Compositions and methods for producing lipids and other biomaterials from grain ethanol stillage and stillage derivatives

Inventors

Jeffries, Thomas W. • Mokry, David Z. • Calvey, Christopher H.

Assignees

Xylome Corp

Abstract

Lipogenic yeasts bioengineered to overexpress genes for lipid production, and methods of use thereof. The yeasts are modified to express, constitutively express, or overexpress an acetyl-CoA carboxylase, an alpha-amylase, an ATP citrate lyase, a diacylglycerol acyltransferase, a fatty acid synthase, a glycerol kinase, a 6-phosphogluconate dehydrogenase, a glycerol-3-phosphate dehydrogenase, a malic enzyme, a fatty acyl-CoA reductase, a delta-9 acyl-CoA desaturase, a glycerol-3-phosphate acyltransferase, a lysophosphatidate acyltransferase, a glucose-6-phosphate dehydrogenase, a beta-glucosidase, a hexose transporter, a glycerol transporter, a glycoside hydrolase enzyme, an auxiliary activity family 9 enzyme, or combinations thereof. The yeasts in some cases are also modified to reduce or ablate activity of certain proteins. The methods include cultivating the yeast to convert low value soluble organic stillage byproducts into lipids suitable for biodiesel production and other higher value uses.

Core Innovation

The invention relates to engineered lipogenic yeasts configured to convert grain ethanol stillage and modified thin stillage into lipids, enzymes, and protein. In an example embodiment, the yeast is a recombinant lipogenic yeast, optionally preferably Lipomyces starkeyi, and is genetically modified to express selected lipid-biosynthesis enzymes and associated metabolic factors to support lipid production.

The engineered yeast expresses one or more recombinant genes configured to encode lipid-metabolism activities, including diacylglycerol acyltransferase together with a protein selected from a malic enzyme and a glycerol-3-phosphate acyltransferase. Additional expressed enzymes are selected from acetyl-CoA carboxylase, glycerol-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, fatty acid synthase subunits, ATP citrate lyase subunits, and glycerol kinase together with glycerol-3-phosphate dehydrogenase, using explicit sequence identity constraints to reference sequences.

The invention further includes genetic design choices that alter competing activities, including optionally reducing or ablating beta-oxidation and other competing pathway activities, and includes screening and engineering rationales tied to acetyl-CoA and NADPH supply and formation of a triglyceride sink. Experimental improvements reported in the document include enzyme-specific sequence changes and specific pathway adjustments that enhance lipid-related fluorescence and lipid titers, and improved utilization of glycerol and pentose phosphate pathway flux, together with identification and engineering of additional genes and secretion-related co-product production.

Claims Coverage

Two independent claims are identified. Across the independent claims, the inventive coverage centers on recombinant yeast engineered to express specific lipid-metabolism enzymes selected and constrained by explicit sequence identity requirements to reference SEQ IDs, including defined structural constraints for at least one enzyme and specification of a particular yeast host.

The independent claims cover recombinant yeast platforms in which the lipid-biosynthesis enzyme set is specified by named enzyme types and by explicit sequence identity constraints to reference SEQ IDs, including a defined residue constraint for acetyl-CoA carboxylase in one independent claim and, in the other independent claim, an additional structural absence constraint for one diacylglycerol acyltransferase, together with specification of Lipomyces starkeyi.

Stated Advantages

Documented Applications