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Engineering of a high lipid producing Yarrowia lipolytica strain.

Friedlander J, Tsakraklides V, Kamineni A, Greenhagen EH, Consiglio AL, MacEwen K, Crabtree DV, Afshar J, Nugent RL, Hamilton MA, Joe Shaw A, South CR, Stephanopoulos G, Brevnova EE - Biotechnol Biofuels (2016)

Bottom Line: These three genetic modifications, selected for their effect on lipid production, enabled a 77 % lipid content and 0.21 g lipid per g glucose yield in batch fermentation.In fed-batch glucose fermentation NS432 produced 85 g/L lipid at a productivity of 0.73 g/L/h.The yields, productivities, and titers reported in this study may further support the applied goal of cost-effective, large -scale microbial lipid production for use as biofuels and biochemicals.

View Article: PubMed Central - PubMed

Affiliation: Total New Energies, 5858 Horton Street, Emeryville, CA 94610 USA.

ABSTRACT

Background: Microbial lipids are produced by many oleaginous organisms including the well-characterized yeast Yarrowia lipolytica, which can be engineered for increased lipid yield by up-regulation of the lipid biosynthetic pathway and down-regulation or deletion of competing pathways.

Results: We describe a strain engineering strategy centered on diacylglycerol acyltransferase (DGA) gene overexpression that applied combinatorial screening of overexpression and deletion genetic targets to construct a high lipid producing yeast biocatalyst. The resulting strain, NS432, combines overexpression of a heterologous DGA1 enzyme from Rhodosporidium toruloides, a heterlogous DGA2 enzyme from Claviceps purpurea, and deletion of the native TGL3 lipase regulator. These three genetic modifications, selected for their effect on lipid production, enabled a 77 % lipid content and 0.21 g lipid per g glucose yield in batch fermentation. In fed-batch glucose fermentation NS432 produced 85 g/L lipid at a productivity of 0.73 g/L/h.

Conclusions: The yields, productivities, and titers reported in this study may further support the applied goal of cost-effective, large -scale microbial lipid production for use as biofuels and biochemicals.

No MeSH data available.


Related in: MedlinePlus

Overexpression of DGA1 genes in Y. lipolytica strain NS18. Nine DGA1 genes (Table 2) under the control of the Y. lipolytica GPD1 promoter were randomly integrated into the NS18 genome and 8 transformants for each gene were analyzed by fluorescence-based lipid assay after 72 h of growth in nitrogen-limited media. The average with standard deviation from triplicate experiments is shown for the parent strain. Fluorescence was measured at excitation 486 nm and emission 510 nm and normalized by cell optical density (OD) at 600 nm
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Fig2: Overexpression of DGA1 genes in Y. lipolytica strain NS18. Nine DGA1 genes (Table 2) under the control of the Y. lipolytica GPD1 promoter were randomly integrated into the NS18 genome and 8 transformants for each gene were analyzed by fluorescence-based lipid assay after 72 h of growth in nitrogen-limited media. The average with standard deviation from triplicate experiments is shown for the parent strain. Fluorescence was measured at excitation 486 nm and emission 510 nm and normalized by cell optical density (OD) at 600 nm

Mentions: In order to evaluate the effect of DGA1 overexpression in NS18, the Y. lipolyticaDGA1 gene (NG15) was introduced into the NS18 genome under the control of the strong constitutive GPD1 promoter to produce strain NS297. Although use of promoters with varied expression levels may allow for a higher degree of metabolic balancing and control, we felt strongly constitutive promoters were suitable for identifying targets for lipid biosynthesis overexpression. To produce engineered strains, 50–200 transformants were screened in 96- or 48-well plates and evaluated by fluorescence-based assay for lipid content. The top isolates were then tested in 50-mL shake flasks and the highest performing strains were grown in 1 L bioreactors using a high cell density fed-batch glucose fermentation process followed by gas chromotograph (GC) lipid content analysis. Lipid content measured by GC is reported as fatty acid methyl ester equivalents, as described in the methods section. NS297 transformants exhibited a twofold increase in lipid content compared to wild-type Y. lipolytica NS18 as evaluated by fluorescence assay (Figs. 2, 4a, b) and GC analysis (Fig. 4b).Fig. 2


Engineering of a high lipid producing Yarrowia lipolytica strain.

Friedlander J, Tsakraklides V, Kamineni A, Greenhagen EH, Consiglio AL, MacEwen K, Crabtree DV, Afshar J, Nugent RL, Hamilton MA, Joe Shaw A, South CR, Stephanopoulos G, Brevnova EE - Biotechnol Biofuels (2016)

Overexpression of DGA1 genes in Y. lipolytica strain NS18. Nine DGA1 genes (Table 2) under the control of the Y. lipolytica GPD1 promoter were randomly integrated into the NS18 genome and 8 transformants for each gene were analyzed by fluorescence-based lipid assay after 72 h of growth in nitrogen-limited media. The average with standard deviation from triplicate experiments is shown for the parent strain. Fluorescence was measured at excitation 486 nm and emission 510 nm and normalized by cell optical density (OD) at 600 nm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4815080&req=5

Fig2: Overexpression of DGA1 genes in Y. lipolytica strain NS18. Nine DGA1 genes (Table 2) under the control of the Y. lipolytica GPD1 promoter were randomly integrated into the NS18 genome and 8 transformants for each gene were analyzed by fluorescence-based lipid assay after 72 h of growth in nitrogen-limited media. The average with standard deviation from triplicate experiments is shown for the parent strain. Fluorescence was measured at excitation 486 nm and emission 510 nm and normalized by cell optical density (OD) at 600 nm
Mentions: In order to evaluate the effect of DGA1 overexpression in NS18, the Y. lipolyticaDGA1 gene (NG15) was introduced into the NS18 genome under the control of the strong constitutive GPD1 promoter to produce strain NS297. Although use of promoters with varied expression levels may allow for a higher degree of metabolic balancing and control, we felt strongly constitutive promoters were suitable for identifying targets for lipid biosynthesis overexpression. To produce engineered strains, 50–200 transformants were screened in 96- or 48-well plates and evaluated by fluorescence-based assay for lipid content. The top isolates were then tested in 50-mL shake flasks and the highest performing strains were grown in 1 L bioreactors using a high cell density fed-batch glucose fermentation process followed by gas chromotograph (GC) lipid content analysis. Lipid content measured by GC is reported as fatty acid methyl ester equivalents, as described in the methods section. NS297 transformants exhibited a twofold increase in lipid content compared to wild-type Y. lipolytica NS18 as evaluated by fluorescence assay (Figs. 2, 4a, b) and GC analysis (Fig. 4b).Fig. 2

Bottom Line: These three genetic modifications, selected for their effect on lipid production, enabled a 77 % lipid content and 0.21 g lipid per g glucose yield in batch fermentation.In fed-batch glucose fermentation NS432 produced 85 g/L lipid at a productivity of 0.73 g/L/h.The yields, productivities, and titers reported in this study may further support the applied goal of cost-effective, large -scale microbial lipid production for use as biofuels and biochemicals.

View Article: PubMed Central - PubMed

Affiliation: Total New Energies, 5858 Horton Street, Emeryville, CA 94610 USA.

ABSTRACT

Background: Microbial lipids are produced by many oleaginous organisms including the well-characterized yeast Yarrowia lipolytica, which can be engineered for increased lipid yield by up-regulation of the lipid biosynthetic pathway and down-regulation or deletion of competing pathways.

Results: We describe a strain engineering strategy centered on diacylglycerol acyltransferase (DGA) gene overexpression that applied combinatorial screening of overexpression and deletion genetic targets to construct a high lipid producing yeast biocatalyst. The resulting strain, NS432, combines overexpression of a heterologous DGA1 enzyme from Rhodosporidium toruloides, a heterlogous DGA2 enzyme from Claviceps purpurea, and deletion of the native TGL3 lipase regulator. These three genetic modifications, selected for their effect on lipid production, enabled a 77 % lipid content and 0.21 g lipid per g glucose yield in batch fermentation. In fed-batch glucose fermentation NS432 produced 85 g/L lipid at a productivity of 0.73 g/L/h.

Conclusions: The yields, productivities, and titers reported in this study may further support the applied goal of cost-effective, large -scale microbial lipid production for use as biofuels and biochemicals.

No MeSH data available.


Related in: MedlinePlus