Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C, Denmark.
BMC Biotechnol. 2011 Jan 31;11:12. doi: 10.1186/1472-6750-11-12.
Metabolic engineering in heterologous organisms is an attractive approach to achieve efficient production of valuable natural products. Glucosinolates represent a good example of such compounds as they are thought to be the cancer-preventive agents in cruciferous plants. We have recently demonstrated that it is feasible to engineer benzylglucosinolate (BGLS) in the non-cruciferous plant Nicotiana benthamiana by transient expression of five genes from Arabidopsis thaliana. In the same study, we showed that co-expression of a sixth Arabidopsis gene, γ-glutamyl peptidase 1 (GGP1), resolved a metabolic bottleneck, thereby increasing BGLS accumulation. However, the accumulation did not reach the expected levels, leaving room for further optimization.
To optimize heterologous glucosinolate production, we have in this study performed a comparative metabolite analysis of BGLS-producing N. benthamiana leaves in the presence or absence of GGP1. The analysis revealed that the increased BGLS levels in the presence of GGP1 were accompanied by a high accumulation of the last intermediate, desulfoBGLS, and a derivative thereof. This evidenced a bottleneck in the last step of the pathway, the transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to desulfoBGLS by the sulfotransferase AtSOT16. While substitution of AtSOT16 with alternative sulfotransferases did not alleviate the bottleneck, experiments with the three genes involved in the formation and recycling of PAPS showed that co-expression of adenosine 5'-phosphosulfate kinase 2 (APK2) alone reduced the accumulation of desulfoBGLS and its derivative by more than 98% and increased BGLS accumulation 16-fold.
Adjusting sulfur metabolism by directing sulfur from primary to secondary metabolism leads to a remarkable improvement in BGLS accumulation and thereby represents an important step towards a clean and efficient production of glucosinolates in heterologous hosts. Our study emphasizes the importance of considering co-substrates and their biological nature in metabolic engineering projects.
在异源生物中进行代谢工程是实现有价值天然产物高效生产的一种有吸引力的方法。硫代葡萄糖苷就是此类化合物的一个很好的例子，因为它们被认为是十字花科植物中的抗癌物质。我们最近证明，通过瞬时表达来自拟南芥的五个基因，在非十字花科植物烟草中构建苄基硫代葡萄糖苷（BGLS）是可行的。在同一研究中，我们表明，共表达第六个拟南芥基因γ-谷氨酰肽酶 1（GGP1）可以解决代谢瓶颈，从而增加 BGLS 的积累。然而，积累并没有达到预期水平，还有进一步优化的空间。
为了优化异源硫代葡萄糖苷的生产，我们在本研究中对存在或不存在 GGP1 的产生 BGLS 的烟草叶片进行了比较代谢分析。分析表明，在 GGP1 存在的情况下，BGLS 水平的增加伴随着最后中间体脱硫 BGLS 和其衍生物的高积累。这表明该途径的最后一步，即 3'-磷酸腺苷-5'-磷酸硫酸（PAPS）中的硫酸盐通过磺基转移酶 AtSOT16 转移到脱硫 BGLS 上，存在瓶颈。虽然用替代磺基转移酶替代 AtSOT16 并不能缓解瓶颈，但涉及 PAPS 形成和循环的三个基因的实验表明，单独共表达腺苷 5'-磷酸硫酸激酶 2（APK2）可使脱硫 BGLS 及其衍生物的积累减少 98%以上，BGLS 积累增加 16 倍。
通过将硫从初级代谢引导到次级代谢来调节硫代谢，可显著提高 BGLS 的积累，从而代表了在异源宿主中清洁高效生产硫代葡萄糖苷的重要一步。我们的研究强调了在代谢工程项目中考虑共底物及其生物学性质的重要性。
