Construction of wild-type Yarrowia lipolytica IMUFRJ 50682 auxotrophic mutants using dual CRISPR/Cas9 strategy for novel biotechnological approaches

A wild strain of Metarhizium anisopliae, an entomopathogenic fungus, was submitted to three mutagenic agents: gamma radiation, ultraviolet light and nitrous acid. Survival curves were obtained and mutants were selected using different mutagenic doses which gave 1 to 5% survival. Morphological and auxotrophic mutants were isolated. Morphological mutants were grouped in a class with yellow conidia and other with pale vinaceous conidia as opposed to the green wild type conidia. Auxotrophic mutants had requirements for vitamin and aminoacid biosynthesis. More than 58% of the total auxotrophk mutants required proline/aipnine. Gamma radiation showed to be the most efficient mutagenic agent giving 0.2% of auxotrophk mutants followed by ultraviolet light (0.12%) and nitrous acid (0.06%).The conidial colour and auxotrophk mutants isolated until now from M. anisopliae were reviewed.

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Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

Applied and Environmental Microbiology ◽

10.1128/aem.02079-13 ◽

2013 ◽

Vol 79 (23) ◽

pp. 7360-7370 ◽

Cited By ~ 54

Author(s):

John Seip ◽

Raymond Jackson ◽

Hongxian He ◽

Quinn Zhu ◽

Seung-Pyo Hong

Keyword(s):

Yarrowia Lipolytica ◽

Lipid Accumulation ◽

Oleaginous Yeast ◽

De Novo ◽

Lipid Synthesis ◽

Omega 3 ◽

Wild Type ◽

Content Type ◽

Reverse Transcription Pcr ◽

Dry Cell Weight

ABSTRACTIn the oleaginous yeastYarrowia lipolytica,de novolipid synthesis and accumulation are induced under conditions of nitrogen limitation (or a high carbon-to-nitrogen ratio). The regulatory pathway responsible for this induction has not been identified. Here we report that the SNF1 pathway plays a key role in the transition from the growth phase to the oleaginous phase inY. lipolytica. Strains with aY. lipolyticasnf1(Ylsnf1) deletion accumulated fatty acids constitutively at levels up to 2.6-fold higher than those of the wild type. When introduced into aY. lipolyticastrain engineered to produce omega-3 eicosapentaenoic acid (EPA),Ylsnf1deletion led to a 52% increase in EPA titers (7.6% of dry cell weight) over the control. Other components of theY. lipolyticaSNF1 pathway were also identified, and their function in limiting fatty acid accumulation is suggested by gene deletion analyses. Deletion of the gene encoding YlSnf4, YlGal83, or YlSak1 significantly increased lipid accumulation in both growth and oleaginous phases compared to the wild type. Furthermore, microarray and quantitative reverse transcription-PCR (qRT-PCR) analyses of theYlsnf1mutant identified significantly differentially expressed genes duringde novolipid synthesis and accumulation inY. lipolytica. Gene ontology analysis found that these genes were highly enriched with genes involved in lipid metabolism. This work presents a new role for Snf1/AMP-activated protein kinase (AMPK) pathways in lipid accumulation in this oleaginous yeast.

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Enlarged Peroxisomes Are Present in Oleic Acid–grown Yarrowia lipolytica Overexpressing the PEX16 Gene Encoding an Intraperoxisomal Peripheral Membrane Peroxin

The Journal of Cell Biology ◽

10.1083/jcb.137.6.1265 ◽

1997 ◽

Vol 137 (6) ◽

pp. 1265-1278 ◽

Cited By ~ 102

Author(s):

Gary A. Eitzen ◽

Rachel K. Szilard ◽

Richard A. Rachubinski

Keyword(s):

Oleic Acid ◽

Yarrowia Lipolytica ◽

Consensus Sequence ◽

Wild Type ◽

Gene Encoding ◽

Peroxisomal Membrane ◽

Peripheral Protein ◽

The Matrix ◽

Peroxisomal Targeting ◽

Targeting Signal

Pex mutants of the yeast Yarrowia lipolytica are defective in peroxisome assembly. The mutant strain pex16-1 lacks morphologically recognizable peroxisomes. Most peroxisomal proteins are mislocalized to a subcellular fraction enriched for cytosol in pex16 strains, but a subset of peroxisomal proteins is localized at, or near, wild-type levels to a fraction typically enriched for peroxisomes. The PEX16 gene was isolated by functional complementation of the pex16-1 strain and encodes a protein, Pex16p, of 391 amino acids (44,479 D). Pex16p has no known homologues. Pex16p is a peripheral protein located at the matrix face of the peroxisomal membrane. Substitution of the carboxylterminal tripeptide Ser-Thr-Leu, which is similar to the consensus sequence of peroxisomal targeting signal 1, does not affect targeting of Pex16p to peroxisomes. Pex16p is synthesized in wild-type cells grown in glucose-containing media, and its levels are modestly increased by growth of cells in oleic acid–containing medium. Overexpression of the PEX16 gene in oleic acid– grown Y. lipolytica leads to the appearance of a small number of enlarged peroxisomes, which contain the normal complement of peroxisomal proteins at levels approaching those of wild-type peroxisomes.

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A set of Yarrowia lipolytica CRISPR/Cas9 vectors for exploiting wild-type strain diversity

Biotechnology Letters ◽

10.1007/s10529-020-02805-4 ◽

2020 ◽

Vol 42 (5) ◽

pp. 773-785 ◽

Cited By ~ 2

Author(s):

Macarena Larroude ◽

Heykel Trabelsi ◽

Jean-Marc Nicaud ◽

Tristan Rossignol

Keyword(s):

Yarrowia Lipolytica ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type ◽

Strain Diversity

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Physiological Characterization of a Novel Wild-Type Yarrowia lipolytica Strain Grown on Glycerol: Effects of Cultivation Conditions and Mode on Polyols and Citric Acid Production

Applied Sciences ◽

10.3390/app10207373 ◽

2020 ◽

Vol 10 (20) ◽

pp. 7373

Author(s):

Seraphim Papanikolaou ◽

Panagiota Diamantopoulou ◽

Fabrice Blanchard ◽

Eleni Lambrinea ◽

Isabelle Chevalot ◽

...

Keyword(s):

Citric Acid ◽

Yarrowia Lipolytica ◽

Acid Production ◽

Unsaturated Fatty Acids ◽

Citric Acid Production ◽

Wild Type ◽

Alcohol Production ◽

Physiological Characterization ◽

Batch Bioreactor ◽

New Yeast

A new yeast wild-type Yarrowia lipolytica isolate presented efficient growth on glycerol. During flask cultures, nitrogen limitation led to the secretion of sugar-alcohols as the major metabolites of the process (mannitol, arabitol and erythritol), whereas insignificant quantities of citrate were synthesized. Although in some instances high initial glycerol concentrations were employed (≈150 g/L), remarkable glycerol assimilation and polyol secretion was observed. Total polyols ≈ 52 g/L (conversion yield on glycerol consumed = 0.43 g/g) was recorded in the flask experiments. The sugar-alcohol production bioprocess was successfully simulated with the aid of a modified Velhlust–Aggelis model that fitted very well with the experimental data, while optimized parameter values seemed to be quite consistent. In bioreactor trials, a noticeable metabolic shift towards citric acid production was observed, while simultaneously insignificant polyol quantities were produced. In fed-batch bioreactor experiments, a total citric acid quantity ≈ 102 g/L was recorded—one of the highest in the literature for wild-type Y. lipolytica strains. This metabolic transition was due to higher oxygen saturation into the medium that occurred in the bioreactor experiments compared with the flasks. Cellular lipids produced in the bioreactor trial contained higher concentrations of unsaturated fatty acids compared with those produced in flasks.

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INCREASED FREQUENCY OF AUXOTROPHIC MUTANTS OF USTILAGO HORDEI AFTER COMBINED UV IRRADIATION AND INOSITOL STARVATION

Canadian Journal of Genetics and Cytology ◽

10.1139/g72-096 ◽

1972 ◽

Vol 14 (4) ◽

pp. 785-788 ◽

Cited By ~ 9

Author(s):

P. L. Thomas

Keyword(s):

Uv Irradiation ◽

Wild Type ◽

Auxotrophic Mutants ◽

Ustilago Hordei

Auxotrophic mutants of Ustilago hordei were recovered after UV irradiation of a wild-type culture, inositol starvation of an inositol-requiring culture and a combination of the two methods. 1.9% of the survivors of UV irradiation followed by inositol starvation were found to carry a requirement additional to inositol. Inositol starvation by itself gave only 0.27% recovery while UV irradiation alone was even less efficient.

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Disruption of PMR1, Encoding a Ca2+-ATPase Homolog in Yarrowia lipolytica, Affects Secretion and Processing of Homologous and Heterologous Proteins

Journal of Bacteriology ◽

10.1128/jb.180.24.6736-6742.1998 ◽

1998 ◽

Vol 180 (24) ◽

pp. 6736-6742 ◽

Cited By ~ 12

Author(s):

Young-Sun Sohn ◽

Cheon Seok Park ◽

Sun-Bok Lee ◽

Dewey D. Y. Ryu

Keyword(s):

Yarrowia Lipolytica ◽

Secretory Pathway ◽

Inhibitory Effect ◽

Extracellular Protease ◽

Heterologous Proteins ◽

Wild Type ◽

Reporter Protein ◽

Recombinant Yeast Strain ◽

Foreign Proteins ◽

Different Characteristics

ABSTRACT The Yarrowia lipolytica PMR1 gene (YlPMR1) is a Saccharomyces cerevisiae PMR1 homolog which encodes a putative secretory pathway Ca2+-ATPase. In this study, we investigated the effects of a YlPMR1 disruption on the processing and secretion of native and foreign proteins in Y. lipolytica and found variable responses by theYlPMR1-disrupted mutant depending on the protein. The secretion of 32-kDa mature alkaline extracellular protease (AEP) was dramatically decreased, and incompletely processed precursors were observed in the YlPMR1-disrupted mutant. A 36- and a 52-kDa premature AEP were secreted, and an intracellular 52-kDa premature AEP was also detected. The acid extracellular protease activity of theYlPMR1-disrupted mutant was increased by 60% compared to that of the wild-type strain. The inhibitory effect of mutations in secretory pathway Ca2+-ATPase genes on the secretion of rice α-amylase was also observed in the Y. lipolytica andS. cerevisiae PMR1-disrupted mutants. Unlike rice α-amylase, the secretion of Trichoderma reeseiendoglucanase I (EGI) was not influenced by the YlPMR1disruption. However, the secreted EGI from theYlPMR1-disrupted mutant had different characteristics than that of the control. While wild-type cells secreted the hyperglycosylated form of EGI, hyperglycosylation was completely absent in the YlPMR1-disrupted mutant. Our results indicate that the effects of the YlPMR1 disruption as manifested by the phenotypic response depend on the characteristics of the reporter protein in the recombinant yeast strain evaluated.

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Inaktivierende und mutagene Wirkung salpetriger Säure auf Zellen von Escherichia coli

Zeitschrift für Naturforschung B ◽

10.1515/znb-1959-8-910 ◽

1959 ◽

Vol 14 (8-9) ◽

pp. 528-537 ◽

Cited By ~ 53

Author(s):

F. Kaudewitz

Keyword(s):

Escherichia Coli ◽

Nitrous Acid ◽

Single Cells ◽

Wild Type ◽

Inactivation Curve ◽

E Coli ◽

Auxotrophic Mutants ◽

Induced Mutants ◽

Mutagene Wirkung ◽

Cellular Dna

Cells of E. coli B incubated with NaNO2 undergo inactivation. In non-metabolizing cells the inactivation follows a two hit curve. In metabolizing cells the rate of inactivation is increased and the inactivation curve does not show two-hit kinetics. The rate of inactivation decreases with rising pH and decreasing NaNO2-concentration. Therefore nitrous acid appears to be the active substance.Nitrous acid proved to be a potent mutagen as shown by isolation of auxotrophic mutants. With an inactivation rate of 10-4 about 1.4 per cent of the surviving cells were auxotrophs. The probability that this increase in mutants may be due to selection during inactivation of auxotrophs present before exposure was excluded experimentally. In none of 559 auxotrophic colonies grown from single cells which had survived contact with nitrous acid wild-type sectors were found. For metabolizing and nonmetabolizing cells the increase of the percentage of auxotrophic mutants with increasing time of exposure to HNO2 followed a two-hit curve. In these experiments the percentage of induced mutants was independent of the different rate of inactivation caused by different states of metabolism and dependent only on the time of incubation with nitrous acid. The results are discussed as being in agreement with the assumption that in non-metabolizing cells nitrous acid acts directly on the cellular DNA leading to inactivation and mutation.

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Golden Gate-based metabolic engineering strategy for wild-type strains of Yarrowia lipolytica

FEMS Microbiology Letters ◽

10.1093/femsle/fnz022 ◽

2019 ◽

Vol 366 (4) ◽

Cited By ~ 13

Author(s):

Michael Egermeier ◽

Michael Sauer ◽

Hans Marx

Keyword(s):

Metabolic Engineering ◽

Yarrowia Lipolytica ◽

Golden Gate ◽

Wild Type ◽

Metabolic Engineering Strategy ◽

Engineering Strategy ◽

Type Strains

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Aspects of glycine and serine biosynthesis during growth of Pseudomonas AM1 on C1 compounds

Biochemical Journal ◽

10.1042/bj1210763 ◽

1971 ◽

Vol 121 (5) ◽

pp. 763-769 ◽

Cited By ~ 20

Author(s):

W. Harder ◽

J. R. Quayle

Keyword(s):

Serine Hydroxymethyltransferase ◽

Aminotransferase Activity ◽

Wild Type ◽

Amino Groups ◽

Auxotrophic Mutants ◽

Wild Type Phenotype ◽

Glyoxylate Aminotransferase ◽

Serine Biosynthesis

1. Methanol or formate can replace serine or glycine as supplements for growth on succinate of the auxotrophic mutants 20S and 82G of Pseudomonas AM1, showing that the organism can synthesize glycine and serine in net fashion from C1 units. 2. Double mutants of Pseudomonas 20S and 82G have been prepared (20ST-1 and 82GT-1) that are unable to grow on succinate+1mm-glyoxylate, succinate+2mm-methanol or methanol alone. 3. Mutants 20ST-1 and 82GT-1 lacked serine–glyoxylate aminotransferase activity, and revertants to the phenotype of 20S and 82G regained serine–glyoxylate aminotransferase activity. A total revertant of 82GT-1 to wild-type phenotype regained activities of serine hydroxymethyltransferase and serine–glyoxylate aminotransferase. 4. The activity of serine–glyoxylate aminotransferase in methanol-grown Pseudomonas AM1 is eightfold higher than in the succinate-grown organism. 5. The combined results show that in Pseudomonas AM1 serine–glyoxylate aminotransferase is necessary for growth on C1 compounds and is involved in the conversion of methanol into glycine via glyoxylate. 6. It is suggested that the phosphorylated pathway of serine biosynthesis from phosphoglycerate replenishes the supply of α-amino groups necessary for the flow of glyoxylate through the main assimilatory pathway during growth on C1 compounds.

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