The use of cyanobacteria and microalgae as cell factories to produce biofuels and added-value bioproducts has received great attention during the last two decades. Important investments have been made by public and private sectors to develop this field. However, it has been a challenge to develop a viable and cost-effective platform for cultivation of cyanobacteria and microalgae under outdoor conditions. Dealing with contamination caused by bacteria, weedy algae/cyanobacteria and other organisms is a major constraint to establish effective cultivation processes. Here, we describe the implementation in the cyanobacterium Synechococcus elongatus PCC 7942 of a phosphorus selective nutrition system to control biological contamination during cultivation. The system is based on metabolic engineering of S. elongatus to metabolize phosphite, a phosphorus source not normally metabolized by most organisms, by expressing a bacterial phosphite oxidoreductase (PtxD). Engineered S. elongatus strains expressing PtxD grow at a similar rate on media supplemented with phosphite as the non-transformed control supplemented with phosphate. We show that when grown in media containing phosphite as the sole phosphorus source in glass flasks, the engineered strain was able to grow and outcompete biological contaminants even when the system was intentionally inoculated with natural competitors isolated from an irrigation canal. The PtxD/phosphite system was successfully used for outdoor cultivation of engineered S. elongatus in 100-L cylindrical reactors and 1000-L raceway ponds, under non-axenic conditions and without the need of sterilizing containers and media. Finally, we also show that the PtxD/phosphite system can be used as selectable marker for S. elongatus PCC 7942 transgenic strains selection, eliminating the need of antibiotic resistance genes. Our results suggest that the PtxD/phosphite system is a stable and sufficiently robust strategy to control biological contaminants without the need of sterilization or other complex aseptic procedures. Our data show that the PtxD/phosphite system can be used as selectable marker and allows production of the cyanobacterium S. elongatus PCC 7942 in non-axenic outdoor reactors at lower cost, which in principle should be applicable to other cyanobacteria and microalgae engineered to metabolize phosphite.

中文翻译：

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Synechococcus elongatus PCC 7942中亚磷酸盐代谢的代谢工程作为控制室外跑道池塘生物污染物的有效措施。

在过去的二十年里，使用蓝藻和微藻作为细胞工厂生产生物燃料和增值生物产品受到了极大的关注。公共和私营部门为发展这一领域进行了重要投资。然而，在室外条件下开发一个可行且具有成本效益的蓝藻和微藻培养平台一直是一项挑战。处理由细菌、杂草藻类/蓝细菌和其他生物引起的污染是建立有效栽培过程的主要限制因素。在这里，我们描述了在蓝藻细长聚球藻 PCC 7942 中实施磷选择性营养系统，以控制培养过程中的生物污染。该系统基于 S. elongatus 的代谢工程来代谢亚磷酸盐，通过表达细菌亚磷酸盐氧化还原酶 (PtxD)，大多数生物体通常不会代谢的磷源。表达 PtxD 的工程化 S. elongatus 菌株在补充有亚磷酸盐的培养基上的生长速度与补充有磷酸盐的非转化对照的生长速度相似。我们表明，当在玻璃烧瓶中含有亚磷酸盐作为唯一磷源的培养基中生长时，工程菌株能够生长并胜过生物污染物，即使系统故意接种了从灌溉渠中分离出来的天然竞争对手。PtxD/亚磷酸盐系统成功地用于在 100-L 圆柱形反应器和 1000-L 水道池中进行工程化 S. elongatus 的户外培养，在非无菌条件下，无需对容器和培养基进行消毒。最后，我们还表明，PtxD/亚磷酸酯系统可用作 S. elongatus PCC 7942 转基因菌株选择的选择性标记，从而消除了对抗生素抗性基因的需要。我们的研究结果表明，PtxD/亚磷酸盐系统是一种稳定且足够稳健的策略来控制生物污染物，无需灭菌或其他复杂的无菌程序。我们的数据表明，PtxD/亚磷酸盐系统可用作选择性标记，并允许在非无菌室外反应器中以较低的成本生产蓝藻 S. elongatus PCC 7942，这原则上应适用于其他蓝藻和微藻工程化代谢亚磷酸盐。我们的研究结果表明，PtxD/亚磷酸盐系统是一种稳定且足够稳健的策略来控制生物污染物，无需灭菌或其他复杂的无菌程序。我们的数据表明，PtxD/亚磷酸盐系统可用作选择性标记，并允许在非无菌室外反应器中以较低的成本生产蓝藻 S. elongatus PCC 7942，这原则上应适用于其他蓝藻和微藻工程化代谢亚磷酸盐。我们的研究结果表明，PtxD/亚磷酸盐系统是一种稳定且足够稳健的策略来控制生物污染物，无需灭菌或其他复杂的无菌程序。我们的数据表明，PtxD/亚磷酸盐系统可用作选择性标记，并允许在非无菌室外反应器中以较低的成本生产蓝藻 S. elongatus PCC 7942，这原则上应适用于其他蓝藻和微藻工程化代谢亚磷酸盐。