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Strain design of Ashbya gossypii for single-cell oil production.

ABSTRACT: Single-cell oil (SCO) represents a sustainable alternative for the oil industry. Accordingly, the identification of microorganisms with either higher lipidogenic ability or novel capacities for the transformation of raw materials constitutes a major challenge for the field of oil biotechnology. With this in mind, here, we were prompted to address the lipidogenic profile of the filamentous hemiascomycete Ashbya gossypii, which is currently used for the microbial production of vitamins. We found that A. gossypii mostly accumulates unsaturated fatty acids (FAs), with more than 50% of the total FA content corresponding to oleic acid. In addition, we engineered A. gossypii strains both lacking the beta-oxidation pathway and also providing ATP-citrate lyase (ACL) activity to block the degradation of FA and to increase the cytosolic acetyl-coenzyme A (CoA) content, respectively. The lipidogenic profile of the newly developed strains demonstrates that the mere elimination of the beta-oxidation pathway in A. gossypii triggers a significant increase in lipid accumulation that can reach 70% of cell dry weight. The use of A. gossypii as a novel and robust tool for the production of added-value oils is further discussed.

SUBMITTER: Ledesma-Amaro R

PROVIDER: S-EPMC3911055 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Strain design of Ashbya gossypii for single-cell oil production.

Ledesma-Amaro Rodrigo R Santos María A MA Jiménez Alberto A Revuelta José Luis JL

Applied and environmental microbiology 20131206 4

Single-cell oil (SCO) represents a sustainable alternative for the oil industry. Accordingly, the identification of microorganisms with either higher lipidogenic ability or novel capacities for the transformation of raw materials constitutes a major challenge for the field of oil biotechnology. With this in mind, here, we were prompted to address the lipidogenic profile of the filamentous hemiascomycete Ashbya gossypii, which is currently used for the microbial production of vitamins. We found t ...[more]

PMID: 24317081

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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.

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.

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Strain design of Ashbya gossypii for single-cell oil production.

Publications

Strain design of Ashbya gossypii for single-cell oil production.

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