Design of an efficient medium for heterologous protein production in Yarrowia lipolytica: case of human interferon alpha 2b

<p>Abstract</p> <p>Background</p> <p>The non conventional yeast <it>Yarrowia lipolytica </it>has aroused a strong industrial interest for heterologous protein production. However most of the studies describing recombinant protein production by this yeast rely on the use of complex media, such media are not convenient for large scale production particularly for products intended for pharmaceutical applications. In addition medium composition can also affect the production yield. Hence it is necessary to design an efficient medium for therapeutic protein expression by this host.</p> <p>Results</p> <p>Five different media, including four minimal media and a complex medium, were assessed in shake flasks for the production of human interferon alpha 2b (hIFN α2b) by <it>Y. lipolytica </it>under the control of POX2 promoter inducible with oleic acid. The chemically defined medium SM4 formulated by Invitrogen for <it>Pichia pastoris </it>growth was the most suitable. Using statistical experimental design this medium was further optimized. The selected minimal medium consisting in SM4 supplemented with 10 mg/l FeCl₃, 1 g/l glutamate, 5 ml/l PTM1 (<it>Pichia </it>Trace Metals) solution and a vitamin solution composed of myo-inositol, thiamin and biotin was called GNY medium. Compared to shake flask, bioreactor culture in GNY medium resulted in 416-fold increase of hIFN α2b production and 2-fold increase of the biological activity.</p> <p>Furthermore, SM4 enrichment with 5 ml/l PTM1 solution contributed to protect hIFN α2b against the degradation by the 28 kDa protease identified by zymography gel in culture supernatant. The screening of the inhibitory effect of the trace elements present in PTM1 solution on the activity of this protease was achieved using a Box-Behnken design. Statistical data analysis showed that FeCl₃and MnSO₄had the most inhibitory effect.</p> <p>Conclusion</p> <p>We have designed an efficient medium for large scale production of heterologous proteins by <it>Y. lipolytica</it>. The optimized medium GNY is suitable for the production of hIFN α2b with the advantage that no complex nitrogen sources with non-defined composition were required.</p

Integrated fermentative process for lipid and β-carotene production from acetogenic syngas fermentation using an engineered oleaginous Yarrowia lipolytica yeast

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG[Abstract] An engineered Yarrowia lipolytica strain was successfully employed to produce β-carotene and lipids from acetic acid, a product of syngas fermentation by Clostridium aceticum. The strain showed acetic acid tolerance up to concentrations of 20 g/L. Flask experiments yielded a peak lipid content of 33.7 % and β-carotene concentration of 13.6 mg/g under specific nutrient conditions. The study also investigated pH effects on production in bioreactors, revealing optimal lipid and β-carotene contents at pH 6.0, reaching 22.9 % and 44 mg/g, respectively. Lipid profiles were consistent across experiments, with C18:1 being the dominant compound at approximately 50 %. This research underscores a green revolution in bioprocessing, showing how biocatalysts can convert syngas, a potentially polluting byproduct, into valuable β-carotene and lipids with a Y. lipolytica strain.This study is financially supported by the Spanish Ministry of Science and Innovation in conjunction with European FEDER funding (Grant No. PID2020-117805RB-I00). Financial support of RR's doctoral scholarship was also provided by the same Ministry (Contract ID: E-15-2019-0344365). Additionally, the research team, part of the BIOENGIN group, is grateful to Xunta de Galicia for its financial contributions to Competitive Reference Research Groups under grant ED431C 2021/55. Funding for open access publication provided by Universidade da Coruña/CISUG.Xunta de Galicia; ED431C 2021/5

Acyl-CoA oxidase is imported as a heteropentameric, cofactor-containing complex into peroxisomes of Yarrowia lipolytica

Five isoforms of acyl-CoA oxidase (Aox), designated Aox1p to Aox5p, constitute a 443-kD heteropentameric complex containing one polypeptide chain of each isoform within the peroxisomal matrix of the yeast Yarrowia lipolytica. Assembly of the Aox complex occurs in the cytosol and precedes its import into peroxisomes. Peroxisomal targeting of the Aox complex is abolished in a mutant lacking the peroxin Pex5p, a component of the matrix protein targeting machinery. Import of the Aox complex into peroxisomes does not involve the cytosolic chaperone Pex20p, which mediates the oligomerization and import of peroxisomal thiolase. Aox2p and Aox3p play a pivotal role in the formation of the Aox complex in the cytosol and can substitute for one another in promoting assembly of the complex. In vitro, these subunits retard disassembly of the Aox complex and increase the efficiency of its reassembly. Neither Aox2p nor Aox3p is required for acquisition of the cofactor FAD by other components of the complex. We provide evidence that the Aox2p- and Aox3p-assisted assembly of the Aox complex in the cytosol is mandatory for its import into peroxisomes and that no component of the complex can penetrate the peroxisomal matrix as a monomer

Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis

AbstractThe role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica

Unraveling fatty acid transport and activation mechanisms in Yarrowia lipolytica

AbstractFatty acid (FA) transport and activation have been extensively studied in the model yeast species Saccharomyces cerevisiae but have rarely been examined in oleaginous yeasts, such as Yarrowia lipolytica. Because the latter begins to be used in biodiesel production, understanding its FA transport and activation mechanisms is essential. We found that Y. lipolytica has FA transport and activation proteins similar to those of S. cerevisiae (Faa1p, Pxa1p, Pxa2p, Ant1p) but mechanism of FA peroxisomal transport and activation differs greatly with that of S. cerevisiae. While the ScPxa1p/ScPxa2p heterodimer is essential for growth on long-chain FAs, ΔYlpxa1 ΔYlpxa2 is not impaired for growth on FAs. Meanwhile, ScAnt1p and YlAnt1p are both essential for yeast growth on medium-chain FAs, suggesting they function similarly. Interestingly, we found that the ΔYlpxa1 ΔYlpxa2 ΔYlant1 mutant was unable to grow on short-, medium-, or long-chain FAs, suggesting that YlPxa1p, YlPxa2p, and YlAnt1p belong to two different FA degradation pathways. We also found that YlFaa1p is involved in FA storage in lipid bodies and that FA remobilization largely depended on YlFat1p, YlPxa1p and YlPxa2p. This study is the first to comprehensively examine FA intracellular transport and activation in oleaginous yeast

Two-stage syngas fermentation into microbial oils and β-carotene with Clostridium carboxidivorans and engineered Yarrowia lipolytica

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG[Abstract] An engineered Yarrowia lipolytica strain was able to simultaneously accumulate microbial oils and β-carotene from both acetic and butyric acids produced from syngas fermentation by Clostridium carboxidivorans, through the Wood-Ljungdahl pathway, with CO and CO2 as carbon sources. It was first determined that Y. lipolytica showed no inhibition at concentrations < 16 g/L of a mixture of VFAs (Volatile Fatty Acids) containing acetic, butyric, and even hexanoic acids. The yeast was then grown in a bioreactor with a culture medium derived from acetogenic syngas (CO, CO2, H2) fermentation containing all three acids. Y. lipolytica consumed a total of 91 g/L VFAs, progressively supplied through eight successive additions of these acids to the reactor. A maximum lipid content of 36.18% g/g cell was achieved at 61 g/L VFAs consumed and with an airflow rate of 2.0 vvm. Besides, a total of 88.4 mg/g cell and 759 mg/L β-carotene content and concentration were achieved at about 80 g/L VFAs consumed and with a 2.0 vvm airflow rate. It was concluded that; a non-limiting air flow favors the metabolic pathway for β-carotene formation against lipid accumulationThis research is part of projects funded by the Spanish Ministry of Science and Innovation and European FEDER funds (PID2020-117805RB-I00; TED2021-130055B-I00). RR thanks that same Ministry for financially supporting his doctoral contract (E-15-2019-0344365). The manuscript is based on research related to COST action Yeast4Bio. The authors, belonging to the BIOENGIN group, thank Xunta de Galicia for financial support to Competitive Reference Research Groups (ED431C 2021/55). Funding for open access charge was covered by Universidade da Coruña/CISUG.Xunta de Galicia; ED431C 2021/5

Unlocking the potential of one-carbon gases (CO2, CO) for concomitant bioproduction of β-carotene and lipids

[Abstract] This study investigates the use of a Yarrowia lipolytica strain for the bioconversion of syngas-derived acetic acid into β-carotene and lipids. A two-stage process was employed, starting with the acetogenic fermentation of syngas by Clostridium aceticum, metabolising CO, CO2, H2, to produce acetic acid, which is then utilized by Y. lipolytica for simultaneous lipid and β-carotene synthesis. The research demonstrates that acetic acid concentration plays a pivotal role in modulating lipid profiles and enhancing β-carotene production, with increased acetic acid consumption leading to higher yields of these compounds. This approach showcases the potential of using one-carbon gases as substrates in bioprocesses for generating valuable bioproducts, providing a sustainable and cost-effective alternative to more conventional feedstocks and substrates, such as sugars.This research is part of projects funded by the Spanish Ministry of Science and Innovation and European FEDER funds (PID2020–117805RB-I00; TED2021–130055B-I00). RRI gratefully acknowledges the same Ministry for supporting his doctoral contract (E-15–2019-0344365). CFB (ED481A-2020/028) thanks Xunta de Galicia for financing her doctoral research. The authors are involved in the European COST action Yeast4Bio. The BIOENGIN group, where this research was undertaken, acknowledges the financial support provided by Xunta de Galicia to Competitive Reference Research Groups (ED431C 2021/55).Xunta de Galicia; ED481A-2020/028Xunta de Galicia; ED431C 2021/5

Development of a cultivation process for the enhancement of human interferon alpha 2b production in the oleaginous yeast, Yarrowia lipolytica

<p>Abstract</p> <p>Background</p> <p>As an oleaginous yeast, <it>Yarrowia lipolytica </it>is able to assimilate hydrophobic substrates. This led to the isolation of several promoters of key enzymes of this catabolic pathway. Less is known about the behavior of <it>Y. lipolytica </it>in large bioreactors using these substrates. There is therefore a lack of established know-how concerning high cell density culture protocols of this yeast. Consequently, the establishment of suitable induction conditions is required, to maximize recombinant protein production under the control of these promoters.</p> <p>Results</p> <p>Human interferon α2b (huIFN α2b) production in <it>Yarrowia lipolytica </it>was used as a model for the enhancement of recombinant protein production under the control of the oleic acid (OA)-inducible promoter POX2. Cell viability and heterologous protein production were enhanced by exponential glucose feeding, to generate biomass before OA induction. The optimal biomass level before induction was determined (73 g L^-1), and glucose was added with oleic acid during the induction phase. Several oleic acid feeding strategies were assessed. Continuous feeding with OA at a ratio of 0.02 g OA per g dry cell weight increased huIFNα2b production by a factor of 1.88 (425 mg L^-1) and decreased the induction time (by a factor of 2.6, 21 h). huIFN α2b degradation by an aspartic protease secreted by <it>Y. lipolytica </it>was prevented by adding pepstatin (10 μM), leading to produce a 19-fold more active huIFN α2b (26.2 × 10⁷IU mg^-1).</p> <p>Conclusion</p> <p><it>Y. lipolytica</it>, a generally regarded as safe (GRAS) microorganism is one of the most promising non conventional yeasts for the production of biologically active therapeutic proteins under the control of hydrophobic substrate-inducible promoter.</p

Transcriptomic Analyses during the Transition from Biomass Production to Lipid Accumulation in the Oleaginous Yeast Yarrowia lipolytica

We previously developed a fermentation protocol for lipid accumulation in the oleaginous yeast Y. lipolytica. This process was used to perform transcriptomic time-course analyses to explore gene expression in Y. lipolytica during the transition from biomass production to lipid accumulation. In this experiment, a biomass concentration of 54.6 gCDW/l, with 0.18 g/gCDW lipid was obtained in ca. 32 h, with low citric acid production. A transcriptomic profiling was performed on 11 samples throughout the fermentation. Through statistical analyses, 569 genes were highlighted as differentially expressed at one point during the time course of the experiment. These genes were classified into 9 clusters, according to their expression profiles. The combination of macroscopic and transcriptomic profiles highlighted 4 major steps in the culture: (i) a growth phase, (ii) a transition phase, (iii) an early lipid accumulation phase, characterized by an increase in nitrogen metabolism, together with strong repression of protein production and activity; (iv) a late lipid accumulation phase, characterized by the rerouting of carbon fluxes within cells. This study explores the potential of Y. lipolytica as an alternative oil producer, by identifying, at the transcriptomic level, the genes potentially involved in the metabolism of oleaginous species

Production and characterization of two medium-chain-length polydroxyalkanoates by engineered strains of Yarrowia lipolytica

Background: The oleaginous yeast Yarrowia lipolytica is an organism of choice for the tailored production of various compounds such as biofuels or biopolymers. When properly engineered, it is capable of producing medium-chain-length polyhydroxyalkanoate (mcl-PHA), a biobased and biodegradable polymer that can be used as bioplastics or biopolymers for environmental and biomedical applications.Results: This study describes the bioproduction and the main properties of two different mcl-PHA polymers. We generated by metabolic engineering, strains of Y. lipolytica capable of accumulating more than 25% (g/g) of mcl-PHA polymers. Depending of the strain genetic background and the culture conditions, we produced (i) a mcl-PHA homopolymer of 3-hydroxydodecanoic acids, with a mass-average molar mass (M-w) of 316,000 g/mol, showing soft thermoplastic properties with potential applications in packaging and (ii) a mcl-PHA copolymer made of 3-hydroxyoctanoic (3HO), decanoic (3HD), dodecanoic (3HDD) and tetradecanoic (3TD) acids with a M-w of 128,000 g/mol, behaving like a thermoplastic elastomer with potential applications in biomedical material.Conclusion: The ability to engineer Y. lipolytica to produce tailored PHAs together with the range of possible applications regarding their biophysical and mechanical properties opens new perspectives in the field of PHA bioproduction