Fernando Valle

A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria

Modifications of microbial genomes often require the use of the antibiotic-resistance (Anb(R))-encoding genes and other easily selectable markers. We have developed a set of such selectable markers (Cm(R), Km(R) and Gm(R)), which could easily be inserted into the genome and subsequently removed by using the Cre/loxP site-specific recombination system of bacteriophage P1. In this manner the same marker could be used more than once in the same background, while the resulting strain could or would remain Anb(R) marker-free. Three plasmids were constructed, each containing a cassette consisting of the Cm(R), Km(R), or Gm(R) gene flanked by two parallel loxP sites and two polylinkers (MCS). To test insertion and excision, cassettes were inserted into the lacZ or galE genes carried on an origamma/pir-dependent suicide plasmid, which contained a dominant Sm(R) gene. The cassettes were crossed into the E. coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Pósfai et al. [Nucl. Acids Res. 22 (1994) 2392-2398], selecting for the Cm(R), Km(R), or Gm(R), for the LacZ(-) or GalE(-) and for the Sm(S) phenotypes (the latter to assure allelic exchange rather than insertion of the entire plasmid). When required, after selecting the strain with the desired modification, the Cm(R), Km(R), or Gm(R) marker was excised by supplying the Cre function. Cre was provided by the thermosensitive plasmid pJW168, which was transformed into the Anb(R) host at 30 degrees C, and was subsequently eliminated at 42 degrees C. Thus the Anb(R) marker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site.

Characterization of sugar mixtures utilization by an Escherichia coli mutant devoid of the phosphotransferase system

Abstract. Due to catabolite repression in microorganisms, sugar mixtures cannot be metabolized in a rapid and efficient manner. Therefore, the development of mutant strains that avoid this regulatory system is of special interest to fermentation processes. In the present study, the utilization of sugar mixtures by an Escherichia coli mutant strain devoid of the phosphotransferase system (PTS) was characterized. This mutant can transport glucose (PTS– Glucose+ phenotype) by a non-PTS mechanism as rapidly as its wild-type parental strain. In cultures grown in minimal medium supplemented with glucose-xylose or glucose-arabinose mixtures, glucose repressed arabinose- or xylose-utilization in the wild-type strain. However, under the same culture conditions with the PTS– Glucose+ mutant, glucose and arabinose were co-metabolized, but glucose still exerted a partial repressive effect on xylose consumption. In cultures growing with a triple mixture of glucose-arabinose-xylose, the wild-type strain sequentially utilized glucose, arabinose and finally, xylose. In contrast, the PTS– Glucose+ strain co-metabolized glucose and arabinose, whereas xylose was utilized after glucose-arabinose depletion. As a result of glucose-arabinose co-metabolism, the PTS– Glucose+ strain consumed the total amount of sugars contained in the culture medium 16% faster than the wild-type strain. [14C]-Xylose uptake experiments showed that in the PTS– Glucose+ strain, galactose permease increases xylose transport capacity and the observed partial repression of xylose utilization depends on the presence of intracellular glucose.

Characterization of the regulatory region of the Escherichia coli penicillin acylase structural gene

Penicillin acylase is utilized in the enzymatic production of semisynthetic penicillins. The enzyme is composed of two different subunits that originate from a common precursor. The partial nucleotide (nt) sequence of the structural gene has been published. This paper reports the nt sequence of the regulatory region of this gene, the identification of a functional promoter, the transcriptional start point, and the description of possible regulatory regions.

Kinetic characterization in batch and continuous culture of Escherichia coli mutants affected in phosphoenolpyruvate metabolism: differences in acetic acid production

The growth kinetics of an Escherichia coli wild type strain and two derivative mutants were examined in batch cultures and in glucose-limited chemostats. One mutant (PB12) had an inactive phosphotranferase transport system and the other (PB25) had interrupted pykA and pykF genes that code for the two pyruvate kinase isoenzymes. In both batch and continuous culture, important differences in acetic acid accumulation and other metabolic activities were found. Compared to the wild type strain, we observed a reduction in acetic acid accumulation of 25 and 80% in PB25 and PB12 strains respectively, in batch culture. Continuous culture experiments revealed that compared to the other two strains, PB25 accumulated less acetic acid as a function of dilution rate. In continuous cultures, oxidoreductase metabolic activities were substantially affected in the two mutant strains. These changes in turn were reflected in different levels of biomass and CO2 production, and in oxygen consumption.

Overexpression of Chromosomal Genes in Escherichia coli

Conversion of some carbon sources into desired compounds by a biological system is the goal of many biotechnologists. The understanding of the mechanisms by which an organism does these conversions permits the improved production of specific metabolites. Pathway engineering involves the strategies to modify cells to overproduce desired molecules. We describe here the methodology to modify chromosomal genes by replacing their native regulatory regions with promoter cassettes to increase or deregulate expression of chromosomal genes.