William Marsh Rice University

Other

Glycos Biotechnologies, Inc.

Ramon Gonzalez

James M Clomburg

Seokjung Cheong

Clementina Dellomonaco

Elliot N Miller

Alexander Chou

Paul Campbell

Jacob E Vick

This disclosure generally relates to the use of microorganisms to make various functionalized polyketides through polyketoacyl-CoA thiolase-catalyzed non-decarboxylative condensation reactions instead of decarboxylative reactions catalyzed by polyketide synthases. Native or engineered polyketoacyl-CoA thiolases catalyze the non-decarboxylative Claisen condensation in an iterative manner (i.e. multiple rounds) between two either unsubstituted or functionalized ketoacyl-CoAs (and polyketoacyl-CoAs) serving as the primers and acyl-CoAs serving as the extender unit to generate (and elongate) polyketoacyl-CoAs. Before the next round of polyketoacyl-CoA thiolase reaction, the &#x3b2;-keto group of the polyketide chain of polyketoacyl-CoA can be reduced and modified step-wise by 3-OH-polyketoacyl-CoA dehydrogenase or polyketoenoyl-CoA hydratase or polyketoenoyl-CoA reductase. Dehydrogenase converts the &#x3b2;-keto group to &#x3b2;-hydroxy group. Hydratase converts the &#x3b2;-hydroxy group to &#x3b1;-&#x3b2;-double-bond. Reductase converts the &#x3b1;-&#x3b2;-double-bond to single bond. Spontaneous or thioesterase catalyzed termination reaction terminates the elongation of polyketide chain of polyketoacyl-CoA at any point through CoA removal and spontaneous reactions rearrange the structure, generating the final functional polyketide products.

Biosynthesis of polyketides

The use of microorganisms to make alpha-functionalized chemicals and fuels, (e.g. alpha-functionalized carboxylic acids, alcohols, hydrocarbons, amines, and their beta-, and omega-functionalized derivatives), by utilizing an iterative carbon chain elongation pathway that uses functionalized extender units. The core enzymes in the pathway include thiolase, dehydrogenase, dehydratase and reductase. Native or engineered thiolases catalyze the condensation of either unsubstituted or functionalized acyl-CoA primers with an alpha-functionalized acetyl-CoA as the extender unit to generate alpha-functionalized &#x3b2;-keto acyl-CoA. Dehydrogenase converts alpha-functionalized &#x3b2;-keto acyl-CoA to alpha-functionalized &#x3b2;-hydroxy acyl-CoA. Dehydratase converts alpha-functionalized &#x3b2;-hydroxy acyl-CoA to alpha-functionalized enoyl-CoA. Reductase converts alpha-functionalized enoyl-CoA to alpha-functionalized acyl-CoA. The platform can be operated in an iterative manner (i.e. multiple turns) by using the resulting alpha-functionalized acyl-CoA as primer and the aforementioned alpha-functionalized extender unit in subsequent turns of the cycle. Termination pathways acting on any of the four alpha-functionalized CoA thioester intermediates terminate the platform and generate various alpha-functionalized carboxylic acids, alcohols and amines with different &#x3b2;-reduction degree.

Iterative platform for the synthesis of alpha functionalized products

Methods of using microorganisms to make chemicals and fuels, including carboxylic acids, alcohols, hydrocarbons, and their alpha-, beta-, and omega-functionalized derivatives are described. Native or engineered thiolases are used condense a growing acyl-ACP and acetyl-ACP in combination with type II fatty acid synthesis. The resulting fatty acid biosynthesis cycle has an ATP yield analogous to the functional reverse &#x3b2;-oxidation cycle.

Modified fatty acid biosynthesis with ACP-dependent thiolases

An engineered microbe that contains a designed platform for the conversion of one-carbon substrates to chemical products is described. The designed platform embodies a new metabolic architecture that consolidates carbon fixation, central metabolism, and product synthesis into a single pathway. This is made possible by the key finding that 2-hydroxyacyl-CoA lyase, an enzyme in the &#x3b1;-oxidation pathway, is capable of catalyzing the C&#x2014;C bond formation between formyl-CoA and aldehydes of different chain lengths, allowing for the elongation of the carbon backbone of said aldehyde by one-carbon units. These novel microbes present an opportunity for the production of chemicals from single-carbon feedstocks such as carbon dioxide, carbon monoxide, formate, formaldehyde, methanol or methane.

Biosynthesis of products from 1-carbon compounds

This disclosure generally relates to the use of enzyme combinations or recombinant microbes comprising same to make isoprenoid precursors, isoprenoids and derivatives thereof including prenylated aromatic compounds. Novel metabolic pathways exploiting Claisen, aldol, and acyioin condensations are used instead of the natural mevalonate (MVA) pathway or 1-deoxy-d-xylulose 5-phosphate (DXP) pathways for generating isoprenoid precursors such as isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), and geranyl pyrophosphate (GPP). These pathways have the potential for better carbon and or energy efficiency than native pathways. Both decarboxylative and non-carboxylative condensations are utilized, enabling product synthesis from a number of different starting compounds. These condensation reactions serve as a platform for the synthesis of isoprenoid precursors when utilized in combination with a variety of metabolic pathways and enzymes for carbon rearrangement and the addition/removal of functional groups. Isoprenoid alcohols are key intermediary products for the production of isoprenoid precursors in these novel synthetic metabolic pathways.

Synthesis of isoprenoids and derivatives

The invention relates to recombinant microorganisms that have been engineered to produce various chemicals using genes that have been repurposed to create a reverse beta oxidation pathway. Generally speaking, the beta oxidation cycle is expressed and driven in reverse by modifying various regulation points for as many cycles as needed, and then the CoA thioester intermediates are converted to useful products by the action of termination enzymes.

Reverse beta oxidation pathway

This disclosure describes enzymes from the type II (a discrete set of enzymes) fatty acid synthesis ('FAS') pathway that can be used in combination with thiolases to operate a functional reversal of the &#x3b2;-oxidation cycle. A combination of thiolases with one or more of 3-oxoacyl-[acyl-carrier-protein] reductase (FabG, others), 3-hydroxyacyl-[acp] dehydratase (FabA, FabZ, others), and enoyl-[acyl-carrier-protein] reductase (FabI, FabK, FabL, FabV, others) yields a functional reversal of the &#x3b2;-oxidation cycle. If only one or two enzymes are used, the remaining enzymes will be traditional beta oxidation enzymes. Once this cycle is coupled with the appropriate priming and termination pathways, the production of carboxylic acids, alcohols, hydrocarbons, amines and their &#x3b1;-, &#x3b2;-, and &#x3c9;-functionalized derivatives from renewable carbon sources can be achieved.

Type II fatty acid synthesis enzymes in reverse &#x3b2;-oxidation

Bacteria that run the beta oxidation cycle in reverse anabolic direction are provided, along with many novel primers to start the reverse cycle, pathways to make such primers, and a large variety of products produced thereby. Methods for making desired product by using such primers in the reverse pathway are also disclosed.

Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation in engineered microbes

Glycerol or other reduced carbon sources may be used as a feedstock for the microbial production of chemical products under certain microaerobic conditions. For example, such production may occur under microaerobic or microrespiratory conditions in which electron acceptors are consumed in the reaction as quickly as they are added. In such reactions, the reaction product is at least as reduced as carbon source. Further, during such a reaction, at least some of the carbon source is used to generate cell mass. In addition, microorganisms with modified genomes are provided for carrying out the methods herein.

Microaerobic cultures for converting glycerol to chemicals

The invention relates to the development of appropriate cultivation conditions for a bacteria to grow anaerobically (fermentatively) on a glycerol substrate. The method requires culturing bacteria having a functional 1,2-propanediol pathway and a functional type II glycerol dehydrogenase-dihydroxyacetone kinase pathway in a culture medium containing high concentrations of glycerol, a neutral to mildly acidic pII, low levels of potassium and phosphate, and high levels of CO2, such that glycerol is thus converted into a desirable product, such as ethanol, hydrogen, formate, succinate, or 1,2-propanediol.

Anaerobic fermentation of glycerol

The invention relates to the development of appropriate cultivation conditions for a bacteria to grow anaerobically (fermentatively) on a glycerol substrate. The method requires culturing bacteria having a functional 1,2-propanediol pathway and a functional type II glycerol dehydrogenase-dihydroxyacetone kinase pathway in a culture medium containing high concentrations of glycerol, a neutral to mildly acidic pH, low levels of potassium and phosphate, and high levels of CO2, such that glycerol is thus converted into a desirable product, such as ethanol, hydrogen, formate, succinate, or 1,2-propanediol.

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Growing community of inventors

Houston, TX, United States of America

Ramon Gonzalez