Programmable probiotic consortium employ an oleic acid-inducible system to sense and degrade cholesterol in high-fat diet mice

Abstract

High cholesterol is a major risk factor for cardiovascular disease (CVD), and current treatment strategies primarily focus on inhibiting cholesterol synthesis and reducing cholesterol absorption. Engineered synthetic consortia are emerging as a promising alternative for lowering cholesterol level and improving cardiovascular health. In this study, Escherichia coli Nissle 1917 (EcN) was engineered to express three genes -IsmA, BSH, and BCoAT, individually. The IsmA-expressing strain achieved a 27.95% reduction in cholesterol, the BSH-expressing strain exhibited a bile salt hydrolase activity of 6.14 U/mL, and the butyric acid production of the BCoAT-expressing strain was 5.81 mmol/L. The synthetic consortium (IsmA-, BSH-, and BCoAT-expressing strains) reduced serum cholesterol level by 43.65% in mice fed high-fat diet. Further, an oleic acid-inducible system was incorporated into the synthetic consortium (GR-Syncon). In contrast to Syncon, GR-Syncon is capable of recognizing fatty acid levels and expressing target genes exclusively in high-fat environments. In high-fat diet mice, administration of GR-Syncon resulted in a 27.60% reduction in serum cholesterol, downregulated the expression of cholesterol synthase HMGCR (31.97%), and upregulated cholesterol hydroxylase Cyp7a1 (86.74%), optimizing lipid metabolism. GR-Syncon also down-regulated inflammatory cytokines (IL-1β, IL-10 and TNF-α), repair liver damage, improved intestinal permeability and maintained the stability of gut microbiota in high-fat diet mice. This study highlights the potential of engineered synthetic consortia as a sustainable and targeted strategy for managing cholesterol and treating CVD.

Keywords: Synthetic biology; cholesterol; engineered probiotics; high-fat diet; lipid metabolism.

Figure 1.

Design and characterization of cholesterol-regulating engineered bacteria. (a) Schematic diagram of cholesterol-lowering engineered bacteria incorporating the IsmA, BSH, and BCoAT genes, respectively. (b) The cholesterol clearance capacity of the IsmA-expressing strain (n = 3). (c) The expression level of the IsmA gene (n = 3). (d) UHPLC detection of cholesterol metabolites produced by the IsmA-expressing strain. (e) Degradation effect of the IsmA-expressing strain on cholesterol levels in an egg environment (n = 3). (f) Bile salt hydrolase produced by the BSH-expressing strain (n = 3). (g) The level of butyrate with the BCoAT-expressing strain intervention. ns, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 2.

Therapeutic effect of the engineered synthetic consortia in mice fed a high-fat diet. (a) Therapeutic schedule of engineered strains administration in a high-fat diet mouse. (b) The body weight. (c) Mouse serum IL-1β level. (d) Mouse serum IL-10 content. (e) Mouse serum TNF-α content. (f) Colonic ZO-1 protein expression levels. (g) The occludin expression level in mouse colonic. (h) H&E staining sections of liver (top) and small intestine (bottom) tissues in each group.

Figure 3.

The Syncon improved the blood lipid imbalance of high-fat diet mice. (a) Schematic diagram of synthetic consortia feed in high-fat diet mice. (b) Mouse serum cholesterol levels (n = 5). (c) Mouse serum triglyceride content. (d) Mouse serum low-density lipoprotein cholesterol (LDL) content. (e) Mouse serum high-density lipoprotein cholesterol (HDL) content. (f) UHPLC detection of cholesterol metabolites produced by the IsmA-expressing strain. (g) UHPLC detection of cholesterol metabolites produced by the Syncon.

Figure 4.

Oleic acid induction system validated in vitro. a. Design of cholesterol-lowering strains combining the oleic acid induction system. b. The ability of the inducible system to be induced by different promoters at 0 mM and 10 mM oleic acid content. c. The binding strength of different amounts of operator fadO on the FadR protein. d. Cholesterol scavenging ability of the IsmA gene under different numbers of operators. e. Bile salt hydrolase-producing capacity of BSH genes under different numbers of operators. f. Butyric acid- producing capacity of BCoAT genes under different numbers of operators.

Figure 5.

GR-Syncon identifies fatty acid levels and regulates lipid balance. a. Schematic diagram of the GR-Syncon treatment programme. b. Serum cholesterol levels in mice in fatty acid recognition experiments (n = 5). c. Serum cholesterol levels in mice treated with GR-Syncon under different numbers of operators. d. Serum cholesterol levels in mice. e. Serum triglyceride levels of mice. f. Serum LDL levels in mice. g. Serum HDL levels in mice. h. Intestinal HMGCR expression levels in mice. i. Intestinal Cyp7a1 expression levels in mice. j. UHPLC detection of cholesterol metabolites produced by the GR-Syncon.

Figure 6.

Gut microbiota analysis of high-fat diet mice treated by GR-Syncon. a. Shannon index of gut microbiota (n = 5). b. NMDS analysis. c. Venn diagram. d. Relative abundance of phylum e. Relative abundance of genus. f. LEfse analysis (LDA ≥2).