Biosynthesis of β-caryophyllene, a novel terpene-based high-density biofuel precursor, using engineered Escherichia coli

Highlights

• Construct a novel efficient metabolic pathway for the β-caryophyllene production.

• The strain YJM59 could accumulate β-caryophyllene up to 1.52 g/L under the fed-batch fermentation conditions.

• Our results provide a new strategy that is green and sustainable for β-caryophyllene production using Escherichia coli.

Abstract

β-caryophyllene is a common sesquiterpene compound currently being studied as a promising precursor for the production of high-density fuels. Acute demand for high-density fuels has provided the impetus to pursue biosynthetic methods to produce β-caryophyllene from reproducible sources. In this study, we produced β-caryophyllene by assembling a biosynthetic pathway in an engineered Escherichia coli strain of which phosphoglucose isomerase gene has been deleted. The 1- deoxy-d-xylulose 5-phosphate (DXP) or heterologous mevalonate (MVA) pathways were employed. Meanwhile, geranyl diphosphate synthase, glucose-6-phosphate dehydrogenase and β-caryophyllene synthase genes were co-overexpressed in the above strain. The final genetically modified strain, YJM59, produced 220 ± 6 mg/L of β-caryophyllene in flask culture. We also evaluated the use of fed-batch fermentation for the production of β-caryophyllene. After induction for 60 h, the YJM59 strain produced β-caryophyllene at a concentration of 1520 mg/L. The volumetric production fermented in the aerobic fed-batch was 0.34 mg/(L·h·OD₆₀₀) and the conversion efficiency of glucose to β-caryophyllene (gram to gram) was 1.69%. Our results are the first successful attempt to produce β-caryophyllene using E. coli BL21(DE3), and provide a new strategy that is green and sustainable for the production of β-caryophyllene.

Introduction

The diminishing petroleum supply and increasingly serious environmental problems are driving a search for alternative and sustainable technologies to produce biofuels and chemical feedstocks generated from renewable sources [1], [2].

Among bio-based fuels, monoterpenes (C10) and sesquiterpenes (C15), which have compact structures and reactive olefin functionality, have great potential for use as feedstocks to produce high-density renewable fuels such as jet fuel [3], [4], [5]. β-Caryophyllene is a common sesquiterpene that is widely distributed among plant species [6], [7] and is being considered as a component of the next generation of aircraft fuel [4], [5], [8]. β-Caryophyllene also possesses anti-inflammatory [9], [10] and anticarcinogenic [11] activities and its derivatives plays a role in plant defense [12], [13].

Sesquiterpene extraction from plants and chemical synthesis are the methods commonly used to produce β-caryophyllene on a large scale; however, both methods have disadvantages. For example, low concentration and poor recovery yield [14], [15] make isolation of β-caryophyllene from plants infeasible and uneconomical, while the complexity of the process and high cost limit the use of chemical synthesis [16]. A case in point is that Larionov and Corey have proposed a chemical method for β-caryophyllene synthesis, which required 8 steps including reduction, dehydration by Mitsunobu activation, diastereoselective reduction, selective tosylation, deprotonation, carbonyl-forming elimination, desilation and Wittig methylenation [17].

Therefore, interest has shifted toward developing technologies to engineer microorganisms to convert renewable resources such as glucose generated from cellulose or hemicellulose into sesquiterpene products [18]. β-Caryophyllene produced by microbial fermentation is expected to be a good alternative to traditional methods because microorganisms grow rapidly and land can be saved for sustainable development, unlike with other methods [19], [20].

Similar to other sesquiterpenes, β-caryophyllene is produced from the common precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which can be synthesized either by the 1-deoxy-d-xylulose 5-phosphate (DXP) or mevalonate (MVA) pathways (Fig. 1) [21]. Although many microorganisms can provide the DMAPP and IPP intermediates via the DXP or MVA pathways, they cannot produce sesquiterpenes due to a lack of sesquiterpene synthase.

Metabolic engineering has developed significantly in the last decade and several microorganisms have been engineered to produce various sesquiterpenes. Yeast has been engineered to produce sesquiterpenes using the MVA pathway. Overexpression of various sesquiterpene synthases and HMG-CoA reductase in yeast in combination with downregulation of squalene synthase resulted in the production of sesquiterpenes including cubebol, patchoulol, and epi-cedrol at concentrations of 10 mg/L, 16.9 mg/L, and 370 μg/L, respectively [22], [23], [24]. Reinsvold et al. introduced a sesquiterpene biosynthetic pathway, using the native DXP pathway to provide DMAPP and IPP, into the cyanobacterium Synechocystis, which produced approximately 464 ± 2.9 ng/(L·week) of β-caryophyllene [25]. Similarly, Martin et al. constructed three Escherichia coli strains overexpressing (+)-δ-cadinene, 5-epi-aristolochene, or vetispiradiene cyclases, which accumulated (+)-δ-cadinene, 5-epi-aristolochene, and vetispiradiene at concentrations of 10.3, 0.24, and 6.4 μg/L, respectively [26]. A variant of the amorphadiene synthase gene (ADS) was expressed in E. coli after its codons were optimized. DXP synthase, IPP isomerase, and farnesyl diphosphate (FPP) synthase were overexpressed to increase the amorphadiene concentration 3.6-fold [27].

Although much progress has been made on sesquiterpene production via microbial fermentation, low productivity remains a bottleneck for large-scale, cost-effective production. In this study, β-caryophyllene yield was improved by employing a multi-step metabolic engineering strategy to increase precursor and cofactor supplies for β-caryophyllene production.