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Limited functional redundancy and oscillation of cyclins in multinucleated Ashbya gossypii fungal cells.

ABSTRACT: Cyclin protein behavior has not been systematically investigated in multinucleated cells with asynchronous mitoses. Cyclins are canonical oscillating cell cycle proteins, but it is unclear how fluctuating protein gradients can be established in multinucleated cells where nuclei in different stages of the division cycle share the cytoplasm. Previous work in A. gossypii, a filamentous fungus in which nuclei divide asynchronously in a common cytoplasm, demonstrated that one G1 and one B-type cyclin do not fluctuate in abundance across the division cycle. We have undertaken a comprehensive analysis of all G1 and B-type cyclins in A. gossypii to determine whether any of the cyclins show periodic abundance across the cell cycle and to examine whether cyclins exhibit functional redundancy in such a cellular environment. We localized all G1 and B-type cyclins and notably found that only AgClb5/6p varies in subcellular localization during the division cycle. AgClb5/6p is lost from nuclei at the meta-anaphase transition in a D-box-dependent manner. These data demonstrate that efficient nuclear autonomous protein degradation can occur within multinucleated cells residing in a common cytoplasm. We have shown that three of the five cyclins in A. gossypii are essential genes, indicating that there is minimal functional redundancy in this multinucleated system. In addition, we have identified a cyclin, AgClb3/4p, that is essential only for sporulation. We propose that the cohabitation of different cyclins in nuclei has led to enhanced substrate specificity and limited functional redundancy within classes of cyclins in multinucleated cells.

SUBMITTER: Hungerbuehler AK

PROVIDER: S-EPMC1828934 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAshbya gossypii is a filamentous Saccharomycete used for the industrial production of riboflavin that has been recently explored as a host system for recombinant protein production. To gain insight into the protein secretory pathway of this biotechnologically relevant fungus, we undertook genome-wide analyses to explore its secretome and its transcriptional responses to protein secretion stress.ResultsA computational pipeline was used to predict the inventory of proteins putatively secreted by A. gossypii via the general secretory pathway. The proteins actually secreted by this fungus into the supernatants of submerged cultures in minimal and rich medium were mapped by two-dimensional gel electrophoresis, revealing that most of the A. gossypii secreted proteins have an isoelectric point between 4 and 6, and a molecular mass above 25 kDa. These analyses together indicated that 1-4% of A. gossypii proteins are likely to be secreted, of which less than 33% are putative hydrolases. Furthermore, transcriptomic analyses carried out in A. gossypii cells under recombinant protein secretion conditions and dithiothreitol-induced secretion stress unexpectedly revealed that a conventional unfolded protein response (UPR) was not activated in any of the conditions, as the expression levels of several well-known UPR target genes (e.g. IRE1, KAR2, HAC1 and PDI1 homologs) remained unaffected. However, several other genes involved in protein unfolding, endoplasmatic reticulum-associated degradation, proteolysis, vesicle trafficking, vacuolar protein sorting, secretion and mRNA degradation were up-regulated by dithiothreitol-induced secretion stress. Conversely, the transcription of several genes encoding secretory proteins, such as components of the glycosylation pathway, was severely repressed by dithiothreitolConclusionsThis study provides the first insights into the secretion stress response of A. gossypii, as well as a basic understanding of its protein secretion potential, which is more similar to that of yeast than to that of other filamentous fungi. Contrary to what has been widely described for yeast and fungi, a conventional UPR was not observed in A. gossypii, but alternative protein quality control mechanisms enabled it to cope with secretion stress. These data will help provide strategies for improving heterologous protein secretion in A. gossypii.

Project description:BackgroundLimonene is a cyclic monoterpene that has applications in the food, cosmetic, and pharmaceutical industries. The industrial production of limonene and its derivatives through plant extraction presents important drawbacks such as seasonal and climate issues, feedstock limitations, low efficiency and environmental concerns. Consequently, the implementation of efficient and eco-friendly bioprocesses for the production of limonene and other terpenes constitutes an attractive goal for microbial biotechnology. In this context, novel biocatalysts with the ability to produce limonene from alternative carbon sources will help to meet the industrial demands of limonene.ResultsEngineered strains of the industrial fungus Ashbya gossypii have been developed to produce limonene from xylose. The limonene synthase (LS) from Citrus limon was initially overexpressed together with the native HMG1 gene (coding for HMG-CoA reductase) to establish a limonene-producing platform from a xylose-utilizing A. gossypii strain. In addition, several strategies were designed to increase the production of limonene. Hence, the effect of mutant alleles of ERG20 (erg20F95W and erg20F126W) were evaluated together with a synthetic orthogonal pathway using a heterologous neryl diphosphate synthase. The lethality of the A. gossypii double mutant erg20F95W-F126W highlights the indispensability of farnesyl diphosphate for the synthesis of essential sterols. In addition, the utilization of the orthogonal pathway, bypassing the Erg20 activity through neryl diphosphate, triggered a substantial increase in limonene titer (33.6 mg/L), without critically altering the fitness of the engineered strain. Finally, the overexpression of the native ERG12 gene further enhanced limonene production, which reached 336.4 mg/L after 96 h in flask cultures using xylose as the carbon source.ConclusionsThe microbial production of limonene can be carried out using engineered strains of A. gossypii from xylose-based carbon sources. The utilization of a synthetic orthogonal pathway together with the overexpression of ERG12 is a highly beneficial strategy for the production of limonene in A. gossypii. The strains presented in this work constitute a proof of principle for the production of limonene and other terpenes from agro-industrial wastes such as xylose-rich hydrolysates in A. gossypii.

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Limited functional redundancy and oscillation of cyclins in multinucleated Ashbya gossypii fungal cells.

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