Transcriptional Regulation Mechanisms in Adaptively Evolved Pichia kudriavzevii Under Acetic Acid Stress

Abstract

Acetic acid, a common weak acid in industrial fermentation processes, occurs naturally in lignocellulosic hydrolysates and is a byproduct of microbial metabolism. As a significant environmental stressor, it triggers the expression of multiple genes involved in various cellular responses, including biological processes, cellular components, and molecular functions. Using the acid-tolerant strain Pichia kudriavzevii PkAC-9, developed through adaptive laboratory evolution under acetic acid stress, we conducted a transcriptional analysis of 70 stress response-associated genes. RT-qPCR analysis revealed significant upregulation of several genes compared with the wild-type strain under acetic acid stress conditions. The most dramatic changes occurred in genes encoding key metabolic enzymes and stress response proteins associated with the TCA cycle (Fum: 18.6-fold, Aco: 17.1-fold, Oxo: 9.0-fold), carbon and energy metabolism (Tdh2: 28.0-fold, Erg2: 2.0-fold), electron transport chain (Gst: 10.6-fold), molecular chaperones (Hsp104: 26.9-fold, Hsp70: 13.0-fold, Sgt2: 10.0-fold), and transcriptional activators. Our findings indicate that the enhanced acetic acid tolerance of P. kudriavzevii PkAC-9 primarily depends on the coordinated upregulation of genes involved in energy metabolism, cellular detoxification mechanisms, and protein quality control systems through heat shock and transcriptional activator proteins.

Keywords: ethanol production; lignocellulosic biomass; real-time PCR; thermotolerant yeast.

Figure 1

Differential expression of genes related to the HOG pathway in P. kudriavzevii PkAC-9 compared with the wild-type strain under normal and acetic acid stress. The relative expression levels were calculated using the comparative critical threshold (2^−ΔΔCT) method, with the wild-type strain serving as the reference strain (control strain) and actin as the internal control gene. Bars represent the fold change in gene expression in the evolved strain (PkAC-9) relative to the wild-type strain under two conditions: control (without acid stress) and acetic acid stress (9 g/L). A fold change of 1 indicates an expression equal to the wild-type strain under the respective condition.

Figure 2

Differential expression of genes related to transcriptional activators in P. kudriavzevii PkAC-9 compared to the wild-type strain under normal and acetic acid stress. The relative expression levels were calculated using the comparative critical threshold (2^−ΔΔCT) method, with the wild-type strain serving as the reference strain (control strain) and actin as the internal control gene. Bars represent the fold change in gene expression in the evolved strain (PkAC-9) relative to the wild-type strain under two conditions: control (without acid stress) and acetic acid stress (9 g/L). A fold change of 1 indicates an expression equal to the wild-type strain under the respective condition.

Figure 3

Differential expression of genes related to carbon and energy metabolism in P. kudriavzevii PkAC-9 compared to the wild-type strain under normal and acetic acid stress. The relative expression levels were calculated using the comparative critical threshold (2^−ΔΔCT) method, with the wild-type strain serving as the reference strain (control strain) and actin as the internal control gene. Bars represent the fold change in gene expression in the evolved strain (PkAC-9) relative to the wild-type strain under two conditions: control (without acid stress) and acetic acid stress (9 g/L). A fold change of 1 indicates an expression equal to the wild-type strain under the respective condition.

Figure 4

Differential expression of genes related to heat shock proteins in P. kudriavzevii PkAC-9 compared to the wild-type strain under normal and acetic acid stress. The relative expression levels were calculated using the comparative critical threshold (2^−ΔΔCT) method, with the wild-type strain serving as the reference strain (control strain) and actin as the internal control gene. Bars represent the fold change in gene expression in the evolved strain (PkAC-9) relative to the wild-type strain under two conditions: control (without acid stress) and acetic acid stress (9 g/L). A fold change of 1 indicates an expression equal to the wild-type strain under the respective condition.

Figure 5

Differential expression of genes related to stress membrane biogenesis (A) and ubiquitin-proteasome (B) in P. kudriavzevii PkAC-9 compared with the wild-type strain under normal and acetic acid conditions. The relative expression levels were calculated using the comparative critical threshold (2^−ΔΔCT) method, with the wild-type strain serving as the reference strain (control strain) and actin as the internal control gene. Bars represent the fold change in gene expression in the evolved strain (PkAC-9) relative to the wild-type strain under two conditions: control (without acid stress) and acetic acid stress (9 g/L). A fold change of 1 indicates an expression equal to the wild-type strain under the respective condition.

Figure 6

Differential expression of genes related to the electron transport chain (oxidative phosphorylation) in P. kudriavzevii PkAC-9 compared with the wild-type strain under normal and acetic acid stress. The relative expression levels were calculated using the comparative critical threshold (2^−ΔΔCT) method, with the wild-type strain serving as the reference strain (control strain) and actin as the internal control gene. Bars represent the fold change in gene expression in the evolved strain (PkAC-9) relative to the wild-type strain under two conditions: control (without acid stress) and acetic acid stress (9 g/L). A fold change of 1 indicates an expression equal to the wild-type strain under the respective condition.

Figure 7

Differential expression of genes related to citrate cycle (TCA cycle) in P. kudriavzevii PkAC-9 compared to wild-type strain under normal and acetic acid stress. The relative expression levels were calculated using the comparative critical threshold (2^−ΔΔCT) method, with the wild-type strain serving as the reference strain (control strain) and actin as the internal control gene. Bars represent the fold change in gene expression in the evolved strain (PkAC-9) relative to the wild-type strain under two conditions: control (without acid stress) and acetic acid stress (9 g/L). A fold change of 1 indicates an expression equal to the wild-type strain under the respective condition.