# Patent

## Title
Production of clavulanic acid by genetic engineering of Streptomyces clavuligerus

## Abstract

Genetically engineered Streptomyces clavuligerus strains with improved capabilities to produce clavulanic acid are provided. The strains are genetically engineered by disrupting newly identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes. This results in an increased intracelluar pool of the clavulanic acid precursor D-glyceraldehyde-3-phosphate (D-G3P), and increased clavulanic acid production. Clavulanic acid production may be further increased by supplying arginine to the medium in which the S. clavuligerus is grown.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. provisional patent application 60/715,999, filed Sep. 9, 2005, the complete contents of which are hereby incorporated by reference.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention generally relates to the improvement of clavulanic acid production in Streptomyces clavuligerus by genetic engineering. In particular, the invention provides genetically engineered S. clavuligerus in which a newly identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene is disrupted, resulting in increased clavulanic acid production.
2. Background of the Invention
The β-lactams penicillin and cephalosporin were among the first useful antibiotics discovered and remain at the forefront of clinical use to combat bacterial infections. The wide-spread use of β-lactam antibiotics for more than 50 years, however, has reduced their effectiveness owing to the emergence of resistance among invading pathogens. As a consequence, strategies aimed at overcoming acquired resistance have become of increasing interest. One of the best examples in broad clinical application is the development of β-lactamase inhibitors. The discovery of clavulanic acid was reported in 1976, (Reading and Cole, 1977) and it has been shown to be a potent inhibitor of β-lactamases produced by staphylococci and plasmid-mediated β-lactamases of E. coli, as well as species from Klebsiella, Proteus, and Hemophilus (Brown et al., 1976). The molecule is produced by the filamentous bacterium Streptomyces clavuligerus, and consists of a β-lactam ring fused to an oxazolidine ring (Howarth et al., 1976; Reading and Cole, 1977). Commercial products such as Augmentin® and Timentin®, which are combinations of clavulanic acid and other established β-lactam antibiotics, are prescribed in more than 150 countries and have attained sales in excess of 2 billion dollars yearly (Elander, 2003).
To date strain improvement of microorganisms to obtain high-titers of secondary metabolites that are more suitable for industrial fermentations has depended largely on random mutagenesis and selection techniques. However, development of a new generation of high production strains with this approach often takes 5 years or more (Nielsen, 1997). A significant drawback is the introduction of a limited spectrum of base-pair substitutions that do not readily solve the specific rate limitations of biosynthetic pathways (Baltz, 1998). In the past few years, as techniques for molecular genetics have become increasingly sophisticated, the ability to modify existing pathways or create non-native pathways has advanced rapidly. Progress in genetics, transcriptional analysis, proteomics, metabolic reconstructions and metabolic flux analysis offer genetic engineering as an alternative approach for strain improvement in a targeted manner (Baltz, 2001). Duplication of specific genes thought to be involved in rate limiting steps can be achieved by inserting the desired gene(s) into a chromosome by homologous recombination or by site-specific integration. In S. clavuligerus, gene dosage constructs of the biosynthetic genes ceas and cs2 resulted in recombinant strains with 60% and 100% higher clavulanic acid production, respectively, compared to the wild-type strain (Perez-Redondo et al., 1999). Disruption of negative regulatory gene(s), or increased expression of positive regulatory gene(s) also can result in the elevated production of secondary metabolites. Paradkar, et al. observed a 2 to 3-fold increase in clavulanic acid production by introducing additional copies of positive regulatory genes in the wild-type (Paradkar et al., 1998; Perez-Llarena et al., 1997; Perez-Redondo et al., 1998). A third approach is to inactivate pathways that compete for key precursors, intermediates, cofactors and energy supply. The inactivation of the clavam pathway, which shares the common intermediate clavaminic acid with the clavulanic acid pathway, has been shown to give an elevated yield of clavulanic acid in S. clavuligerus (Paradkar et al., 2001).
The primary metabolic precursors of clavulanic acid are D-glyceraldehyde-3-phosphate (G3P) (Khaleeli et al., 1999) and L-arginine (Valentine et al., 1993). The observation of arginase and ornithine carbamoyltransferase activities are strongly suggestive of a functional urea cycle in S. clavuligerus (Bascaran et al., 1989; Ives and Bushell, 1997; Romero et al., 1986). The prokaryotic urea cycle is unusual and provides a very effective pathway for arginine biosynthesis such that the pool size of this amino acid could remain sufficient to support an increased rate of clavulanic acid production. Supplemented fermentations of S. clavuligerus with arginine increases only the intracellular pool size of this precursor, but not the production of clavulanic acid (Chen et al., 2002; Chen et al., 2003). Metabolic flux analysis has further suggested that a limiting factor for clavulanic acid biosynthesis is the C3 precursor, G3P (Ives and Bushell, 1997). This deduction was supported by the observation of a stimulatory effect on clavulanic acid production by supplementing cultures of S. clavuligerus with glycerol (Chen et al., 2003). G3P is an intermediate of the glycolytic pathway and also the entry point in the gluconeogenesis pathway for the synthesis of glucose. Metabolic analysis has further shown that in wild-type S. clavuligerus the favored direction of G3P flux ( ̃80%) is consistently towards the glycolytic pathway, and the rest ( ̃20%) enters the gluconeogenesis and clavulanic acid pathways (Kirk et al., 2000) (see FIG. 1). G3P is converted into 1,3-bisphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the glycolytic pathway and finally enters the Krebs cycle through pyruvate.
While these observations suggest that increasing the intracellular pool of G3P could result in enhanced clavulanic acid production in S. clavuligerus, the prior art has thus far failed to exploit this potential, even though there is an ongoing need to develop additional strains with improved capacity to produce high yields of clavulanic acid.
SUMMARY OF THE INVENTION
The present invention is based on the identification of a new S. clavuligerus glyceraldehyde-3-phosphate dehydrogenase gene (gap1), and on the discovery that disruption of gap1 by genetic engineering results in S. clavuligerus mutants in which clavulanic acid production is significantly elevated. Without being bound by theory, it appears that, in gap1 disruption mutants, the C3 pool available for clavulanic acid synthesis is increased dramatically, likely by diversion of the C3 flux away from the glycolytic pathway, which requires a functional GAPDH activity. The C3 pool increases to such an extent that L-arginine becomes the rate-limiting precursor for clavulanic acid biosynthesis. Thus, in a fed-batch culture of the S. clavuligerus gap1 mutants of the invention, a further improvement in clavulanic acid production is observed when the media is supplemented with arginine.
The invention provides Streptomyces clavuligerus genetically modified such that at least one sequence encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity is missing, not functioning, or functioning at a reduced rate, or progeny thereof.
The invention further provides a Streptomyces genetically modified such that at least one sequence encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity is missing, not functioning, or functioning at a reduced rate, or progeny thereof.
The invention further provides a host cell genetically modified such that at least one sequence encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity is missing, not functioning, or functioning at a reduced rate, or progeny thereof.
The invention also provides a method for producing clavulanic acid. The method comprises the steps of 1) growing Streptomyces clavuligerus genetically modified such that at least one sequence encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity is missing, not functioning, or functioning at a reduced rate, or progeny thereof; and 2) recovering clavulanic acid produced by said genetically modified S. clavuligerus or progeny thereof. In some embodiments, the step of growing includes providing arginine.
The invention further provides a method for producing clavulanic acid. The method comprises the steps of 1) growing to Streptomyces genetically modified such that at least one sequence encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity is missing, not functioning, or functioning at a reduced rate, or progeny thereof; and 2) recovering clavulanic acid produced by said genetically modified S. clavuligerus, or progeny thereof. In some embodiments, the step of growing includes providing arginine.
The invention further provides a method for producing clavulanic acid that comprises the steps of 1) growing a host cell genetically modified such that at least one sequence encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity is missing, not functioning, or functioning at a reduced rate, or progeny thereof; and 2) recovering clavulanic acid produced by said genetically modified S. clavuligerus, or progeny thereof. In some embodiments, the step of growing includes providing arginine.
The invention also provides a gap1 gene, a Gap1 protein, a gap2 gene, and a Gap2 protein. Further, a DNA sequence as represented by SEQ ID NO: 50; an amino acid sequence as represented by SEQ ID NO: 43; a DNA sequence as represented by SEQ ID NO: 53; and an amino acid sequence as represented by SEQ ID NO: 56 are also provided.
The invention further provides a process of: 1) impeding a primary metabolic pathway by genetically modifying a host cell such that at least one DNA sequence coding for at least one activity in said primary metabolic pathway is missing, not functioning, or functioning at a reduced rate, or encodes a gene product that is not functioning, or functioning at a reduced rate, said impeding step producing a genetically modified host cell; and 2) providing nutrients to said genetically modified host cell or progeny thereof, whereby said impeding step increases production of one or more chemical species in one or more secondary pathways. In one embodiment of the invention, the primary metabolic pathway is the glycolytic pathway. In one embodiment of the invention, the secondary pathway is a clavulanic acid biosynthetic pathway. In yet another embodiment of the invention, the secondary pathway is a non-mevalonate biosynthetic pathway. In some embodiments of the invention, the impeding step eliminates, prevents the function of, or causes functioning at a reduced rate of Gap1. In other embodiments, the impeding and providing steps are performed in a host cell which is a plant cell. In yet other embodiments, the impeding and providing steps are performed in a host cell which is a Streptomyces. 
The invention further provides a diagnostic for identifying the presence or absence of gap1 or gap2, or both, in a host.
The invention thus provides, in a host cell, a process of impeding at least a portion of a “primary” metabolic pathway (i.e., a pathway considered essential for life such as the glycolytic pathway, Krebs cycle, etc.) to benefit a “secondary” pathway (i.e., a pathway not considered essential for life such as the pathway for clavulanic acid production and the non-mevalonate pathway) while maintaining the viability (and preferably normal functioning) of the host cell. For example, the glycolytic pathway in a host cell can be impeded by genetically modifying said host to either lack at least one gene coding for GADPH activity, or to have a non-functioning or reduced functioning gene, or to encode a non-functioning or reduced functioning gene product. The host cell can be provided with nutrients (e.g., glycerol, arginine, ornithine, vitamins, proteins, carbohydrates, lipids, etc.) that might otherwise be used in the “primary” metabolic pathway. Having the primary pathway “impeded” results in the buildup of precursor molecules which can then be used in a secondary pathway. This invention is distinct from prior schemes in that the “primary” pathway is being impeded to stimulate a “secondary” pathway, yet the host (which can be a plant or microbe, with Streptomyces being a specific example) remains viable and preferably grows normally. With respect to impeding the glycolytic pathway in a host cell, by providing nutrients to the host cell, the impeding pathway results in increases in the production of one or more chemical species in one or more secondary pathways that include D-glyceraldehyde-3-phosphate as a precursor. In one embodiment of the invention, the one or more secondary pathways that include D-glyceraldehyde-3-phosphate as a precursor includes without limitation the clavulanic acid biosynthetic pathway and the non-mevalonate biosynthetic pathway.
By “impeding” it should be understood that the invention contemplates eliminating or disrupting at least one gene coding for at least one enzyme or activity used in a primary pathway, but that the host cell remains viable and preferably functioning (e.g. growing) normally since other genes of the organism (including those in the same pathway coding for the same activity) remain operative. The process results in the accumulation of a precursor that benefits one or more secondary pathways, but does not result in host death (and preferably allows the host cell to function normally) as the host cell maintains genes coding for alternative enzymes or activities which perform the function of the protein coded by the eliminated or disrupted gene.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Clavulanic acid biosynthetic scheme and its precursors showing the carbon flux (some intermediates are omitted). Heavy arrows indicate the C3 precursor flux channeled to the clavulanic acid pathway by targeted gene disruption of gap1 .
FIG. 2: (a) The three conserved motifs of GAPDHs. The accession numbers are as following: S. coelicolor (NP—626211.1), T. tengcongensis (NP—62335, T. fusca (ZP—00059379), S. aureofacien (Q59800), M. tuberculosis (NP—215952), B. megaterium (CAA38376), M. avium (P94915), S. arenae (P54226), O. iheyensis (NP—693359), S. avermitilis (NP—827472), C. tetani (NP—781078), B. cereus (NP—834805). The numbers at two ends indicate the position of amino acids in the proteins, and the numbers between the motifs indicate the separation in amino acid residues. (b) Primers designed for the M1, M2, and M3 motifs according to the CODEHOP program (Rose et al., 1998). N, A/T/G/C; Y, C/T; M, A/C; R, A/G.
FIG. 3A-D. a) Alignment of the deduced S. clavuligerus Gap1 (SEQ ID NO: 43) with homologous GAPDH proteins from other organisms, S. arenae (P54226), S. aureofacien (Q59800), S. coelicolor (NP—626211.1), T. fusca (ZP—00059379), B. halodurans (NP—244427), and C. diphtheriae (NP—939663). Amino acids identical in all aligned proteins are indicated by asterisks (*), and the similar amino acids are indicated by dots (.). The amino acids that have been identified to be important in NAD+−binding and the catalytic mechanism (Skarzynski et al., 1987) are shown in gray and double-underlined; b) nucleic acid sequence encoding Gap1 (SEQ ID NO: 50); c) nucleic acid sequence encoding Gap2 (SEQ ID NO: 53); d) amino acid sequence of Gap2 (SEQ ID NO: 56).
FIG. 4A-C. (a) The organization of gap1 and pgk in clone 1-95 and construction of the gap1 disruption mutant. The restriction maps of the wild-type gap1 gene and its disrupted copy in the mutant are shown, and the expected band sizes in the Southern hybridization. The solid boxes and arrows represent gap1, the cross-hatched arrows represent tsr cassette, and the dark grey arrows represent the pgk gene. (b) Southern analysis of gap1::tsr mutant, showing the hybridizing bands to the gap probe. (c) Southern analysis of gap 1::tsr mutant, showing the hybridizing bands to the tsr probe.
FIG. 5A-C. (a) The organization of gap2 and construction of the gap2 disruption mutant. Restriction maps of the wild-type gap2 gene and its disrupted copy in Gap2-4-14. The solid boxes and arrows represent gap2, the cross-hatched arrows represent the apr cassette. (b) Southern analysis of the gap2::apr mutant, showing the hybridizing bands to the gap2 probe. (c) Southern analysis of the gap2::apr mutant, showing the hybridizing bands to the apr probe.
FIG. 6. DNA sequence of the PCR product of the gap1::tsr region in gap1 mutant (SEQ ID NO: 54). Sequences of gap1 are in bold and underlined, tsr gene is in capitals, and the sequences from the vector are in lower case.
FIG. 7. DNA sequence of the PCR product of the gap2::apr region in gap2 mutant (SEQ ID NO: 55). Sequences of gap2 are in bold and underlined, and apr gene is in capitals
FIG. 8: (a) Time course of clavulanic acid production and biomass in batch cultures of PES medium. (b) HPLC analysis of clavulanic acid production (as its imidazole derivative, tR=5.4 min.) in wild-type, Gap15-7-30, and Gap15-7-30 (1-95).



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
The present invention provides significant improvements in the production of clavulanic acid by the bacterium S. clavuligerus. To accomplish this, the primary metabolism of S. clavuligerus was manipulated by targeted inactivation of the newly discovered gap1 gene, which encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the resulting gap1 mutants, clavulanic acid production is dramatically increased in comparison to the non-mutant control. Clavulanic acid production is elevated during the entire fermentation process, and experiments described in the Examples section below demonstrate that this elevation is attributable to the inactivation of gap1. Without being bound by theory, it appears that disruption of gap1 prevents the normal flow of the C3 pool into the glycolytic pathway, resulting in channeling or redirecting of the C3 precursor G3P towards the clavulanic acid pathway instead. Evidence is also provided that, as a result of gap1 inactivation, the C3 pool size in S. clavuligerus is increased to such an extent that L-arginine (not G3P) becomes the rate-limiting precursor for clavulanic acid biosynthesis. This finding led to a further improvement in clavulanic acid production by the intermittent addition of arginine to cultures of S. clavuligerus gap1 mutants. The present invention thus provides a non-standard way of generating higher yields of clavulanic acid, previous methods focusing principally on control mechanisms, e.g. via mutations of regulatory genes.
While the present invention was first demonstrated in S. clavuligerus, those of skill in the art will appreciate that the methodology disclosed herein is applicable to many other organisms. For example, the methodology disclosed herein is readily applicable to other species of bacteria that produce clavulanic acid, for example, Actinomycetes such as various Streptomyces spp., e.g. S. jumonjinensis, S. katsurahamanus, S. lipmanii, etc. (see for example, the review by Jensen and Paradkar, 1999); Pseudomonas spp., etc. Clavulanic acid may be increased in any such species by inactivation of a gene encoding GADPH activity. In addition, the organism that produces clavulanic acid need not be a native or natural producer of this product. Rather, the genes for clavulanic acid synthesis may be inserted into a naive, heterologous host organism (e.g. Escherichia coli, S. coelicolor, S. lividans, S. albus, S. venezuelae, etc.) and the methods of the invention may be carried out in the genetically altered host.
Many varieties of S. clavuligerus have been, are being, or will be developed commercially, especially to increase clavulanic acid production. Typically, these are S. clavuligerus that contain mutations in regulatory genes (e.g. promoter alterations) and that, as a result, have some desired feature such as the ability to synthesize increased amounts of clavulanic acid, increased stability, etc. Such mutant S. clavuligerus strains may also be further genetically engineered by the methodology described herein, to further improve the yield of clavulanic acid from these organisms. Alternatively, S. clavuligerus (and other organisms) that are genetically engineered as described herein to inactivate or attenuate GAPDH may be further engineered in other ways, e.g. by mutations of regulatory genes. All such genetically engineered organisms are intended to be encompassed by the present invention.
Further, inactivation of the gap1 gene in S. clavuligerus to increase clavulanic acid production is illustrative of a broader inventive approach to increasing production of biosynthetic products in living organisms. Accordingly, the present invention provides methodology for increasing production of a biosynthetic product that is produced in an organism by a biosynthetic pathway (“Pathway A”), by inactivating a gene of interest in another pathway (“Pathway B”). The two pathways utilize precursors from a common precursor pool, and compete for precursors from the pool. Inactivation of the gene of interest in Pathway B leads to inhibition of Pathway B, and decreases the channeling of precursors through Pathway B. A buildup of precursors in the pool ensues, making more precursors available for Pathway A. Preferably, Pathway B is non-essential for the organism, or there is a compensatory mechanism in the organism for the activity that is inhibited, so the organism remains viable. For example, another gene that encodes a gene product with the activity may be present and allow Pathway B to function at least partially. Alternatively, another different pathway may be present in the organism (e.g. a “bypass”, compensatory, or supplemental pathway) that fulfills the same or a similar function. However, in organisms where Pathway B is essential and there is no compensatory mechanism, the method of the invention can be carried out by partial inhibition of Pathway B. For example, the gene of interest can be genetically modified to be transcribed at a lower level, or so that the transcribed mRNA is translated at a lower level, or so that the gene product is less active. Thus, Pathway B would not be totally eliminated but would utilize less precursor. Excess precursor would then be available for use in Pathway A. Pathway B may, like Pathway A, be a biosynthetic pathway. Alternatively, Pathway B may be a pathway of another type (e.g. catabolic, energy generating, gluconeogenesis, etc.). Further, the methodology need not be confined to a single product produced by a single pathway, and the blocking of a only one gene in only one competing pathway. Rather, according to the methods of the invention, the production of several products (or intermediates) may be increased in one or more pathways, and multiple genes may be inactivated in one or more competing pathways.
Particular examples of other organisms to which the methods disclosed herein apply include but are not limited to S. coelicolor and S. avermitilis. Whole genome sequences have been published for S. coelicolor and S. avermitilis, and each of these organisms contains a gap1 and a gap2 gene. The cloning strategy that was used to isolate gap1 and gap2 from S. clavuligerus (or other known cloning strategies) could readily be carried out in these organisms, and the methods of the invention could then be applied. Further, as all Streptomyces, must have a functioning glycolytic pathway and Krebs cycle to live, it is likely that at least one GADPH-encoding gene is present in other Streptomyces (both naive hosts and those producing clavulanic acid) and the principles and methodology taught herein will be widely applicable to such organisms. Further, most if not all microorganisms must have a functioning glycolytic pathway and Krebs cycle to live, and likely possess at least one GADPH-encoding gene that can be inactivated by the methods disclosed herein, in order to augment production of a product or products from competing pathways.
The mevalonate and non-mevalonate biosynthetic pathways are examples of two biosynthetic pathways to which the methods of the present invention can be applied. The mevalonate pathway is responsible for the synthesis of universal terpenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) and is essential in plants, many eubacteria and apicomplexan parasites, but not in archaea and animals. The details of this pathway are well-known. However, another mevalonate-independent pathway for IPP and DMAPP synthesis is also known, although it has been less well characterized (see Eisenreich et al., 2004). The non-mevalonate pathway utilizes glyceraldehyde 3-phosphate (G3P) as a precursor, whereas the mevalonate pathway does not. Thus, inhibiting GADPH activity would lead to a buildup of G3P, which would then be available for use in, e.g, the non-mevalonate pathway.
Those of skill in the art will recognize that ultimately, it is the activity of a gene product of interest (e.g. the gap1 gene product, Gap 1) that is eliminated or attenuated by the methods of the invention. In a preferred embodiment of the invention, this is done by preventing or attenuating expression of the gene that encodes the gene product (e.g. the gap1 gene). As is demonstrated herein, this may be accomplished by disrupting or replacing all or some of the nucleotide sequences of the gene with nucleotide sequences that do not encode a gene product that functions in the pathway, e.g. by targeted gene replacement. In this case, a sufficient portion of the gene sequence must be replaced so that the gene is either not translated at all, or so that the translated gene product is unable to carry out its usual biological function in the pathway that is being blocked.
However, those of skill in the art will recognize that other means are also available to preclude or decrease transcription of a gene. For example, various mutations of the gene sequence, or of sequences necessary for the transcription of the gene sequence, may be carried out, resulting in no or low production of the gene product (e.g. a protein or polypeptide). Examples include but are not limited to point mutations, deletions, additions, replacements, insertions of stop codons, rearrangements, etc. Alternatively, translation of the mRNA encoding the gene product may be prevented or attenuated, e.g. by expressing antisense RNA, or inhibitory or small inhibitory RNA (i.e. RNAi or siRNA), or by inhibiting the translation process in some other manner. In addition, mutations may be introduced into the gene which allow the production of a gene product, but which render the gene product non-functional or of low activity. As a further alternative, the gene product itself may be inhibited from normal functioning, e.g. by exposure to an inhibitor. In all cases, the normal activity of the gene product is prevented or impaired, and the biological pathway in which the gene product normally functions is, as a result, fully or partially inactive. In a preferred embodiment of the invention, the gene of interest is disrupted/inactivated by targeted gene replacement.
By “inactivated” or “attenuated” or “impaired” or “inhibited” or “impeded”, we mean that the level or rate of functioning (e.g. transcription and/or translation of the gene of interest, or activity of the gene product) is prevented or altered so that detectable activity of the gene product is decreased by at least about 5%, or about 10%, or about 20%, or about 30%, or about 40%, or preferably by about 50%, more preferably by about 75% to 80%, and most preferably by about 90 or even 95 to 100%, with the caveat that preferably normal growth of the organism is maintained (e.g. growth compared to wild type or a reference strain, or compared to growth of the organism before genetic modification according to the present invention). The decrease in the rate or level of functioning is determined by comparison to the rate or level in, e.g. a wild type or reference strain, or compared to the organism before genetic modification according to the present invention. Those of skill in the art will recognize that the desired level or rate of inactivation may vary from one organism to another, depending on several factors (e.g. whether or not the activity is essential to the organism, whether or not compensatory activities exist, the level of inactivation that is useful to achieve a desired level of production of a biosynthetic product, etc.) Further, this inactivation results in a concomitant inhibition of the pathway in which the gene product normally participates. Therefore, “inactivation” (attenuation, inhibition, impairment, etc.) may be also be used to refer to the pathway that is inhibited. The level of inactivation (disruption, impairment, attenuation, inhibition, etc.) may be detected or measured by any suitable method, e.g. by detecting the gene of interest or mutant forms thereof, by detecting mRNA transcribed from the gene, by detecting an attenuated gene product, by detecting an activity known to be associated with the gene product, by detecting a substance normally produced by the gene product, by detecting the buildup of a substrate or precursor of the gene product, by detecting metabolites or catabolites of the pathway that is inhibited, etc. All such detection or measuring is carried out using suitable controls for comparison, as is well known in the art.
According to the invention, an increase in the production of a biosynthetic product by one biochemical pathway is caused by inhibition of a competing pathway, e.g. by inactivation of a gene of interest in the competing pathway. This results in an “increase” or “elevation” in the amount of the biosynthetic product, by which we mean that at least about 1 to 10%, or about 10 to 25%, or about 25-50%, or even about 50 to 100% more product is produced; or preferably about 100% (i.e. 2-fold) or even more (e.g. 5-10 fold or more) additional product is produced as a result of inhibition of the competing pathway. Those of skill in the art will recognize that such increases may be measured in any of many suitable ways, e.g. by measuring the product itself, by measuring an activity of the product, etc.
Many biosynthetic pathways require one or more precursors (and/or components such as cofactors) in order to carry out the biosynthesis of a single biosynthetic product. For example, the primary metabolic precursors of clavulanic acid are G3P and L-arginine. Thus, when the available pool of one precursor for a biosynthetic reaction is increased by the methods of the invention, it may also be beneficial to supply other precursors at an increased concentration. This may be accomplished by any available means (e.g. by genetic engineering of the host organism to increase production of the other precursors; by addition of the precursor to the media in which the host organism is grown; by increasing the concentration of a substance that, in turn, increases precursor production, etc.). In one embodiment of the invention, the level of G3P available to the clavulanic acid biosynthetic pathway is increased by the methods of the invention, and the concentration of L-arginine is increased by supplementing the media in which the host organism (e.g. S. clavuligerus) is grown. Those of skill in the art will recognize that the level and frequency of supplementation will vary depending on the biosynthetic pathway and the precursor that is being supplemented. For supplementing S. clavuligerus mutants as described herein with L-arginine, in one embodiment, the media in which the S. clavuligerus is grown is supplemented with L-arginine at a concentration of from about 0.1 to about 100 mM, and preferably from about 0.1 to about 50 mM, and most preferably from about 1 to about 20 mM. In one embodiment, 13.5 mM of L-arginine is used. In addition, the frequency of supplementation may vary, but for bacteria, will generally be carried out in the range of from about every 1 to 24 hours or longer. In addition, supplementation may be continuous, or the additional supplement may be included in the original media, i.e. all at once at the beginning of culture. In one embodiment, supplementation is once every 24 hours.
In some embodiments of the invention, the production of one biosynthetic product is increased by the methods of the invention. However, this need not be the case. Manipulation of a biochemical pathway may result in increased production of more than one biosynthetic product, from one or more different biosynthetic pathways. In addition, the invention is not limited to inhibiting one pathway in an organism. Rather, several biochemical pathways in an organism may be inhibited in order to increase production of a product or products, so long as the organism remains viable.
In one embodiment of the invention, the gap1 gene disclosed herein can also be used as a diagnostic tool to identify related genes in other organisms. Those of skill in the art are acquainted with techniques for doing so, e.g. by designing primers based on the gap1 sequence, for example, primers that are unique to gap1, and using the primers to amplify DNA from other organisms suspected of containing a related GAPDH gene by polymerase chain reaction (PCR), e.g. via the CODEHOP technology. The invention thus also provides the sequence of gap1 (SEQ ID NO: 50) and encompasses nucleic acid sequences that are at least about 50, 60, 70, 80, 90 or even 95% homologous to gap1. Complementary DNA sequences are also contemplated, as are RNA sequences that can be transcribed from the gap1 gene, and primer sequences that can be used to specifically amplify gap1 by PCR, or to otherwise detect gap1. In a preferred embodiment, the invention also provides DNA and/or RNA sequences that encode the Gap1 protein (SEQ ID NO: 43), or protein sequences that display at least about 30, 40, 50, 60, 70, 75, 80, 85, 90, or even 95% or more identity with SEQ ID NO: 43. Those of skill in the art are familiar with techniques for establishing levels of identity when comparing protein/polypeptide sequences, and would be able to readily ascertain whether or not a protein meets this criteria.
The gap1 gene or portions thereof may be used to identify homologous genes in other organisms, or for comparisons to other gene sequences, for example, to identify whether or not a functional gap1 (or homologous) gene is present in an organism. The Gap 1 protein may also be useful for similar purposes.
EXAMPLES
Clavulanic acid is a potent β-lactamase inhibitor used to combat resistance to penicillin and cephalosporin antibiotics. There is a demand for high-yielding fermentation strains for industrial production of this valuable product. Clavulanic acid biosynthesis is initiated by the condensation of L-arginine and D-glyceraldehyde-3-phosphate (G3P). To overcome the limited G3P pool and improve clavulanic acid production, the glycolytic pathway in Streptomyces clavuligerus was genetically engineered. Two genes (gap1 and gap2) whose protein products are distinct glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) were inactivated in S. clavuligerus by targeted gene disruption. A doubled production of clavulanic acid was consistently obtained when gap1 was disrupted, and reversed by complementation. Addition of arginine to the cultured mutant further improved clavulanic acid production giving a greater than 2-fold increase over wild-type, suggesting that arginine became limiting for biosynthesis.
2. Materials and Methods
2.1. Bacterial Strains, Plasmids, Media, and Bacteriological Techniques.
The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli and Streptomyces strains were grown as described by Sambrook, Kieser, and Li (Kieser et al., 2000; Li et al., 2000; Sambrook et al., 1989). For clavulanic acid production in batch cultures, mycelia from the seed cultures were transferred into either starch-aspargine (SA) (Paradkar and Jensen, 1995), PES medium (per liter: glycerol 20 g; protein extract from soybean 5.5 g; K2HPO4 0.8 g; MOPS 21 g; pH 7.0) or PES2 medium (per liter: soluble starch 20 g; asparagine 2 g; protein extract from soybean 5.5 g; K2HPO4 0.8 g; MOPS 21 g; pH 7.0) at the ratio of 1:20. The fermentation cultures were grown under the same conditions as the seed culture (Li et al., 2000).







TABLE 1







Bacterial strains and plasmids









Strain or plasmid
Relevant characteristics
Reference or source





Strains




E. coli


DH5α
F- recA1 gyrA96 thi-1 hsdR17 supE44 relA1
Invitrogen



deoR (lacZYA- argF)U196 φdlacZΔM15


JM110
dam dcm supE44 thi leu rpsL lacY galK galT
Stratagene



ara tonA thr Tsx Δ(lac-proAB) F'[traD36



proAB+lacZΔM15


Gap-195
DH5α transformant containing 3.5 kb gap1
This study



cluster


ESS
β-lactam-supersensitive indicator strain
(Aoki et al., 1976)


K. pneumoniae
Indicator strain for clavulanic acid bioassay
(Romero et al.,


Subsp. pneumoniae

1984)


ATCC 29665


S. clavuligerus


ATCC 27064
clavulanic acid producer, wild-type strain
ATCCa


Gap15-7-30
gap insertional disruption mutant (gap::tsr)
This study


Gap15-7-30
Gap15-7-30 with plasmid pSET152
This study


(pSET152)


Gap15-7-30 (1-95)
Gap15-7-30 with plasmid pSET152/1-95
This study


S. coelicolor
Strain used as control for PCR cloning of
ATCC


CH999
gap


Bacillus sp. ATCC
Indicator strain used for clavam bioassay
(Pruess and Kellett,


27860

1983)


Plasmids


pBluescript II SK
Phagemid; AmpR
Stratagene


(−)


pKC1139
Streptomyces-E. coli bifunctional vector,
(Bierman et al.,



AmR
1992)


pIJ680
High-copy-number Streptomyces cloning
(Kieser et al., 2000)



vector, ThioR, NeoR


pWHM3
High-copy-number Streptomyces - E. coli
(Vara et al., 1989)



bifunctional



Vector derived from pIJ486, ThioR


pWHM3Am
High-copy-number Streptomyces-E. coli
This study



bifunctional vector, AprR


pSET152
Streptomyces-E. coli bifiinctional integrative
(Bierman et al.,



vector, AmR
1992)


pBSGAP
pBluescript II SK (−) containing the 510 bp
This study



gap PCR product


pBSGAPT
pBSGAP containing tsr-disrupted gap PCR
This study



product


pGAPT
pWHM3Am containing the tsr-disrupted gap
This study



PCR product


pBS/1-95
pBluescript II SK (−) containing the 3.5 kb
This study



fragment hybridizing to 510-bp gap probe


pSET152/1-95
pSET152 containing the 3.5-kb gap
This study



fragment


pBSGAP2
pBluescript II SK (−) containing the 510 bp
This study



gap2 PCR product


pBAGAP2Am
pBSGAP2 containing apr-disrupted gap2
This study



PCR product


pGAP2Am
pWHM3 containing the apr-disrupted gap2
This study



PCR product





aATCC, American Type Culture Collection






2.2. Cloning the Gap Genes

PCR primers were designed by the CODEHOP strategy (Rose et al., 1998) according to the highly conserved motifs found in putative GAPDH proteins that showed highest homology to S. coelicolor GAPDH (website located at www.sanger.ac.uk/Projects/S_coelicolor/) in a BLASTP analysis (FIG. 2). The components of reaction mixture were as described by Stratagene. The PCR reaction was performed using a “touchdown” program which consisted of (1) denaturation at 98° C. for 2 min, (2) 7 cycles of 40 sec at 98° C., 1 min at 63-57° C. (decreasing 1° C. every cycle) and 40 sec at 72° C., (3) 30 cycles of 40 sec at 98° C., 1 min at 56° C. and 40 sec at 72° C., and (4) 10 min at 72° C. The 510-bp products, amplified with the Gap-Deg-5/Gap-Deg-3 primers, were cloned into pBluescript II SK(−) (pBSG510). To clone the whole gap1 gene, the genomic DNA of S. clavuligerus was digested with BamHI, BglII, EcoRI, PstI, XbaI-HindIII, or XbaI-XhoI, and probed with the 510-bp partial gap1 (May, 1998; Sambrook et al., 1989). A sub-genomic library was constructed by ligating the 3-5 kb BglII genomic DNA fragments of S. clavuligerus with pBluescript II SK(−). The positive clones were identified by colony hybridization (Sambrook et al., 1989). To clone the whole gap2 gene, genomic DNA of S. clavuligerus was digested with BglII, ClaI, EcoRI-HindIII, KpnI, NcoI, NotI, PstI, SacI, ScaI, or XbaI-XhoI, and probed with the 510-bp gap2 PCR. A sub-library was constructed by ligating the 3-5 kb fragments of NcoI digested genomic DNA with pGEM®-5Zf(+). Positive clones were selected by colony hybridization using the 510-bp gap2 probe.
2.3. Recombinant DNA Procedures
All plasmids and genomic DNAs were isolated and purified using standard methods (Kieser et al., 2000; Sambrook et al., 1989) or as described by the manufacturer.
To construct pWHM3 Am, a replicationally unstable bifunctional vector in Streptomyces, the apramycin resistance gene was recovered as a 1.5-kb EcoRI-PstI fragment from pKC1139. A 1.1-kb fragment containing about 600-bp corresponding to the thiostrepton resistance gene (tsr) was deleted from pWHM3 (Vara et al., 1989), and the remaining part of the plasmid was blunt-ended with Klenow DNA polymerase and ligated into the apr cassette.
pGAPT was constructed as follows. The 1.1-kb tsr fragment was excised by digestion of pIJ680 with BclI. The blunt-ended tsr fragment was ligated into the unique BlpI internal site of the 510-bp gap PCR product in pBSGAP to give the pBSGAPT. The 1.6-kb tsr-disrupted gap fragment was excised and inserted into the unique ClaI site of pWHM3Am by blunt ligation to generate the disruption vector pGAPT. pGAPT was transformed into the methylation-deficit strain of E. coli JM110 before transforming into S. clavuligerus. 
To complement the gap1::tsr mutant, pSET152/1-95 was constructed as follows. The 3.5-kb fragment containing the whole gap gene and the upstream regulatory elements was excised from a positive hybridizing clone of the sub-library pBS/1-95 by XbaI-EcoRV and ligated into the site-specific integrative vector pSET 152. The resulting vector, pSET152/1-95 was transformed into E. coli JM110, from whence the plasmid DNA was prepared and used for the transformation of the gap1 disruption mutant.
To construct pGAP2 Am, the 1.5-kb blunt-ended apramycin resistance cassette (apr) was inserted into the unique NruI site in the 510-bp gap2 fragment in pBSGAP2 to give pBSGAP2 Am. The 2.1-kb apr disrupted gap2 fragment was excised and ligated into the blunt-ended XbaI site of pWHM3 to generate pGAP2 Am. To overcome the restriction barrier, pGAP2 Am re-isolated from E. coli JM110 was used to transform S. clavuligerus. 
2.4. Transformation of S. clavuligerus and Gene Disruption
Protoplast formation, DNA transformation, and selection and confirmation of double-crossover mutants of S. clavuligerus were carried out as described previously (Li et al., 2000). Two primers were designed according to the 510-bp gap sequence to confirm the double crossover event in gap1 disruption mutants by PCR. CLAGAP5, CACGCCTCCTGTACCACCAACTGTG (SEQ ID NO: 51), and CLAGAP3, TGGAGTAGCCCCACTCGTTTCATACC (SEQ ID NO: 52). The PCR reaction was carried out using the standard protocol (Sambrook et al., 1989).
2.5. DNA and Protein Analyses
All DNA sequencing was carried out in the Peptide/Protein Facility, The Johns Hopkins University School of Medicine (Baltimore, Md.). PSIPRED Protein Structure Prediction Server (website located at bioinf.cs.ucl.ac.uk/psipred/) was used for secondary structure prediction, and the Swiss-Model server (website located at swissmodel.expasy.org/SWISS-MODEL.html) was used for homology modeling.
2.6. Fed-batch Culture
Chemical feeding experiments were preformed as described by Chen et al. (Chen et al., 2003). Starting after 60 h of batch culture, arginine or glycerol was fed every 24 h to a final concentration of 13.5 mmol and 0.5 mmol, respectively. Samples were withdrawn and analyzed for clavulanic acid titer at intervals of 24 h to 192 h of fermentation.
2.7. Analysis of β-lactam Antibiotics
A 2-ml sample was taken every 24 h from each culture during the fermentation process. The cell pellet was used to determine the wet weight as the biomass, and the supernatant was used for assays of clavulanic acid, cephamycin C or antipodal clavams (Aoki et al., 1976; Pruess and Kellett, 1983; Romero et al., 1984). Clavulanic acid and its bicyclic β-lactam-containing co-metabolites were also analyzed by reaction with imidazole (Bird et al., 1982). Filtered fermentation supernatant (90 μl) was reacted with 30 μl of 3 M imidazole solution (pH 6.8) at 37° C. for 40 min. The imidazole derivative was diluted 15-fold with ddH2O and its absorbance was measured at 312 nm. Clavulanic acid yields in S. clavuligerus strains were determined by comparison to a calibration curve generated from a pure standard of clavulanic acid. Clavulanic acid was also analyzed by high-pressure liquid chromatography (HPLC) using a Hewlett-Packard Series 1050 HPLC system. A 50-111 imidazole derivative was analyzed on a Phenomenex Prodigy ODS(3) column (250 mm×4.6 mm) (Torrance, Calif.) at 312 nm. The mobile phase consisted of 0.1 M KH2PO4 (pH 3.3 adjusted with H3PO4) and 6% CH3OH (flow rate 1 ml/min).
2.8. Nucleotide Sequence Accession Numbers
The GenBank accession number for gap1 is DQ178995, gap2 is DQ178997, and pgk is DQ178996. The amino acid sequence of Gap1 protein (SEQ ID NO: 43) is shown in FIG. 3A, and the nucleic acid sequence of gap1 (SEQ ID NO: 50) is shown in FIG. 3B.
3. Results
3.1. Cloning of the Gap Genes from S. clavuligerus 
The GAPDHs involved in glycolysis in prokaryotic organisms belong to the phosphorylating GAPDH family (Mateos and Serrano, 1992), whose primary amino acid sequences show considerable homology (Mateos and Serrano, 1992). BLASTP analysis revealed a group of GAPDHs from different organisms having high homology, including proteins from four Streptomyces strains. The similarities among these organisms were reduced to three short motifs: (H/Q)(H/T/N)(V/I)S NASCTTN (motif M1, SEQ ID NO: 57), (S/K/R)D(L/Y/P)RRARAAA (motif M2, SEQ ID NO: 58), and WYDNE(S/W/T) G(Y/F)S(H/A/H/T) (motif M3, SEQ ID NO: 59) (FIG. 2a). Using the recently developed CODEHOP program (Rose et al., 1998), we designed two forward primers for the M1 and M2 motifs and one reverse primer for the M3 motif (FIG. 2b). A distinct band with the expected size of 510 bp was obtained from both S. clavuligerus and S. coelicolor chromosomal DNA with Gap-Deg-5/Gap-Deg-3 primers, while no PCR product was detected when Gap-Deg-5-2/Gap-Deg-3 primers were used, presumably due to the high Tm value of the Gap-Deg-5-2 primer. The 510-bp product was cloned and nine randomly selected clones were sequenced.
DNA sequence analysis revealed that two PCR products were indeed present in the 510-bp PCR band. BLASTX analysis of the first PCR product, which was obtained from seven clones, showed that the amino acid sequence from one translated frame had high homology to the M1 and M2 motifs conserved in GAPDHs. A positive band of approximately 3.5 kb was identified in a Southern hybridization from BglII digested chromosomal DNA by using the 510-bp PCR product as probe. A sub-genomic library generated from the 3-5 kb BglII gDNA fragments was screened by colony hybridization. Of 320 colonies screened, 4 positive clones were identified. Restriction mapping revealed a 4.0-kb insert in these clones. One of them, clone Gap-195, was sequenced on both strands.
FramePlot (Ishikawa and K. Hotta, 1999) analysis of the DNA sequence of clone1-95 revealed one incomplete and two complete open reading frames (ORF). The first ORF, gap1, encodes a 355-amino acid protein with a predicted molecular weight (MW) of 36 009 Da and the isoelectric point (pI) of 4.83. A BLASTP search showed that Gap1 has significantly high similarity (94 to 57% identity and 97 to 72% similarity) to GAPDHs from a number of microorganisms. The greatest similarities are to the putative GAPDHs from S. avermitilis MA-4680 (92% identity and 97% similarity) and S. coelicolor A3(2) (94% and 96%, respectively). Gap 1 possesses all three conserved motifs and almost all the amino acid residues thought to be required for NAD+−binding (Gly11, Arg12, Asp35, Phe101, and Ala122) and catalysis (Cys153 and His180) (FIG. 3a) (Skarzynski et al., 1987). Homology modeling demonstrated a significantly high structural similarity between Gap1 and other GAPDHs (not shown). These comparative analyses strongly suggested that gap1 encodes GAPDH in S. clavuligerus. 
Downstream of gap1 lies the second ORF encoding a protein of 403 amino acids with the calculated MW of 41 923 Da and pI of 4.89. A BLASTP search showed that the deduced amino acid sequence of the encoded protein is highly homologous to 3-phosphoglycerate kinases (Pgk) from several Streptomyces strains and many other organisms. The greatest similarity was found to the Pgk from S. coelicolor A3(2) (85% identity and 92% similarity). Multiple sequence alignment and secondary structure prediction between S. clavuligerus Pgk and homologous proteins revealed a high degree of conservation in 14 β-sheets and adjacent peptide segments that are involved in the formation of inner loops in the substrate-binding cleft present in all Pgk proteins (Hong et al., 2000).
A second PCR product was present in two of the nine clones sequenced. Southern hybridization of genomic DNA digested with restriction enzymes revealed that the gene was located in a 4 ̃5-kb NcoI gDNA region. 900 clones in a sub-library constructed and screened by colony hybridization, and two positive clones were identified. FramePlot analysis showed one complete ORF (gap2) was present in the sequenced region. The encoded protein is composed of 481 amino acids with a predicted molecular weight (MW) of 52 354 Da and pI of 6.43. The deduced Gap2 protein contains the conserved M1 and M3 motifs and shows significant similarity to a group of putative glyceraldehyde-3-phosphate dehydrogenases, including Gap2 in S. coelicolor (89% identity and 95% similarity), and GapX in S. roseofulvus (86% identity and 92% similarity). The residues from 135 to 300 show similarity to NAD+binding domains that usually encompasses amino acids 1-150 in GAPDHs. Residues 300-460 show similarity to C-terminal catalytic sites in GAPDHs (Skarzynski et al., 1987). Interestingly, the overall homology between Gap1 and Gap2 is significantly low (25% identity and 37% similarity) except within the conserved motifs.
3.2. Disruption of Gap1 and Gap2 in S. clavuligerus 
To investigate whether gap1 and gap2 are essential for S. clavuligerus survival and if an increased intracellular pool of G3P can increase precursor flux to clavulanic acid, two mutants were generated by targeted gene replacement. The 510-bp gap1 PCR product was disrupted in vitro by insertion of the thiostrepton resistance cassette (tsr); the disrupted gene was then inserted into pWHM3AM, and the resulting vector, pGAPT, was introduced into S. clavuligerus. The primary transformants were subjected to two rounds of sporulation and the progeny were screened for the AmS/ThioR phenotype expected for a double-crossover event between the disrupted gene and its chromosomal counterpart. Five hundred colonies were screened and two showed the AmS/ThioR phenotype. One of them, Gap15-7-30, was used for further investigation.
S. clavuligerus protoplasts were transformed with the recombinant plasmid pGAP2Am, which confers thiostrepton resistance and also carries the 510-bp gap2 disrupted by an apramycin resistance gene. Because the transformants showed poor sporulation on the tested media, they were subjected to protoplast formation and regeneration to allow homologous recombination and vector elimination. Progeny screened for the loss of thiostrepton resistance yielded strains with the ThioS/AmR phenotype. 350 clones were screened for double crossover in this case and three ThioS/AmR strains were obtained. One of them, Gap2-4-14 was used for the subsequent studies.
The replacement of wild-type gap1 and gap2 in Gap15-7-30 and Gap2-4-14 and subsequent plasmid elimination were confirmed by both PCR amplification and sequencing of the disrupted region, and analysis by Southern hybridization. A specific 1.5-kb PCR product was obtained from Gap15-7-30 genomic DNA, corresponding to the sum of the gap1 fragment and the tsr cassette, whereas under the same conditions the wild-type genomic DNA gave a 0.5-kb PCR product. DNA sequencing confirmed that the PCR product contained the gap1 fragment disrupted by the tsr cassette at a BlpI site (data not shown). Chromosomal DNA isolated from the wild type and Gap15-7-30 were separately digested with NarI-NcoI and hybridized to gap1-specific and tsr-specific probes. As expected, the gap1 probe gave a 0.4-kb hybridization band in the wild-type genome, and this band was replaced by a 1.4-kb band in the Gap15-7-30 genomic hybridization. Gap15-7-30 gave exactly the same hybridization pattern to the tsr probe as the gap1 probe, but no hybridization band was seen from the wild-type genomic DNA (FIG. 4). Thus, the wild-type gap1 had been replaced by the tsr-disrupted copy in Gap15-7-30 in a double-crossover event. To confirm the double-crossover event in Gap2-4-14, genomic DNA isolated from wild-type and Gap2-4-14 were digested with AhdI and hybridized with gap2-specific and apr-specific probes. Gap2-4-14 consistently gave a 2.4-kb positive band to both gap2 and apr probes, corresponding to the predicted size of gap2::apr, whereas the wild-type gave a 0.9-kb hybridization band to gap2 probe, and no hybridization occurred to apr probe. These results demonstrated that the wild-type gap2 has been replaced by an apr-disrupted copy in Gap2-4-14 (FIG. 5).
3.3. Analysis of the Gap15-7-30 and Gap2-4-14 Mutants
The Gap 15-7-30 and Gap2-4-14 mutants were then characterized to determine the effect of the mutations on clavulanic acid production. The mutants were cultivated on PES, PES2 and SA media as batch cultures, and samples were withdrawn every 24 h during incubation to assess secondary metabolite yields by bioassay, imidazole derivatization, and HPLC analysis. The fermentation was repeated four times, and the performance of the Gap 15-7-30 culture was observed to be highly reproducible. All mutant cultures were compared to similarly grown wild-type S. clavuligerus control cultures. Cell density was measured as wet weight. As shown in FIG. 6a, the biomass of Gap 15-7-30, Gap2-4-14 and the wild-type strain were quite similar in all media tested. Microscopic observation of mycelia of both wild-type and two mutants did not show any distinct morphological differences, indicating that inactivation of gap1 and gap2 had no detectable effects on the morphology of S. clavuligerus. 
Bioassay with the β-lactam supersensitive E. coli ESS showed that wild-type and both mutants produced similar levels of cephamycin, indicating the disruption of gap1 and gap2 had no effect on cephamycin biosynthesis (data not shown). The time course of clavulanic acid production in PES batch cultures in shake-flasks was measured by both imidazole derivatization followed by UV and HPLC chromatographic analyses. In PES medium the wild-type and two mutants produced similar levels of clavulanic acid in the first 48 h of fermentation. Over several fermentations, the maximal production of clavulanic acid in wild-type S. clavuligerus was between 120 and 135 mg/L. The production in Gap15-7-30 started increasing from 48 h and reached a maximum at a range of 260 to 282 mg/L, a 110% increase over the wild-type strain. The higher production was maintained between 80% and 110% over the wild-type strain until the end of the fermentation (216 h) (FIG. 8a). Enhanced clavulanic acid production in Gap15-7-30 has been consistently obtained from repeated fermentations under identical conditions. Bioassay on K. pneumoniae plates showed an increased inhibition zone from the Gap 15-7-30 supernatant, consistent with the increased production of clavulanic acid. When culture supernatants were reacted with imidazole, HPLC analysis revealed that the peak corresponding to the chromophoric derivative of clavulanic acid (tR 5.4 min) was increased in Gap 15-7-30, quantifying the elevated production (FIG. 8b). The increased clavulanic acid production was also observed in PES2 and SA media. In contrast to the PES medium, growth in PES2 medium showed no onset of production occurring in the late exponential phase of growth. In contrast clavulanic acid was produced throughout the growth phase, but only about 20% more was obtained over the wild-type in PES2 medium at its peak (data not shown). In SA medium, the maximal clavulanic acid production in Gap15-7-30 was also only about 20% greater than the wild-type (data not shown).
We tested the stability of the gap1::tsr mutant by passing Gap 15-7-30 through five generations without selective antibiotic pressure. The progeny still showed the same level of thiostrepton resistance and enhanced production of clavulanic acid in PES medium. Thus, the double-crossover in Gap15-7-30 is genetically stable (data not shown).
The effect of inactivation of gap2 on clavulanic acid productivity was similarly investigated in both PES, and PES2 media. The mutant produced similar amounts of cephamycin and clavams as the wild-type strain as indicated by bioassays on E. coli ESS and Bacillus sp. 27860 plates (data not shown). In PES medium, a consistent 20-30% decreased clavulanic acid production in Gap2-4-14 was observed after 72 h of fermentation (FIG. 8a), whereas in PES2 medium the disruptant produced a similar level clavulanic acid as the wild-type strain (data not shown).
3.4. Complementation of the Gap15-7-30 Disruption Mutant
We assumed that increased clavulanic acid production was due to the build up of G3P in the gap1 disruption mutant. To confirm this, an in trans complementation was carried out. A 55-bp A/T rich (44%) region, which could form a stem-loop structure with a calculated free energy of −49.5 kcal/mol, is present in the 221-bp intergenic region between gap1 and an upstream partial carboxypeptidase gene. Similar gap1-pgk-tpi operons were also found in S. coelicolor and S. avertimilis (Bentley et al., 2002) (Ikeda et al., 2003). These analyses suggested a promoter could be present in this intergenic region (Strohl, 1992). The recombinant plasmid pSET152/1-95 was constructed by insertion of the 4.0-kb fragment excised from pBS/1-95 into pSET152. The resulting vector was transformed into Gap15-7-30, and the transformants were selected and named Gap15-7-30 (1-95). Three independent transformants were fermented in PES medium. UV and HPLC analyses of imidazole derivatives showed that the yield of clavulanic acid in all three Gap15-7-30 (1-95) strains was consistently reduced during fermentation, whereas the control strain Gap 15-7-30 (pSET152) still produced an undimished level clavulanic acid. Gap15-7-30 (1-95), as shown in FIG. 8b, produced 70% less clavulanic acid than the gap1 mutant at 72 h of fermentation, which is only 25% more than the wild-type strain. These results confirmed that the targeted disruption of gap1 correlated directly with the increased production of clavulanic acid in this mutant.
3.5. Feeding of Gap15-7-30 with Arginine and Glycerol
The availability of arginine is not limiting in wild-type S. clavuligerus, so arginine supplementation has no effect on clavulanic acid levels, whereas additions of glycerol resulted in the increased production of clavulanic acid (Chen et al., 2003). To investigate if this is the case in Gap15-7-30, the mutant was fed glycerol or arginine every 24 h at the final concentration of 13.5 mmol and 0.5 mmol, respectively. Surprisingly, no further increase in clavulanic acid production was observed in the cultures fed glycerol, but more than tripled production (422 mg/L, 310%) from cultures fed arginine (FIG. 8a). The effect of arginine started after 60 h of exposure, and clavulanic acid production continuously increased to its maximum at the 192 h of fermentation. Enhanced production was also obtained when Gap15-7-30 was fed both arginine and glycerol, confirming that only arginine was needed to stimulate clavulanic acid production in the mutant. Feeding the mutant with higher concentrations of arginine did not result in additional increases in clavulanic acid production (data not shown).
4. Discussion
Metabolic engineering has become a rational alternative to classical strain improvement for the optimization of metabolite production. The introduction of directed genetic modifications through recombinant DNA technology can be visualized to improve the cellular properties of production strains and result in substantial increases in existing β-lactam antibiotic fermentation processes (Malmberg et al., 1993; Theilgaard et al., 2001). Augmentin® has been available for clinical use for over 20 years and its high effectiveness, safety, and tolerance profiles continue to make it an important agent in the treatment of bacterial infections (White et al., 2004). The development of high-dose formulations of Augmentin®, Augmentin ES-600® and Augmentin XR® (White et al., 2004), requires higher industrial production of clavulanic acid. Increasing the pool size of the rate-limiting precursor is a fundamental approach to strain improvement. This could be achieved by either increasing the gene dosage of key enzymes(s) involved in precursor biosynthesis (Malmberg et al., 1993), or direct feeding of the desired precursor(s) or its closely related derivatives (Chen et al., 2003). We report here a third approach to increasing the intracellular G3P pool by using homologous recombination technology to partially block the downstream portion of the glycolytic pathway that competes with clavulanic acid biosynthesis in S. clavuligerus. Thus, the C3-carbohydrate flux that would have normally gone through glycolysis instead would be diverted into clavulanic acid biosynthesis, thus accounting for an overall increase in clavulanic acid yield in the mutant strain. Our findings clearly demonstrate that clavulanic acid production in the gap-disrupted mutant is enhanced up to 282 mg/L (210% of the wild-type strain) in batch cultures in the absence of additives and without changing any components in the fermentation medium. An even higher yield of 422 mg/L (310% of wild-type strain) was achieved in fed-batch cultures in which L-arginine was fed to Gap15-7-30 at low concentration.
4.1. The Glycolytic Pathways in S. clavuligerus 
Although the biochemical activities of the glycolytic enzymes have been studied in detail, no information for S. clavuligerus has so far been described. Sequence comparisons of GAPDH and PGK from various species have shown that they are among the most-conserved proteins in eubacteria and eukaryotes where large stretches of amino acid residues have changed little or not at all (Jones and Harris, 1972). The genes gap and pgk have been reported to be linked to the triosephosphate isomerase gene (tpi) in many organisms (Bentley et al., 2002) (Ikeda et al., 2003) suggesting that gap1, pgk, and tpi could be also clustered as a tricistronic operon in S. clavuligerus. 
Although Gap1 and Gap2 have some conserved motifs and amino acid residues that are involved in NAD+−binding and substrate catalysis in GAPDHs, their overall homology is relatively low (25% identity). BLASTP and phylogenetic analyses clearly revealed that Gap1 and Gap2 are homologous to two distinct groups of GAPDHs. The same phenomenon has been observed in Gap1 and Gap2 of S. coelicolor (Bentley et al., 2002), indicating the evolutionary distance between these two groups.
4.2. Gap1 is Involved in Metabolism of Glycerol
Unlike gap mutants in most other bacteria in which a glucose assimilation deficiency requires an additional carbon source for survival (Hillman and Fraenkel, 1975; Valverde et al., 1997), the growth of Gap15-7-30 on a variety of media, including PES and PES2 medium (complex) and SA medium (minimal), is similar to the parental strain without any additional carbon source(s). This could be due to in-trans complementation from gap2. The gap2 mutant does not show enhanced clavulanic acid production in either PES or PES2 media, indicating that gap2 might be involved in the metabolism of carbon sources other than glycerol. On the other hand, glycerol can also be metabolized through glycerol hydrogenase, aldehyde dehydrogenase and glycerate kinase to form 2-phosphoglycerate, and then pyruvate (FIG. 1). A gene encoding a putative aldehyde dehydrogenase has been recently identified in S. clavuligerus, whose product showed 87% identity to aldehyde dehydrogenases in S. coelicolor and S. avermitilis, indicating that this glycerol-utilizing pathway is present in S. clavuligerus. Thus, in Gap15-7-30 most glycerol is still metabolized through glycolysis, but the buildup of G3P is then channeled to the clavulanic acid pathway. Meanwhile, the alternative glycerol-utilizing pathway produces pyruvate to by-pass the blocked glycolytic pathway.
Gap15-7-30 produces only slightly higher levels of clavulanic acid than the wild-type strain in SA and PES2 media, indicating that, due to the absence of glycerol, the level of carbon flux in the glycolytic pathway is inefficiently low that even the disruption mutant cannot accumulate more G3P. It has been demonstrated that most glycolytic enzymes are substrate inducible (Smith and Chater, 1988). The clavulanic acid production profiles of Gap15-7-30 and Gap2-4-14 in glycerol-containing (PES) and glycerol-absent (PES2 and SA) medium are strongly suggestive that the gap1-pgk-tpi cluster is substrate (glycerol) inducible in S. clavuligerus. 
4.3. Disruption of Gap1 Relieves the 3-carbon Precursor Limitation
An important finding in this study is that the C3 precursor became saturating after diversion of G3P towards the clavulanic acid biosynthetic pathway, so that feeding the mutant with glycerol no longer resulted in elevated clavulanic acid production. On the contrary, the C5 precursor became rate-limiting, and the administration of arginine apparently alleviated its lack of availability and enhanced clavulanic acid production. This indicates that the increased pool of G3P is so significant that even the very effective urea cycle and a paralogous gene encoding OAT in the clavulanic acid gene cluster (Kershaw et al., 2002) can not provide sufficient arginine to fully supply the Gap 15-7-30 mutant. When glycerol and arginine were both fed, the production of clavulanic acid was close to that with added arginine alone. This might be because the increased intracellular concentrations of C3 and C5 precursors have saturated CEAS, the first dedicated biosynthetic enzyme of the pathway, and introduced a new bottleneck downstream in the overall pathway to clavulanic acid.
4.4. Additional Exemplary Applications in Industry
Because mutants generated by double-crossover through homologous recombination are genetically stable, the metabolic switch is permanent. Industrial production of clavulanic acid uses a similar fermentation medium that also contains glycerol (Elander, 2003), suggesting that our technology could be easily applied for strain/method improvement. Clavulanic acid and the antipodal clavams use the same primary metabolic precursors and share the early part of the biosynthetic pathway up to the common intermediate clavaminic acid (Townsend, 2002). Thus, some of the increased G3P pool likely flows into the clavam pathway and would be anticipated to result in an increased yield of clavams. It has been shown that disruption of clavam biosynthesis has a positive effect on clavulanic acid production (Mosher et al., 1999; Paradkar et al., 2001). Therefore, the yield of clavulanic acid could be further improved if clavam biosynthesis were also blocked in Gap15-7-30. This approach would in addition eliminate clavam contamination so that the subsequent extraction and purification of clavulanic acid from the fermentation broth would be simplified.
Improved production of secondary metabolites involves significant changes in precursor flux, energy, and cofactors from primary to secondary metabolism (Drew and Demain, 1977). Based on understanding the biosynthetic pathway characterized so far, the increased clavulanic acid productivity in Gap15-7-30 would raise the competition for arginine, ATP (β-LS), α-KG (CS2), and NADPH (CAD) between primary and secondary metabolism, and introduce new metabolic bottlenecks.
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While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

## Claims

1. Streptomyces clavuligerus genetically modified such that a nucleic acid sequence encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of SEQ ID NO: 43 is deleted or mutated so that GAPDH activity is missing or reduced, or progeny thereof.

2. The Streptomyces clavuligerus of claim 1, wherein said nucleic acid sequence encoding a GAPDH enzyme is SEQ ID NO: 50.