Triggering respirofermentative metabolism in the crabtree-negative yeast Pichia guilliermondii by disrupting the CAT8 gene

Abstract

Pichia guilliermondii is a Crabtree-negative yeast that does not normally exhibit respirofermentative metabolism under aerobic conditions, and methods to trigger this metabolism may have applications for physiological study and industrial applications. In the present study, CAT8, which encodes a putative global transcriptional activator, was disrupted in P. guilliermondii. This yeast's ethanol titer increased by >20-fold compared to the wild type (WT) during aerobic fermentation using glucose. A comparative transcriptional analysis indicated that the expression of genes in the tricarboxylic acid cycle and respiratory chain was repressed in the CAT8-disrupted (ΔCAT8) strain, while the fermentative pathway genes were significantly upregulated. The respiratory activities in the ΔCAT8 strain, indicated by the specific oxygen uptake rate and respiratory state value, decreased to one-half and one-third of the WT values, respectively. In addition, the expression of HAP4, a transcriptional respiratory activator, was significantly repressed in the ΔCAT8 strain. Through disruption of HAP4, the ethanol production of P. guilliermondii was also increased, but the yield and titer were lower than that in the ΔCAT8 strain. A further transcriptional comparison between ΔCAT8 and ΔHAP4 strains suggested a more comprehensive reprogramming function of Cat8 in the central metabolic pathways. These results indicated the important role of CAT8 in regulating the glucose metabolism of P. guilliermondii and that the regulation was partially mediated by repressing HAP4. The strategy proposed here might be applicable to improve the aerobic fermentation capacity of other Crabtree-negative yeasts.

FIG 1

(A) Plasmid pCatIN constructed for CAT8 disruption. (B) Outline of the disruption of CAT8. (C) Yeast colony PCR (ΔCol.) and genome PCR (ΔGen.) confirmation of the correct insertion mutant. (D) Verification of the stability of the mutant by PCR using P1 and P2 as primers. Lane B, using deionized water as a template; lanes Δ1 to Δ5, using the genome of mutant from the first to the fifth passage as a template; lane WT, using the genome of the WT (wild-type) strain as the template.

FIG 2

Glucose fermentation of wild-type P. guilliermondii (WT, closed symbols) and its CAT8-disrupted mutant (ΔCAT8, open symbols) at 30°C and 200 rpm in 100-ml Erlenmeyer flasks with 20 ml of MM medium. (A) Time courses of medium residual glucose (squares) and DCW (circles). (B) Time courses of pyruvate (triangles) and acetate (squares) formation. (C) Time courses of ethanol (squares) and glycerol (circles) formation. The values are the averages of three independent experiments, and the error bars represent the standard derivations. Statistically significant differences (Student t test [paired, two tailed], P < 0.05) were determined by using the Student t test and are indicated with an asterisk.

FIG 3

Aerobic glucose fermentation of ΔCAT8 (closed symbols), ΔHAP4 (open symbols), and WT (dash lines with closed gray symbols) strains at 30°C and 200 rpm in 100-ml Erlenmeyer flasks with 20 ml of MM medium. (A) Time courses of medium residual glucose (squares) and DCW (circles). (B) Time courses of pyruvate (triangles) and acetate (squares) formation. (C) Time courses of ethanol (squares) and glycerol (circles) formation. The values are the averages of three independent experiments, and the error bars represent the standard derivation,. Statistically significant differences (Student t test [paired, two tailed], P < 0.05) were determined by using the Student t test and are indicated with an asterisk.

FIG 4

Expression of genes in central metabolism pathways during aerobic glucose fermentation of the ΔCAT8 and ΔHAP4 mutants at 30°C and 200 rpm in 100-ml Erlenmeyer flasks with 20 ml of MM medium. The data were relative value to that of the WT. The upper values indicate the relative value of the ΔHAP4 mutant to the WT; the lower value indicates the relative value of the ΔCAT8 mutant to the WT. The values are the averages of three independent experiments, and the standard derivations are indicated. Statistically significant differences (Student t test [paired, two tailed], P < 0.05) were determined by using the Student t test and are indicated with an asterisk.