A Brevibacterium lactofermentum 16S rRNA gene used as target site for homologous recombination

Amador, Eladio

doi:10.1016/s0378-1097(00)00103-8

Cited by 5 publications

(6 citation statements)

References 13 publications

(20 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The multiple copies of ribosomal DNA would provide more chances for genes to integrate and prevent the disruption of vital gene loci. Ribosomal DNA has frequently been used for recombination sites of transformation vectors for yeasts, fungi, and plants (3,13,14) to achieve high and stable expression of target genes. However, we found that DNA fragments of selectable markers (neo r expression cassettes designated Ubi-Neo r ) were efficiently integrated into the chromosomal DNA of four species of thraustochytrids at random positions.…”

Section: Discussionmentioning

confidence: 99%

Versatile Transformation System That Is Applicable to both Multiple Transgene Expression and Gene Targeting for Thraustochytrids

Sakaguchi

Matsuda

Kobayashi

et al. 2012

Appl Environ Microbiol

View full text Add to dashboard Cite

A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neo r ), driven with an ubiquitin or an EF-1␣ promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neo r marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neo r mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single-or double-crossover homologous recombination. Finally, the fatty acid ⌬5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-␥-linolenic acid (C 20:3n-6 ) and eicosatetraenoic acid (C 20:4n-3 ), substrates for ⌬5 desaturase, and a decrease of arachidonic acid (C 20:4n-6 ) and eicosapentaenoic acid (C 20:5n-3 ), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.

show abstract

Section: Discussionmentioning

confidence: 99%

Versatile Transformation System That Is Applicable to both Multiple Transgene Expression and Gene Targeting for Thraustochytrids

Sakaguchi

Matsuda

Kobayashi

et al. 2012

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…In past decades, constructions of integrative plasmids which carry a homologous chromosomal fragment and drug resistance marker have made it possible to insert a gene into the chromosome. The homologous fragment in a plasmid consists of multiple copies in the chromosome, such as IS13869 (Amador et al, 2000) and rRNA gene (Correia et al, 1996). The integration is carried out by single-crossover recombination between the integrative plasmid and the recipient genome occurring in the homologous region (Ikeda and Katsumata, 1998).…”

Section: Strategies For Gene Over-expressionmentioning

confidence: 99%

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

Zhang

2016

J. Zhejiang Univ. Sci. B

View full text Add to dashboard Cite

Abstract:With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.

show abstract

“…Thus, the rDNA sequence gene can be used as homologous sequences to direct HR. Successful experiments have been reported in yeast (Wei et al 2009;Wery et al 1997), plant (Barzel and kupiec 2008;Liu et al 2003) and a bacterium (Amador et al 2000). The multiple target sites of HR provide more integration chance.…”

Section: Introductionmentioning

confidence: 99%

Integrated expression of the α-amylase, dextranase and glutathione gene in an industrial brewer’s yeast strain

Wang

et al. 2011

World J Microbiol Biotechnol

View full text Add to dashboard Cite

Genetic engineering is widely used to meliorate biological characteristics of industrial brewing yeast. But how to solve multiple problems at one time has become the bottle neck in the genetic modifications of industrial yeast strains. In a newly constructed strain TYRL21, dextranase gene was expressed in addition of α-amylase to make up α-amylase's shortcoming which can only hydrolyze α-1,4-glycosidic bond. Meanwhile, 18s rDNA repeated sequence was used as the homologous sequence for an effective and stable expression of LSD1 gene. As a result, TYRL21 consumed about twice much starch than the host strain. Moreover TYRL21 speeded up the fermentation which achieved the maximum cell number only within 3 days during EBC tube fermentation. Besides, flavor evaluation comparing TYRL21 and wild type brewing strain Y31 also confirmed TYRL21's better performances regarding its better saccharides utilization (83% less in residual saccharides), less off-flavor compounds (57% less in diacetyl, 39% less in acetaldehyde, 67% less in pentanedione), and improved stability index (increased by 49%) which correlated with sensory evaluation of final beer product.

show abstract

A Brevibacterium lactofermentum 16S rRNA gene used as target site for homologous recombination

Cited by 5 publications

References 13 publications

Versatile Transformation System That Is Applicable to both Multiple Transgene Expression and Gene Targeting for Thraustochytrids

Versatile Transformation System That Is Applicable to both Multiple Transgene Expression and Gene Targeting for Thraustochytrids

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression

Integrated expression of the α-amylase, dextranase and glutathione gene in an industrial brewer’s yeast strain

Contact Info

Product

Resources

About