Metabolic engineering for increased isoprenoid production often benefits from the simultaneous expression of the two naturally available isoprenoid metabolic routes, namely the 2-methyl-D-erythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. Quantification of the contribution of these pathways to the overall isoprenoid production can help to obtain a better understanding of the metabolism within a microbial cell factory. Such type of investigation can benefit from ¹³C metabolic flux ratio studies. Here, we designed a method based on parallel labeling experiments (PLEs), using [1-¹³C]- and [4-¹³C]glucose as tracers to quantify the metabolic flux ratios in the glycolytic and isoprenoid pathways. By just analyzing a reporter isoprenoid molecule and employing only four equations, we could describe the metabolism involved from substrate catabolism to product formation. These equations infer ¹³C atom incorporation into the universal isoprenoid building blocks, isopentenyl-pyrophosphate (IPP) and dimethylallyl-pyrophosphate (DMAPP). Therefore, this renders the method applicable to the study of any of isoprenoid of interest. As proof of principle, we applied it to study amorpha-4,11-diene biosynthesis in the bacterium Rhodobacter sphaeroides. We confirmed that in this species the Entner-Doudoroff pathway is the major pathway for glucose catabolism, while the Embden-Meyerhof-Parnas pathway contributes to a lesser extent. Additionally, we demonstrated that co-expression of the MEP and MVA pathways caused a mutual enhancement of their metabolic flux capacity. Surprisingly, we also observed that the isoprenoid flux ratio remains constant under exponential growth conditions, independently from the expression level of the MVA pathway. Apart from proposing and applying a tool for studying isoprenoid biosynthesis within a microbial cell factory, our work reveals important insights from the co-expression of MEP and MVA pathways, including the existence of a yet unclear interaction between them.

用于增加类异戊二烯产生的代谢工程学通常得益于两种天然可用类异戊二烯代谢途径（即2-甲基-D-赤藓糖醇4-磷酸（MEP）途径和甲羟戊酸（MVA）途径）的同时表达。量化这些途径对类异戊二烯总产量的贡献可以帮助人们更好地了解微生物细胞工厂内的新陈代谢。此类研究可受益于¹³ C代谢通量比研究。在这里，我们设计了一种基于并行标记实验（PLEs）的方法，使用[1-1 ¹³ C]-和[4- ¹³C]葡萄糖作为示踪剂，用于量化糖酵解和类异戊二烯途径中的代谢通量比。仅通过分析报告者类异戊二烯分子并仅使用四个方程式，我们就可以描述从底物分解代谢到产物形成的代谢过程。这些方程式推论出¹³ C原子掺入通用类异戊二烯结构单元，异戊烯基焦磷酸盐（IPP）和二甲基烯丙基焦磷酸盐（DMAPP）中。因此，这使得该方法可用于研究任何感兴趣的类异戊二烯。作为原理证明，我们将其用于研究球形球形红细菌中的amorpha-4,11-diene生物合成。我们证实，在该物种中，Entner-Doudoroff途径是葡萄糖分解代谢的主要途径，而Embden-Meyerhof-Parnas途径的贡献程度较小。此外，我们证明了MEP和MVA途径的共表达引起其代谢通量的相互增强。出人意料的是，我们还观察到类异戊二烯通量比在指数增长条件下保持恒定，与MVA途径的表达水平无关。除了提出并应用一种用于研究微生物细胞工厂中类异戊二烯生物合成的工具外，我们的工作还揭示了MEP和MVA途径共表达的重要见解，包括它们之间尚存在不清楚的相互作用。