Zcf24, a zinc-finger transcription factor, is required for lactate catabolism and inhibits commensalism in Candida albicans.

Candida albicans is a normal resident of humans and also a prevalent fungal pathogen. Lactate, a nonfermentative carbon source available in numerous anatomical niches, can be used by C. albicans as a carbon source. However, the key regulator(s) involved in this process remain unknown. Here, through a genetic screen, we report the identification of a transcription factor Zcf24 that is specifically required for lactate utilization in C. albicans. Zcf24 is responsible for the induction of CYB2, a gene encoding lactate dehydrogenase that is essential for lactate catabolism, in response to lactate. Chromatin immunoprecipitation showed a significantly higher signal of Zcf24 on the CYB2 promoter in lactate-grown cells than that in glucose-grown cells. Genome-wide transcription profiling indicates that, in addition to CYB2, Zcf24 regulates genes involved in the ß-oxidation of fatty acids, iron transport, and drug transport. Surprisingly, deleting ZCF24 confers enhanced commensal fitness. This could be attributed to Crz1-activated ß-glucan masking in the zcf24 mutant. The orthologs of Zcf24 are distributed in species most closely to C. albicans and some filamentous fungal species. Altogether, Zcf24 is the first transcription factor identified to date that regulates lactate catabolism in C. albicans and it is also involved in the regulation of commensalism.

Flavocytochrome b2 of the Methylotrophic Yeast Ogataea polymorpha: Construction of Overproducers, Purification, and Bioanalytical Application.

Flavocytochrome b2 (EC 1.1.2.3; L-lactate cytochrome: c oxidoreductase, FC b2) from the thermotolerant methylotrophic yeast Ogataea polymorpha is a thermostable enzyme-prospective for a highly selective L-lactate analysis in the medicine, nutrition sector, and quality control of commercial products. Here we describe the construction of FC b2 producers by overexpression of the CYB2 gene O. polymorpha, encoding FC b2, under the control of a strong alcohol oxidase promoter in the frame of plasmid for multicopy integration with the next transformation of recipient strain O. polymorpha C-105 (gcr1 catX) impaired in the glucose repression and devoid of catalase activity. The selected recombinant strain O. polymorpha "tr1" (gcr1 catX CYB2), characterized by eightfold increased FC b2 activity compared to the initial strain, was used as a source of the enzyme. For purification of FC b2 a new method of affinity chromatography was developed and purified preparations of the enzyme were used for the construction of the highly selective enzymatic kits and amperometric biosensor for L-lactate analysis in human liquids and foods.

Culture-based analysis of fungi in leaves after the primary and secondary fermentation processes during Ishizuchi-kurocha production and lactate assimilation of P. kudriavzevii.

Ishizuchi-kurocha is a Japanese traditional fermented tea that is produced by primary aerobic and secondary fermentation steps. The secondary fermentation step of Ishizuchi-kurocha is mainly mediated through lactic acid bacteria. Here, we performed quantitative analyses of the culturable fungal communities at each step and identified several morphologically representative fungal isolates. While filamentous fungi (median, 3.2 × 107 CFU/g sample) and yeasts (median, 3.7 × 107 CFU/g) were both detected after the primary fermentation step, only yeasts (median, 1.6 × 107 CFU/g) were detected in the end of the secondary fermentation step, suggesting that the fungal community in tea leaves are dramatically changed between the two steps. Pichia kudriavzevii and Pichia manshurica, the prevalent fungal species at the end of the secondary fermentation step, grew well in exudate from the secondary fermentation step. P. kudriavzevii also grew well in media containing d- or l-lactate as the sole carbon source. The growth of the disruptant of cyb2A encoding a cytochrome b2 lactate dehydrogenase in P. kudriavzevii was severely impaired on medium supplemented with l-lactate, but not d-lactate, suggesting that Cyb2Ap plays a crucial role in the use of l-lactate, and P. kudriavzevii efficiently uses both l- and d-lactate as carbon sources. Thus, lactate assimilation seems to be a key phenotype to become a prevalent species in the secondary fermentation step, and Cyb2Ap has a pivotal role in l-lactate metabolism in P. kudriavzevii. Further understanding and engineering of P. kudriavzevii and P. manshurica will contribute to the control of lactic acid bacteria fermentation during the fermented tea production and also to other industrial uses.