Engineering Escherichia coli BL21 (DE3) for high‐yield production of germacrene A, a precursor of β‐elemene via combinatorial metabolic engineering strategies

Abstract

Abstractβ‐elemene is one of the most commonly used antineoplastic drugs in cancer treatment. As a plant‐derived natural chemical, biologically engineering microorganisms to produce germacrene A to be converted to β‐elemene harbors great expectations since chemical synthesis and plant isolation methods come with their production deficiencies. In this study, we report the design of an Escherichia coli cell factory for the de novo production of germacrene A to be converted to β‐elemene from a simple carbon source. A series of systematic approaches of engineering the isoprenoid and central carbon pathways, translational and protein engineering of the sesquiterpene synthase, and exporter engineering yielded high‐efficient β‐elemene production. Specifically, deleting competing pathways in the central carbon pathway ensured the availability of acetyl‐coA, pyruvate, and glyceraldehyde‐3‐phosphate for the isoprenoid pathways. Adopting lycopene color as a high throughput screening method, an optimized NSY305N was obtained via error‐prone polymerase chain reaction mutagenesis. Further overexpression of key pathway enzymes, exporter genes, and translational engineering produced 1161.09 mg/L of β‐elemene in a shake flask. Finally, we detected the highest reported titer of 3.52 g/L of β‐elemene and 2.13 g/L germacrene A produced by an E. coli cell factory in a 4‐L fed‐batch fermentation. The systematic engineering reported here generally applies to microbial production of a broader range of chemicals. This illustrates that rewiring E. coli central metabolism is viable for producing acetyl‐coA‐derived and pyruvate‐derived molecules cost‐effectively.

Improved Membrane Permeability via Hypervesiculation for In Situ Recovery of Lycopene in Escherichia coli

“…Hence, we established an exogenous lycopene production platform by expressing the lycopene gene cluster crtEBI from C. glutamicum ATCC 13032 in E. coli BL21 (DE3). , The resulting strain LYC-1, while overexpressing the efficient MVA pathway, produced 13.61 mg/L of lycopene after 72 h shake-flask fermentation (Figure A, B). Although an MVA pathway was introduced, lycopene production did not increase much.…”

Catalytic Mechanism and Heterologous Biosynthesis Application of Sesquiterpene Synthases

“…coli (4 L bioreactor). The substitution from Y to N in the 305th residue shortened the hydrogen bond between the residues and the ligand, thereby reducing the binding affinity between the receptor and ligand …”

Two-Phase Fermentation Systems for Microbial Production of Plant-Derived Terpenes

“…Microbial cells in the culture medium synthesize products, which are then extracted or adsorbed by the SP, disrupting the equilibrium and promoting product release. For instance, in β-elemene biosynthesis by E. coli , strategies like efflux protein enhancement and the use of n-dodecane as an organic phase in fermentation increased the β-elemene yield to 3.52 g/L [ 64 ].…”

“…However, this yield is insufficient for industrial production. Recent studies have identified efficient synthases from algae and integrated key pathway enzymes, export genes, and translational engineering to implement TPF technology in bioreactors with n-dodecane as the SP, achieving a β-elemene yield of 3.52 g/L [ 64 ]. Y. lipolytica , serving as an exceptional cell factory, has been engineered to reconstruct the endogenous mevalonate pathway and regulate lipid metabolism, resulting in a β-elemene titer of 39 g/L in a bioreactor containing an isopropyl myristate organic phase [ 78 ].…”