Enhancement of β-Caryophyllene Biosynthesis in Saccharomyces cerevisiae via Synergistic Evolution of β-Caryophyllene Synthase and Engineering the Chassis

Abstract

β-Caryophyllene is a plant-derived bicyclic sesquiterpene with multiple biological functions. β-Caryophyllene production by engineered Saccharomyces cerevisiae represents a promising technological route. However, the low catalytic activity of β-caryophyllene synthase (CPS) is one of the main restrictive factors for β-caryophyllene production. Here, directed evolution of the Artemisia annua CPS was performed, and variants of CPS enhancing the β-caryophyllene biosynthesis in S. cerevisiae were obtained, in which an E353D mutant enzyme presented large improvements in V max and K cat . The K cat /K m of the E353D mutant was 35.5% higher than that of wild-type CPS. Moreover, the E353D variant exhibited higher catalytic activity in much wider pH and temperature ranges. Thus, both the higher catalytic activity and the robustness of the E353D variant contribute to the 73.3% increase in β-caryophyllene production. Furthermore, the S. cerevisiae chassis was engineered by overexpressing genes related to β-alanine metabolism and MVA pathway to enhance the synthesis of the precursor, and ATP-binding cassette transporter gene variant STE6 T1025N to improve the transmembrane transport of β-caryophyllene. The combined engineering of CPS and chassis resulted in 70.45 mg/L of β-caryophyllene after 48 h of cultivation in a test tube, which was 2.93-fold of that of the original strain. Finally, a βcaryophyllene yield of 594.05 mg/L was obtained by fed-batch fermentation, indicating the potential of β-caryophyllene production by yeast.

An Update on Microbial Biosynthesis of β-Caryophyllene, a Sesquiterpene with Multi-Pharmacological Properties

“…The novel S. cerevisiae chassis, showing higher metabolic flux of FPP synthesis and a higher capacity for transmembrane transport of β-caryophyllene, was constructed by overexpressing β-alanine-metabolism-related genes GAD1, PAN6, and CAB1; MVA-pathway-related genes EfHMGR, ERG12, ERG8, CUP1 (promoter), ERG19, IDI1, and ERG20; and the ATP-binding cassette transporter gene variant STE6T1025N. After 48 h of cultivation in a test tube, the final strain, YEH4-ECPSE353D, produced 70.45 mg/L BCP, 2.93 times more than the strain SQ3-4-CPS [58,59]. Ultimately, a 96 h fed-batch fermentation process yielded a BCP yield of 594.05 mg/L at a synthesis rate of 6.19 mg/L/h [59].…”

“…Overexpression of the A. annua QHS1 gene in S. cerevisiae SQ3-4 resulted in the strain SQ3-4-CPS, which produced 11.86 ± 0.09 mg/L BCP in a shake flask. In a subsequent study, Lu et al employed directed evolution to optimize A. annua β-caryophyllene synthase [59]. One of the resulting variants, the E353D mutant, exhibited 35% higher catalytic activity than the wild type and operated in significantly wider pH and temperature ranges.…”

Catalytic Mechanism and Heterologous Biosynthesis Application of Sesquiterpene Synthases

“…There is an urgent need to develop a genuine high-throughput screening method for STSs. Currently, random mutagenesis is mainly combined with low- to medium-throughput screening strategies, primarily involving vanillin-sulfuric acid and the measurement of specific PPi levels measured by the PiPER assay. ,, In addition, β-caryophyllene synthase mutants with high catalytic activity were obtained by adaptive evolution using NaCl stress as a screening condition . Some reports suggest indirect screening of target terpenes by reverse high-throughput screening of competitive products with color .…”

“…54,82,90 In addition, βcaryophyllene synthase mutants with high catalytic activity were obtained by adaptive evolution using NaCl stress as a screening condition. 82 Some reports suggest indirect screening of target terpenes by reverse high-throughput screening of competitive products with color. 97 In the future, researchers may explore utilizing the natural reaction mechanism of sesquiterpenes to develop STS biosensors for high-throughput screening.…”

“…Its V max and k cat / K m were increased by 34.8% and 35.5%, respectively, resulting in a 73.3% increase of β-caryophyllene production (594.05 mg/L) in engineered S. cerevisiae, which is the highest production of β-caryophyllene heterologous biosynthesis . E353 is close to the predicted magnesium ion binding sites, so the substitution may influence the catalytic activity by changing its binding state with metal ions.…”