Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-15
2002-12-03
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S072000, C435S108000, C435S105000, C435S200000, C536S023700, C536S024100, C536S023200
Reexamination Certificate
active
06489100
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the biosynthetic production of organic chemical compounds. In particular, the present invention relates to methods for increasing the yield of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) in microorganisms through genetic alterations. The present invention also relates to methods of enhancing the production of cyclic and aromatic metabolites derived from DAHP in microorganisms through genetic alterations. For example, the biosynthesis of DAHP is the first step in the common aromatic pathway from which tyrosine, tryptophan, phenylalanine, and other aromatic metabolites are formed. Also, pathways branching from the common aromatic pathway provide such useful chemical products as catechol and quinoid organics such as quinic acid, benzoquinone, and hydroquinone. In addition, aspartame and indigo can be produced from products derived from the common aromatic pathway.
BACKGROUND OF THE INVENTION
Production of chemicals from microorganisms has long been an important application of biotechnology. Typically, the steps involved in developing a microorganism production strain include (i) selection of a proper host microorganism, (ii) elimination of metabolic pathways leading to by-products, (iii) deregulation of such pathways at both the enzyme activity level and the transcriptional level, and (iv) overexpression of appropriate enzymes in the desired pathways.
The last three steps can now be achieved by use of a variety of in vivo and in vitro methods. These methods are particularly user-friendly in well-studied microorganisms such as
Escherichia coli
(
E. coli
). Therefore, many examples of engineered microorganisms for physiological characterization and metabolite production have been published.
In most cases, the first target for engineering is the terminal pathway leading to the desired product, and the results are usually successful. However, further improvements of productivity (product formation rate) and yield (percent conversion) of desired products call for the alteration of central metabolic pathways which supply necessary precursors and energy for the desired biosyntheses of those products.
Cyclic and aromatic metabolites such as tryptophan, phenylalanine, tyrosine, quinones, and the like trace their biosynthesis to the condensation reaction of phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) to form DAHP. DAHP biosynthesis is the first committed step in the common aromatic pathway. DAHP biosynthesis is mediated by three DAHP synthases or isoenzymes. These isoenzymes are coded by genes aroF, aroG, and aroH, whose gene products are feed-back inhibited by tyrosine, phenylalanine and tryptophan, respectively.
After DAHP biosynthesis, some DAHP is converted to chorismate. Chorismate is an intermediate in biosynthetic pathways that ultimately leads to the production of aromatic compounds such as phenylalanine, tryptophan, tyrosine, folate, melanin, ubiquinone, menaquinone, prephenic acid (used in the production of the antibiotic bacilysin) and enterochelin. Because of the large number of biosynthetic pathways that depend from chorismate, the biosynthetic pathway utilized by organisms to produce chorismate is often known as the “common aromatic pathway”.
Besides its use in chorismate production, DAHP can also be converted to quinic acid, hydroquinone, benzohydroquinone, or catechol as described by Draths et al. (Draths, K. M., Ward, T. L., Frost, J. W., “Biocatalysis and Nineteenth Century Organic Chemistry: conversion of D-Glucose into Quinoid Organics,”
J. Am. Chem. Soc.,
1992, 114, 1925-26). These biosynthetic pathways branch off from the common aromatic pathway before shikimate is formed.
The efficient production of DAHP by a microorganism is important for the production of aromatic metabolites because DAHP is the precursor in major pathways that produce the aromatic metabolites. The three aromatic amino acids, besides being essential building blocks for proteins, are useful precursor chemicals for other compounds such as aspartame, which requires phenylalanine. Additionally, the tryptophan pathway can be genetically modified to produce indigo.
The production of tryptophan and phenylalanine by
E. coli
has been well documented. For example, Aiba et al. (Aiba, S., H. Tsunekawa, and T. Imanaka, “New Approach to Tryptophan Production by
Escherichia coli
: Genetic Manipulation of Composite Plasmids In Vitro,”
Appl. Env. Microbiol.
1982, 43:289-297) have reported a tryptophan overproducer that contains overexpressed genes in the tryptophan operon in a host strain that is trpR and tna (encoding tryptophanase) negative. Moreover, various enzymes, such as the trpE gene product, have been mutated to resist feedback inhibition. Similar work has been reported for phenylalanine production.
In the past, the enhanced commitment of cellular carbon sources entering and flowing through the common aromatic pathway has been accomplished with only modest success (i.e., such attempts have fallen far below the theoretical yield). Typically, the enhancements were accomplished, by transferring into host cells, genetic elements encoding enzymes that direct carbon flow into and/or through the common aromatic pathway. Such genetic elements can be in the form of extrachromosomal plasmids, cosmids, phages, or other replicons capable of transforming genetic elements into the host cell.
U.S. Pat. No. 5,168,056 to Frost described the use of a genetic element containing an expression vector and a. gene coding for transketolase (Tkt), the tkt gene. This genetic element can be integrated into the microorganisms chromosome to provide overexpression of the Tkt enzyme.
Additional examples include: Miller et al. (Miller, J. E., K. C. Backman, J. M. O'Connor, and T. R. Hatch, “Production of phenylalanine and organic acids by PEP carboxylase-deficient mutants of
Escherichia coli,”J. Ind. Microbiol.,
1987, 2:143-149) who attempted to direct more carbon flux into the amino acid pathway by use of a phosphoenolpyruvate carboxylase (coded by ppc) deficient mutant; Draths et al. (Draths, K. M., D. L. Pompliano, D. L. Conley, J. W. Frost, A. Berry, G. L. Disbrow, R. J. Staversky, and J. C. Lievense, “Biocatalytic synthesis of aromatics from D-glucose: The role of transketolase,”
J. Am. Chem. Soc.,
1992, 114:3956-3962) who reported that overexpression of transketolase (coded by tktA) and a feed-back resistant DAHP synthase (coded by aroG
fbr
) resulted in improved production of DAHP from glucose.
The overproduction of transketolase in tkt transformed cells has been found to provide an increased flow of carbon resources into the common aromatic pathway relative to carbon resource utilization in whole cells that do not harbor such genetic elements. However, the increased carbon flux may be further enhanced by additional manipulation of the host strain.
Thus, it is desirable to develop genetically engineered strains of microorganisms that are capable of enhancing the production of DAHP to near theoretical yield. Such genetically engineered strains can then be used for selective production of DAHP or in combination with other incorporated genetic material for selective production of desired metabolites. Efficient and cost-effective biosynthetic production of chorismate, quinic acid, hydroquinone, benzohydroquinone, catechol, or derivatives of these chemicals requires that carbon sources such as glucose, lactose, galactose, xylose, ribose, or other sugars be converted to the desired product in high yields. Accordingly, it is valuable from the standpoint of industrial biosynthetic production of metabolites to increase the influx of carbon sources for cell biosynthesis of DAHP and its derivatives.
SUMMARY OF THE INVENTION
The present invention provides genetically engineered strains of microorganisms that overexpress the pps gene for increasing the production of DAHP to near theoretical yields. The present invention also provides genetically engineered strains of microorganisms where at least one of the plasmids pPS341, pPSL706, pPS706, or der
Bracewell & Patterson L.L.P.
Jones W. Gary
Taylor Janell E.
Texas A & M University System
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