Stability of Integrated Plasmids in the Chromosome of
            <i>Lactococcus lactis</i>

Leenhouts, Kees; Kok, Jan; Venema, Gerhardus

doi:10.1128/aem.56.9.2726-2735.1990

Cited by 148 publications

(73 citation statements)

References 31 publications

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“…However, it was found that the copy number of genes integrated at delta sites may decrease by gene loop-out events due to homologous recombination during long-term nonselective cultivation (Lee and Silva, 1997;Oliveira et al, 2007;Wang et al, 1996). Genetic instability under nonselective conditions was also reported after classical chromosomal integration of the target gene at a specific site (Leenhouts et al, 1990). To avoid this problem, improvements may be achieved by changing culture conditions, which may however not be feasible in industrial applications.…”

Section: Discussionmentioning

confidence: 99%

Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters

Shi

Valle-Rodríguez

Siewers

et al. 2014

Biotech & Bioengineering

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In recent years, significant advances have been made to engineer robust microbes for overproducing biochemical products from renewable resources. These accomplishments have to a large extend been based on plasmid based methods. However, plasmid maintenance may cause a metabolic burden on the host cell and plasmid-based overexpression of genes can result in genetically unstable strains, which contributes to loss in productivity. Here, a chromosome engineering method based on delta integration was applied in Saccharomyces cerevisiae for the production of fatty acid ethyl esters (FAEEs), which can be directly used as biodiesel and would be a possible substitute for conventional petroleum-based diesel. An integration construct was designed and integrated into chromosomal delta sequences by repetitive transformation, which resulted in 1-6 copies of the integration construct per genome. The corresponding FAEE production increased up to 34 mg/L, which is an about sixfold increase compared to the equivalent plasmid-based producer. The integrated cassette in the yeast genome was stably maintained in nonselective medium after deletion of RAD52 which is essential for efficient homologous recombination. To obtain a further increase of FAEE production, genes encoding endogenous acyl-CoA binding protein (ACB1) and a bacterial NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN) were overexpressed in the final integration strain, which resulted in another 40% percent increase in FAEE production. Our integration strategy enables easy engineering of strains with adjustable gene copy numbers integrated into the genome and this allows for an easy evaluation of the effect of the gene copy number on pathway flux. It therefore represents a valuable tool for introducing and expressing a heterologous pathway in yeast.

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Section: Discussionmentioning

confidence: 99%

Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters

Shi

Valle-Rodríguez

Siewers

et al. 2014

Biotech & Bioengineering

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“…Afterwards, the counterstained cells were observed for microscopic assessment (Paik 1980). The total genomic DNA was extracted by the method described by Leenhouts et al (1990) with some modifications. For molecular identification, the amplification was carried by designing the genus specific primer pairs (Lactobacillus genus) that were; F: 5 0 AGAGTTTGATCMTG GCTCAG-3 0 and R: 5 0 -TACCTTGTTAGGACTTCACC-3 0 for direct amplification of 16S ribosomal RNA gene containing 1500 bp.…”

Section: Isolation Biochemical/molecular Identification and Probiotimentioning

confidence: 99%

Microencapsulation of probiotic bacteria Lactobacillus plantarum 15HN using alginate-psyllium-fenugreek polymeric blends

et al. 2015

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“…Extraction of chromosomal DNA was achieved using the protocol of Leenhouts et al (1990). The primers LacAll-F (5 0 -TGCCTAATACATGCAAGTC-3 0 ) and LacAll-R (5 0 -CCTTGTTACGACTTCACC-3 0 ) were used to amplify nearly the full length of the 16S rRNA gene, corresponding to the conserved 16S rRNA gene regions of four type strains of vaginal Lactobacillus: Lactobacillus crispatus ATCC 33197 (AF257096); L. crispatus ATCC 33820 (AF257097); Lactobacillus jensenii ATCC 25258 (AF243176); and Lactobacillus vaginalis ATCC 49540 (AF243177).…”

Section: Taxonomic Identification Of Lactobacillus By 16s Rrna Gene Smentioning

confidence: 99%

Selection and identification of anaerobic lactobacilli producing inhibitory compounds against vaginal pathogens

Kaewsrichan

Peeyananjarassri

Kongprasertkit

2006

FEMS Immunology &Amp; Medical Microbiology

134

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Two strains of Lactobacillus crispatus (15L08 and 21L07) and one strain of Lactobacillus jensenii (5L08) were selected from amongst 100 isolates from the vaginas of healthy premenopausal women for properties relevant to mucosal colonization and the production of H2O2 and/or bacteriocin-like compound. All three strains self-aggregated and adhered to vaginal epithelial cells, displacing well-known vaginal pathogens, such as Gardnerella vaginalis and Candida albicans. Lactobacillus crispatus 15L08 was characterized as a potential H2O2 producer. A high level of bacteriocin-like compound was synthesized by L. jensenii 5L08, with a bactericidal mode of action for G. vaginalis, C. albicans and Escherichia coli. However, H2O2-dependent activity alone was not sufficient to inhibit the growth of C. albicans. Simultaneous actions of H2O2 and bacteriocin-like compound produced by lactobacilli may be important for antagonizing pathogenic bacteria. These strains of lactobacilli may be excellent candidates for eventual use as probiotics to restore the normal microbial communities in the vaginal ecosystem.

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Stability of Integrated Plasmids in the Chromosome of Lactococcus lactis

Cited by 148 publications

References 31 publications

Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters

Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters

Microencapsulation of probiotic bacteria Lactobacillus plantarum 15HN using alginate-psyllium-fenugreek polymeric blends

Selection and identification of anaerobic lactobacilli producing inhibitory compounds against vaginal pathogens

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