Characterization of Met25 as a color associated genetic marker in Yarrowia lipolytica

Harley Edwards; Zhiliang Yang; Peng Xu

doi:10.1016/j.mec.2020.e00147

Characterization of Met25 as a color associated genetic marker in Yarrowia lipolytica

Harley Edwards, Zhiliang Yang, Peng Xu^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

6 Scopus citations

Abstract

Yarrowia lipolytica offers an ideal host for biosynthesis of high value natural products and oleochemicals through metabolic engineering despite being restricted to a limited number of selective markers, and counter-selection achieved primarily with URA3. In this work, we investigate MET25, a locus encoding sulfide housekeeping gene within the cell, to be exploited as a standard genetic marker. Divalent lead supplemented in media induces lead sulfide (PbS) aggregation in MET25-deficient cells such that deficient cells grow brown/black, and cells with functional copies of MET25 grow white. Loss of MET25 did not induce strict auxotrophic requirements for methionine in Y. lipolytica, indicating MET25 deficiency could be rescued by alternative pathways. Plasmid and chromosomal-based complementation of MET25 in the met25 deficient cells on a double layer agar plate with nutrient gradients demonstrates delayed phenotype (white morphology) restoration, indicating post-transcriptional feedback regulation of methionine biosynthesis in this yeast. MET25 deficient Y. lipolytica could be used as an efficient whole-cell lead sensor with detection limit as low as 10 ppm of lead in drinking water. We further tested whether MET25 deficiency can be exploited to confer resistance to methyl-mercury through chemical neutralization and detoxification. Kinetic growth curves of wild type and MET25-deficient cells were obtained under varying concentrations of methylmercury and cellular toxicity to methyl mercury was calculated from the Hill equation. Our results indicate that methylmecury may not be used as the counter-selectable marker due to insignificant changes of growth fitness. This work demonstrates the utility of using MET25 as a sensitive lead sensor and the challenges of using MET25 as a counter-selectable genetic marker, as well as the complex regulation of methionine biosynthesis in Y. lipolyitca, which may shed lights for us to develop valuable biotechnological applications centering around the sulfur house-keeping metabolism of the nonconventional yeast.

Original language	English
Article number	e00147
Journal	Metabolic Engineering Communications
Volume	11
DOIs	https://doi.org/10.1016/j.mec.2020.e00147
State	Published - Dec 2020
Externally published	Yes

Keywords

Color associated screening
Counter selection
Genetic marker
Methionine biosynthesis
Yarrowia lipolytica

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.mec.2020.e00147

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@article{c34d9f0d71184f32aa9b57ac567138e8,

title = "Characterization of Met25 as a color associated genetic marker in Yarrowia lipolytica",

abstract = "Yarrowia lipolytica offers an ideal host for biosynthesis of high value natural products and oleochemicals through metabolic engineering despite being restricted to a limited number of selective markers, and counter-selection achieved primarily with URA3. In this work, we investigate MET25, a locus encoding sulfide housekeeping gene within the cell, to be exploited as a standard genetic marker. Divalent lead supplemented in media induces lead sulfide (PbS) aggregation in MET25-deficient cells such that deficient cells grow brown/black, and cells with functional copies of MET25 grow white. Loss of MET25 did not induce strict auxotrophic requirements for methionine in Y. lipolytica, indicating MET25 deficiency could be rescued by alternative pathways. Plasmid and chromosomal-based complementation of MET25 in the met25 deficient cells on a double layer agar plate with nutrient gradients demonstrates delayed phenotype (white morphology) restoration, indicating post-transcriptional feedback regulation of methionine biosynthesis in this yeast. MET25 deficient Y. lipolytica could be used as an efficient whole-cell lead sensor with detection limit as low as 10 ppm of lead in drinking water. We further tested whether MET25 deficiency can be exploited to confer resistance to methyl-mercury through chemical neutralization and detoxification. Kinetic growth curves of wild type and MET25-deficient cells were obtained under varying concentrations of methylmercury and cellular toxicity to methyl mercury was calculated from the Hill equation. Our results indicate that methylmecury may not be used as the counter-selectable marker due to insignificant changes of growth fitness. This work demonstrates the utility of using MET25 as a sensitive lead sensor and the challenges of using MET25 as a counter-selectable genetic marker, as well as the complex regulation of methionine biosynthesis in Y. lipolyitca, which may shed lights for us to develop valuable biotechnological applications centering around the sulfur house-keeping metabolism of the nonconventional yeast.",

keywords = "Color associated screening, Counter selection, Genetic marker, Methionine biosynthesis, Yarrowia lipolytica",

author = "Harley Edwards and Zhiliang Yang and Peng Xu",

note = "Publisher Copyright: {\textcopyright} 2020 The Author(s)",

year = "2020",

month = dec,

doi = "10.1016/j.mec.2020.e00147",

language = "英语",

volume = "11",

journal = "Metabolic Engineering Communications",

issn = "2214-0301",

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TY - JOUR

T1 - Characterization of Met25 as a color associated genetic marker in Yarrowia lipolytica

AU - Edwards, Harley

AU - Yang, Zhiliang

AU - Xu, Peng

PY - 2020/12

Y1 - 2020/12

N2 - Yarrowia lipolytica offers an ideal host for biosynthesis of high value natural products and oleochemicals through metabolic engineering despite being restricted to a limited number of selective markers, and counter-selection achieved primarily with URA3. In this work, we investigate MET25, a locus encoding sulfide housekeeping gene within the cell, to be exploited as a standard genetic marker. Divalent lead supplemented in media induces lead sulfide (PbS) aggregation in MET25-deficient cells such that deficient cells grow brown/black, and cells with functional copies of MET25 grow white. Loss of MET25 did not induce strict auxotrophic requirements for methionine in Y. lipolytica, indicating MET25 deficiency could be rescued by alternative pathways. Plasmid and chromosomal-based complementation of MET25 in the met25 deficient cells on a double layer agar plate with nutrient gradients demonstrates delayed phenotype (white morphology) restoration, indicating post-transcriptional feedback regulation of methionine biosynthesis in this yeast. MET25 deficient Y. lipolytica could be used as an efficient whole-cell lead sensor with detection limit as low as 10 ppm of lead in drinking water. We further tested whether MET25 deficiency can be exploited to confer resistance to methyl-mercury through chemical neutralization and detoxification. Kinetic growth curves of wild type and MET25-deficient cells were obtained under varying concentrations of methylmercury and cellular toxicity to methyl mercury was calculated from the Hill equation. Our results indicate that methylmecury may not be used as the counter-selectable marker due to insignificant changes of growth fitness. This work demonstrates the utility of using MET25 as a sensitive lead sensor and the challenges of using MET25 as a counter-selectable genetic marker, as well as the complex regulation of methionine biosynthesis in Y. lipolyitca, which may shed lights for us to develop valuable biotechnological applications centering around the sulfur house-keeping metabolism of the nonconventional yeast.

AB - Yarrowia lipolytica offers an ideal host for biosynthesis of high value natural products and oleochemicals through metabolic engineering despite being restricted to a limited number of selective markers, and counter-selection achieved primarily with URA3. In this work, we investigate MET25, a locus encoding sulfide housekeeping gene within the cell, to be exploited as a standard genetic marker. Divalent lead supplemented in media induces lead sulfide (PbS) aggregation in MET25-deficient cells such that deficient cells grow brown/black, and cells with functional copies of MET25 grow white. Loss of MET25 did not induce strict auxotrophic requirements for methionine in Y. lipolytica, indicating MET25 deficiency could be rescued by alternative pathways. Plasmid and chromosomal-based complementation of MET25 in the met25 deficient cells on a double layer agar plate with nutrient gradients demonstrates delayed phenotype (white morphology) restoration, indicating post-transcriptional feedback regulation of methionine biosynthesis in this yeast. MET25 deficient Y. lipolytica could be used as an efficient whole-cell lead sensor with detection limit as low as 10 ppm of lead in drinking water. We further tested whether MET25 deficiency can be exploited to confer resistance to methyl-mercury through chemical neutralization and detoxification. Kinetic growth curves of wild type and MET25-deficient cells were obtained under varying concentrations of methylmercury and cellular toxicity to methyl mercury was calculated from the Hill equation. Our results indicate that methylmecury may not be used as the counter-selectable marker due to insignificant changes of growth fitness. This work demonstrates the utility of using MET25 as a sensitive lead sensor and the challenges of using MET25 as a counter-selectable genetic marker, as well as the complex regulation of methionine biosynthesis in Y. lipolyitca, which may shed lights for us to develop valuable biotechnological applications centering around the sulfur house-keeping metabolism of the nonconventional yeast.

KW - Color associated screening

KW - Counter selection

KW - Genetic marker

KW - Methionine biosynthesis

KW - Yarrowia lipolytica

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U2 - 10.1016/j.mec.2020.e00147

DO - 10.1016/j.mec.2020.e00147

M3 - 文章

AN - SCOPUS:85092302931

SN - 2214-0301

VL - 11

JO - Metabolic Engineering Communications

JF - Metabolic Engineering Communications

M1 - e00147

ER -

Characterization of Met25 as a color associated genetic marker in Yarrowia lipolytica

Abstract

Keywords

UN SDGs

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