Recombinant Saccharomyces cerevisiae expressing P450 in artificial digestive systems: a model for biodetoxication in the human digestive environment

Abstract

The use of genetically engineered microorganisms such as bacteria or yeasts as live vehicles to carry out bioconversion directly in the digestive environment is an important challenge for the development of innovative biodrugs. A system that mimics the human gastrointestinal tract was combined with a computer simulation to evaluate the survival rate and cinnamate 4-hydroxylase activity of a recombinant model of Saccharomyces cerevisiae expressing the plant P450 73A1. The yeasts showed a high level of resistance to gastric and small intestinal secretions (survival rate after 4 h of digestion, 95.6% +/- 10.1% [n = 4]) but were more sensitive to the colonic conditions (survival rate after 4 h of incubation, 35.9% +/- 2.7% [n = 3]). For the first time, the ability of recombinant S. cerevisiae to carry out a bioconversion reaction has been demonstrated throughout the gastrointestinal tract. In the gastric-small intestinal system, 41.0% +/- 5.8% (n = 3) of the ingested trans-cinnamic acid was converted into p-coumaric acid after 4 h of digestion, as well as 8.9% +/- 1.6% (n = 3) in the stomach, 13.8% +/- 3.3% (n = 3) in the duodenum, 11.8% +/- 3.4% (n = 3) in the jejunum, and 6.5% +/- 1.0% (n = 3) in the ileum. In the large intestinal system, cinnamate 4-hydroxylase activity was detected but was too weak to be quantified. These results suggest that S. cerevisiae may afford a useful host for the development of biodrugs and may provide an innovative system for the prevention or treatment of diseases that escape classical drug action. In particular, yeasts may provide a suitable vector for biodetoxication in the digestive environment.

FIG. 1.

The recombinant model of S. cerevisiae expresses the plant cytochrome P450 73A1 and catalyzes the bioconversion of trans-cinnamic acid into p-coumaric acid (CA4H activity). (a) Schematic representation of the P450 73A1 and its yeast-associated proteins, the NADPH CPR, the cytochrome (Cyt.) b₅, and the NADH cytochrome b₅ reductase bound to the endoplasmic reticulum (ER) membrane. (b) Genetic construction of the recombinant model of S. cerevisiae. The YeDP60/CA4H plasmid was used to transform the S. cerevisiae W303-1B strain overproducing yeast CPR [W(R) strain]. ORF, open reading frame; PGK, phosphoglycerate kinase; ter, terminator; ori, origin of replication.

FIG. 2.

Diagram of the TNO Nutrition and Food Research Institute (Zeist, The Netherlands) gastric-small intestinal system TIM 1 (a) (17) and large intestinal system TIM 2 (b) (18).

FIG. 3.

Survival rate of the recombinant model of S. cerevisiae in TIM 1. Panel a represents the mean cumulative ileal delivery of viable cells ± standard deviation (n = 4). The curves obtained in each digestive compartment for the recombinant yeasts (blue circles) and for a nonabsorbable marker, blue dextran (green triangles), are shown in panel b. Values are expressed as mean percentages ± standard deviation (n = 4) of the initial intake.

FIG. 4.

Survival rate of the recombinant model of S. cerevisiae in TIM 2. Values are expressed as mean percentages ± standard deviations (n = 3) of viable yeasts relative to the total amount introduced in the large intestine (a) or mean number of viable cells ± standard deviation (n = 3) with a logarithmic ladder (b).

FIG. 5.

CA4H activity of the recombinant model of S. cerevisiae in TIM 1. The trans-cinnamic acid conversion was evaluated in the overall TIM 1 (a) and, thanks to the computer simulation, in each compartment of the TIM 1 (b). Values are expressed as mean cumulative percentages ± standard deviations (n = 3) of ingested trans-cinnamic acid converted into p-coumaric acid.

FIG. 6.

Specific CA4H activity of the recombinant model of S. cerevisiae in each compartment of TIM 1. Values are expressed as mean percentages ± standard deviations (n = 3) of micromoles of p-coumaric acid produced per yeast cell and per minute (10¹⁰).