2,3-butanediol production, methyl ethyl ketone production, and induction of drought tolerance in plants

Inventors

Jin, Yong-Su • Lee, Jae Won • Cho, Young B. • LEE, Ye-Gi

Assignees

University of Illinois System

Abstract

Provided herein are compositions and methods for the fermentative production of 2,3-butanediol (2,3-BDO), compositions and methods for making methyl ethyl ketone (MEK), and methods of inducing drought tolerance in plants.

Core Innovation

The patent describes engineered Saccharomyces yeast for fermentative production of 2,3-butanediol (2,3-BDO) using extensive metabolic reprogramming. The approach reduces or eliminates expression of glyceraldehyde-3-phosphate dehydrogenase encoded by GPD1 and GPD2, pyruvate decarboxylase encoded by PDC1, and alcohol dehydrogenase encoded by ADH1, and it introduces heterologous nucleic acid molecules encoding acetolactate synthase (alsS) and acetolactate decarboxylase (alsD).

To route metabolism toward 2,3-BDO and improve pyruvate utilization, the yeast increases expression of pyruvate decarboxylase encoded by PYC1 and PYC2 as compared to expression in a wild-type yeast cell of the same species. The patent also introduces heterologous nucleic acids encoding malate dehydrogenase (Mdh3) and malic enzyme (Me1), and in additional disclosures malate dehydrogenase activity is implemented using truncated MDH3 (tMdh3) that lacks the SKL sequence, with a pyruvate-malate (PM) NAD+/NAD+ regeneration cycle used for NAD+ regeneration.

The patent also discloses converting 2,3-BDO into methyl ethyl ketone (MEK). It includes collecting and purifying 2,3-BDO and performing catalytic dehydration on the purified 2,3-BDO to generate MEK, including use of tricalcium phosphate as a dehydration catalyst. The document further discloses use of unpurified yeast 2,3-BDO fermentation broth as a biostimulant to induce drought tolerance in plants, with roots contacted with fermentation broth.

Claims Coverage

The partial content provides one independent claim. It covers an engineered recombinant yeast cell defined by multiple coordinated genetic modifications, including reductions or eliminations of native enzyme expressions, addition of heterologous genes, increased pyruvate decarboxylase expression, and heterologous Mdh3 and Me1.

The inventive coverage is directed to a recombinant yeast cell that suppresses specific native glycerol- and ethanol-forming activities, introduces heterologous alsS and alsD, increases PYC1/PYC2-associated pyruvate decarboxylase expression relative to wild type, and adds heterologous Mdh3 and Me1 for engineered fermentative 2,3-BDO production.

Stated Advantages

Documented Applications