Engineering of metabolic control

Details Engineering of metabolic control Engineering of metabolic control

2000-07-27

2004-03-16

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Bacteria or actinomycetales; media therefor

C435S252300, C435S320100, C435S166000, C435S167000, C536S024100

Reexamination Certificate

active

06706516

ABSTRACT:

BACKGROUND OF THE INVENTION
The use of recombinant DNA technology has allowed the engineering of host cells to produce desired compounds, such as polypeptides and secondary metabolites. The large scale production of polypeptides in engineered cells allows for the production of proteins with pharmaceutical uses and enzymes with industrial uses. Secondary metabolites are products derived from nature that have long been known for their biological and medicinal importance. Because of the structural complexity inherent in such molecules, traditional chemical synthesis often requires extensive effort and the use of expensive precursors and cofactors to prepare the compound. In recent years, the expression of heterologous proteins in cells has facilitated the engineering of heterologous biosynthetic pathways in microorganisms to produce metabolites from inexpensive starting materials. In this manner, a variety of compounds have been produced, including polyketides, &bgr;-lactam antibiotics, monoterpenes, steroids, and aromatics.
SUMMARY OF THE INVENTION
The invention is based, in part, on the discovery that production of heterologous polypeptides and metabolites can be enhanced by the regulated expression of the polypeptide (e.g., a biosynthetic enzyme) using a promoter which is regulated by the concentrations of a second metabolite, e.g. acetyl phosphate. The term “heterologous” refers to a polypeptide or metabolite which is introduced by artifice. A heterologous polypeptide or metabolite can be identical to endogenous entity that is naturally present. The term “metabolite” refers to a organic compound which is the product of one or more biochemical reactions A metabolite may itself be a precursor for other reactions. A secondary metabolite is a metabolite derived from another.
Accordingly, in one aspect, the invention features a bacterial host cell containing a nucleic acid sequence comprising a promoter and a nucleic acid sequence encoding a heterologous polypeptide. Examples of bacterial host cells include
Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Agrobacterium tumefaciens, Thermus thermophilus
, and
Rhizobium leguminosarum
cells. The nucleic acid sequence is operably linked to the promoter which is controlled by a response regulator protein. In other words, the nucleic acid sequence is linked to the promoter sequence in a manner which allows for expression of the nucleotide sequence in vitro and in vivo. “Promoter” refers to any DNA fragment which directs transcription of genetic material. The promoter is controlled by a response regulator protein, for example, ntrC, phoB, phoP, ompR, cheY, creB, or torR, of
E. coli
or its homologs from other bacterial species. Further, the response regulator protein can be another member of the cluster orthologous group (COG) COG0745 as defined by http//www.ncbi.nlm.nih.gov/COG/(Tatusov et al.
Nucleic Acids Res
. (2000); 28:33-36). In one implementation, the promoter is bound by
E. coli
ntrC. The term “ntrC” refers to both the
E. coli
ntrC protein (SWISSPROT: P06713, http://www.expasy.ch/) and its homologs in other bacteria as appropriate. As used herein, “bound” refers to a physical association with a equilibrium binding constant (K
D
) of less than 100 nM, preferably less than 1 nM. An example of the promoter is the
E. coli
glnAp
2
promoter, e.g. a region between positions about 93 and about 323 in the published DNA sequence, GenBank accession no. M10421(Reitzer & Magasanik (1985)
Proc Nat Acad Sci USA
82:1979-1983). This region includes untranslated sequences from the glnA gene. Further, a translational fusion can be constructed between coding sequences for glnA and coding sequences for the heterologous polypeptide.
The host cell is genetically modified such that the promoter is regulated by acetyl phosphate in the absence of nitrogen starvation. For example, the host cell can genetically modified by deletion or mutation of a gene encoding a histidine protein kinase, e.g., a member of COG0642 as defined by (http://www.ncbi.nlm.nih.gov/COG/; Tatusov et al. supra.), e.g., glnL, phoR, phoQ, creC, or envZ. In another example, the histidine protein kinase has specificity for the response regulator protein which controls the promoter. The histidine protein kinase can be encoded by glnL, e.g.,
E coli
glnL (SWISSPROT P06712; http://www.expasy.ch/).
Whereas the host cell is genetically modified such that the promoter is regulated by acetyl phosphate in the absence of nitrogen starvation, for heterologous polypeptide or metabolite expression, the host cell can be propagated in any desired condition, e.g., in nitrogen starvation conditions, nitrogen poor conditions, or nitrogen rich conditions.
The heterologous polypeptide encoded by the nucleic acid sequence can be a biosynthetic enzyme required for production of a metabolite. It can be a mammalian protein, e.g., a secreted growth factor, a monoclonal antibody, or an extracellular matrix component. In yet another example, the heterologous polypeptide can be a desired antigen for use in a vaccine, e.g., a surface protein from a viral, bacterial, fungal, or protist pathogen.
Another aspect of the invention features a kit containing a nucleic acid sequence which includes a promoter controlled by a response regulator protein. The kit further optionally contains a bacterial host cell which is genetically modified such that the promoter is regulated by acetyl phosphate in the absence of nitrogen starvation. The kit can also provide instructions for their use. The nucleic acid sequence can contain a restriction enzyme polylinker located 3′ of the promoter such that a sequence inserted into the polylinker is operably linked to the promoter which is controlled by a response regulator protein. In one implementation of the kit, the promoter is the
E. coli
glnAp
2
promoter and the bacterial host cell is an
E. coli
cell containing a mutation or deletion of the glnL gene.
Another aspect of the invention features a host cell containing a first expression cassette. The first expression cassette includes a promoter, such as any of those described above, and a nucleic acid sequence encoding an enzyme required for biosynthesis of a heterologous metabolite. As used herein, “enzyme” refers to a polypeptide having ability to catalyze a chemical reaction or multiple reactions. The nucleic acid sequence is operably linked to the promoter which is regulated by acetyl phosphate in the absence of nitrogen starvation. The host cell also contains additional nucleic acid sequences for expressing other enzymes required for biosynthesis of the metabolite. Such additional sequences may be endogenous sequences expressing endogenous enzymes, or introduced sequences expressing heterologous enzymes.
In one example, the heterologous metabolite is an isoprenoid, a polyhydroxyalkanoate, a polyketide, a &bgr;-lactamn antibiotic, an aromatic, or a precursor, e.g., an upstream metabolite, or a derivative, e.g., a downstream metabolite, thereof. For instance, the isoprenoid can be a carotenoid, a sterol, a taxol, a diterpene, a gibberellin, and a quinone. Specific examples of isoprenoids include isopentyl diphosphate, dimethylallyl diphosphate, geranyl diphosphate, farnesyl diphosphate, geranylgeranyl diphosphate, and phytoene. Specific examples of carotenoids include &bgr;-carotene, &zgr;-carotene, astaxanthin, zeaxanthin, zeaxanthin-&bgr;-glucoside, phytofluene, neurosporene, lutein, and torulene. When the desired heterologous metabolite is an isoprenoid, the heterologous enzyme can be isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase, or 1-deoxyxylulose 5-phosphate synthase. When the desired heterologous metabolite is an polyhydroxyalkanoate, the heterologous enzyme can be 3-ketoacyl reductase, or poly-3-hydroxyalkanoate polymerase.
The host cell can be a bacterial cell, e.g., an
E. coli
cell. The host cell is optionally genetically modified by deletion or mutation of a gene, e.g., a gene encoding a histidine protein kinase, as described above. In one specific example, th

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