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

Author:

Fordjour Eric123,Liu Chun‐Li123ORCID,Hao Yunpeng123,Sackey Isaac4,Yang Yankun123ORCID,Liu Xiuxia123,Li Ye123,Tan Tianwei5,Bai Zhonghu123ORCID

Affiliation:

1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology Jiangnan University Wuxi China

2. National Engineering Research Center of Cereal Fermentation, and Food Biomanufacturing Jiangnan University Wuxi China

3. Jiangsu Provincial Research Centre for Bioactive Product Processing Technology Jiangnan University Wuxi China

4. Department of Biological Sciences, Faculty of Biosciences University for Development Studies Tamale Ghana

5. College of Life Science and Technology Beijing University of Chemical Technology Beijing China

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.

Funder

National Natural Science Foundation of China

Publisher

Wiley

Subject

Applied Microbiology and Biotechnology,Bioengineering,Biotechnology

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