scholarly journals Construction of a Vitreoscilla Hemoglobin Promoter-Based Tunable Expression System for Corynebacterium glutamicum

Catalysts ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 561 ◽  
Author(s):  
Kei-Anne Baritugo ◽  
Hee Taek Kim ◽  
Mi Na Rhie ◽  
Seo Young Jo ◽  
Tae Uk Khang ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Fluorescent Protein ◽  
Expression System ◽  
Vitreoscilla Hemoglobin ◽  
Aminobutyric Acid ◽  
Vector System ◽  
Efficient Production ◽  
Gamma Aminobutyric Acid ◽  
Recombinant Strains ◽  
Gaba Production

Corynebacterium glutamicum is an industrial strain used for the production of valuable chemicals such as L-lysine and L-glutamate. Although C. glutamicum has various industrial applications, a limited number of tunable systems are available to engineer it for efficient production of platform chemicals. Therefore, in this study, we developed a novel tunable promoter system based on repeats of the Vitreoscilla hemoglobin promoter (Pvgb). Tunable expression of green fluorescent protein (GFP) was investigated under one, four, and eight repeats of Pvgb (Pvgb, Pvgb4, and Pvgb8). The intensity of fluorescence in recombinant C. glutamicum strains increased as the number of Pvgb increased from single to eight (Pvgb8) repeats. Furthermore, we demonstrated the application of the new Pvgb promoter-based vector system as a platform for metabolic engineering of C. glutamicum by investigating 5-aminovaleric acid (5-AVA) and gamma-aminobutyric acid (GABA) production in several C. glutamicum strains. The profile of 5-AVA and GABA production by the recombinant strains were evaluated to investigate the tunable expression of key enzymes such as DavBA and GadBmut. We observed that 5-AVA and GABA production by the recombinant strains increased as the number of Pvgb used for the expression of key proteins increased. The recombinant C. glutamicum strain expressing DavBA could produce higher amounts of 5-AVA under the control of Pvgb8 (3.69 ± 0.07 g/L) than the one under the control of Pvgb (3.43 ± 0.10 g/L). The average gamma-aminobutyric acid production also increased in all the tested strains as the number of Pvgb used for GadBmut expression increased from single (4.81–5.31 g/L) to eight repeats (4.94–5.58 g/L).

scholarly journals A Newly Designed EGFP-2A Peptide Monocistronic Baculoviral Vector for Concatenating the Expression of Recombinant Proteins in Insect Cells

Processes ◽  
2019 ◽  
Vol 7 (5) ◽  
pp. 291 ◽  
Author(s):  
Chih-Yu Wu ◽  
Chao-Wei Huang ◽  
Yu-Shin Nai ◽  
Pei-Yu Chu ◽  
Chung-Hsiung Wang ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Fluorescent Protein ◽  
Expression System ◽  
Recombinant Baculovirus ◽  
New Combination ◽  
Vector System ◽  
Baculovirus Expression Vector

Recombinant proteins produced by the baculovirus expression vector system (BVES) have been widely applied in the agricultural and medical fields. However, the procedure for protein expression is inefficient and needs to be improved. Herein, we propose a simple construct that incorporates a selectable marker (enhanced green fluorescent protein, EGFP) and a picorna viral-derived “self-cleaving” 2A-like peptide to separate the EGFP and target proteins in a monocistronic baculovirus vector to facilitate isolation of the recombinant baculovirus in the BVES. In this study, porcine adiponectin (ADN), a secreted, multimeric protein with insulin-sensitizing properties, was used to demonstrate its utility in our EGFP-2A-based expression system. EGFP and ADN were simultaneously expressed by a recombinant alphabaculovirus. Co-expression of EGFP facilitates the manipulation of the following processes, such as determining expression kinetics and harvesting ADN. The results showed that the 2A “self-cleaving” process does not interfere with EGFP activity or with signal peptide removal and the secretion of recombinant ADN. Posttranslational modifications, including glycosylation, of the recombinant ADN occurred in insect cells, and the formation of various multimers was further verified. Most importantly, the insect-produced ADN showed a similar bioactivity to that of mammalian cells. This concept provides a practical and economic approach that utilizes a new combination of alphabaculovirus/insect cell expression systems for future applications.

scholarly journals Development of a Dual-Vector System Utilizing MicroRNA Mimics of the Autographa californica miR-1 for an Inducible Knockdown in Insect Cells

2019 ◽  
Vol 20 (3) ◽  
pp. 533
Author(s):  
Krisztina Koczka ◽  
Wolfgang Ernst ◽  
Dieter Palmberger ◽  
Miriam Klausberger ◽  
Lisa Nika ◽  
...  
Keyword(s):  
Fluorescence Intensity ◽  
Insect Cell ◽  
Fluorescent Protein ◽  
Expression System ◽  
Yellow Fluorescent Protein ◽  
Mrna Level ◽  
Vector System ◽  
Sf9 Cells ◽  
Artificial Microrna ◽  
T7 Promoter

The baculovirus-insect cell expression system is a popular tool for the manufacturing of various attractive recombinant products. Over the years, several attempts have been made to engineer and further improve this production platform by targeting host or baculoviral genes by RNA interference. In this study, an inducible knockdown system was established in insect (Sf9) cells by combining an artificial microRNA precursor mimic of baculoviral origin and the bacteriophage T7 transcription machinery. Four structurally different artificial precursor constructs were created and tested in a screening assay. The most efficient artificial microRNA construct resulted in a 69% reduction in the fluorescence intensity of the target enhanced yellow fluorescent protein (eYFP). Next, recombinant baculoviruses were created carrying either the selected artificial precursor mimic under the transcriptional control of the T7 promoter or solely the T7 RNA polymerase under a baculoviral promoter. Upon co-infecting Sf9 cells with these two viruses, the fluorescence intensity of eYFP was suppressed by ~30–40% on the protein level. The reduction in the target mRNA level was demonstrated with real-time quantitative PCR. The presented inducible knockdown system may serve as an important and valuable tool for basic baculovirus-insect cell research and for the improvement of production processes using this platform.

scholarly journals Gamma-aminobutyric acid-producing lactobacilli positively affect metabolism and depressive-like behaviour in a mouse model of metabolic syndrome

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
E. Patterson ◽  
P. M. Ryan ◽  
N. Wiley ◽  
I. Carafa ◽  
E. Sherwin ◽  
...  
Keyword(s):  
Metabolic Syndrome ◽  
Mouse Model ◽  
Plasma Cholesterol ◽  
Forced Swim Test ◽  
Lactobacillus Brevis ◽  
Aminobutyric Acid ◽  
Metabolic Dysfunction ◽  
Gamma Aminobutyric Acid ◽  
Gaba Production ◽  
Glucose Challenge

Abstract Metabolic and neuroactive metabolite production represents one of the mechanisms through which the gut microbiota can impact health. One such metabolite, gamma-aminobutyric acid (GABA), can modulate glucose homeostasis and alter behavioural patterns in the host. We previously demonstrated that oral administration of GABA-producing Lactobacillus brevis DPC6108 has the potential to increase levels of circulating insulin in healthy rats. Therefore, the objective of this study was to assess the efficacy of endogenous microbial GABA production in improving metabolic and behavioural outcomes in a mouse model of metabolic dysfunction. Diet-induced obese and metabolically dysfunctional mice received one of two GABA-producing strains, L. brevis DPC6108 or L. brevis DSM32386, daily for 12 weeks. After 8 and 10 weeks of intervention, the behavioural and metabolic profiles of the mice were respectively assessed. Intervention with both L. brevis strains attenuated several abnormalities associated with metabolic dysfunction, causing a reduction in the accumulation of mesenteric adipose tissue, increased insulin secretion following glucose challenge, improved plasma cholesterol clearance and reduced despair-like behaviour and basal corticosterone production during the forced swim test. Taken together, this exploratory dataset indicates that intervention with GABA-producing lactobacilli has the potential to improve metabolic and depressive- like behavioural abnormalities associated with metabolic syndrome in mice.

A new metabolic route for the production of gamma-aminobutyric acid by Corynebacterium glutamicum from glucose

Amino Acids ◽  
2016 ◽  
Vol 48 (11) ◽  
pp. 2519-2531 ◽  
Author(s):  
João M. P. Jorge ◽  
Christian Leggewie ◽  
Volker F. Wendisch
Keyword(s):  
Corynebacterium Glutamicum ◽  
Aminobutyric Acid ◽  
Gamma Aminobutyric Acid ◽  
Metabolic Route

scholarly journals Optimisation of culture conditions for gamma-aminobutyric acid production in rice bran extracts

2021 ◽  
Vol 63 (1) ◽  
pp. 42-48
Author(s):  
Dai Hung Ngo ◽  
◽  
Quoc Tuan Tran ◽  
Thi Nhat Hang Nguyen ◽  
Dai Nghiep Ngo ◽  
...  
Keyword(s):  
Rice Bran ◽  
Monosodium Glutamate ◽  
Carbon Sources ◽  
Nitrogen Sources ◽  
Culture Conditions ◽  
High Potential ◽  
Aminobutyric Acid ◽  
Process Conditions ◽  
Gamma Aminobutyric Acid ◽  
Gaba Production

Gamma-aminobutyric acid (GABA) is a potent bioactive component that widely exists in both plants and animals, has numerous health benefits. This study aimed to optimise the fermentation process conditions for the growth of Lactobacillus fermentum from rice bran extracts that have high potential to produce GABA. GABA content was assessed by thin-layer chromatography (TLC) method. In this study, fermenting conditions for medium production of GABA by L. fermentum from rice bran extracts were optimised. L. fermentum showed high potential for GABA-producing ability. Some factors influencing the GABA production such as carbon sources, nitrogen sources, mineral salt sources, substrate concentration of monosodium glutamate (MSG), pH, and the time of fermentation were investigated. When the L. fermentum is cultivated in the rice bran extracts medium supplemented with 1.5% lactose, 2% yeast extract, and 1% MSG with pH 6.0 in 48 h, this strain showed high GABA at a concentration of 736 mg/l.

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