Improvement of protein production in lactic acid bacteria using 5′-untranslated leader sequence of slpA from Lactobacillus acidophilus

Self Cite

102

In order to achieve direct and efficient fermentation of optically pure D-lactic acid from raw corn starch, we constructed L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 ␣-amylase (AmyA). The resulting strain produced only D-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct D-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct D-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct D-lactic acid fermentation from raw starch.

Section: Methodsmentioning

confidence: 99%

Efficient Production of Optically Pure d -Lactic Acid from Raw Corn Starch by Using a Genetically Modified l -Lactate Dehydrogenase Gene-Deficient and α-Amylase-Secreting Lactobacillus plantarum Strain

Okano¹,

Zhang²,

Shinkawa³

et al. 2009

Self Cite

102

“…There might be other, dominant factors that influence transcription efficiency in L. lactis. In the P slpA1 promoter, the 5Ј untranslated leader sequence of the slpA gene contributes to mRNA stabilization by producing a 5Ј stem and loop structure (37). In this work, the PCR primers amplified the P slpA1 promoter region, including the 5Ј untranslated leader sequence, and high-level extracellular production of recombinant LZ-8 was achieved.…”

Section: Vol 74 2008 Extracellular Expression Of Bioactive Recombinmentioning

confidence: 99%

“…Two other, more-efficient L. lactis recombinant LZ-8 expression vectors, pNZLZ and pNZSLZ, were constructed by serial subcloning. The recombinant LZ-8-ter or SP YaB -recombinant LZ-8-ter fragments of pOA-LZ8 and pOAS-LZ8 were obtained by NdeI and XbaI digestion and ligated with pHYSA1-1ATT treated with the same enzymes, resulting in pHYLZ and pHYSLZ, in which the recombinant LZ-8-ter or SP YaB -recombinant LZ-8-ter fragment was expressed from P slpA1 , an efficient promoter in L. lactis (3,37). pHYLZ or pHYSLZ was digested with XbaI and BglII, and the P slpA1 -recombinant LZ-8-ter or P slpA1 -SP YaB -recombinant LZ-8-ter fragments were ligated with pNZter treated with the same enzymes.…”

Section: Methodsmentioning

confidence: 99%

Extracellular Expression of a Functional Recombinant Ganoderma lucidium Immunomodulatory Protein by Bacillus subtilis and Lactococcus lactis

Yeh

Hsu

et al. 2008

Bacillus subtilis and Lactococcus lactis are ideal hosts for the production of extracellular heterologous proteins of major commercial importance. A recombinant gene for the novel Ganoderma lucidium immunomodulatory protein LZ-8, recombinant LZ-8, was designed encoding the same amino acid sequence but using the preferred codons for both strains and was synthesized by overlapping extension PCR. Using the signal peptide (SP) from subtilisin YaB (SP YaB ), recombinant LZ-8 was expressed extracellularly in Bacillus subtilis and Lactococcus lactis. In the absence of SP YaB , recombinant LZ-8 was expressed extracellularly in B. subtilis, but not in L. lactis. The three expressed recombinant LZ-8s showed different capacities for modulating the production of Th1 and Th2 cytokines by peripheral blood mononuclear cells and of tumor necrosis factor alpha by a macrophage cell line.

“…Native regulatory systems naturally inducible in vivo, or constitutive promoters, could ensure constant production of therapeutic peptides at the site of colonization. Several constitutive promoters have previously been used for heterologous expression in lactobacilli, including the P ldhL (20,26), P slpA (7,26,29,31), P 144 (15), and lactococcal P 23 (10) promoters. Recently, several groups have described the use of homologous promoters for highly efficient gene expression (11,16,30).…”

mentioning

confidence: 99%

Transformation of, and Heterologous Protein Expression in, Lactobacillus agilis and Lactobacillus vaginalis Isolates from the Chicken Gastrointestinal Tract

Stephenson

Moore

Allison

2011

Lactobacilli are naturally found in the gastrointestinal tract of chickens, and there is interest in utilizing autochthonous strains for the delivery of therapeutic proteins. Previously we identified three chicken-derived Lactobacillus strains, Lactobacillus agilis La3, Lactobacillus vaginalis Lv5, and Lactobacillus crispatus Lc9, which persist in the gastrointestinal tract of chickens fed either a commercial or high-protein diet. In the current study, we investigated the ability to electrotransform these strains, determined plasmid vector stability, and compared reporter gene expression directed by several different promoters. The La3 and Lv5 strains were reproducibly transformed with efficiencies of 10 8 and 10 6 transformants per microgram of plasmid DNA, respectively. The third strain tested, L. crispatus Lc9, was recalcitrant to all transformation protocols examined. The plasmid vectors pTRK563 and pTRKH2 were maintained over 100 generations in La3 and Lv5, respectively. The ability of La3 and Lv5 to express the heterologous reporter gene gfp was analyzed using heterologous and homologous promoters. Transformants of both La3 and Lv5 containing the La3 ldhL promoter were the most fluorescent. To our knowledge, this is the first report of successful transformation and heterologous protein expression in L. agilis and L. vaginalis. The ability of these strains to express heterologous proteins in vitro indicates their potential utility as in vivo delivery vectors for therapeutic peptides to the chicken gastrointestinal tract.Lactobacilli are autochthonous inhabitants of the chicken gastrointestinal tract (GIT), where they predominate the upper GIT microbiota (53). Their dominance of the upper GIT makes lactobacilli excellent candidates for development as live vectors for the delivery of therapeutic proteins targeting bacterial pathogens such as Clostridium perfringens, Campylobacter jejuni, Listeria monocytogenes, or Salmonella enterica. Additionally, lactobacillus delivery vectors could be used to immunize against avian viruses, such as infectious bursal disease, Marek's disease, Newcastle disease, or avian influenza virus. We have recently identified several Lactobacillus strains, Lactobacillus agilis La3, Lactobacillus vaginalis Lv5, and Lactobacillus crispatus Lc9, which are able to colonize and persist within the GIT of chickens fed either a commercial or a highprotein diet (45). These strains show great potential as vectors for the in situ delivery of therapeutic proteins targeting GIT pathogens.Lactobacilli have several advantages as mucosal delivery vectors (54), including "generally regarded as safe" status, survival within the GIT, stimulation of immune responses in the mucosa, and the potential to be engineered to express therapeutic peptides. In regard to the latter, lactobacilli are notoriously recalcitrant to transformation, representing an important limitation in realizing the vectoring potential of persistent strains.Several studies have investigated the genetic transformation of chicken Lactobac...