Effectively Improve the Astaxanthin Production by Combined Additives Regulating Different Metabolic Nodes in Phaffia rhodozyma

Astaxanthin is an important natural resource that is widely found in marine environments. Metabolic regulation is an effective method for improving astaxanthin production in Phaffia rhodozyma. Most studies have focused on single regulators, which have limited effects. In this study, 16 metabolic regulators were screened to improve astaxanthin production in high-yield and wild-type strains. Fluconazol and glutamic acid increased astaxanthin volumetric yield in MVP14 by 25.8 and 30.9%, respectively, while ethanol increased astaxanthin volumetric yield in DSM626, 29.3%. Furthermore, six additives that inhibit the competing pathways and promote the main pathway for astaxanthin synthesis were selected for combination treatment. We found that the optimal combination was penicillin, ethanol, triclosan, and fluconazol, which increased astaxanthin cell yield by 51%. Therefore, we suggest that simultaneously promoting the master pathways (mevalonate) and inhibiting competing pathways (fatty acid synthesis and ergosterol) is the best strategy to improve astaxanthin cell yield. Moreover, regulators of the biomass pathway should be avoided to improve cell yield. This study provides a technical basis for the utilisation of astaxanthin in P. rhodozyma.

Aprimoramento da etapa downstream para a extração de carotenoides produzidos pela levedura Phaffia rhodozyma

Os carotenoides são a classe de pigmentos mais difundida na natureza. Possuem alta demanda de mercado devido as suas propriedades benéficas a saúde, resultando em aumento nas pesquisas referentes à sua produção via biotecnológica, podendo estes, serem produzidos por diferentes micro-organismos como a levedura Phaffia rhodozyma. Para tanto, pesquisas são necessárias para avaliar métodos e técnicas de extração e recuperação mais eficientes destes produtos. Neste contexto, este estudo propõe avaliar diferentes pré-tratamentos na biomassa de P. rhodozyma e diferentes solventes para a recuperação de carotenoides. Para tal, três pré-tratamentos na biomassa foram avaliados (secagem 24 h – congelamento 24 h; secagem 48 h – congelamento 48 h e liofilização). A utilização de hexano e éter de petróleo como solvente para a obtenção do extrato carotenogênico também foi verificada. Com o aprimoramento da metodologia, obteve-se máxima concentração específica (245,71 ± 6,08 µg g-1) e volumétrica (1803,50 ± 36,46 µg L-1) de carotenoides utilizando éter de petróleo no pré-tratamento com secagem 24 h (35 °C) e congelamento 24 h (-18 °C). Este resultado mostra que no ensaio 24 h a quantidade de carotenoides extraído da biomassa é 23,3% mais eficiente que o método usualmente utilizado (secagem 48 h - congelamento 48 h), reduzindo o tempo de processo em 48 h.

Multiple Pathways to Homothallism in Closely Related Yeast Lineages in the Basidiomycota

ABSTRACT Sexual reproduction in fungi relies on proteins with well-known functions encoded by the mating type (MAT) loci. In the Basidiomycota, MAT loci are often bipartite, with the P/R locus encoding pheromone precursors and pheromone receptors and the HD locus encoding heterodimerizing homeodomain transcription factors (Hd1/Hd2). The interplay between different alleles of these genes within a single species usually generates at least two compatible mating types. However, a minority of species are homothallic, reproducing sexually without an obligate need for a compatible partner. Here, we examine the organization and function of the MAT loci of Cystofilobasidium capitatum, a species in the order Cystofilobasidiales, which is unusually rich in homothallic species. We determined MAT gene content and organization in C. capitatum and found that it resembles a mating type of the closely related heterothallic species Cystofilobasidium ferigula. To explain the homothallic sexual reproduction observed in C. capitatum, we examined HD protein interactions in the two Cystofilobasidium species and determined C. capitatum MAT gene expression both in a natural setting and upon heterologous expression in Phaffia rhodozyma, a homothallic species belonging to a clade sister to that of Cystofilobasidium. We conclude that the molecular basis for homothallism in C. capitatum appears to be distinct from that previously established for P. rhodozyma. Unlike in the latter species, homothallism in C. capitatum may involve constitutive activation or dispensability of the pheromone receptor and the functional replacement of the usual Hd1/Hd2 heterodimer by an Hd2 homodimer. Overall, our results suggest that homothallism evolved multiple times within the Cystofilobasidiales. IMPORTANCE Sexual reproduction is important for the biology of eukaryotes because it strongly impacts the dynamics of genetic variation. In fungi, although sexual reproduction is usually associated with the fusion between cells belonging to different individuals (heterothallism), sometimes a single individual is capable of completing the sexual cycle alone (homothallism). Homothallic species are unusually common in a fungal lineage named Cystofilobasidiales. Here, we studied the genetic bases of homothallism in one species in this lineage, Cystofilobasidium capitatum, and found it to be different in several aspects from those of another homothallic species, Phaffia rhodozyma, belonging to the genus most closely related to Cystofilobasidium. Our results strongly suggest that homothallism evolved independently in Phaffia and Cystofilobasidium, lending support to the idea that transitions between heterothallism and homothallism are not as infrequent as previously thought. Our work also helps to establish the Cystofilobasidiales as a model lineage in which to study these transitions.