Production of Rhizopus oryzae lipase using optimized Yarrowia lipolytica expression system.

Yarrowia lipolytica is an alternative yeast for heterologous protein production. Based on auto-cloning vectors, a set of 18 chromogenic cloning vectors was developed, each containing one of the excisable auxotrophic selective markers URA3ex, LYS5ex, and LEU2ex, and one of six different promoters: the constitutive pTEF, the phase dependent hybrid pHp4d, and the erythritol-inducible promoters from pEYK1 and pEYL1 derivatives. These vectors allowed to increase the speed of cloning of the gene of interest. In parallel, an improved new rProt recipient strain JMY8647 was developed by abolishing filamentation and introducing an auxotrophy for lysine (Lys-), providing an additional marker for genetic engineering. Using this cloning strategy, the optimal targeting sequence for Rhizopus oryzae ROL lipase secretion was determined. Among the eight targeting sequences, the SP6 signal sequence resulted in a 23% improvement in the lipase activity compared to that obtained with the wild-type ROL signal sequence. Higher specific lipase activities were obtained using hybrid erythritol-inducible promoters pHU8EYK and pEYL1-5AB, 1.9 and 2.2 times, respectively, when compared with the constitutive pTEF promoter. Two copy strains produce a 3.3 fold increase in lipase activity over the pTEF monocopy strain (266.7 versus 79.7 mU/mg).

How do engineered Yarrowia lipolytica strains secrete free fatty acids: hints from comparative transcriptomics.

Yarrowia lipolytica has been considered one of the most promising platforms for the microbial production of fatty acids and derived products. The deletion of the faa1 gene coding for an acyl-CoA synthetase leads to the accumulation and secretion of free fatty acids (FFAs) into the extracellular space. The secretion of products is beneficial for the development of microbial cell factories to avoid intracellular inhibitory effects and reduce downstream processing costs. However, the mechanism behind the secretion of fatty acids is not well known. As a starting point, we compared the transcriptome of this mutant showing FFA secretion to a wildtype-like strain not showing this phenotype. The 12 most upregulated genes were evaluated for involvement in FFA secretion by the creation of deletion and overexpression mutants, among them MCH2, YMOH, three cell wall proteins CWP3, CWP4, and CWP11, M12B, and three proteins with unknown functions YUP1, YUP2, and YUP3. None of these proteins take a clear or isolated role in FFA export. As the transcriptomic data revealed an overrepresentation of cell wall-related proteins, some of them were further examined on a theoretical and experimental way. Surprisingly, overexpression of Ygpi led to the production of FFAs in the wildtype-like genetic background. Finally, some of the evaluated genes showed involvement in resistance to FFA toxicity.

Bidirectional hybrid erythritol-inducible promoter for synthetic biology in Yarrowia lipolytica.

BACKGROUND: The oleaginous yeast Yarrowia lipolytica is increasingly used as a chassis strain for generating bioproducts. Several hybrid promoters with different strengths have been developed by combining multiple copies of an upstream activating sequence (UAS) associated with a TATA box and a core promoter. These promoters display either constitutive, phase-dependent, or inducible strong expression. However, there remains a lack of bidirectional inducible promoters for co-expressing genes in Y. lipolytica. RESULTS: This study built on our previous work isolating and characterizing the UAS of the erythritol-induced genes EYK1 and EYD1 (UAS-eyk1). We found an erythritol-inducible bidirectional promoter (BDP) located in the EYK1-EYL1 intergenic region. We used the BDP to co-produce YFP and RedStarII fluorescent proteins and demonstrated that the promoter's strength was 2.7 to 3.5-fold stronger in the EYL1 orientation compared to the EYK1 orientation. We developed a hybrid erythritol-inducible bidirectional promoter (HBDP) containing five copies of UAS-eyk1 in both orientations. It led to expression levels 8.6 to 19.2-fold higher than the native bidirectional promoter. While the BDP had a twofold-lower expression level than the strong constitutive TEF promoter, the HBDP had a 5.0-fold higher expression level when oriented toward EYL1 and a 2.4-fold higher expression level when oriented toward EYK1. We identified the optimal media for BDP usage by exploring yeast growth under microbioreactor conditions. Additionally, we constructed novel Golden Gate biobricks and a destination vector for general use. CONCLUSIONS: In this research, we developed novel bidirectional and hybrid bidirectional promoters of which expression can be fine-tuned, responding to the need for versatile promoters in the yeast Y. lipolytica. This study provides effective tools that can be employed to smoothly adjust the erythritol-inducible co-expression of two target genes in biotechnology applications. BDPs developed in this study have potential applications in the fields of heterologous protein production, metabolic engineering, and synthetic biology.

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

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.

Bioproducts generation from carboxylate platforms by the non-conventional yeast Yarrowia lipolytica.

In recent years, there has been a growing interest in the use of renewable sources for bio-based production aiming at developing sustainable and feasible approaches towards a circular economy. Among these renewable sources, organic wastes (OWs) can be anaerobically digested to generate carboxylates like volatile fatty acids (VFAs), lactic acid, and longer-chain fatty acids that are regarded as novel building blocks for the synthesis of value-added compounds by yeasts. This review discusses on the processes that can be used to create valuable molecules from OW-derived VFAs; the pathways employed by the oleaginous yeast Yarrowia lipolytica to directly metabolize such molecules; and the relationship between OW composition, anaerobic digestion, and VFA profiles. The review also summarizes the current knowledge about VFA toxicity, the pathways by which VFAs are metabolized and the metabolic engineering strategies that can be employed in Y. lipolytica to produce value-added biobased compounds from VFAs.

Macrothrombocytopenia and dense granule deficiency associated with FLI1 variants: ultrastructural and pathogenic features.

Congenital macrothrombocytopenia is a family of rare diseases, of which a significant fraction remains to be genetically characterized. To analyze cases of unexplained thrombocytopenia, 27 individuals from a patient cohort of the Bleeding and Thrombosis Exploration Center of the University Hospital of Marseille were recruited for a high-throughput gene sequencing study. This strategy led to the identification of two novel FLI1 variants (c.1010G>A and c.1033A>G) responsible for macrothrombocytopenia. The FLI1 variant carriers' platelets exhibited a defect in aggregation induced by low-dose adenosine diphosphate (ADP), collagen and thrombin receptor-activating peptide (TRAP), a defect in adenosine triphosphate (ATP) secretion, a reduced mepacrine uptake and release and a reduced CD63 expression upon TRAP stimulation. Precise ultrastructural analysis of platelet content was performed using transmission electron microscopy and focused ion beam scanning electron microscopy. Remarkably, dense granules were nearly absent in the carriers' platelets, presumably due to a biogenesis defect. Additionally, 25-29% of the platelets displayed giant α-granules, while a smaller proportion displayed vacuoles (7-9%) and autophagosome-like structures (0-3%). In vitro study of megakaryocytes derived from circulating CD34+ cells of the carriers revealed a maturation defect and reduced proplatelet formation potential. The study of the FLI1 variants revealed a significant reduction in protein nuclear accumulation and transcriptional activity properties. Intraplatelet flow cytometry efficiently detected the biomarker MYH10 in FLI1 variant carriers. Overall, this study provides new insights into the phenotype, pathophysiology and diagnosis of FLI1 variant-associated thrombocytopenia.

Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors.

Variants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants, and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to corepressors. We also observed a large expansion of megakaryocyte colony-forming units derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34+ cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton CDC42 and RHOA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, and spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34+ cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts, while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production, and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels.

Lipid Readjustment in Yarrowia lipolytica Odd-Chain Fatty Acids Producing Strains.

Yarrowia lipolytica is a promising oleaginous yeast for producing unusual lipids, such as odd-chain fatty acids (OCFA). Their diverse applications and low natural production make OCFA particularly interesting. In recent studies, inhibiting the catabolic pathway of precursor, boosting precursor pools, and optimizing substrate combination greatly improved the production of OCFA in Y. lipolytica. We explored the lipid readjustment of OCFA in engineered Y. lipolytica strains. NPLC-Corona-CAD® evidenced a time-dependent overproduction of free fatty acids, diglycerides, and phosphatidylcholine (PC) in obese LP compared to obese L. Phosphatidylethanolamine (PE) and phosphatidylinositol, largely overproduced in obese LP at 72 h compared to obese L, vanished at 216 h. The fatty acyls (FAs) composition of glycero- and glycerophospholipids was determined by NPLC-APPI+-HRMS from in-source generated monoacylglycerol-like fragment ions. C18:1 and C17:1 were predominant acylglycerols in obese L and obese LP, respectively. Phosphatidic acid, PE, and PC exhibited similar FAs composition but differed in their molecular species distributions. Cardiolipin (CL) is known to contain mostly C18:2 FAs corresponding to the composition in obese L, 50% of C18:2, and 35% of C18:1. In obese LP, both FAs dropped to drop to 20%, and C17:1 were predominant, reaching 55%. We hypothesize that CL-modified composition in obese LPs may alter mitochondrial function and limit lipid production.