School of Life Sciences and Technology, Xinxiang Medical University, Xinxiang, 453003, Henan, China.
Synthetic Biology Engineering Lab of Henan Province, Xinxiang, 453003, Henan, China.
Microb Cell Fact. 2021 Feb 8;20(1):38. doi: 10.1186/s12934-021-01518-1.
Violaceins have attracted much attention as potential targets used in medicines, food additives, insecticides, cosmetics and textiles, but low productivity was the key factor to limit their large-scale applications. This work put forward a direct RBS engineering strategy to engineer the violacein biosynthetic gene cluster cloned from Chromobacterium violaceum ATCC 12,472 to efficiently improve the fermentation titers.
Through four-rounds of engineering of the native RBSs within the violaceins biosynthetic operon vioABCDE, this work apparently broke through the rate-limiting steps of intermediates conversion, resulting in 2.41-fold improvement of violaceins production compared to the titers of the starting strain Escherichia coli BL21(DE3) (Vio12472). Furthermore, by optimizing the batch-fermentation parameters including temperature, concentration of IPTG inducer and fermentation time, the maximum yield of violaceins from (BCDE)m (tnaA) reached 3269.7 µM at 2 mM tryptophan in the medium. Interestingly, rather than previous reported low temperature (20 ℃), we for the first time found the RBS engineered Escherichia coli strain (BCDE)m worked better at higher temperature (30 ℃ and 37 ℃), leading to a higher-level production of violaceins.
To our knowledge, this is the first time that a direct RBS engineering strategy is used for the biosynthesis of natural products, having the potential for a greater improvement of the product yields within tryptophan hyperproducers and simultaneously avoiding the costly low temperature cultivation for large-scale industrial production of violaciens. This direct RBS engineering strategy could also be easily and helpfully used in engineering the native RBSs of other larger and value-added natural product biosynthetic gene clusters by widely used site-specific mutagenesis methods represented by inverse PCR or CRISPR-Cas9 techniques to increase their fermentation titers in the future.
由于色烯具有作为药物、食品添加剂、杀虫剂、化妆品和纺织品的潜在目标的吸引力，但低生产率是限制其大规模应用的关键因素。本工作提出了一种直接 RBS 工程策略，用于工程改造从 Chromobacterium violaceum ATCC 12,472 克隆的色烯生物合成基因簇，以有效地提高发酵产量。
通过对色烯生物合成操纵子 vioABCDE 内的天然 RBS 进行四轮工程改造，本工作明显突破了中间产物转化的限速步骤，与出发菌株 Escherichia coli BL21(DE3)（Vio12472）的产量相比，色烯产量提高了 2.41 倍。此外，通过优化包括温度、IPTG 诱导剂浓度和发酵时间在内的分批发酵参数，在培养基中 2mM 色氨酸的条件下，（BCDE）m（tnaA）的色烯最大产量达到 3269.7µM。有趣的是，与之前报道的低温（20℃）不同，我们首次发现经过 RBS 工程改造的大肠杆菌菌株（BCDE）m 在较高温度（30℃和 37℃）下表现更好，导致色烯产量更高。
据我们所知，这是首次将直接 RBS 工程策略用于天然产物的生物合成，有可能在色烯高产菌株中进一步提高产物产量，同时避免大规模工业生产色烯所需的昂贵低温培养。这种直接 RBS 工程策略也可以通过广泛使用的定点突变方法（如反向 PCR 或 CRISPR-Cas9 技术），轻松且有助于工程改造其他更大、更有价值的天然产物生物合成基因簇的天然 RBS，以提高它们的发酵产量。
