Efficient production of oxidized terpenoids via engineering fusion proteins of terpene synthase and cytochrome P450

Highlights

• The fusion of terpene synthase and P450 leads to more efficient terpene oxidation.

• Structure-based prediction and evaluation of fusion protein activity was performed.

• CS-P450 fusion led up to 5.4-fold titer improvement in 1,8-cineole hydroxylation.

• Implementing fusion strategy significantly increased in sesquiterpene oxidation.

• SEC-SAXS characterized the proximal orientation of domains in CS-P450 fusion.

Abstract

The functionalization of terpenes using cytochrome P450 enzymes is a versatile route to the production of useful derivatives that can be further converted to value-added products. Many terpenes are hydrophobic and volatile making their availability as a substrate for P450 enzymes significantly limited during microbial production. In this study, we developed a strategy to improve the accessibility of terpene molecules for the P450 reaction by linking terpene synthase and P450 together. As a model system, fusion proteins of 1,8-cineole synthase (CS) and P450_cin were investigated and it showed an improved hydroxylation of the monoterpenoid 1,8-cineole up to 5.4-fold. Structural analysis of the CS-P450_cin fusion proteins by SEC-SAXS indicated a dimer formation with preferred orientations of the active sites of the two domains. We also applied the enzyme fusion strategy to the oxidation of a sesquiterpene epi-isozizaene and the fusion enzymes significantly improved albaflavenol production in engineered E. coli. From the analysis of positive and negative examples of the fusion strategy, we proposed key factors in structure-based prediction and evaluation of fusion enzymes. Developing fusion enzymes for terpene synthase and P450 presents an efficient strategy toward oxidation of hydrophobic terpene compounds. This strategy could be widely applicable to improve the biosynthetic titer of the functionalized products from hydrophobic terpene intermediates.

Introduction

Terpenes are a large class of natural products, primarily produced by plants and constitute the main components of essential oils. A typical monoterpene (C₁₀), such as limonene, is a cyclic hydrocarbon molecule (C₁₀H₁₆) and can be used as a precursor of fuel additives, fragrances, insecticides, and pharmaceuticals (Aharoni et al., 2005). Production of terpenes in the microbial system is considered a more sustainable and stable alternative to the isolation from plants or via chemical synthesis. Functionalization of the terpene carbon backbone by enzymes such as cytochrome P450s could further expand the range of bio-based compounds which frequently can be converted to additional products of commercial interest (Bernhardt, 2006; Chang et al., 2007; Pateraki et al., 2015; Renault et al., 2014; Urlacher and Girhard, 2019). For example, limonene can be oxidized by P450 (CYP153) to perillyl alcohol, a precursor of promising anti-cancer agents (van Beilen et al., 2005). While P450s play an important role in the decoration and modification of terpenes essential for the new bioactivities, the hydrophobicity and volatility of terpene molecules might limit the availability of the substrate around the enzyme and result in low enzymatic conversion during microbial production, especially when a solvent overlay is used to reduce the loss of these volatile compounds via extraction of hydrophobic terpenes to the overlay (Alonso-Gutierrez et al., 2013). This makes the subsequent enzymatic reaction (which uses terpenes as substrates) less efficient and eventually lowers the titer of the final product.

To overcome the low availability of hydrophobic substrates for downstream enzymes such as P450s, one popular strategy is to create a spatial constraint that improves the proximity between the enzyme and the substrate (Conrado et al., 2008). Engineering of fusion proteins (Kourtz et al., 2005; Meynial Salles et al., 2007), protein scaffolds (Dueber et al., 2009), and compartmentalization of metabolic pathways (Avalos et al., 2013) have been explored to achieve the proximity effect. Among these approaches, engineering synthetic fusion proteins have been extensively used to modify enzymes toward efficient metabolic catalysis due to their simplicity and effectiveness (Yu et al., 2015). Using a short peptide linker sequence, two or more enzymes are combined and generate a single polypeptide that exhibits more than one activity or increases the reaction rate for consecutive enzymes. In the microbial production of isoprenoids, a higher pinene production level was reported by linking terpene synthase with geranyl pyrophosphate (GPP) synthase to overcome product inhibition from GPP (Sarria et al., 2014). Similarly, an engineered fusion of isopentenyl diphosphate (IPP) isomerase and isoprene synthase showed a 3.3-fold increase of isoprene titer (Gao et al., 2016). For P450 enzymes, fusions of P450 with a heterologous cytochrome P450 reductase have also proven successful in various instances. For example, a P450 TxtE was linked to the reductase domain of P450BM3 for improved activity and regio-promiscuity in aromatic nitration (Zuo et al., 2017).

Although engineering a fusion of P450 with a cytochrome P450 reductase is widely studied, there are fewer reports for engineering a fusion between P450 and a terpene synthase. Given that the considerable loss of the terpene substrate from the cell is a critical limitation for the subsequent P450 reaction during the microbial production (Alonso-Gutierrez et al., 2013), engineering a fusion protein by linking terpene synthase and P450 to form a chimeric protein could improve the proximity of P450 and the terpene substrate, which in turn would improve the substrate availability for P450. In this study, we selected the hydroxylation of monoterpene 1,8-cineole as a model system to demonstrate this approach in the microbial system via engineering the recombinant enzyme fusion between terpene synthase-P450 enzyme fusion (Fig. 1). Guided by structural modeling, we engineered a series of fusion proteins between 1,8-cineole synthase and P450_cin (CYP176A1) to investigate the hydroxylation of 1,8-cineole in both in vitro and in vivo conditions in comparison to non-fused enzymes. Structural analysis of fusion proteins revealed that different linker length changes the flexibility of the fusion enzymes. We also applied this enzyme fusion strategy for the oxidation of several other terpenes, and the data from both experiments and the modeling analysis, of positive and negative results suggested key factors (e.g. linker length, enzyme orientation, etc.) for designing a fusion protein. Our results demonstrated that engineering fusion enzyme is a feasible strategy for the efficient production of functionalized products from hydrophobic terpene intermediates.