Design of microaerobically inducible miniR1 plasmids.

Plasmid DNA manufacture is an essential step to produce gene therapy agents and next-generation vaccines. However, little attention has been paid toward developing alternative replicons that can be coupled with large-scale production conditions. Our results demonstrate that the miniR1 replicon can be efficiently induced by oxygen limitation when a copy of the regulatory protein RepA under control of a microaerobic promoter is used. The results are potentially attractive for industrial applications.

Physiological and transcriptional characterization of Escherichia coli strains lacking interconversion of phosphoenolpyruvate and pyruvate when glucose and acetate are coutilized.

Phosphoenolpyruvate (PEP) is a precursor involved in the biosynthesis of aromatics and other valuable compounds in Escherichia coli. The PEP:carbohydrate phosphotransferase system (PTS) is the major glucose transport system and the largest PEP consumer. To increase intracellular PEP availability for aromatics production purposes, mutant strains of E. coli JM101 devoid of the ptsHIcrr operon (PB11 strain) have been previously generated. In this derivative, transport and growth rate on glucose decreased significantly. A laboratory evolved strain derived from PB11 that partially recovered its growth capacity on glucose was named PB12. In the present study, we blocked carbon skeletons interchange between PEP and pyruvate (PYR) in these ptsHIcrr(-) strains by deleting the pykA, pykF, and ppsA genes. The PB11 pykAF(-) ppsA(-) strain exhibited no growth on glucose or acetate alone, but it was viable when both substrates were consumed simultaneously. In contrast, the PB12 pykAF(-) ppsA(-) strain displayed a low growth rate on glucose or acetate alone, but in the mixture, growth was significantly improved. RT-qPCR expression analysis of PB11 pykAF(-) ppsA(-) growing with both carbon sources showed a downregulation of all central metabolic pathways compared with its parental PB11 strain. Under the same conditions, transcription of most of the genes in PB12 pykAF(-) ppsA(-) did not change, and few like aceBAK, sfcA, and poxB were overexpressed compared with PB12. We explored the aromatics production capabilities of both ptsHIcrr(-) pykAF(-) ppsA(-) strains and the engineered PB12 pykAF(-) ppsA(-) tyrR(-) pheA(ev2+) /pJLBaroG(fbr) tktA enhanced the yield of aromatic compounds when coutilizing glucose and acetate compared with the control strain PB12 tyrR(-) pheA(ev2+) /pJLBaroG(fbr) tktA.

A novel plasmid vector designed for chromosomal gene integration and expression: use for developing a genetically stable Escherichia coli melanin production strain.

Recombinant Escherichia coli strains for the production of valuable products are usually generated by transformation with plasmid expression vectors. However, in spite of their usefulness, common problems associated with plasmid use include segregrational and structural instability as well as undesired copy-number effects. A viable alternative to plasmid use is chromosomal gene integration. With the purpose of facilitating the process of stable strain generation, a novel chromosomal integration vector was developed and tested. We describe the construction and use of novel expression vector pLoxGentrc that contains the strong trc promoter (P(trc)), a multiple cloning site, the T1 and T2 rrnB terminator sequences, the lacI(q) gene and the aacC1 gene conferring gentamicin resistance flanked by two loxP sites. As a demonstration of utility, melanin-producing strains of E. coli were generated employing this vector. Melanin is a polymer synthesized by the enzyme tyrosinase using l-tyrosine as substrate. The melA gene encoding a tyrosinase from Rhizobium etli was ligated to pLoxGentrc to generate pLoxGentrcmelA. This plasmid was transformed into E. coli W3110 to generate a melanin-producing strain. A region from this plasmid including P(trc)melA, T1 and T2 rrnB and the aacC1 gene was amplified by PCR employing primers with 45 b regions of homology to the lacZ gene. The PCR product was electroporated into strain W3110 that expressed the λ-Red enzymes. From this experiment, strain W3110P(tr)(c)melA, was obtained having the melA gene inserted in the lacZ locus. Fermentor cultures with strain W3110/pLoxGentrcmelA grown in the presence and absence of gentamicin as well as W3110P(tr)(c)melA without antibiotic revealed that the latter displays high genetic stability as well as the highest melanin titer. Vector pLoxGentrc should be useful during strain generation processes, enabling direct comparison of plasmid and chromosome-based production systems.