In this work, genome hunting strategy was adopted in screening for reductases from Candida glabrata . A total of 37 putative reductases were successfully expressed in E. coli BL21(DE3). A substrate library containing 32 substrates was established for characterization of each reductase by average specific activity and Shannon-Wiener index. Among them, Cg26 was identified with the highest efficiency and wider substrate spectrum in the reduction of prochiral ketones, with average activity and Shannon-Wiener index of 8.95 U·mg −1 and 2.82. Cg26 is a member of ‘extended’ short chain dehydrogenase/reductase superfamily. Ni 2+ could improve its activity. As much as 150 g·L −1 ethyl 2-oxo-4-phenylbutyrate could be completely converted by 10 g·L −1 Cg26. This study provides evidence for this newly identified Cg26 in the preparation of chiral secondary alcohols.
Yanyu Su,Yi Li,Meng Xiao,Dan Guo,David S. Perlin,Erika Shor,Yingchun Xu,Yingxing Li
Antifungal heteroresistance has emerged as a clinical challenge across diverse species. In Candida glabrata , however, it has rarely been reported. Here, we characterized heteroresistance in a multicenter collection of 156 C. glabrata isolates, revealing a 25% prevalence with caspofungin specificity and 37°C dependent phenotypes. Transcriptomic profiling of the heteroresistant subpopulation under escalating drug pressure revealed an adaptive program centered on cell cycle and cell wall integrity, including several members of the calcineurin pathway. Mechanically, we identified that the phenotype was abolished by pharmacological inhibition or Δ cnb1 , Δ crz1 genetic deletion, confirming the regulatory role of calcineurin. Crucially, heteroresistance functioned as a reservoir for resistance, with in vitro descendant strains spanning a spectrum of MIC alterations. Mechanistically, we found the heteroresistance phenotype mostly independent of aneuploidy. To decipher the genetic basis, we applied an unbiased machine-learning framework to genomic data, which not only identified the canonical FKS2_F659del mutation but also prioritized novel candidate PIR2_G149_I167del , demonstrating its power to uncover drivers of resistance from complex datasets. In summary, our study established a stepwise model of heteroresistance in C. glabrata , wherein calcineurin serves as a master regulator that promotes a resistance reservoir, revealing a potential vulnerability that could be exploited to prevent treatment failure.
d -(+)-pantothenic acid (DPA) has been widely used in feed and food additives and pharmaceutical industries. d -(−)-pantolactone ( D PL) is a key intermediate in the production of DPA . The bio-asymmetric synthesis of D PL from ketopantoyl lactone (KPL) by conjugated polyketone reductases is an attractive route. In this study, high expression of a conjugated polyketone reductase from Candida glabrata ( Cgl CPR) in Escherichia coli was achieved after optimization of vectors. The optimum temperature and pH of purified Cgl CPR were 40 °C and 4.5, respectively. The Michaelis-Menten constant K m and V max of purified Cgl CPR for KPL are 73.09 μM and 2.29 μmol mg −1 s −1 , respectively. To circumvent the spontaneous degradation of KPL in biocatalytic reaction of synthesizing DPL, the reaction was performed by a strategy combing fed-batch addition of whole cell catalyst with substrate semi-continuous feeding. At the final KPL loading of 1000 mM, DPL with an isolated yield of 94.24 % was obtained after 13-h reaction and enantiomeric excess ( e.e. ) achieved > 99 %. The space-time yield of DPL reached to 228 g L −1 d −1 .
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