The 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.

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.

Yarrowia lipolytica has emerged as a well-established platform for producing a wide range of biomolecules, including recombinant proteins (rProteins). Its robust metabolism and resistance to various environmental stressors make it particularly well-suited as a microbial cell factory. However, additional physiological modifications are still required to fully meet industrial demands. Over years of strain development, Y. lipolytica has been engineered to carry auxotrophic markers, streamline the secretory pathway via deletion of native secretory proteins, prevent filamentation, and enable inducible gene expression systems. In this study, we continued the fine-tuning of Y. lipolytica as a platform for rProtein synthesis, building on previous work. Specifically, we: (i) introduced a third auxotrophy to facilitate more complex genetic engineering strategies, (ii) removed bacterial vector elements (including antibiotic resistance genes) from previous constructs, and (iii) carried out extensive deletions of extracellular proteases and a peroxidase gene. The newly constructed chassis strains, JMY9438 and JMY9451/9452, both bear triple auxotrophies. The latter strain additionally lacks proteolytic activity due to the deletion of five protease genes. We evaluated the rProtein production efficiency of these strains harboring one, two or three integrated copies of the target gene. rProtein expression levels increased with copy number up to two; however, no further improvement was observed with three copies. Notably, the strain with protease deletions and a single gene copy showed the highest rProtein production per cell, while the strain retaining proteases but harboring two copies yielded the highest absolute rProtein levels. We present a new generation of Y. lipolytica chassis strains specifically optimized for recombinant protein production. Our results demonstrate that extensive protease deletions can provide a high-performance genetic background, enabling high-level rProtein production without relying on multi-copy expression strategies.