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Metabolic engineering strategies applied over the last two decades to produce shikimate (SA) in Escherichia coli have resulted in a battery of strains bearing many expression systems. However, the effects that these systems have on the host physiology and how they impact the production of SA are still not well understood. In this work we utilized an engineered E. coli strain to determine the consequences of carrying a vector that promotes SA production from glucose with a high-yield but that is also expected to impose a significant cellular burden. Kinetic comparisons in fermentors showed that instead of exerting a negative effect, the sole presence of the plasmid increased glucose consumption without diminishing the growth rate. By constitutively expressing a biosynthetic operon from this vector, the more active glycolytic metabolism was exploited to redirect intermediates toward the production of SA, which further increased the glucose consumption rate and avoided excess acetate production. Fluxomics and metabolomics experiments revealed a global remodeling of the carbon and energy metabolism in the production strain, where the increased SA production reduced the carbon available for oxidative and fermentative pathways. Moreover, the results showed that the production of SA relies on a specific setup of the pentose phosphate pathway, where both its oxidative and non-oxidative branches are strongly activated to supply erythrose-4-phosphate and balance the NADPH requirements. This work improves our understanding of the metabolic reorganization observed in E. coli in response to the plasmid-based expression of the SA biosynthetic pathway. Biotechnol. Bioeng. 2017;114: 1319-1330. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

A pBRINT-Ts family of integrative vectors for Escherichia coli was constructed by using a temperature-sensitive replicon derived from pSC101, a region of homology to the lacZ gene, and various antibiotic resistance markers (kanamycin, chloramphenicol and gentamycin) for selection of the integrants. The gene or group of genes to be integrated can be inserted into a multiple cloning site, flanked by an antibiotic resistance marker and lacZ sequences. A simple and rapid procedure was developed for the selection of cells where allelic exchange had occurred. With this procedure, the efficiency of integration of around 10-3 was observed, using several E. coli strains. From colonies with an integrated pBRINT-Ts plasmid, we detected an average allelic exchange event frequency of 7.5%. As a test for this system, we integrated the amy gene that codes for the alpha-amylase from B. stearothermophilus, into the lacZ gene of E. coli W3110. Production of alpha-amylase was found to be proportional to copy number; at up to 10 copies per chromosome.

Plasmid pBRINT is an efficient vector for chromosomal integration of cloned DNA into the lacZ gene of Escherichia coli [Balbás et al., Gene 136(1993) 211-213]. A family of related plasmids containing different antibiotic-resistance markers (CmR or GmR or KmR) and a larger multiple cloning site (MCS) has been constructed. This set of plasmids, whose integration efficiencies are as good as those obtained with the prototype plasmid pBRINT, constitutes a collection of tools that allow rapid and easy integration of cloned DNA, at the chromosomal level. Their functionality as integration vectors has been ascertained by integrating the Vitreoscilla sp. hemoglobin-encoding gene and the Photobacterium leiognathi lux genes. To evaluate the level of expression obtained after chromosomal integration, we constructed strains carrying one or two copies of the cat gene integrated in the chromosome, and compared their enzymatic activities with those obtained from a strain carrying cat on a multicopy plasmid.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.

In vitro recombinant DNA experiments involving restriction endonuclease fragments derived from the plasmids pBR322 and pBR325 resulted in the construction of two new cloning vehicles. One of these plasmids, designated pBR327, was obtained after an EcoRII partial digestion of pBR322. The plasmid pBR327 confers resistance to tetracycline and ampicillin, contains 3273 base pairs (bp) and therefore is 1089 bp smaller than pBR322. The other newly constructed vector, which has been designated pBR328, confers resistance to chloramphenicol as well as the two former antibiotics. This plasmid contains unique HindIII, BamHI and SalI sites in the tetracycline resistance gene, unique PvuI and PstI sites in the ampicillin resistance gene and unique EcoRI, PvuII and BalI sites in the chloramphenicol resistance gene. The pBR328 plasmid contains approx. 4900 bp.

The 4150-bp plasmid pBR329 was constructed by the the insertion into pBR327 of an 877-bp DNA fragment carrying the Cmr gene from pBR328. This new cloning vector does not contain the 482-bp inverted duplication that has been reported to be present in pBR325 and pBR328 (Prentki et al., 1981). In pBR329 the Cmr gene lacks its original promoter but is transcribed counterclockwise toward the Apr gene by a promoter located to the right of the HindIII site in the Tcr gene.

The plasmid pBR322 was one of the first EK2 multipurpose cloning vectors to be designed and constructed (ten years ago) for the efficient cloning and selection of recombinant DNA molecules in Escherichia coli. This 4363-bp DNA molecule has been extensively used as a cloning vehicle because of its simplicity and the availability of its nucleotide sequence. The widespread use of pBR322 has prompted numerous studies into its molecular structure and function. These studies revealed two features that detract from the plasmid's effectiveness as a cloning vector: plasmid instability in the absence of selection and, the lack of a direct selection scheme for recombinant DNA molecules. Several vectors based on pBR322 have been constructed to overcome these limitations and to extend the vector's versatility to accommodate special cloning purposes. The objective of this review is to provide a survey of these derivative vectors and to summarize information currently available on pBR322.

By employing a system of RNA polymerase binding and restriction endonuclease digestion, we demonstrate that the region in and around the Hind III site of pBR313 and pBR322 is the promoter for the tetracycline (Tc) resistance gene(s). Furthermore, it is shown that this region was transferred intact from pSC101 during the construction of the latter plasmids. The in vitro insertion of a few base pairs at the Hind III site produces a series of "down" promoter mutations in which the level of in vivo Tc resistance is reduced. Sequence analysis of the various promoter mutations revealed significant base pair rearrangements in the region between -40 and -12 of the promoter. While these base alterations do not appear to affect the firm binding of RNA polymerase, they do affect the ability of mutant promoters to initiate transcription. These observations suggest that the region from -40 to -12, previously designated as the "recognition region", is actually involved in the process of initiation of transcription.

A DNA sequence essential for the R64drd11 + ColK-mediated conjugal transfer of pBR322 has been located in a 540 bp HaeIII fragment (HaeIII-2) between the vegetative origin of replication and the tetracycline resistance (Tcr) gene of this vector. The pBR322 derivatives pBR327 and pBR328 lack this DNA sequence and are not mobilized by conjugation. Two derivatives of pBR328 were constructed by re-inserting the HaeIII-2 fragment in both orientations into the chloramphenicol-resistance gene of the same vector. One orientation of the HaeIII-2 fragment permitted mobilization by conjugation while the opposite orientation prevented mobilization. Further examination of pBR322 and derivatives revealed that the region between the origin of replication and Tcr gene also plays a role in regulating plasmid copy number.

In vitro recombinant DNA experiments, using plasmid pBR327 and a DNA fragment derived from plasmid pSC101 containing the par region, resulted in the construction of plasmid pBR327par. This new cloning vehicle has all the cloning properties of the parental plasmid, and is more stable than pBR327. Since the nucleotide sequence of the par region has been determined, this new vector is completely characterized. Some features of the sequence with possible functional significance are discussed.

The gene encoding a heat-stable enterotoxin of Escherichia coli was cloned as a 960-bp fragment from a plasmid isolated from a Mexican strain of human origin. Deoxyribonucleotide sequencing unveiled a 216-bp open reading frame similar to that of a previously sequenced ST-toxin gene. The gene is preceded by a proposed binding site for the cAMP-mediated positive regulator (CAP) that is part of a 23-bp inverted repeat. The proposed CAP site is followed by a 6A, 1T, and 6A deoxyribonucleotides. Minicells containing the toxin gene, which were isolated from Ficoll gradients, shown to preserve the localization of intracellular and periplasmic enzymes, allowed the detection of a biosynthetically radiolabeled polypeptide with an apparent Mr 8400. The data suggest that the enterotoxin genes estA2, estA3, and estA4 are very similar, even in clinical strains isolated from distinct geographical locations; that the transcription of heat-stable enterotoxin genes is controlled by the cAMP-mediated positive regulatory system, and that the heat-stable enterotoxins are initially synthesized as 72 amino acid precursors to yield the extracellular active 18-19 amino acid polypeptides.

Plasmid pRSP1, isolated from the Escherichia coli gene bank of Clarke and Carbon (1975), has been shown to complement the gdh-1 mutation that affects the synthesis of glutamate dehydrogenase (GDH) in the E. coli strain PA340. The GDH structural gene of E. coli is present on a 2.3-Md DNA fragment from the hybrid plasmid pRSP1 and certain of its derivatives. Polypeptides synthesized by minicells carrying some of these plasmids, enabled us to make a preliminary determination of the direction of transcription of the gdh gene on the cloned fragments.

A small (approximately 1100 base pairs) ColE1-type plasmid, pBR345, was constructed from plasmid pMB1 by a series of in vitro recombinant manipulations. Approximately 9% of the supercoiled pBR345 DNA obtained from cultures amplified with chloramphenicol appears to be replicative intermediates with replicating "eye" structures of uniform size. Results obtained from electron microscopy and biochemical analyses have enabled us to localize the origin of replication at the same position as that reported for ColE1. A sequence of 420 nucleotides surrounding this origin has been determined. A comparison between this sequence and the one determined for the origin of replication of ColE1 is presented.

In vitro recombination techniques were used to construct a new cloning vehicle, pBR322. This plasmid, derived from pBR313, is a relaxed replicating plasmid, does not produce and is sensitive to colicin E1, and carries resistance genes to the antibiotics ampicillin (Ap) and tetracycline (Tc). The antibiotic-resistant genes on pBR322 are not transposable. The vector pBR322 was constructed in order to have a plasmid with a single PstI site, located in the ampicillin-resistant gene (Apr), in addition to four unique restriction sites, EcoRI, HindIII, BamHI and SalI. Survival of Escherichia coli strain X1776 containing pBR313 and pBR322 as a function of thymine and diaminopimelic acid (DAP) starvation and sensitivity to bile salts was found to be equivalent to the non-plasmid containing strain. Conjugal transfer of these plasmids in bi- and triparental matings were significantly reduced or undetectable relative to the plasmid ColE1.