A Yarrowia lipolytica Strain Engineered for Pyomelanin Production

International audienceThe yeast Yarrowia lipolytica naturally produces pyomelanin. This pigment accumulates in the extracellular environment following the autoxidation and polymerization of homogentisic acid, a metabolite derived from aromatic amino acids. In this study, we used a chassis strain optimized to produce aromatic amino acids for the de novo overproduction of pyomelanin. The gene 4HPPD, which encodes an enzyme involved in homogentisic acid synthesis (4-hydroxyphenylpyruvic acid dioxygenase), was characterized and overexpressed in the chassis strain with up to three copies, leading to pyomelanin yields of 4.5 g/L. Homogentisic acid is derived from tyrosine. When engineered strains were grown in a phenylalanine-supplemented medium, pyomelanin production increased, revealing that the yeast could convert phenylalanine to tyrosine, or that the homogentisic acid pathway is strongly induced by phenylalanine

Exon junction complex components Y14 and Mago still play a role in budding yeast

Abstract Since their divergence from Pezizomycotina, the mRNA metabolism of budding yeasts have undergone regressive evolution. With the dramatic loss of introns, a number of quality control mechanisms have been simplified or lost during evolution, such as the exon junction complex (EJC). We report the identification of the core EJC components, Mago, Y14, and eIF4A3, in at least seven Saccharomycotina species, including Yarrowia lipolytica. Peripheral factors that join EJC, either to mediate its assembly (Ibp160 or Cwc22), or trigger downstream processes, are present in the same species, forming an evolutionary package. Co-immunoprecipitation studies in Y. lipolytica showed that Mago and Y14 have retained the capacity to form heterodimers, which successively bind to the peripheral factors Upf3, Aly/REF, and Pym. Phenotypes and RNA-Seq analysis of EJC mutants showed evidence of Y14 and Mago involvement in mRNA metabolism. Differences in unspliced mRNA levels suggest that Y14 binding either interferes with pre-mRNA splicing or retains mRNA in the nucleus before their export and translation. These findings indicate that yeast could be a relevant model for understanding EJC function

A unique, newly discovered four-member protein family involved in extracellular fatty acid binding in Yarrowia lipolytica

International audienceAbstract Background Yarrowia lipolytica , a nonconventional oleaginous yeast species, has attracted attention due to its high lipid degradation and accumulation capacities. Y. lipolytica is used as a chassis for the production of usual and unusual lipids and lipid derivatives. While the genes involved in the intracellular transport and activation of fatty acids in different cellular compartments have been characterized, no genes involved in fatty acid transport from the extracellular medium into the cell have been identified thus far. In this study, we identified secreted proteins involved in extracellular fatty acid binding. Results Recent analysis of the Y. lipolytica secretome led to the identification of a multigene family that encodes four secreted proteins, preliminarily named UP1 to UP4. These proteins were efficiently overexpressed individually in wild-type and multideletant strain (Q4: Δup1Δup2Δup3Δup4 ) backgrounds. Phenotypic analysis demonstrated the involvement of these proteins in the binding of extracellular fatty acids. Additionally, gene deletion and overexpression prevented and promoted sensitivity to octanoic acid (C8) toxicity, respectively. The results suggested binding is dependent on aliphatic chain length and fatty acid concentration. 3D structure modeling supports the proteins’ role in fatty acid assimilation at the molecular level. Conclusions We discovered a family of extracellular-fatty-acid-binding proteins in Y. lipolytica and have proposed to name its members eFbp1 to eFbp4. The exact mode of eFbps action remains to be deciphered individually and synergistically; nevertheless, it is expected that the proteins will have applications in lipid biotechnology, such as improving fatty acid production and/or bioconversion

Additional file 1 of A unique, newly discovered four-member protein family involved in extracellular fatty acid binding in Yarrowia lipolytica

Additional file 1: Figure S1. Schematic representation of the destination vector JMP4230, a variant of JMP62 shuttle vector series, bearing the strong Erythritol-inducible promoter pHU8EYK1, tLip2 terminator, the excisable URA3ex auxotrophy selection marker, and zeta integration sites, flanking the expression cassette. Bacterial part bearing ori and kanamycin resistance marker was removed, prior to the yeast transformation, by NotI restriction enzyme digestion. Main unique restriction sites are indicated; ClaI-BamH1 for promotor exchange, BamH1-AvrII for gene cloning and I-SceI for marker exchange. Figure S2. The rank 1 to rank 5 models of UP1 structure independently predicted by AlphaFold were superimposed. Each chain is colored in blue to red from N-end to C end. The five long helices (residues 50-200 in matured protein) are consistently predicted in the same relative positions. The predicted structure of residue 1 to 50 are more variable between predictions. Table S1. List of primers used in this study

Functional classification of the metatranscriptome during surface-ripened cheese maturation.

<p>Functional classes were determined according to KEGG annotations. Read counts corresponding to all species were cumulated. Read numbers were normalized (according to the library size) to 50,000 reads per sampling day.</p

Reference genomes used for mapping of the sequence reads.

<p>Reference genomes used for mapping of the sequence reads.</p

Selection of genes showing a differential abundance between Day 1 and Day 14 and/or Day 14 and Day 31.

<p>The log2 fold change and the average number of reads (mean) are indicated only if the gene revealed a significant difference between the two ripening times (adjusted p-value < 0.05).</p><p>Selection of genes showing a differential abundance between Day 1 and Day 14 and/or Day 14 and Day 31.</p

Number of differentially expressed genes according to ripening time.

<p>Number of differentially expressed genes according to ripening time.</p

Sequencing coverage (C) and percentage of genes (P) with at least an average of five uniquely mapped reads in the DNA-Seq dataset across the three replicates for each microbial genome during ripening.

<p>^aAA = <i>Arthrobacter arilaitensis</i>; BA = <i>Brevibacterium aurantiacum</i>; CC = <i>Corynebacterium casei</i>; DH = <i>Debaryomyces hansenii</i>; GC = <i>Geotrichum candidum</i>; HA = <i>Hafnia alvei</i>; KL = <i>Kluyveromyces lactis</i>; LL = <i>Lactococcus lactis</i>; SE = <i>Staphylococcus equorum</i></p><p>Sequencing coverage (C) and percentage of genes (P) with at least an average of five uniquely mapped reads in the DNA-Seq dataset across the three replicates for each microbial genome during ripening.</p

Protein degradation during surface-ripened cheese maturation.

<p>(A) Proteolysis and free amino acid concentration. Expression data observed for genes encoding proteases (B) and peptidases (C). Read numbers were normalized (according to the library size) to 50,000 reads per sampling day. SE: <i>Staphylococcus equorum</i>. BA: <i>Brevibacterium aurantiacum</i>. AA: <i>Arthrobacter arilaitensis</i>. HA: <i>Hafnia alvei</i>. CC: <i>Corynebacterium casei</i>. LL: <i>Lactococcus lactis</i>. KL: <i>Kluyveromyces lactis</i>. DH: <i>Debaryomyces hansenii</i>. GC: <i>Geotrichum candidum</i>.</p