Abstract
Flavonoids are one of the predominant groups of plant polyphenols, and these compounds have significant effects on human health and nutrition. Sulfated flavonoids have more favorable attributes compared to their parent compounds such as increased solubility, stability, and bioavailability. In this research, we developed a microbial system to produce sulfated naringenin using Escherichia coli expressing a sulfotransferase (ST) from Arabidopsis thaliana (At2g03770). This wild-type strain was used as a model system for testing clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) metabolic engineering strategies. Using synthetic sgRNA to mediate transcriptional repression of cysH, a gene encoding 3′-phosphoadenosine-5′-phosphosulfate (PAPS) ST, which is involved in sulfur metabolism, resulted in an increase in intracellular PAPS accumulation by over 3.28-fold without impairing cell growth. Moreover, naringenin 7-sulfate production by engineering E. coli with its cysH gene repressed in the open reading frame through CRISPRi was enhanced by 2.83-fold in compared with the wild-type control. To improve the efficiency of biotransformation, the concentration of , glucose, and substrate were optimized. The bioproductivity of naringenin 7-sulfate was 135.49 μM [∼143.1 mg (47.7 mg L-1)] in a 3-L fermenter at 36 h. These results demonstrated that the CRISPRi system was successfully applied for the first time in E. coli to develop an efficient microbial strain for production of a sulfated flavonoid. In addition, antibacterial and anticancer activities of naringenin 7-sulfate were investigated and found to be higher than the parent compound.
Highlights
Flavonoids are major natural phenolic compounds found in plants (Buer et al, 2010)
To optimize conditions for the expression of recombinant ST At2g03770 from A. thaliana, we induced with various IPTG
The intracellular phosphoadenosine5 -phosphosulfate (PAPS) was detected at tR ∼ 1.27 min in the cytosol of wild-type as well as mutant strains (Supplementary Figure S5). These peaks were confirmed by HR-QTOF ESI/MS and the results were shown in Supplementary Table S3 and Supplementary Figures S6, S7. These results suggested that UHPLC-PDA coupled HRQTOF ESI/MS the distinguishing between PAPS from potential interferents adenosine -diphosphate (ADP) and adenosine -triphosphate disodium salt hydrate (ATP) and the detection of the intracellular PAPS in the cytosol of E. coli
Summary
Flavonoids are major natural phenolic compounds found in plants (Buer et al, 2010). One of the most predominant citrus flavonoids is naringenin and it was found to exhibit various biological effects on human health and nutrition. CRISPRi has been applied to down-regulation of genes in certain pathways via metabolic engineering of E. coli for production of value natural compounds (Wu et al, 2015; Liang et al, 2016; Gao et al, 2017). We employed a CRISPRi system as a tool for improving PAPS availability by knockdown PAPS ST (cysH) in the sulfur metabolism (Figure 1), resulted in enhancement the final products. This is the first research that a metabolic engineering approach for conjugating a sulfate moiety generated in the cytoplasm of E. coli. The biosynthesized compound was tested for its potential bioactivities against various pathogens and cancer cell lines
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