Metabolically engineered E. coli for enhanced production of...

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using a micro-organism to make a protein or polypeptide

Reexamination Certificate

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C435S041000, C435S045000, C435S071100

Reexamination Certificate

active

06455284

ABSTRACT:

BACKGROUND OF THE INVENTION
Tremendous commercial potential exists for producing oxaloacetate-derived biochemicals via aerobic or anaerobic bacterial fermentation processes. Aerobic fermentation processes can be used to produce oxaloacetate-derived amino acids such as asparagine, aspartate, methionine, threonine, isoleucine, and lysine. Lysine, in particular, is of great commercial interest in the world market. Raw materials comprise a significant portion of lysine production cost, and hence process yield (product generated per substrate consumed) is an important measure of performance and economic viability. The stringent metabolic regulation of carbon flow (described below) can limit process yields. Carbon flux towards oxaloacetate (OAA) remains constant regardless of system perturbations (J. Vallino et al.,
Biotechnol. Bioeng
., 41, 633-646 (1993)). In one reported fermentation, to maintain this rigid regulation of carbon flow at the low growth rates desirable for lysine production, the cells converted less carbon to oxaloacetate, thereby limiting the lysine yield (R. Kiss et al.,
Biotechnol. Bioeng
., 39, 565-574 (1992)). Hence, a tremendous opportunity exists to improve the process by overcoming the metabolic regulation of carbon flow.
Anaerobic fermentation processes can be used to produce oxaloacetate-derived organic acids such as malate, fumarate, and succinate. Chemical processes using petroleum feedstock can also be used, and have historically been more efficient for production of these organic acids than bacterial fermentations. Succinic acid in particular, and its derivatives, have great potential for use as specialty chemicals. They can be advantageously employed in diverse applications in the food, pharmaceutical, and cosmetics industries, and can also serve as starting materials in the production of commodity chemicals such as 1,4-butanediol and tetrahydrofuran (L. Schilling,
FEMS Microbiol. Rev
., 16, 101-110 (1995)). Anaerobic rumen bacteria have been considered for use in producing succinic acid via bacterial fermentation processes, but these bacteria tend to lyse during the fermentation. More recently, the strict anaerobe
Anaerobiospirillum succiniciproducens
has been used, which is more robust and produces higher levels of succinate (R. Datta, U.S. Pat. No. 5,143,833 (1992); R. Datta et al., Eur. Pat. Appl.
405707
(1991)).
Commercial fermentation processes use crop-derived carbohydrates to produce bulk biochemicals. Glucose, one common carbohydrate substrate, is usually metabolized via the Embden-Meyerhof-Pamas (EMP) pathway, also known as the glycolytic pathway, to phosphoenolpyruvate (PEP) and then pyruvate. All organisms derive some energy from the glycolytic breakdown of glucose, regardless of whether they are grown aerobically or anaerobically. However, beyond these two intermediates, the pathways for carbon metabolism are different depending on whether the organism grows aerobically or anaerobically, and the fates of PEP and pyruvate depend on the particular organism involved as well as the conditions under which metabolism is taking place.
In aerobic metabolism, the carbon atoms of glucose are oxidized fully to carbon dioxide in a cyclic process known as the tricarboxylic acid (TCA) cycle or, sometimes, the citric acid cycle, or Krebs cycle. The TCA cycle begins when oxaloacetate combines with acetyl-CoA to form citrate. Complete oxidation of glucose during the TCA cycle ultimately liberates significantly more energy from a single molecule of glucose than is extracted during glycolysis alone. In addition to fueling the TCA cycle in aerobic fermentations, oxaloacetate also serves as an important precursor for the synthesis of the amino acids asparagine, aspartate, methionine, threonine, isoleucine and lysine. This aerobic pathway is shown in
FIG. 1
for
Escherichia coli
, the most commonly studied microorganism. Anaerobic organisms, on the other hand, do not fully oxidize glucose. Instead, pyruvate and oxaloacetate are used as acceptor molecules in the reoxidation of reduced cofactors (NADH) generated in the EMP pathway. This leads to the generation and accumulation of reduced biochemicals such as acetate, lactate, ethanol, formate and succinate. This anaerobic pathway for
E. coli
is shown in FIG.
2
.
Intermediates of the TCA cycle are also used in the biosynthesis of many important cellular compounds. For example, &agr;-ketoglutarate is used to biosynthesize the amino acids glutamate, glutamine, arginine, and proline, and succinyl-CoA is used to biosynthesize porphyrins. Under anaerobic conditions, these important intermediates are still needed. As a result, succinyl-CoA, for example, is made under anaerobic conditions from oxaloacetate in a reverse reaction; i.e., the TCA cycle runs backwards from oxaloacetate to succinyl-CoA.
Oxaloacetate that is used for the biosynthesis of these compounds must be replenished if the TCA cycle is to continue unabated and metabolic functionality is to be maintained. Many organisms have thus developed what are known as “anaplerotic pathways” that regenerate intermediates for recruitment into the TCA cycle. Among the important reactions that accomplish this replenishing are those in which oxaloacetate is formed from either PEP or pyruvate. These pathways that resupply intermediates in the TCA cycle can be utilized during either aerobic or anaerobic metabolism.
PEP occupies a central position, or node, in carbohydrate metabolism. As the final intermediate in glycolysis, and hence the immediate precursor in the formation of pyruvate via the action of the enzyme pyruvate kinase, it can serve as a source of energy. Additionally, PEP can replenish intermediates in the TCA cycle via the anaplerotic action of the enzyme PEP carboxylase, which converts PEP directly into the TCA intermediate oxaloacetate. PEP is also often a cosubstrate for glucose uptake into the cell via the phosphotransferase system (PTS) and is used to biosynthesize aromatic amino acids. In many organisms, TCA cycle intermediates can be regenerated directly from pyruvate. For example, pyruvate carboxylase (PYC), which is found in some bacteria but not
E. coli
, mediates the formation of oxaloacetate by the carboxylation of pyruvate utilizing carboxybiotin. As might be expected, the partitioning of PEP is rigidly regulated by cellular control mechanisms, causing a metabolic “bottleneck” which limits the amount and direction of carbon flowing through this juncture. The enzyme-mediated conversions that occur between PEP, pyruvate and oxaloacetate are shown in FIG.
3
.
TCA cycle intermediates can also be regenerated in some plants and microorganisms from acetyl-CoA via what is known as the “glyoxylate shunt,” “glyoxylate bypass” or glyoxylate cycle (FIG.
4
). This pathway enables organisms growing on 2-carbon substrates to replenish their oxaloacetate. Examples of 2-carbon substrates include acetate and other fatty acids as well as long-chain n-alkanes. These substrates do not provide a 3-carbon intermediate such as PEP which can be carboxylated to form oxaloacetate. In the glyoxylate shunt, isocitrate from the TCA cycle is cleaved into glyoxylate and succinate by the enzyme isocitrate lyase. The released glyoxylate combines with acetyl-CoA to form malate through the action of the enzyme malate synthase. Both succinate and malate generate oxaloacetate through the TCA cycle. Expression of the genes encoding the glyoxylate bypass enzymes is tightly controlled, and normally these genes are repressed when 3-carbon compounds are available. In
E. coli
, for example, the genes encoding the glyoxylate bypass enzymes are located on the aceBAK operon and are controlled by several transcriptional regulators: ic/R (A. Sunnarborg et al.,
J. Bacteriol
., 172, 2642-2649 (1990)), fad/R (S. Maloy et al.,
J. Bacteriol
. 148 83-90 (1981)), fruR (A. Chia et al.,
J. Bacteriol
., 171, 2424-2434 (1989)), and arcAB (S. Iuchi et al.,
J. Bacteriol
. 171 868-873 (1989); S. Iuchi et al.,
Proc. Natl. Acad. Sci. USA
, 85, 1888-1892 (1988)). The glyoxylate bypass enzyme

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