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Microorganisms and processes for enhanced production of pantothenate

Microorganisms and processes for enhanced production of pantothenate description/claims

This application claims the benefit of prior-filed provisional Patent Application Ser. No. 60/______, entitled “Microorganisms and Processes for Enhanced Production of Pantothenate”, filed Jan. 11, 2002 (pending), to prior-filed provisional Patent Application Ser. No. 60/263,053, filed Jan. 19, 2001 (pending), and to prior-filed provisional Patent Application Ser. No. 60/262,995, filed Jan. 19, 2001 (pending). The present invention is also related to U.S. patent application Ser. No. 09/667,569, filed Sep. 21, 2000 (pending), which is a continuation-in-part of U.S. patent application Ser. No. 09/400,494, filed Sep. 21, 1999 (abandoned). U.S. patent application Ser. No. 09/667,569 also claims the benefit of prior-filed provisional Patent Application Ser. No. 60/210,072, filed Jun. 7, 2000 (expired), provisional Patent Application Ser. No. 60/221,836, filed Jul. 28, 2000 (expired), and provisional Patent Application Ser. No. 60/227,860, filed Aug. 24, 2000 (expired). The entire content of each of the above-referenced applications is incorporated herein by this reference.

Pantothenate, also known as pantothenic acid or vitamin B5, is a member of the B complex of vitamins and is a nutritional requirement for mammals, including livestock and humans (e.g., from food sources, as a water soluble vitamin supplement or as a feed additive). In cells, pantothenate is used primarily for the biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP). These coenzymes function in the metabolism of acyl moieties which form thioesters with the sulfhydryl group of the 4′-phosphopantetheine portion of these molecules. These coenzymes are essential in all cells, participating in over 100 different intermediary reactions in cellular metabolism.

The conventional means of synthesizing pantothenate (in particular, the bioactive D isomer) is via chemical synthesis from bulk chemicals, a process which is hampered by excessive substrate cost as well as the requirement for optical resolution of racemic intermediates. Accordingly, researchers have recently looked to bacterial or microbial systems that produce enzymes useful in pantothenate biosynthesis processes (as bacteria are themselves capable of synthesizing pantothenate). In particular, bioconversion processes have been evaluated as a means of favoring production of the preferred isomer of pantothenic acid. Moreover, methods of direct microbial synthesis have recently been examined as a means of facilitating D-pantothenate production.

There is still, however, significant need for improved pantothenate production processes, in particular, for microbial processes optimized to produce higher yields of desired product.

The present invention relates to improved processes (e.g., microbial syntheses) for the production of pantothenate. Pantothenate production processes have been described in related applications which feature, for example, microbes engineered to overexpress key enzymes of the pantothenate biosynthetic pathway and the isoleucine-valine biosynthetic pathway (see e.g., FIG. 1). Strains have been engineered that are capable of producing >50 g/l of pantothenate in standard fermentation processes (see e.g., International Public. No. WO 01/21772 and U.S. Patent Application No. 60/262,995). In particular, increasing the expression of the panB, panC, panD and panE1 genes and increasing the expression of the ilvBNC and ilvD genes results in strains that convert glucose (pyruvate) to commercially attractive quantities of pantothenate.

In order to enhance production levels of for example, pantothenate, various improvements on the above-described methods have now been developed. For example, U.S. patent application Ser. No. 09/667,569 describes production strains having modified (e.g., deleted or decreased-activity) pantothenate kinase enzymes. In such strains, the pantothenate levels are effectively increased by decreasing utilization of pantothenate for coenzyme A (“CoA”) synthesis. U.S. Patent Application Ser. No. 60/262,995 further describes improved pantothenate-productions strains that have been engineered to minimize utilization of various pantothenate biosynthetic enzymes and/or isoleucine-valine biosynthetic enzymes and/or their respective substrates from being used to produce an alternative product identified as HMBPA.

The present invention features methods to further enhance pantothenate production by modulating a biosynthetic pathway that supplies a substrate for the pantothenate biosynthetic pathway, namely the methylenetetrahydrofolate (“MTF”) biosynthetic pathway. In particular, it has been discovered that increasing levels of MTF by modification of the MTF biosynthetic pathway results in enhanced levels of the key pantothenate biosynthetic pathway intermediate, ketopantoate. Enhanced ketopantoate levels, in turn, result in significantly enhanced pantothenate production levels in appropriately engineered strains. In essence, the present inventors have identified a limiting step in the production of panto-compounds (e.g., pantothenate) by strains engineered to overexpress, for example, the panB, panC, panD, panE1, ilvBNC and ilvD genes, and describe herein a means for overcoming this limitation by modification of the MTF biosynthetic pathway.

At least three effective means of modifying the MTF biosynthetic pathway are described herein. In one aspect, it has been demonstrated that increasing serine levels in the culture medium of pantothenate-producing microorganisms results in enhanced panto-compound production. It has also been demonstrated that increasing the synthesis or activity of 3-phosphoglycerate dehydrogenase (the serA gene product), or the synthesis or activity of serine hydroxymethyl transferase (the glyA gene product), thereby enhancing serine and methylenetetrahydrofolate biosynthesis in appropriately engineered microorganisms, increases panto-compound production.

Accordingly, in one aspect the invention features processes for the enhanced production of pantoate and pantothenate that involve culturing microorganisms having modified pantothenate biosynthetic enzyme activities and having modified methylenetetrahydrofolate (MTF) biosynthetic enzyme activities under conditions such that pantothenate production is enhanced. In another aspect the invention features processes for the enhanced production of pantoate and pantothenate that involve culturing microorganisms having modified pantothenate biosynthetic enzyme activities, having modified isoleucine-valine (ilv) biosynthetic enzymes, and having modified methylenetetrahydrofolate (MTF) biosynthetic enzyme activities under conditions such that pantothenate production is enhanced. In particular, the invention features methods for enhancing production of desired products (e.g., pantoate and/or pantothenate) by increasing the levels of a key intermediate, ketopantoate, by enzymes that contribute to its synthesis. Preferred methods result in production of pantothenate at levels greater than 50, 60, 70 or more g/L after 36 hours of culturing the microorganisms, or such that at least 60, 70, 80, 90 or more g/L pantothenate is produced after 36 hours of culturing the microorganisms. Recombinant microorganisms and conditions for culturing same are also are featured. Also featured are compositions produced by such microorganisms.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

FIG. 1 is a schematic representation of the pantothenate and isoleucine-valine (ilv) biosynthetic pathways. Pantothenate biosynthetic enzymes are depicted in bold and their corresponding genes indicated in italics. Isoleucine-valine (ilv) biosynthetic enzymes are depicted in bold italics and their corresponding genes indicated in italics.

FIG. 2 is a schematic representation of the methylenetetrahydrofolate (“MTF”) biosynthetic pathway in E. coli (and presumably in B. subtilis).

FIG. 3 is a schematic representation of the construction of the plasmid pAN665.

FIG. 4 is a schematic representation of the construction of the plasmid pAN670.

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