Polynucleotide portions of the biotin operon from B....

Details Polynucleotide portions of the biotin operon from B.... Polynucleotide portions of the biotin operon from B....

2000-08-08

2003-12-02

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S252310, C435S254110, C435S325000, C435S419000, C435S320100, C536S023100, C536S023200

Reexamination Certificate

active

06656721

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a process for producing a target fermentation product. More particularly, the present invention relates to a process for over-producing a target fermentation product in a microorganism having a mutation which causes auxotrophic growth of the microorganism, but that does not compromise its ability to over-produce the target fermentation product. Host cells and polynucleotide sequences used in the process are also provided.
BACKGROUND OF THE INVENTION
Many commercially valuable products are produced by fermentation reactions. For example, riboflavin, which is an essential vitamin that is required by all bacteria, animals, and plants, is synthesized by plants and bacteria, however, it cannot be produced by higher animals, which must acquire it from their diet.
Riboflavin is produced commercially for use as a food and feed additive by, for example, fermentation reactions using
Ashbya govsypii, Eremothecium ashbyii
, or
Candida flareri
cells. (See e.g., Ainsworth, G. C. and Sussman, A. S., The Fungi, Academic Press, New York (1965); Heefner, D. L., et al., WO 88/09822; Hickey, R. J., Production of Riboflavin by Fermentation, in Industrial Fermentation (Underkofler, L. A. and Hickey, R. J., eds.) pp. 157-190, Chemical Publishing Co., New York (1954); and Perlman, D., et al., Fermentation Ind. Eng. Chem. 44:1996-2001 (1952).
The enzymes required to catalyze the biosynthesis of riboflavin from guanosine triphosphate (GTP) and ribulose-5-phosphate are encoded by four genes (ribG, ribB, ribA, and ribH) in
B. subtilis
. See, FIG.
1
A. These genes are located in an operon, the gene order of which differs from the order of the enzymatic reactions catalyzed by the enzymes. For example, GTP cyclohydrolase II, which catalyzes the first step in riboflavin biosynthesis is encoded by the third gene in the operon, ribA. See, FIG.
2
. The ribA protein also encodes a second enzymatic activity, i.e., DHB synthase, which catalyzes the conversion of ribulose-5-phosphate to the four-carbon unit DHB. Deaminase and reductase are encoded by the first gene of the operon, ribG. The bi-functionality of the ribA and ribG gene products may facilitate a coordinated riboflavin precursor flux. The penultimate step in riboflavin biosynthesis is catalyzed by lumazine (Lum) synthase, the product of the last rib gene, ribH. Riboflavin synthase, which controls the last step of the pathway, is encoded by the second gene of the operon, ribB. The function of the gene X (
FIG. 1
) located at the 3′ end of the rib operon is, at present, unclear, however, its gene product is not required for riboflavin synthesis.
Transcription of the riboflavin operon from the ribP
1
promoter is controlled by an attenuation mechanism involving a regulatory leader region located between ribP
1
and ribG. RibO mutations within this leader region result in deregulated expression of the riboflavin operon. Deregulated expression is also observed in strains containing missense mutations in the ribC gene. The ribC gene has recently been shown to encode the flavin kinase/FAD synthase of
B. subtilis
. See, Mack, M., et al.,
J. Bact
., 180:950-55 (1998). Deregulating mutations reduce the flavokinase activity of the ribC gene product resulting in reduced intracellular concentrations of flavinmononucleotide (FMN), the effector molecule of the riboflavin regulatory system.
Recently, a
Bacillus subtilis
microorganism was genetically engineered to produce high yields of riboflavin during a short fermentation cycle. See, Perkins, J. B., U.S. Pat. No. 5,837,528 (“Perkins '528”), which is hereby incorporated by reference as if recited in full herein. This approach combined classical genetic mutant selection and fermentation improvement with genetic engineering of the riboflavin biosynthetic genes by deregulating and increasing the level of gene expression. In this system, the expression of the rib genes was increased by mutating the flavokinase encoding ribC gene, by linking the rib genes to strong, constitutive promoters, and by increasing the copy number of the rib genes.
For example, in the engineered rib operon present in plasmid pRF69 disclosed by Perkins '528, the entire ribP
1
promoter and most of the regulatory leader region were deleted and replaced with a constitutive phage SPO1promoter, P
15
. See, FIG.
1
B. In addition, the phage promoter was introduced between the ribB and ribA genes to further increase the transcription of the corresponding downstream genes. Finally, pRF69 was provided with a chloramphenicol resistance gene downstream of the rib genes. pRF69 was targeted by single cross-over transformation into the rib operon of the host microorganism RB50, which contained mutations deregulating purine biosynthesis and which contained a mutation in the ribC gene deregulating riboflavin biosynthesis.
The genomic structure resulting from single crossover transformation of RB50 with pRF69 includes a chloramphenicol resistance gene flanked by the wild type rib operon at one end and by the engineered rib operon of pRF69 at the other end. The iterative elements within this structure originate increased copy numbers of the resistance gene and of the flanking rib operon upon selection of the pRF69 transformed bacteria for increased chloramphenicol resistance.
Enhanced transcription of the rib genes in RB50 containing multiple (n) copies of the modified rib operon of pRF69 (i.e., RB50::[pRF69]
n
) has been confirmed by Northern blot analysis. Unlike wild-type
B. subtilis
, which accumulated very small amounts of RNA transcript that covered the entire rib operon, RB50::[pRF69]
n
accumulated large amounts of shorter transcripts that covered primarily the first two genes of the operon. The second P
15
promoter engineered upstream of ribA gave rise to significant accumulation of RNA transcripts that covered the three downstream genes of the rib operon. See, Perkins, J. B., et al.,
J. Ind. Microbiol Biotechnol
., 22:8-18 (1999).
In a riboflavin fed batch fermentation reactor containing, for example,
B. subtilis
RB50::[pRF69]
n
, biomass and riboflavin are produced from a common fermentation substrate, glucose. The rate by which glucose is pumped into the reactor (“glucose feeding rate”) is critical to its utilization in the production of biomass and riboflavin, respectively. A fast glucose feeding rate allows the culture to grow at elevated rates causing an excess of biomass formation and a reduction of the riboflavin yield. Glucose feeding rates that are too slow, however, while lowering biomass production, result in low riboflavin productivity. Because low yield, low productivity, or both increase riboflavin production costs, a balance must be struck between biomass and riboflavin production by carefully regulating the glucose feeding rate in commercial riboflavin fermentation reactors.
SUMMARY OF THE INVENTION
In view of the deficiencies noted above, it would be desirable to optimize production of a target fermentation product, such as riboflavin, while concurrently maintaining biomass production at a level that is most efficient for the size and type of reactor used.
One embodiment of the present invention is a process for producing a target fermentation product. This process includes providing a fermentation medium containing a recombinantly-produced microorganism that over-produces a target fermentation product and that contains a mutation which causes auxotrophic growth of the microorganism, wherein the auxotrophy within the microorganism does not compromise the ability of the microorganism to produce the target fermentation product. The medium is then supplied with all substrates required for production of the fermentation product, a substrate for the target fermentation product, and a substrate complementing the auxotrophy. The former substrate(s) is/are provided in excess, ensuring maximal productivity. The latter substrate (i.e., substrate complementing the auxotrophy) is supplied in limited amounts to maintain biomass formatio

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