De Novo Production of Xanthohumol by a Metabolically Engineered Escherichia coli

Abstract

Xanthohumol is a prenylflavonoid from hops with relevant bioactivities. Microbial production has emerged as a sustainable and potentially economic solution to produce it. Herein, we constructed a pathway for the de novo production of xanthohumol in Escherichia coli. Since the xanthohumol pathway depends on the availability of dimethylallyl pyrophosphate (DMAPP) and S-adenosylmethionine (SAM), SAM synthase (metK) was integrated into the genome of E. coli strains with previously engineered DMAPP pathways. Eleven prenyltransferases (PT) and the O-methyltransferase (OMT) from Humulus lupulus (HlOMT1) were tested. E. coli M-PAR-121:BlIDI:metK, constructed by integrating metK into the E. coli strain with integration of isopentenyl diphosphate isomerase (IDI) from Bacillus licheniformis (E. coli M-PAR-121:BlIDI) and expressing CdpC3PT from Neosartorya fischeri and HlOMT1 in combination with the naringenin chalcone pathway, was the best producer. This strain was able to produce 7.3 mg/L of desmethylxanthohumol and 5.3 mg/L of xanthohumol in the bioreactor, representing the first report of de novo production of xanthohumol in E. coli.

Keywords: CRISPR-Cas12a; Escherichia coli; heterologous production; metabolic engineering; synthetic biology; xanthohumol.

1

Xanthohumol biosynthetic pathway. The pathway is composed of tyrosine-ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), prenyltransferase (PT), and O-methyltransferase (OMT). Malonyl-CoA, dimethylallyl diphosphate (DMAPP), and S-adenosylmethionine (SAM) are required by CHS, PT, and OMT, respectively.

2

De novo production of desmethylxanthohumol (DMX) and xanthohumol (XAN) by E. coli M-PAR-121:BlIDI:metK expressing pRSFDuet_FjTAL_CmCHS_At4CL and pCDFDuet_CdpC3PT_HlOMT1 in shake flask experiments using different production media. The combination of LB+M9 and the use of only M9-modified medium and TB medium were tested. The production experiments were carried out using glucose as the sole substrate. Results correspond to the average of three independent experiments ± the standard deviation.

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Profile of p-coumaric acid, naringenin chalcone, desmethylxanthohumol (DMX), and xanthohumol (XAN) production by E. coli M-PAR-121:BlIDI:metK expressing pRSFDuet_FjTAL_CmCHS_At4CL and pCDFDuet_CdpC3PT_HlOMT1. Shake flask experiments were performed by using LB+M9 (A), M9-modified medium (B), and TB medium (C). Results correspond to the average of three independent experiments ± standard deviation.

4

Evaluation of metabolites accumulation, glucose consumption, and cell growth in the bioreactor batch experiment by E. coli M-PAR-121:BlIDI:metK expressing pRSFDuet_FjTAL_CmCHS_At4CL and pCDFDuet_CdpC3PT_HlOMT1. (A) Profile of p-coumaric acid, naringenin chalcone, desmethylxanthohumol (DMX), and xanthohumol (XAN) production and accumulation. (B) Glucose consumption and optical density at 600 nm (OD_600nm) of the strain during the experiment. Results correspond to the average of two independent experiments ± the standard deviation.

5

Evaluation of metabolites accumulation, glucose consumption, and cell growth in the batch experiment with two additional glucose pulses (10 g/L) in a bioreactor by E. coli M-PAR-121:BlIDI:metK expressing pRSFDuet_FjTAL_CmCHS_At4CL and pCDFDuet_CdpC3PT_HlOMT1. (A) Profile of p-coumaric acid, naringenin chalcone, desmethylxanthohumol (DMX), and xanthohumol (XAN) production and accumulation. (B) Glucose consumption and optical density at 600 nm (OD_600nm) of the strain during the experiment. Two additional pulses of 10 g/L glucose, represented by a red arrow, were added at 24 and 48 h. Results correspond to the average of two independent experiments ± standard deviation.