Materials and methods for the efficient production of acetate and other products

Abstract

The subject invention provides materials and methods wherein unique and advantageous combinations of gene mutations are used to direct carbon flow from sugars to a single product. The techniques of the subject invention can be used to obtain products from native pathways as well as from recombinant pathways. In preferred embodiments, the subject invention provides new materials and methods for the efficient production of acetate and pyruvic acid.

Description

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 60 / 424,372, filed Nov. 6, 2002.BACKGROUND OF INVENTION[0003] Recent trends toward the production of "green" chemicals will require development of innovative synthesis techniques that are highly efficient and cost effective.[0004] Throughout the past decade, a number of traditional chemical companies in the United States and Europe have begun to develop infrastructures for the production of compounds using biocatalytic processes. Considerable progress has been reported toward new processes for commodity chemicals such as ethanol (Ingram, L. O., H. C. Aldrich, A. C. C. Borges, T. B. Causey, A. Martinez, F. Morales, A. Saleh, S. A. Underwood, L. P. Yomano, S. W. York, J. Zaldivar, and S. Zhou, 1999 "Enteric bacterial catalyst for fuel ethanol production" Biotechnol. Prog. 15:855-866; Underwood, S. A., S. Zhou, T. B. Causey, L. P. Yomano, K. T. Shanmugam, and L. O. Ingram, 2002 "Genetic changes to opti...

AI Technical Summary

Benefits of technology

[0027] Further embodiments of the subject invention provide additional derivatives of Escherichia coli W3110 as new biocatalysts for the production of homo-acetate. In one embodiment, homo-acetate production by the new strain, TC36, approaches the theoretical maximum of two acetates per glucose. Eliminating the fermentation pathways of W3110 resulted in the new strain SZ47 and doubled the loss of carbon as volatile products. While the rate of acetate production decreased in SZ47 as compared to W3110, the cell yield increased. The inactivation of oxidative phosphorylation (.DELTA.atpFH) in SZ47 to produce TC24 resulted in a 5-fold increase in acetate yield and a 3-fold improvement in carbon recovery.

[0028] In accordance with the subject invention, competing pathways are eliminated by chromosomal inactivation of genes encoding lactate dehydrogenase, pyruvate formatelyase, and fumarate reductase (.DELTA.(focA-pflB)::FRT .DELTA.frdBC .DELTA.ldhA), (F.sub.1F.sub.0)H.sup.+-ATP synthase (atpFH), alcohol / aldehyde dehydrogenase (adhE), and 2-ketoglutarate dehydrogenase (sucA), which increases the production of acetate.

[0029] Using a simple two-step batch feeding strategy can increase acetate production. Specifically, a second addition of 3% glucose added at the end of the growth phase (12 h) and metabolized to completion results in 78% of the theoretical maximum. A further increase in acetate production can be obtained by combining the two-step batch feeding strategy with a nitrogen limitation, which results in 86% of the theoretical maximum.

[0030] The subject invention provides a method to reduce the loss of substrate carbon into cell mass and / or into carbon dioxide. Also, the subject invention provides a method to reduce oxygen demand during bioconversion process.

[0031] The use of mineral salts medium, lack of antibiotic resistance genes or plasmids, high yield of homo-acetate, and high product purity achieved according to the subject invention are advantageous because of reduced costs associated with nutrients, purification, containment, BOD, and waste treatment.

[0032] In an alternative embodiment, the subject invention provides a new biocatalyst for the efficient production of pyruvate from glucose that requires only simple mineral salts as nutrients.

Problems solved by technology

Acetic acid has been produced using microbial systems; however, the production of acetic acid in microbial systems competes with the production of CO.sub.2 and cell mass.

All three of these current microbial acetic acid production systems require complex nutrients, which increase the cost of materials, acetate purification, and waste disposal.

Due to the competing production of dicarboxylic acids and cell mass from glucose, the level of acetate production using these methods has been relatively low.

Indeed, none of these methods have been reported to grow and produce acetate efficiently in mineral salts media containing sugar.

In fact, each of these methods requires the use of complex nutrients, which ultimately increases the cost of materials, acetate purification, and waste disposal.

This process involves toxic solvents and is energy intensive with an estimated production cost of $8,650 per ton of pyruvate.

As noted above, T. glabrata strains used in the commercial process are multivitamin auxotrophs requiring tight regulation of vitamin concentrations which result in complex vitamin feeding strategies during fermentation (Li, Y., J. Chen, and S.-Y.

Previous E. coli strains constructed for pyruvate production were cultured in complex media and have been plagued by low titers and yields (Tomar, A. et al.

Nutrients in culture medium often represent a major cost associated with commercial fermentations.

Images