A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example, overexpression was achieved by introducing an additional gene copy into a phage attachment site on the chromosome. This resulted in a series of strains with phosphofructokinase activities from 1.4 to 11 times the wild-type activity level. In this example, the pfk gene was cloned upstream of a gusA gene encoding ␤-glucuronidase, resulting in an operon structure in which both genes are transcribed from a common promoter. We show that there is a linear correlation between the expressions of the two genes, which facilitates screening for mutants with suitable enzyme activities. In a second example, we show that the method can be applied to modulating the expression of native genes on the chromosome. We constructed a series of strains in which the expression of the las operon, containing the genes pfk, pyk, and ldh, was modulated by integrating a truncated copy of the pfk gene. Importantly, the modulation affected the activities of all three enzymes to the same extent, and enzyme activities ranging from 0.5 to 3.5 times the wild-type level were obtained.Microorganisms are used for numerous purposes in industry, including for bulk production of chemicals and enzymes and for food fermentations. These applications have stimulated a great interest in trying to improve the properties of the production organisms through genetic manipulations. Most often, the strategy has been to overproduce (by many fold) an enzyme presumed to be rate limiting or to eliminate a branching pathway flux by deleting a gene. The outcomes of such attempts have often been disappointing (16,28,29), and the interest in modulating or tuning enzyme activities instead and thus to perform metabolic optimization (18) is therefore increasing.Genetic tools for modulating enzyme activities by changing gene expression have long been available and have been employed for fundamental studies of cell physiology, e.g., with inducible systems like the lac-type promoters (31,15,26) or the nisin promoter (6). These methods, however, are less well suited for modulating gene expression on an industrial scale. In addition, according to metabolic control analysis (11,19), truly rate-limiting steps will rarely be found in a metabolic pathway, and it will therefore frequently be necessary to increase the levels of many enzymes to achieve an increased flux.We recently reported a new approach for obtaining promoter libraries (17, 18), which is based on randomization of the DNA sequences (spacers) that separate the individual consensus sequences of promoters. The method takes advantage of the fact that actual sequences of bases in these spacer areas are less important for the strength of a particular promoter than the resulting DNA structure. By randomizing many base pairs simultane...

Metabolic cofactors such as NADH and ATP play important roles in a large number of cellular reactions, and it is of great interest to dissect the role of these cofactors in different aspects of metabolism. Toward this goal, we overexpressed NADH oxidase and the soluble F1-ATPase in Escherichia coli to lower the level of NADH and ATP, respectively. We used a global interaction network, comprising of protein interactions, transcriptional regulation, and metabolic networks, to integrate data from transcription profiles, metabolic fluxes, and the metabolite levels. We identified high-scoring networks for the two strains. The results revealed a smaller, but denser network for perturbations of ATP level, compared with that of NADH level. The action of many global transcription factors such as ArcA, Fnr, CRP, and IHF commonly involved both NADH and ATP, whereas others responded to either ATP or NADH. Overexpressing NADH oxidase invokes response in widespread aspects of metabolism involving the redox cofactors (NADH and NADPH), whereas ATPase has a more focused response to restore ATP level by enhancing proton translocation mechanisms and repressing biosynthesis. Interestingly, NADPH played a key role in restoring redox homeostasis through the concerted activity of isocitrate dehydrogenase and UdhA transhydrogenase. We present a reconciled network of regulation that illustrates the overlapping and distinct aspects of metabolism controlled by NADH and ATP. Our study contributes to the general understanding of redox and energy metabolism and should help in developing metabolic engineering strategies in E. coli.Microbial metabolic networks function in a coherent fashion to convert the available substrates into biomass and products. Often, intermediates of metabolism are not balanced with respect to their redox and/or energy content. Metabolic cofactors such as NADH and ATP serve in overcoming these constraints. Indeed, these cofactors rank among the most highly connected metabolites in the metabolic networks of most microorganisms (1). A direct consequence of this metabolic structure is that a small change in the concentration of these cofactors is likely to propagate to widespread aspects of metabolism. For example, the synthesis of proteins, lipids, and nucleotides is energetically expensive and would drain cellular ATP and also require NADPH (2). ATP is primarily produced in the electron transport chain, which is fueled by NADH. NADH is produced in the catabolism, relating substrate utilization to biosynthesis and product formation. Therefore, these cofactors hold the potential to serve as targets for altering cellular metabolism.To achieve the desired metabolic changes, it is important to understand the metabolic processes that are specifically controlled by cofactors. NADPH primarily drives anabolic reactions, whereas NADH is the result of catabolism. To fulfill their distinct roles, these two redox couples are generally not in thermodynamic equilibrium. NADPH is primarily produced in the oxidative branch of the pento...

In this paper we describe the new selection/counterselection vector pCS1966, which is suitable for both sequence-specific integration based on homologous recombination and integration in a bacteriophage attachment site. This plasmid harbors oroP, which encodes a dedicated orotate transporter, and can replicate only in Escherichia coli. Selection for integration is performed primarily by resistance to erythromycin; alternatively, the ability to utilize orotate as a pyrimidine source in a pyrimidine auxotrophic mutant could be utilized. Besides allowing the cell to utilize orotate, the transporter renders the cell sensitive to 5-fluoroorotate. This sensitivity is used to select for loss of the plasmid. When expressed from its own promoter, oroP was toxic to E. coli, whereas in Lactococcus lactis the level of expression of oroP from a chromosomal copy was too low to confer 5-fluoroorotate sensitivity. In order to obtain a plasmid that confers 5-fluoroorotate sensitivity when it is integrated into the chromosome of L. lactis and at the same time can be stably maintained in E. coli, the expression of the oroP gene was controlled from a synthetic promoter conferring these traits. To demonstrate its use, a number of L. lactis strains expressing triosephosphate isomerase (tpiA) at different levels were constructed.Construction of tailor-made strains is dependent on efficient genetic methods, and in order to obtain genetically stable strains, chromosomal integration is often desirable. This calls for techniques that allow efficient selection of both chromosomal integration and excision. For many years, plasmids unable to replicate but expressing antibiotic resistance in Lactococcus lactis have been used to obtain strains in which the plasmid has been integrated into the chromosome. Whereas the isolation of integrants was straightforward, strains that had lost the plasmid were not as easy to obtain. A genetic tool based on a plasmid whose replication is impaired at high temperatures has been used extensively to obtain chromosomal insertions and deletions in L. lactis (2). While the integration is dependent on selection for antibiotic resistance at a nonpermissive temperature, the excision step relies on lowering the temperature to the permissive temperature, taking advantage of the growth inhibition resulting from initiation of rolling circle replication from the plasmid origin located on the chromosome (2). Besides inducing genetic instability, a disadvantage of this system is that the nonpermissive temperature is 37°C, which is at the limit for growth of a number of lactococcal strains. A gene involved in nucleotide metabolism has been demonstrated to work as a counterselection marker; loss of the upp gene encoding uracil phosphoribosyltransferase results in resistance to 5-fluorouracil (3, 14, 15). The main drawbacks of using upp are that this gene is found in almost every organism and that 5-fluorouracil may be toxic even in a upp mutant (14, 15).L. lactis synthesizes pyrimidines de novo, but it is also able to metabo...

We studied how the introduction of an additional ATP-consuming reaction affects the metabolic fluxes in Lactococcus lactis. Genes encoding the hydrolytic part of the F 1 domain of the membrane-bound (F 1 F 0 ) H ؉ -ATPase were expressed from a range of synthetic constitutive promoters. Expression of the genes encoding F 1 -ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F 1 -ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase activity did not shift metabolism from homolactic to mixed-acid fermentation, which indicated that a low energy state is not the signal for such a change. The effect of uncoupled ATPase activity on the glycolytic flux depended on the growth conditions. The uncoupling stimulated the glycolytic flux threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions or in sugar transport.Lactic acid bacteria are used extensively in the dairy industry, where the production of lactic acid is important for texture, flavor, and preservation purposes. In addition, lactic acid bacteria are also used for industrial lactate production, which has numerous applications, such as cosmetics, cleaning agents, and biodegradable polylactic acid polymers. From an industrial point of view there is great interest in improving the performance of these organisms with respect to both the rate and the yield of lactate production.In spite of the importance of glycolysis for fermentation purposes, it is still not known what controls the glycolytic flux in microbial bioreactors. It has been suggested that the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has a high level of control (estimated to be 90% of the control) over the glycolytic flux in nonproliferating cells of Lactococcus lactis (33). However, it has recently been shown that GAPDH has no control over the glycolytic flux in steadily growing L. lactis cells (Solem, Koebmann, and Jensen, unpublished data). The control over the glycolytic flux exerted by lactate dehydrogenase was also reported to be close to zero (2).According to metabolic control theory (16, 25), flux control can reside in any of the steps in a system; i.e., it can reside in the numerous processes that consume the ATP generated in glycolysis (8,17). Indeed, we have recently shown that at least 75% of the control over glycolysis in aerobic Escherichia coli cultures occurs in the ATP-consuming reactions (26). This result was obtained by overexpression of genes encoding part of the F 1 unit of the (F 1 F 0 ) H ϩ -ATPase, which resulted in uncoupling of glycolysis from biomass production and a 70% increase in the glycolytic flux.In this paper we show that expression of genes encoding...

The ability to modulate gene expression is an important genetic tool in systems biology and biotechnology. Here, we demonstrate that a previously published easy and fast PCR-based method for modulating gene expression in lactic acid bacteria is also applicable to Corynebacterium glutamicum. We constructed constitutive promoter libraries based on various combinations of a previously reported C. glutamicum -10 consensus sequence (gngnTA(c/t)aaTgg) and the Escherichia coli -35 consensus, either with or without an AT-rich region upstream. A promoter library based on consensus sequences frequently found in low-GC Gram-positive microorganisms was also included. The strongest promoters were found in the library with a -35 region and a C. glutamicum -10 consensus, and this library also represents the largest activity span. Using the alternative -10 consensus TATAAT, which can be found in many other prokaryotes, resulted in a weaker but still useful promoter library. The upstream AT-rich region did not appear to affect promoter strength in C. glutamicum. In addition to the constitutive promoters, a synthetic inducible promoter library, based on the E. coli lac-promoter, was constructed by randomizing the 17-bp spacer between -35 and -10 consensus sequences and the sequences surrounding these. The inducible promoter library was shown to result in β-galactosidase activities ranging from 284 to 1,665 Miller units when induced by IPTG, and the induction fold ranged from 7-59. We find that the synthetic promoter library (SPL) technology is convenient for modulating gene expression in C. glutamicum and should have many future applications, within basic research as well as for optimizing industrial production organisms.