CgMED3 Changes Membrane Sterol Composition To Help Candida glabrata Tolerate Low-pH Stress

Abstract

is a promising microorganism for organic acid production. The present study aimed to investigate the role of Mediator complex subunit 3 (Med3p) in protecting under low-pH conditions. To this end, genes and were deleted, resulting in the double-deletionΔ strain. The final biomass and cell viability levels of Δ decreased by 64.5% and 35.8%, respectively, compared to the wild-type strain results at pH 2.0. In addition, lack ofMed3ABp resulted in selective repression of a subset of genes in the lipid biosynthesis and metabolism pathways. Furthermore, C18:1, lanosterol, zymosterol, fecosterol, and ergosterol were 13.2%, 80.4%, 40.4%, 78.1%, and 70.4% less abundant, respectively, in the Δ strain. In contrast, the concentration of squalene increased by about 44.6-fold. As a result, membrane integrity, rigidity, and H-ATPase activity in the Δ strain were reduced by 62.7%, 13.0%, and 50.3%, respectively. In contrast, overexpression of increased the levels of C18:0, C18:1, and ergosterol by 113.2%, 5.9%, and 26.4%, respectively. Moreover, compared to the wild-type results, dry cell weight and pyruvate production increased, irrespective of pH buffering. These results suggest that regulates membrane composition, which in turn enables cells to tolerate low-pH stress. We propose that regulation ofMed3ABp may provide a novel strategy for enhancing low-pH tolerance and increasing organic acid production by The objective of this study was to investigate the role of Mediator complex subunit 3 (Med3ABp) and its regulation of gene expression at low pH in We found thatMed3ABp was critical for cellular survival and pyruvate production during low-pH stress. Measures of the levels of plasma membrane fatty acids and sterol composition indicated that Med3ABp could play an important role in regulating homeostasis in We propose that controlling membrane lipid composition may enhance the robustness of for the production of organic acids.

Engineering of membrane complex sphingolipids improves osmotic tolerance of Saccharomyces cerevisiae

“…For example, when sugar and ion transporter, OmpF, was deleted, and the long chain fatty acids transporter, FadL, was overexpressed in E. coli , membrane integrity was enhanced and the fatty acid titer improved (39); (ii) Phospholipids: Membrane integrity can also be altered by modifying the distribution of phospholipid head groups, by adjusting phospholipid saturation, and by altering phospholipid acyl chain length (14, 40). For instance, when pssA, a phosphatidylserine synthase, was overexpressed, PE content increased and membrane integrity was enhanced, as a result of which bio-renewable fuel tolerance and titer was improved (12); (iii) Sterols: Sterol serves as an essential cell membrane component, where deletion of CgMed3AB decreased lanosterol, zymosterol, fecosterol, and ergosterol content, thereby reducing the membrane integrity of Candida glabrata (19); (iv) Sphingolipids: Sphingolipid metabolism plays an important role in membrane integrity (41). When sphingolipid biosynthesis genes were deleted in S. cerevisiae , the resultant strains exhibited resistance to amphipathic peptidomimetic LTX109, which decreased membrane integrity (41).…”

“…For example, through expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa , the tufa were incorporated into the E. coli membrane, decreasing membrane fluidity, as a result of which robustness and the bio-renewable fuel titer were improved (16). The content and compositions of sterols can be changed by engineering key enzymes associated with sterol biosynthesis or by changing the transcription levels of sterol biosynthesis enzymes, which are affected by global transcription factors, such as Upc2 and Ecm22, and mediators, such as CgMed3AB (17-19). For example, the expression of a key sterol C-5 desaturase FvC5SD from an edible mushroom in fission yeast, improved the ergosterol and oleic acid contents, which resulted in enhanced tolerance to ethanol and high temperature (20).…”

Candida glabrata Med3 Plays a Role in Altering Cell Size and Budding Index To Coordinate Cell Growth

“…ScMed3 is related to various stress responses, including H 2 O 2 (12), rapamycin (12), and osmotic stress (26), whereas CaMed3 determines the nuclear localization of TLOs and correlates with virulence of C. albicans (21). In addition, CgMed3 helps cells tolerate acid stress (13). Although ScMed3, CaMed3, and CgMed3 are orthologues, the amino acid sequence of CgMed3 shares only 35.7% and 30.7% similarity with those of S. cerevisiae and C. albicans, respectively.…”

“…ScMed3 responds to stress by modulating subunit composition (12) and stress-related gene expression. CgMed3 regulates the acid stress response by changing the membrane lipid composition (13). The difference between ScMed3 and CgMed3 stress response mechanisms may be attributed to a discrepancy between their amino acid sequences.…”

“…The strains used in this study are listed in Table 1, and all the strains were manipulated by standard genetic techniques under a C. glabrata ATCC 55 background. The temperaturesensitive strain acs2-ts was constructed following a previously described protocol (56), and the other knockout strains were constructed by homologous recombination (13). The gene expression strengths of Cgmed3ab were divided into three levels: strong in the TEF promoter of plasmid pY26, middle in the GPD promoter of plasmid pY26, and low in the native promoter of Cgmed3ab.…”