Business Unit Bioscience, Wageningen Plant Research, Wageningen University & Research, Wageningen, The Netherlands.
Axxence Aromatic GmbH, Emmerich am Rhein, Germany.
Microb Cell Fact. 2022 Mar 24;21(1):45. doi: 10.1186/s12934-022-01771-y.
The nitrogen containing aromatic compound indole is known for its floral odor typical of jasmine blossoms. Due to its characteristic scent, it is frequently used in dairy products, tea drinks and fine fragrances. The demand for natural indole by the flavor and fragrance industry is high, yet, its abundance in essential oils isolated from plants such as jasmine and narcissus is low. Thus, there is a strong demand for a sustainable method to produce food-grade indole.
Here, we established the biotechnological production of indole upon L-tryptophan supplementation in the bacterial host Corynebacterium glutamicum. Heterologous expression of the tryptophanase gene from E. coli enabled the conversion of supplemented L-tryptophan to indole. Engineering of the substrate import by co-expression of the native aromatic amino acid permease gene aroP increased whole-cell biotransformation of L-tryptophan to indole by two-fold. Indole production to 0.2 g L was achieved upon feeding of 1 g L L-tryptophan in a bioreactor cultivation, while neither accumulation of side-products nor loss of indole were observed. To establish an efficient and robust production process, new tryptophanases were recruited by mining of bacterial sequence databases. This search retrieved more than 400 candidates and, upon screening of tryptophanase activity, nine new enzymes were identified as most promising. The highest production of indole in vivo in C. glutamicum was achieved based on the tryptophanase from Providencia rettgeri. Evaluation of several biological aspects identified the product toxicity as major bottleneck of this conversion. In situ product recovery was applied to sequester indole in a food-grade organic phase during the fermentation to avoid inhibition due to product accumulation. This process enabled complete conversion of L-tryptophan and an indole product titer of 5.7 g L was reached. Indole partitioned to the organic phase which contained 28 g L indole while no other products were observed indicating high indole purity.
The bioconversion production process established in this study provides an attractive route for sustainable indole production from tryptophan in C. glutamicum. Industrially relevant indole titers were achieved within 24 h and indole was concentrated in the organic layer as a pure product after the fermentation.
含氮芳香化合物吲哚以其典型的茉莉花香而闻名。由于其独特的气味，它经常被用于乳制品、茶饮料和香精中。香精和香料行业对天然吲哚的需求很高，但从茉莉花和水仙花等植物中分离得到的精油中吲哚的含量却很低。因此，人们强烈需要一种可持续的方法来生产食品级吲哚。
在这里，我们在细菌宿主谷氨酸棒杆菌中补充 L-色氨酸的情况下建立了吲哚的生物技术生产。大肠杆菌色氨酸酶基因的异源表达使补充的 L-色氨酸转化为吲哚。通过共表达天然芳香族氨基酸转运蛋白基因 aroP 来工程化底物导入，使 L-色氨酸全细胞生物转化为吲哚的转化率提高了两倍。在生物反应器培养中，当补充 1 g/L 的 L-色氨酸时，达到了 0.2 g/L 的吲哚产量，同时没有观察到副产物的积累或吲哚的损失。为了建立一个高效和稳健的生产过程，通过挖掘细菌序列数据库来招募新的色氨酸酶。该搜索检索到 400 多个候选者，经过色氨酸酶活性筛选，确定了 9 种新的酶作为最有前途的酶。在谷氨酸棒杆菌中，基于 Providencia rettgeri 的色氨酸酶，体内吲哚的产量最高。对几种生物学方面的评估表明，产物毒性是该转化的主要瓶颈。采用原位产物回收技术，在发酵过程中将吲哚隔离在食品级有机相中，以避免因产物积累而导致的抑制。该过程使 L-色氨酸完全转化，达到了 5.7 g/L 的吲哚产物浓度。吲哚分配到有机相中，其中含有 28 g/L 的吲哚，而没有观察到其他产物，表明吲哚的纯度很高。
本研究建立的生物转化生产工艺为谷氨酸棒杆菌中从色氨酸可持续生产吲哚提供了一条有吸引力的途径。在 24 小时内达到了工业相关的吲哚浓度，并在发酵后将吲哚浓缩在有机层中作为纯产物。
