A Food-Grade System for Inducible Gene Expression in <i>Lactobacillus plantarum</i> Using an Alanine Racemase-Encoding Selection Marker

Nguyen, Tien‐Thanh; Mathiesen, Geir; Fredriksen, Lasse; Kittl, Roman; Nguyen, Thu‐Ha; Eijsink, Vincent G. H.; Haltrich, Dietmar; Peterbauer, Clemens K.

doi:10.1021/jf104755r

Cited by 66 publications

(75 citation statements)

References 43 publications

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“…Although Oenococcus oeni, the preferred species for MLF, has often been reported to be nonsusceptible to DNA transformation (6), an electroporation protocol for O. oeni has been established recently (2). Further, recent developments showed promising results in the direction of true "food grade expression" by establishing selection markers that are not dependent on antibiotics (30,40).…”

Section: Discussionmentioning

confidence: 99%

Characterization of Two Distinct Glycosyl Hydrolase Family 78 α- l -Rhamnosidases from Pediococcus acidilactici

Michlmayr

Brandes

Eder

et al. 2011

Appl Environ Microbiol

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␣-L-Rhamnosidases play an important role in the hydrolysis of glycosylated aroma compounds (especially terpenes) from wine. Although several authors have demonstrated the enological importance of fungal rhamnosidases, the information on bacterial enzymes in this context is still limited. In order to fill this important gap, two putative rhamnosidase genes (ram and ram2) from Pediococcus acidilactici DSM 20284 were heterologously expressed, and the respective gene products were characterized. In combination with a bacterial ␤-glucosidase, both enzymes released the monoterpenes linalool and cis-linalool oxide from a muscat wine extract under ideal conditions. Additionally, Ram could release significant amounts of geraniol and citronellol/ nerol. Nevertheless, the potential enological value of these enzymes is limited by the strong negative effects of acidity and ethanol on the activities of Ram and Ram2. Therefore, a direct application in winemaking seems unlikely. Although both enzymes are members of the same glycosyl hydrolase family (GH 78), our results clearly suggest the distinct functionalities of Ram and Ram2, probably representing two subclasses within GH 78: Ram could efficiently hydrolyze only the synthetic substrate p-nitrophenyl-␣-L-rhamnopyranoside (V max ‫؍‬ 243 U mg ؊1 ). In contrast, Ram2 displayed considerable specificity toward hesperidin (V max ‫؍‬ 34 U mg ؊1 ) and, especially, rutinose (V max ‫؍‬ 1,200 U mg ؊1 ), a disaccharide composed of glucose and rhamnose. Both enzymes were unable to hydrolyze the flavanone glycoside naringin. Interestingly, both enzymes displayed indications of positive substrate cooperativity. This study presents detailed kinetic data on two novel rhamnosidases, which could be relevant for the further study of bacterial glycosidases.␣-L-Rhamnosidases (EC 3.2.1.40) catalyze the hydrolysis of a wide spectrum of natural glycosides containing terminal Lrhamnose. The biotechnological interest in rhamnosidases includes the debittering of citrus fruit juices (naringinase and hesperidinase), the release of flavonoids from rhamnosylated precursors, and the production of L-rhamnose as starting material for chemical synthesis (52). Further, rhamnosidases may play a crucial role in wine making due to their contributions to releasing grape-derived aroma compounds (mainly terpenes and terpenic alcohols). Depending on the grape variety, rutinosides (␣-L-rhamnopyranosyl-␤-D-glucopyranoside) constitute 6 to 13% of the terpenic glycosides found in wine (29). The combination of ␤-D-glucosidase and ␣-L-rhamnosidase allows the sequential release of volatile compounds from these precursors (18). Several authors (9, 32, 46) have demonstrated the enological value of fungal (Aspergillus sp.) rhamnosidases. Based on their adaptation to the harsh wine milieu and their general hydrolytic abilities toward different glycosides (16,17,50), wine-related lactic acid bacteria (LAB) have attracted much interest as a potential source of novel glycosidases (34).Consequently, several (intracellular) glycosid...

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Section: Discussionmentioning

confidence: 99%

Characterization of Two Distinct Glycosyl Hydrolase Family 78 α- l -Rhamnosidases from Pediococcus acidilactici

Michlmayr

Brandes

Eder

et al. 2011

Appl Environ Microbiol

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“…22 Available inducible expression systems 23 are based on quorum-sensing mechanisms involved in the production of sakacins A and P. 24−26 More recently, the erythromycin antibiotic resistance gene ( erm ) was replaced by the homologous alanine racemase gene ( alr ), and an alanine racemase-deficient host strain was constructed that obviates the undesirable fermentation in the presence of antibiotics and is suitable for applications in the food industry. 19 …”

Section: Introductionmentioning

confidence: 99%

l-Arabinose Isomerase and d-Xylose Isomerase from Lactobacillus reuteri: Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion of d-Galactose and d-Glucose

Staudigl

Haltrich

Peterbauer

2014

J. Agric. Food Chem.

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The l-arabinose isomerase (l-AI) and the d-xylose isomerase (d-XI) encoding genes from Lactobacillus reuteri (DSMZ 17509) were cloned and overexpressed in Escherichia coli BL21 (DE3). The proteins were purified to homogeneity by one-step affinity chromatography and characterized biochemically. l-AI displayed maximum activity at 65 °C and pH 6.0, whereas d-XI showed maximum activity at 65 °C and pH 5.0. Both enzymes require divalent metal ions. The genes were also ligated into the inducible lactobacillal expression vectors pSIP409 and pSIP609, the latter containing a food grade auxotrophy marker instead of an antibiotic resistance marker, and the l-AI- and d-XI-encoding sequences/genes were coexpressed in the food grade host Lactobacillus plantarum. The recombinant enzymes were tested for applications in carbohydrate conversion reactions of industrial relevance. The purified l-AI converted d-galactose to d-tagatose with a maximum conversion rate of 35%, and the d-XI isomerized d-glucose to d-fructose with a maximum conversion rate of 48% at 60 °C.

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“…That enological LAB, such as O. oeni, can be genetically manipulated is of biotechnological interest, although at present this approach raises societal dilemmas. Moreover, our study implements a self-cloning strategy using a food-grade expression system that could be used instead of adding bacterial enzymes, a technique that consumer preferences preclude in the food industry at present (Maischberger et al, 2010;Nguyen et al, 2011;Peterbauer et al, 2011;Sørvig et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

Genetically engineered Oenococcus oeni strains to highlight the impact of estA2 and estA7 esterase genes on wine ester profile

Darsonval¹,

Alexandre²,

Grandvalet³

2016

Food Microbiology

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A Food-Grade System for Inducible Gene Expression in Lactobacillus plantarum Using an Alanine Racemase-Encoding Selection Marker

Cited by 66 publications

References 43 publications

Characterization of Two Distinct Glycosyl Hydrolase Family 78 α- l -Rhamnosidases from Pediococcus acidilactici

Characterization of Two Distinct Glycosyl Hydrolase Family 78 α- l -Rhamnosidases from Pediococcus acidilactici

l-Arabinose Isomerase and d-Xylose Isomerase from Lactobacillus reuteri: Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion of d-Galactose and d-Glucose

Genetically engineered Oenococcus oeni strains to highlight the impact of estA2 and estA7 esterase genes on wine ester profile

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