Promoter knock-in: a novel rational method for the fine tuning of genes

Background  

Metabolic engineering aims at channeling the metabolic fluxes towards a desired compound. An important strategy to achieve this is the modification of the expression level of specific genes. Several methods for the modification or the replacement of promoters have been proposed, but most of them involve time-consuming screening steps. We describe here a novel optimized method for the insertion of constitutive promoters (referred to as "promoter knock-in") whose strength can be compared with the native promoter by applying a promoter strength predictive (PSP) model.     

Development of a trpE promoter-strength measuring system and its use in comparison of the trpEDCBA, trpR and aroH promoters

An expression system was developed for measuring in vivo promoter strength at the single copy level and this system was used to compare the trp, aroH and trpR promoters. This system employs trpE enzyme activity as a measure of promoter strength and lacZ expression for internal copy number reference. Promoter-containing fragments are inserted into a cloning vector and subsequently recombined on to phage lambda by genetic exchange. Single lysogens are then prepared and used in promoter-strength analyses. The strength of several promoters was determined using this system. Among the promoters tested, the Escherichia coli trpEDCBA promoter was the strongest; it was four times more active than the lacUV5 promoter and about ten times stronger than the trpR and aroH promoters. To validate measurement of trpE enzyme activity as an indicator of promoter strength, trpE enzyme activity was compared with the level of trpE mRNA. There was excellent correspondence between the two, suggesting that with this system trpE enzyme activity accurately reflects promoter strength. We also examined a homologous promoter-strength measuring system in which the promoter-cloning plasmid lacked a 104 base-pair DNA spacer that was present immediately downstream from the promoter-cloning site in our preferred system. We found that the spacer was essential; the transcribed region accompanying a cloned promoter apparently affected trpE translational efficiency and/or trpE mRNA stability.     

Comparative activity of several promoters in driving NIS expression in melanoma cells

Targeted drug delivery systems are special importance for developing gene therapeutic drugs that recognize and eliminate tumor cells. It is desirable that therapeutic genes be expressed predominantly in tumor cells after their targeted delivery into the tumor. Hence, the distribution of the expression product through various tissues should be studied when testing a therapeutic gene in vivo. The sodium iodide symporter (NIS) is attractive as a reporter because its tissue level is easy to quantify by noninvasive imaging methods. Therapeutic gene expression in tumor cells is achieved using various promoters, including strong nonspecific promoters; moderately active tissue-specific promoters; and tumor-specific promoters, which function in a broad range of tumor cells, but have low activity. The relationship between the promoter strength and reporter NIS activity is still unclear. The reporter gene was used to test three promoters types for activity in melanoma cells. The functional activity of NIS expressed from a cloned gene was compared for the three promoters types. Although the promoters greatly varied in strength, only minor changes were observed for NIS functional activity. A relatively weak melanoma-specific promoter ensured a high NIS activity in melanoma cells. Weaker tumorspecific promoters determined a high NIS activity only in some cells of the melanoma origin.     

Novel Promoters Derived from Chinese Hamster Ovary Cells via In Silico and In Vitro Analysis

For the industrial production of recombinant proteins in mammalian cell lines, a high rate of gene expression is desired. Therefore, strong viral promoters are commonly used. However, these have several drawbacks as they override cellular responses, are not integrated into the cellular network, and thus can induce stress and potentially epigenetic silencing. Endogenous promoters potentially have the advantage of a better response to cellular state and thus a lower stress level by uncontrolled overexpression of the transgene. Such fine‐tuning is typically achieved by endogenous enhancers and other regulatory elements, which are difficult to identify purely based on the genomic sequence. Here, Chinese hamster ovary (CHO) endogenous promoters and enhancers are identified using histone marks and chromatin states, ranked based on expression level and tested for normalized promoter strength. Successive truncation of these promoters at the 5′‐ and 3′‐end as well as the combination with enhancers are identified in the vicinity of the promoter sequence further enhance promoter activity up to threefold. In an initial screen within stable cell lines, the strongest CHO promoter appears to be more stable than the human cytomegalovirus promoter with enhancer, making it a promising candidate for recombinant protein production and cell engineering applications. A deeper understanding of promoter functionality and response elements will be required to take full advantage of such promoters for cell engineering, in particular, for multigene network engineering applications.     

Functional Analysis and Randomization of the Nisin-Inducible Promoter for Tuning Gene Expression in Lactococcus lactis

Three putative promoter regions were identified preceding the nisZ gene in Lactococcus lactis HSM-22. To investigate their function in the control of nisZ biosynthesis, green fluorescence protein (GFP) was adopted as probe to determine activities of the three promoters. The results showed that PnisZ-0 containing two sets of the ?35 and ?10 regions exhibited the same maximum activity as promoter PnisZ-2 containing the putative promoter region near the start codon. However, the GFP expression level directed by PnisZ-0 was twofold higher than that found with PnisZ-2 under low-dose nisin, indicating that promoter PnisZ-1 distant from the start codon could be important in response to the inducer nisin. Then, Pnis-2 was randomized to develop functional promoters through the degenerate oligonucleotide approach in L. lactis. 35 inducible promoters and 14 constitutive promoters were obtained, covering 3–5 logs of expression levels in small increments of activity. Sequence analysis revealed that base changes in both consensus sequence and spacing sequence resulted in remarkable decrease of promoter activity, while the sequence outside ?35 and ?10 regions would influence the promoter function radically. The functional promoters were evaluated for the efficiency and stability to control β-galactosidase (Gal) expression in L. lactis. High correlation was obtained between the Gal activity and promoter strength, suggesting that promoters developed here have the potential for fine tuning gene expression in L. lactis.     

Optimization of phage λ promoter strength for synthetic small regulatory RNA-based metabolic engineering

Synthetic small regulatory RNAs (sRNAs) are gene-silencing tools that can be used to tune gene expression in prokaryotes. A recent study by our group proposed rational design principles, introduced a regulatory system that may be used to implement synthetic sRNAs, and showed their utility in metabolic engineering. The regulatory system employed the strong phage λ P_R promoter to tightly control synthetic sRNA production. Here, we fine-tuned the strength of the P_R promoter via mutagenesis in order to optimize the level of synthetic sRNAs while maintaining the ability of the promoter to be regulated by CI proteins. Five mutant promoters of different strengths, ranging from 24 to 87% of that of the wild-type P_R promoter, were identified and confirmed to be repressed by CI proteins. A mutated promoter with only 40% of the original strength still produced enough synthetic sRNAs to inhibit the translation of the target mRNA to ~10% of the original level. As a practical application, we tested our promoters as drivers for a synthetic anti-murE sRNA, which was used to adjust the production of cadaverine. As the promoter strength decreased, the cadaverine titer first increased and then dropped. A mutated promoter with 39% of the original strength achieved the improved cadaverine titer of 2.15 g/L. The mutant promoters developed in this study should prove useful for tuning the expression levels of synthetic sRNAs for metabolic engineering.     

Comparison of promoter activity in Aleutian mink disease parvovirus, minute virus of mice, and canine parvovirus: possible role of weak promoters in the pathogenesis of Aleutian mink disease parvovirus infection.

Aleutian mink disease parvovirus (ADV) infection causes both acute and chronic disease in mink, and we have previously shown that it is the level of viral gene expression that determines the disease pattern. To study the gene regulation of ADV, we have cloned the P3 ADV and P36 ADV promoters in front of a reporter gene, the chloramphenicol acetyltransferase (CAT) gene, and analyzed these constructs by transient transfection in a feline kidney cell line and mouse NIH 3T3 cells. The genes for ADV structural proteins (VP1 and VP2) and the nonstructural proteins (NS-1, NS-2, and NS-3) were cloned into a eukaryotic expression vector, and their functions in regulation of the P3 ADV and P36 ADV promoters were examined in cotransfection experiments. The ADV NS-1 protein was able to transactivate the P36 ADV promoter and, to a lesser degree, the P3 ADV promoter. Constitutive activities of the P3 ADV and P36 ADV promoters were weaker than those of the corresponding promoters from the prototypic parvovirus minute virus of mice (MVM) and canine parvovirus (CPV). Also, the level of transactivation of the P36 ADV promoter was much lower than those of the corresponding P38 MVM and P38 CPV promoters transactivated with MVM NS-1. Moreover, the ADV NS-1 gene product could transactivate the P38 MVM promoter to higher levels than it could transactivate the P36 ADV promoter, while the P36 ADV promoter could be transactivated by MVM NS-1 and ADV NS-1 to similar levels. Taken together, these data indicated that cis-acting sequences in the P36 ADV promoter play a major role in determining the low level of transactivation observed. The P3 ADV and P4 MVM promoters could be transactivated to some degree by their respective NS-1 gene products. However, in contrast to the situation for the late promoters, switching NS-1 proteins between the two viruses was not possible. This finding may indicate a different mechanism of transactivation of the early promoters (P3 ADV and P4 MVM) compared with the late (P36 ADV and P38 MVM) promoters. In summary, the constitutive levels of expression from the ADV promoters are weaker than the levels from the corresponding promoters of MVM and CPV. Moreover, the level of NS-1-mediated transactivation of the late ADV promoter is impaired compared with the level of transactivation of the late promoters of MVM and CPV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activity of native vs. synthetic promoters in Brucella

Brucellosis caused by Brucella species is reportedly the most common zoonotic infection worldwide. The bacterial pathogen is also classified by the Centers for Disease Control and Prevention as a category (B) pathogen that has the potential for development as a bioweapon. Although eight genomes of Brucella have been sequenced, little information is available regarding the regulation of gene expression and promoter activity in Brucella spp. We therefore constructed a set of broad-host-range vectors expressing the lacZ reporter gene from various promoters. Four groups of promoters (Brucella native, antibiotic resistant, bacteriophage and synthetic promoters) were tested in vivo and in vitro in Brucella suis. The highest level of heterologous gene expression was achieved with synthetic hybrid trc promoter carrying the adenine-rich upstream element. Furthermore, this demonstrates the usefulness of synthetic promoters for enhanced level of gene expression in Brucella spp.     

Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping

The cauliflower mosaic virus 35S (35S) promoter has been extensively used for the constitutive expression of transgenes in dicotyledonous plants. The repetitive use of the same promoter is known to induce transgene inactivation due to promoter homology. As a way to circumvent this problem, we tested two different strategies for the development of synthetic promoters that are functionally equivalent but have a minimum sequence homology. Such promoters can be generated by (a) introducing known cis-elements in a novel or synthetic stretch of DNA or (b) "domain swapping," wherein domains of one promoter can be replaced with functionally equivalent domains from other heterologous promoters. We evaluated the two strategies for promoter modifications using domain A (consisting of minimal promoter and subdomain A1) of the 35S promoter as a model. A set of modified 35S promoters were developed whose strength was compared with the 35S promoter per se using beta-glucuronidase as the reporter gene. Analysis of the expression of the reporter gene in transient assay system showed that domain swapping led to a significant fall in promoter activity. In contrast, promoters developed by placing cis-elements in a novel DNA context showed levels of expression comparable with that of the 35S. Two promoter constructs Mod2A1T and Mod3A1T were then designed by placing the core sequences of minimal promoter and subdomain A1 in divergent DNA sequences. Transgenics developed in tobacco (Nicotiana tabacum) with the two constructs and with 35S as control were used to assess the promoter activity in different tissues of primary transformants. Mod2A1T and Mod3A1T were found to be active in all of the tissues tested, at levels comparable with that of 35S. Further, the expression of the Mod2A1T promoter in the seedlings of the T1 generation was also similar to that of the 35S promoter. The present strategy opens up the possibility of creating a set of synthetic promoters with minimum sequence homology and with expression levels comparable with the wild-type prototype by modifying sequences present between cis-elements for transgene expression in plants.     

Sequence elements determining ampC promoter strength in E. coli

A number of spontaneous up-promoter mutations have been isolated in the ampC beta-lactamase gene of Escherichia coli. The mutants were analyzed by DNA sequencing, and the level of ampC gene expression was determined. Six mutants with a 21-fold increase in promoter strength compared with the wild-type were mutated in the -35 promoter region from TTGTCA to the consensus sequence TTGACA . The -10 region sequence TACAAT was mutated to the consensus sequence TATAAT in three mutants exhibiting an ampC promoter seven times stronger than the wild-type. We have previously described a 1-bp insertion mutant ( Jaurin et al., 1981) that changes the inter-region distance to the consensus 17 bp. Thus, all the up-mutations found in the ampC promoter represent corrections of the three major discrepancies between the ampC promoter and the consensus E. coli promoter. We conclude that the three consensus elements of E. coli promoters, the -35 and -10 regions and an optimal inter-region distance of 17 bp, are the main elements determining the promoter strength.     

Novel baculovirus DNA elements strongly stimulate activities of exogenous and endogenous promoters.

A DNA sequence upstream from the polyhedrin gene of baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) was found to activate strongly the expression of full or minimal promoters derived from AcMNPV and other sources. Promoters tested included the minimal CMV (CMVm) promoter from human cytomegalovirus, the full heat shock 70 promoter from Drosophila, and the minimal p35 promoter from baculovirus. Deletion and mutagenesis analyses showed that this functional polyhedrin upstream (pu) activator sequence contains three open reading frames (ORFs), ORF4, ORF5, and lef2. In plasmid transfection assays, the pu sequence was able to confer high level luciferase expression driven by all of these full or minimal promoters in insect Sf21 cells. A known baculovirus enhancer, the homologous region (hr) of AcMNPV, further enhanced the expression of these promoters. Experiments showed that although multiple hr sequences function in an additive manner, pu and hr together function synergistically, resulting in as much as 18,000-fold promoter activation. Furthermore, a modified CMVm promoter containing pu and/or hr was inserted into the baculovirus genome to drive the luciferase coding region. The CMVm promoter expressed luciferase much earlier, and although it expressed a bit less than did the p10 promoter, the CMVm promoter gave rise to greater luciferase activity. Therefore, we have uncovered a cryptic viral sequence capable of activating a diverse group of promoters. Finally, these experiments demonstrate that synthetic sequences containing pu, hr, and different full or minimal promoters can generate a set of essentially unlimited novel promoters for weak to very strong expression of foreign proteins using baculovirus.     

Quantitative transient gene expression: Comparison of the promoters for maize polyubiquitin1, rice actin1, maize-derivedEmu andCaMV 35S in cells of barley,maize and tobacco

The particle gun approach was used for the quantification of promoter efficiency in a test system for transient gene expression. β-Glucuronidase was used as reporter gene for determining promotote strength. The variability inherent in this gene transfer system was considerably reduced by calculating a transformation efficiency factor given by the expression of a cotransferred second reporter gene (firefly luciferase). The calibration of β-glucuronidase activity by the transformation efficiency factor caused a lower statistical variance of the values and allowed reliable results to be obtained with a smaller set of repetitions. The CaMV 35S promoter (as a control) and the monocot-specific promoters for maize polyubiquitin1, rice actin 1 and the maize-derivedEmu were characterized and compared with respect to expression strength, as tested under identical conditions in suspension cell cultures of maize, barley and tobacco. Compared to the 35S promoter, the monocot-specific promoters show up to 15-fold higher expression in maize and barley but give only weak expression in tobacco. No expression was found for the rice actin 1 promoter in tobacco. The level of reporter gene expression is influenced by the osmotic potential in the agar medium. For theEmu promoter, the calibrated β-glucuronidase activities remained mearly constant at low sucrose concentrations. Above 8% sucrose, the calibrated activities increased steadily with increasing osmotic conditions, reaching a three-to four-fold higher level at the highest sucrose concentration (32%) as compared to the standard concentration (4% sucrose) in the medium.     

Manifestation of the Kanagawa phenomenon, the virulence-associated phenotype, of Vibrio parahaemolyticus depends on a particular single base change in the promoter of the thermostable direct haemolysin gene

Thermostable direct haemolysin of Vibrio parahaemolyticus has been shown to be a major virulence factor. The Kanagawa phenomenon (KP), haemolysis induced by this haemolysin on a special blood agar medium, is strongly associated with clinical strains. We have been studying the expressions of various tdh genes encoding this haemolysin to elucidate the significance of the tdh genes possessed by KP-negative strains isolated from patients. We examined the importance of the promoter sequence variation for expression level of the tdh gene in this study. Only the tdh 2 gene, one of the two tdh genes ( tdh 1 and tdh 2) present in a KP-positive strain, was previously shown to be responsible for the haemolytic activity of the KP-positive strain. The tdh 1– and tdh 2– lacZ fusions were used to determine and analyse the promoter sequence by primer extension and site-directed mutagenesis methods. Two bases (positions −24 and −34) within the determined tdh 2 promoter sequence were shown to be mostly responsible for the difference in the promoter strength between the tdh 2 and tdh 1 genes both in Escherichia coli and in V. parahaemolyticus backgrounds. Representative tdh promoters of KP-negative strains are close to the tdh 2 promoter; they differ at position −34 but have the same base at position −24 as the tdh 2 promoter. We demonstrated that base substitution of the tdh promoters of KP-negative strains only at position −34 is sufficient to increase the expression of these genes to the KP-positive level. Therefore, the tdh genes of KP-negative strains are considered to be potentially important because they can generate a KP-positive subclone by a point mutation in their promoters.