scholarly journals Streamlined, recombinase-free genome editing with CRISPR-Cas9 inLactobacillus plantarumreveals barriers to efficient editing

2018 ◽  
Author(s):  
Ryan T. Leenay ◽  
Justin M. Vento ◽  
Malay Shah ◽  
Maria Elena Martino ◽  
François Leulier ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Genome Editing ◽  
Lactobacillus Plantarum ◽  
Point Mutation ◽  
Stop Codon ◽  
Different Strains ◽  
Unexpected Outcomes ◽  
Complete Deletion ◽  
Sequence Modification

ABSTRACTLactic-acid bacteria such asLactobacillus plantarumare commonly used for fermenting foods and as probiotics, where increasingly sophisticated genome-editing tools are currently being employed to elucidate and enhance these microbes’ beneficial properties. The most advanced tools to-date require heterologous single-stranded DNA recombinases to integrate short oligonucleotides followed by using CRISPR-Cas9 to eliminate cells harboring unedited sequences. Here, we show that encoding the recombineering template on a replicating plasmid allowed efficient genome editing with CRISPR-Cas9 in multipleL. plantarumstrains without a recombinase. This strategy accelerated the genome-editing pipeline and could efficiently introduce a stop codon inribB, silent mutations inackA, and a complete deletion oflacM. In contrast, oligo-mediated recombineering with CRISPR-Cas9 proved far less efficient in at least one instance. We also observed unexpected outcomes of our recombinase-free method, including an ~1.3-kb genomic deletion when targetingribBin one strain, and reversion of a point mutation in the recombineering template in another strain. Our method therefore can streamline targeted genome editing in different strains ofL. plantarum, although the best means of achieving efficient editing may vary based on the selected sequence modification, gene, and strain.

scholarly journals Microbiological and Physicochemical Qualities of Moo Som (Traditional Thai Fermented Meat) Inoculated with Lactic Acid Bacteria Starter

2020 ◽  
Vol 17 (8) ◽  
pp. 788-800
Author(s):  
Pussadee TANGWATCHARIN ◽  
Jiraroj NITHISANTAWAKHUP ◽  
Supaluk SORAPUKDEE
Keyword(s):  
Staphylococcus Aureus ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Production Rate ◽  
Lactobacillus Plantarum ◽  
Sensory Evaluation ◽  
Acid Production ◽  
Starter Cultures ◽  
Control Growth ◽  
Different Strains

The effects of different strains of lactic acid bacteria (LAB) inoculation on the fermentation rates and qualities of moo som, a traditional Thai fermented pork, were evaluated. Lactobacillus plantarum KL102 (spontaneous starter) and L. plantarum TISIR543 (commercial starter) were used as starter cultures in the production of moo som. The decreased amounts of Staphylococcus aureus and coliforms in moo som inoculated with L. plantarum KL102 were at faster rates than those in Moo som inoculated with L. plantarum TISIR543. However, the final products of moo som inoculated with both LAB starters did not find S. aureus or and coliform loadings. Inoculation of both LAB starters could control growth of yeast in samples during fermentation. Furthermore, both starter cultures exhibited a higher rate of fermentation than the control (without inoculum), as demonstrated by the faster rate pH drop and acid production (p < 0.05) during fermentation, while the fermentation of all samples were completed within 3 d. Due to higher acid production rate, texture, and especially hardness, gumminess and chewiness of inoculated moo som were higher than control moo som in the final products (p < 0.05). From the results, the inoculation of LAB starter was more beneficial in color and in overall sensory evaluation (p < 0.05). The overall quality was positively correlated with the color, odor, and texture of moo som (p < 0.01). Based on microbiological and physicochemical qualities and sensory evaluation, KL102 is a potential LAB starter for moo som production.

scholarly journals CRISPR/Cas9-Assisted Seamless Genome Editing in Lactobacillus plantarum and Its Application in N-Acetylglucosamine Production

2019 ◽  
Vol 85 (21) ◽  
Author(s):  
Ding Zhou ◽  
Zhennan Jiang ◽  
Qingxiao Pang ◽  
Yuan Zhu ◽  
Qian Wang ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Genome Editing ◽  
Lactobacillus Plantarum ◽  
Strain Engineering ◽  
Single Stranded Dna ◽  
Content Type ◽  
Recombination Efficiency ◽  
Double Stranded Dna ◽  
Exogenous Genes

ABSTRACT Lactobacillus plantarum is a potential starter and health-promoting probiotic bacterium. Effective, precise, and diverse genome editing of Lactobacillus plantarum without introducing exogenous genes or plasmids is of great importance. In this study, CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering was established in L. plantarum WCFS1 to seamlessly edit the genome, including gene knockouts, insertions, and point mutations. To optimize our editing method, phosphorothioate modification was used to improve the dsDNA insertion, and adenine-specific methyltransferase was used to improve the ssDNA recombination efficiency. These strategies were applied to engineer L. plantarum WCFS1 toward producing N-acetylglucosamine (GlcNAc). nagB was truncated to eliminate the reverse reaction of fructose-6-phosphate (F6P) to glucosamine 6-phosphate (GlcN-6P). Riboswitch replacement and point mutation in glmS1 were introduced to relieve feedback repression. The resulting strain produced 797.3 mg/liter GlcNAc without introducing exogenous genes or plasmids. This strategy may contribute to the available methods for precise and diverse genetic engineering in lactic acid bacteria and boost strain engineering for more applications. IMPORTANCE CRISPR/Cas9-assisted recombineering is restricted in lactic acid bacteria because of the lack of available antibiotics and vectors. In this study, a seamless genome editing method was carried out in Lactobacillus plantarum using CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering, and recombination efficiency was effectively improved by endogenous adenine-specific methyltransferase overexpression. L. plantarum WCFS1 produced 797.3 mg/liter N-acetylglucosamine (GlcNAc) through reinforcement of the GlcNAc pathway, without introducing exogenous genes or plasmids. This seamless editing strategy, combined with the potential exogenous GlcNAc-producing pathway, makes this strain an attractive candidate for industrial use in the future.

scholarly journals Genetic and Technological Characterisation of Vineyard- and Winery-Associated Lactic Acid Bacteria

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Aspasia A. Nisiotou ◽  
Dimitra Dourou ◽  
Maria-Evangelia Filippousi ◽  
Ellie Diamantea ◽  
Petros Fragkoulis ◽  
...  
Keyword(s):  
Species Richness ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactobacillus Plantarum ◽  
Strain Distribution ◽  
Oenococcus Oeni ◽  
Low Ph ◽  
Region Of Origin ◽  
Different Strains ◽  
Associated Species

Vineyard- and winery-associated lactic acid bacteria (LAB) from two major PDO regions in Greece, Peza and Nemea, were surveyed. LAB were isolated from grapes, fermenting musts, and winery tanks performing spontaneous malolactic fermentations (MLF). Higher population density and species richness were detected in Nemea than in Peza vineyards and on grapes than in fermenting musts.Pediococcus pentosaceusandLactobacillus graminiswere the most abundant LAB on grapes, whileLactobacillus plantarumdominated in fermenting musts from both regions. No particular structure ofLactobacillus plantarumpopulations according to the region of origin was observed, and strain distribution seems random. LAB species diversity in winery tanks differed significantly from that in vineyard samples, consisting principally ofOenococcus oeni. Different strains were analysed as per their enological characteristics and the ability to produce biogenic amines (BAs). Winery-associated species showed higher resistance to low pH, ethanol, SO2, and CuSO4than vineyard-associated isolates. The frequency of BA-producing strains was relatively low but not negligible, considering that certain winery-associatedLactobacillus hilgardiistrains were able to produce BAs. Present results show the necessity of controlling the MLF by selected starters in order to avoid BA accumulation in wine.

scholarly journals Optimization of the Bioactivation of Isoflavones in Soymilk by Lactic Acid Bacteria

Processes ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 963
Author(s):  
Jon Kepa Izaguirre ◽  
Leire Barañano ◽  
Sonia Castañón ◽  
Itziar Alkorta ◽  
Luis M. Quirós ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactobacillus Plantarum ◽  
Inoculum Size ◽  
Process Time ◽  
Medical Applications ◽  
Bacterial Strains ◽  
Food Industries ◽  
Glucosidase Activity ◽  
Interesting Candidate

Soybeans and soy-based products contain isoflavones which can be used for nutraceutical and medical applications. In soybeans and in unfermented soy foods, isoflavones are normally present as glycosides. Isoflavone glycosides can be enzymatically converted to isoflavone aglycones, thus releasing the sugar molecule. The effective absorption of isoflavones in humans requires the bioconversion of isoflavone glycosides to isoflavone aglycones through the activity of the enzyme β-glucosidase. The objective was to assess the capacity of 42 bacterial strains (belonging to Lactobacillus, Streptococcus and Enterococcus) to produce β-glucosidase activity. The strain that showed the highest β-glucosidase activity (Lactobacillus plantarum 128/2) was then used for the optimization of the bioconversion of genistin and daidzin present in commercial soymilk to their aglycone forms genistein and daidzein. The contribution of process parameters (temperature, inoculum size, time) to the efficiency of such bioactivation was tested. Lactobacillus plantarum 128/2 was able to completely bioactivate soymilk isoflavones under the following conditions: 25 °C temperature, 2% inoculum size and 48 h process time. These results confirm the suitability of lactic acid bacteria for the bioactivation of isoflavones present in soymilk and provide an interesting candidate (L. plantarum 182/2) for food industries to perform this transformation.

scholarly journals Probiotic Potential of a Novel Vitamin B2-Overproducing Lactobacillus plantarum Strain, HY7715, Isolated from Kimchi

2021 ◽  
Vol 11 (13) ◽  
pp. 5765
Author(s):  
Joo-Yun Kim ◽  
Eun-Jung Choi ◽  
Jae-Ho Lee ◽  
Myeong-Seok Yoo ◽  
Keon Heo ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactobacillus Plantarum ◽  
Probiotic Strain ◽  
Optimal Growth ◽  
Control Group ◽  
High Survival ◽  
Vitamin B2 ◽  
Riboflavin Deficiency

Vitamin B2, also known as riboflavin, is essential for maintaining human health. The purpose of this study was to isolate novel lactic acid bacteria that overproduce vitamin B2 and to validate their potential as probiotics. In this study, Lactobacillus plantarum HY7715 (HY7715) was selected among lactic acid bacteria isolated from Kimchi. HY7715 showed a very high riboflavin-producing ability compared to the control strain due to the high expression of ribA, ribB, ribC, ribH, and ribG genes. HY7715 produced 34.5 ± 2.41 mg/L of riboflavin for 24 h without consuming riboflavin in the medium under optimal growth conditions. It was able to produce riboflavin in an in vitro model of the intestinal environment. In addition, when riboflavin deficiency was induced in mice through nutritional restriction, higher levels of riboflavin were detected in plasma and urine in the HY7715 administration group than in the control group. HY7715 showed high survival rate in simulated gastrointestinal conditions and had antibiotic resistance below the cutoff MIC value suggested by the European Food Safety Authority; moreover, it did not cause hemolysis. In conclusion, HY7715 could be considered a beneficial probiotic strain for human and animal applications, suggesting that it could be a new alternative to address riboflavin deficiency.

Enzymatic and antioxidant activity of camel milk fermented with different strains of lactic acid bacteria

2020 ◽  
Vol 27 (2) ◽  
pp. 193-200
Author(s):  
Vinay Kumar Mehra ◽  
Raghvendar Singh ◽  
Devendra Kumar ◽  
Rakesh Poonia
Keyword(s):  
Antioxidant Activity ◽  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Camel Milk ◽  
Different Strains

Influence of Lactobacillus brevis 15 and Lactobacillus plantarum 13 on blood glucose and body weight in rats after high-fructose diet

2015 ◽  
Vol 6 (4) ◽  
pp. 505-512 ◽  
Author(s):  
M. Yakovlieva ◽  
T. Tacheva ◽  
S. Mihaylova ◽  
R. Tropcheva ◽  
K. Trifonova ◽  
...  
Keyword(s):  
Body Weight ◽  
Lactic Acid ◽  
Blood Glucose ◽  
Lactic Acid Bacteria ◽  
Lactobacillus Plantarum ◽  
Lactobacillus Brevis ◽  
Control Group ◽  
Standard Diet ◽  
Glucose Levels ◽  
Blood Glucose Levels

In recent years, many authors have investigated the possible antidiabetic effect of lactic acid bacteria. Lactobacillus species constitute a major part of the lactic acid bacteria group and have been found to exhibit beneficial effects on the development of diabetes and its complications. In the current study, we investigated the effects of newly characterised Bulgarian Lactobacillus strains, Lactobacillus brevis 15 and Lactobacillus plantarum 13, on blood glucose levels and body weight of rats fed a fructose-enriched diet. An experiment was conducted over a period of 8 weeks with 24 2-month-old Wistar rats randomly assigned to receive a standard diet (Con, control group), fructose-enriched diet (Fr group), standard diet with probiotics given twice a week (Pro group), and fructose-enriched diet with probiotics given twice a week (Pro+Fr group). At the end of the experimental period, a statistically significant increase in body weight was observed in all experimental groups (P<0.0001). The highest rise was seen in the fructose group (Fr, 169±19 g), followed by the Pro+Fr group (153±15 g), Pro group (149±13 g), and Con group (141±5 g). Moreover, the final blood glucose levels had risen significantly in the groups receiving fructose either without (Fr; P<0.0001) or with lactobacilli (Pro+Fr; P=0.002), while the rise was insignificant in the group of rats given probiotic supplementation only (Pro, P=0.071) and inexistent in the Con group (P=0.999). The highest elevation of blood glucose levels was observed in the Fr group (3.18 mmol/l), followed by the Pro+Fr group (2.00 mmol/l) whereas the Pro group showed the lowest levels (0.60 mmol/l). The results of our study suggest that the newly characterised Bulgarian Lactobacillus strains, L. brevis 15 and L. plantarum 13, could be considered as possible probiotics and might be able to prevent some metabolic disturbances.

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