以下问题：生长周期长、生长速度慢、藻密度较低、占地面积广，而且易受到细菌和杂藻污染。

为了提供另一种虾青素可用的来源，将集胞藻 PCC6803用作底盘细胞，延伸类胡萝卜素生物合成途径以生产虾青素。集胞藻PCC6803具有培养条件较简单、生长速度较快、不会产生毒素、细胞结构较简单、遗传背景较清楚、方便于分子操作等有利于在光合生物反应器进行大规模生产的条件，是理想的蓝藻基因工程的受体。集胞藻PCC6803能够合成虾青素的上游底物海胆酮和玉米黄质，和雨生红球藻相比，缺乏合成虾青素的关键酶β-胡萝卜素酮化酶(BKT)和类胡萝卜素羟化酶(CRTR-B)，不能将海胆酮和玉米黄质进一步转化为虾青素。

将来自雨生红球藻的β-胡萝卜素酮化酶基因(bkt)和类胡萝卜素羟化酶基因(crtR-B)根据集胞藻PCC6803的密码子偏好性进行了密码子优化，然后在集胞藻 PCC6803中进行异源表达，之后通过基因组PCR，Southern blot，RT-PCR，Western blot验证bkt和crtR-B基因的表达，高效液相色谱检测发现转入bkt和 crtR-B基因的突变株中产生了虾青素，虾青素的总量为0.68 ± 0.01 mg/g (干重)。之后通过胁迫提高虾青素的产量，发现集胞藻 PCC6803突变株在10% NaNO₃胁迫14天后虾青素总量达到4.81 ± 0.06 mg/g (DW)。且游离虾青素占总虾青素的比例为30％以上，3S,3'S虾青素占总虾青素的比例为82.61％。这些结果证明了集胞藻PCC6803用于代谢工程生产虾青素的可用性和行业应用潜力。

Astaxanthin (3,3'-dihydroxy-4,4'-dione-β, β'-carotene, C₄₀H₅₂O₄) is a secondary carotenoid that has been found in nature. Astaxanthin is the strongest antioxidant, its antioxidant activity is 1000 times that of vitamin E. Astaxanthin is mainly extracted from algae sources (Haematococcus pluvialis), Phaffia rhodozyma, and synthetic methods by various methods. The source of algae containing astaxanthin is mainly Haematococcus pluvialis. The structure of algae astaxanthin is 3S, 3'S, its structure is stable. After human or animal ingestion, it can exert some important protective functions and maximize its biological effects. The astaxanthin in Phaffia rhodozyma is 3R, 3'R, while the synthetic astaxanthin contains 3S, 3'S; 3S, 3'R; 3R, 3'R.

H. pluvialis is the preferred source for natural astaxanthin because of its high content. In various stress conditions, H. pluvialis rapidly synthesizes and accumulates astaxanthin to 5% of the cell dry weight (DW). This process is affected by environmental factors such as light, temperature, pH, and nutrient availability. High light, C/N ratio, and salinity all contribute to the accumulation of astaxanthin. However, the production of astaxanthin by H. pluvialis on a large scale is hindered by the long growth cycle of this species; it is affected by the seasons and the weather; it is susceptible to bacterial and algal contamination; and it cannot take into account both cells growth and accumulation of astaxanthin. These factors severely limit astaxanthin production using H. pluvialis on an industrial scale. Microbial production of astaxanthin via metabolic engineering has emerged as an attractive alternative.

Synechocystis sp. PCC6803 is a prokaryote capable of photosynthesis. It is the phototrophic organism to have its genome completely sequenced and has the efficient and mature genetic transformation systems. Synechocystis sp. PCC6803 is a model organism for the study of metabolic engineering and also an excellent chassis for constructing cell factories by using synthetic biology. It is both autotrophic and heterotrophic, grows rapidly, and is suitable for large-scale cultivation; thus, it has various potential applications. Synechocystis sp. PCC6803 can synthesize echinenone and zeaxanthin, which are precursors of astaxanthin. Based on the above characteristics, we hypothesized that Synechocystis sp. PCC6803 would be an excellent chassis for astaxanthin production. However, compared with H. pluvialis, Synechocystis sp. PCC6803 lacks the enzymes β-carotene ketolase (BKT) and carotenoid hydroxylase (CRTR-B), encoded by the genes bkt and crtR-B respectively.

Here, we extended the carotenoid synthesis pathway in Synechocystis sp. PCC6803 to explore an alternative to H. pluvialis for astaxanthin production. First, the accumulation of astaxanthin was increased by choosing efficient promoters. Through codon optimization, the bkt and crtR-B coding sequences from H. pluvialis were adapted for expression in Synechocystis sp. PCC6803. Then, culture conditions were optimized to increase astaxanthin production. Consequently, the highest amount of astaxanthin produced was 4.81 ± 0.06 mg/g DW.