Engineered P450 BM3 and cpADH5 coupled cascade reaction for ß-oxo fatty acid methyl ester production in whole cells.

Hydroxy- or ketone- functionalized fatty acid methyl esters (FAMEs) are important compounds for production of pharmaceuticals, vitamins, cosmetics or dietary supplements. Biocatalysis through enzymatic cascades has drawn attention to the efficient, sustainable, and greener synthetic processes. Furthermore, whole cell catalysts offer important advantages such as cofactor regeneration by cell metabolism, omission of protein purification steps and increased enzyme stability. Here, we report the first whole cell catalysis employing an engineered P450 BM3 variant and cpADH5 coupled cascade reaction for the biosynthesis of hydroxy- and keto-FAMEs. Firstly, P450 BM3 was engineered through the KnowVolution approach yielding P450 BM3 variant YE_M1_2, (R47S/Y51W/T235S/N239R/I401 M) which exhibited boosted performance toward methyl hexanoate. The initial oxidation rate of YE_M1_2 toward methyl hexanoate was determined to be 23-fold higher than the wild type enzyme and a 1.5-fold increase in methyl 3-hydroxyhexanoate production was obtained (YE_M1_2; 2.75 mM and WT; 1.8 mM). Subsequently, the whole cell catalyst for the synthesis of methyl 3-hydroxyhexanoate and methyl 3-oxohexanoate was constructed by combining the engineered P450 BM3 and cpADH5 variants in an artificial operon. A 2.06 mM total product formation was achieved by the whole cell catalyst including co-expressed channel protein, FhuA and co-solvent addition. Moreover, the generated whole cell biocatalyst also accepted methyl valerate, methyl heptanoate as well as methyl octanoate as substrates and yielded ω-1 ketones as the main product.

Iron Metabolism, Pseudohypha Production, and Biofilm Formation through a Multicopper Oxidase in the Human-Pathogenic Fungus Candida parapsilosis.

Among all the essential micronutrients, iron plays an important role in mammalian biology. It is also essential for pathogens infecting mammalian hosts, including bacteria, fungi, and protozoans. As the availability of accessible iron is limited within the mammalian host, several human-pathogenic fungal pathogens, such as Candida albicans, Cryptococcus neoformans, Candida glabrata, and Aspergillus fumigatus, have developed various iron uptake mechanisms. Although Candida parapsilosis is the second or third most common non-albicans Candida species associated with systemic and superficial Candida infections in immunocompromised patients, the mechanisms of iron uptake and homoeostasis remain unknown in this fungus. In the current report, we show that a homologue of the multicopper oxidase gene FET3 is present in the genome of C. parapsilosis (CPAR2_603600) and plays a significant role in iron acquisition. We found that homozygous deletion mutants of CPAR2_603600 showed defects under low-iron conditions and were also sensitive to various stressors. Our results also revealed that the levels of pseudohypha formation and biofilm formation were reduced in the null mutants compared to the wild type. This phenotypic defect could be partially rescued by supplementation with excess iron in the growth medium. The expression levels of the orthologues of various iron metabolism-related genes were also altered in the mutants compared to the parental strain. In conclusion, our report describes the role of CPAR2_603600 in iron homoeostasis maintenance as well as morphology and biofilm formation regulation in this pathogenic fungus.IMPORTANCEC. parapsilosis is the second or third most common opportunistic human-pathogenic Candida species, being responsible for severe fungal infections among immunocompromised patients, especially low-birth-weight infants (0 to 2 years of age). Among the major virulence factors that pathogenic fungi possess is the ability to compete with the host for essential micronutrients, including iron. Accessible iron is required for the maintenance of several metabolic processes. In order to obtain accessible iron from the host, pathogenic fungi have developed several iron acquisition and metabolic mechanisms. Although C. parapsilosis is a frequent cause of invasive candidiasis, little is known about what iron metabolic processes this fungus possesses that could contribute to the species' virulent behavior. In this study, we identified the multicopper oxidase FET3 gene that regulates iron homeostasis maintenance and also plays important roles in the morphology of the fungus as well as in biofilm formation, two additional factors in fungal virulence.

Molecular Identification, Genotypic Diversity, Antifungal Susceptibility, and Clinical Outcomes of Infections Caused by Clinically Underrated Yeasts, Candida orthopsilosis, and Candida metapsilosis: An Iranian Multicenter Study (2014-2019).

Despite the increasing occurrence of Candida orthopsilosis and Candida metapsilosis in clinical settings, little is known about their microbiological and clinical properties. Herein, we conducted a national retrospective study (2014-2019) from multiple centers in Iran. Among the 1,770 Candida isolates collected, we identified 600 Candida parapsilosis species complex isolates. Isolate identification was performed by 9-plex PCR, matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS), and rDNA sequencing, and antifungal susceptibility testing (AFST) followed CLSI M27-A3/S4; genotyping was performed by amplified fragment length polymorphism (AFLP) analysis; and clinical information was mined. Thirty-one isolates of C. orthopsilosis from various clinical sources, one mixed sample (blood) concurrently containing C. orthopsilosis and C. parapsilosis and one isolate of C. metapsilosis from a nail sample were identified. Although both 9-plex PCR and MALDI-TOF successfully identified all isolates, only 9-plex PCR could identify the agents in a mixed sample. For the C. orthopsilosis isolates, resistance (non-wild type) was noted only for itraconazole (n = 4; 12.5%). Anidulafungin and fluconazole showed the highest and voriconazole had the lowest geometric mean values. AFLP analysis showed three main and four minor genotypes. Interestingly, 90% of nail isolates clustered with 80% of the blood isolates within two clusters, and four blood isolates recovered from four patients admitted to a hospital clustered into two genotypes and showed a high degree of similarity (>99.2%), which suggests that C. orthopsilosis disseminates horizontally. Supported by our data and published case studies, C. orthopsilosis and C. metapsilosis can be linked to challenging clinical failures, and successful outcomes are not always mirrored by in vitro susceptibility. Accordingly, conducting nationwide studies may provide more comprehensive data, which is required for a better prognosis and clinical management of patients.

Repurposing of Ribavirin as an Adjunct Therapy against Invasive Candida Strains in an In Vitro Study.

The use of antifungal agents in clinical settings is limited by the appearance of drug resistance and adverse side effects. There is, therefore, an urgent need to develop new drugs to strengthen the treatment of invasive fungal diseases. The aim of this study is to describe the potential repurposing of ribavirin as an adjunct therapy against Candida spp. Primary screening of a Prestwick Chemical library against Candida albicans ATCC 90028 and fluconazole-resistant Candida albicans strains was performed. Subsequently, we evaluated the responses of 100 Candida sp. strains to ribavirin, an antiviral agent, using the broth microdilution method as recommended by CLSI. We checked the involvement of efflux pump activity in the development of ribavirin resistance. We studied time-kill curves and performed a checkerboard assay for a ribavirin-antifungal combination study. Twenty-one nonstandard antifungal compounds were identified, including ribavirin. Ribavirin had antifungal activity in vitro against 63 Candida strains, including strains of C. albicans, C. parapsilosis, and C. tropicalis, with MICs ranging from 0.37 to 3.02 µg/ml, while MICs for C. krusei, C. glabrata, C. lusitaniae, and some C. albicans strains remained high (≥24.16 µg/ml). No relation was observed between efflux pump activity and ribavirin resistance. Ribavirin exhibited fungistatic activity against multidrug-resistant (MDR) C. albicans and fungicidal activity against a C. parapsilosis strain. In addition, ribavirin acted synergistically with azoles against Candida strains for which ribavirin MICs were <24.4 µg/ml. This study highlights the potential clinical application of ribavirin, alone or in association with other antifungal agents, as an adjunct anti-Candida drug.