scholarly journals A Myo-Inositol-Inducible Expression System for Corynebacterium glutamicum and Its Application

2021 ◽  
Vol 9 ◽  
Author(s):  
Nan Lu ◽  
Chenglin Zhang ◽  
Wenjie Zhang ◽  
Haoran Xu ◽  
Yuhong Li ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Large Scale ◽  
Expression Vector ◽  
Aminolevulinic Acid ◽  
Expression System ◽  
Value Added ◽  
Inducible Promoter ◽  
Inducible Expression ◽  
Expression Vectors ◽  
Chloramphenicol Resistance

Corynebacterium glutamicum is one of the important industrial microorganisms for production of amino acids and other value-added compounds. Most expression vectors used in C. glutamicum are based on inducible promoter (Ptac or Ptrc) activated by isopropyl-β-D-thiogalactopyranoside (IPTG). However, these vectors seem unsuitable for large-scale industrial production due to the high cost and toxicity of IPTG. Myo-inositol is an ideal inducer because of its non-toxicity and lower price. In this study, a myo-inositol-inducible expression vector pMI-4, derived from the expression vector pXMJ19, was constructed. Besides the original chloramphenicol resistance gene cat, multiple cloning sites, and rrnB terminator, the pMI-4 (6,643 bp) contains the iolRq cassette and the myo-inositol-inducible promoter PiolT1. The pMI-4 could stably replicate in the C. glutamicum host. Meanwhile, the non-myo-inositol degradation host strain C. glutamicumΔiolGΔoxiCΔoxiDΔoxiE for maintaining the pMI-4 was developed. Overexpression of hemAM and hemL using pMI-4 resulted in a significant accumulation of 5-aminolevulinic acid, indicating its potential application in metabolic engineering and industrial fermentation.

scholarly journals Construction and Characterization of a Gradually Inducible Expression Vector for Halobacterium salinarum, Based on thekdpPromoter

2012 ◽  
Vol 78 (7) ◽  
pp. 2100-2105 ◽  
Author(s):  
Dorthe Kixmüller ◽  
Jörg-Christian Greie
Keyword(s):  
Gene Expression ◽  
Expression Vector ◽  
Expression System ◽  
Expression Patterns ◽  
Inducible Expression ◽  
Halobacterium Salinarum ◽  
Expression Vectors ◽  
Content Type ◽  
Inducible Expression System

ABSTRACTGradually inducible expression vectors which are governed by variations of growth conditions are powerful tools for gene expression of conditionally lethal mutants. Furthermore, controlled expression allows monitoring of overproduction of proteins at various stages in their expressing hosts. ForHalobacterium salinarum, which is often used as a paradigm for halophilic archaea, such an inducible expression system is not available to date. Here we show that thekdppromoter (Pkdp), which facilitates gene expression upon K+limitation, can be used to establish such a system for molecular applications. Pkdpfeatures a rather high expression rate, with an approximately 50-fold increase that can be easily varied by K+concentrations in the growth medium. Besides the construction of an expression vector, our work describes the characterization of expression patterns and, thus, offers a gradually inducible expression system to the scientific community.

scholarly journals Light-induced production of isobutanol and 3-methyl-1-butanol by metabolically engineered cyanobacteria

2022 ◽  
Vol 21 (1) ◽  
Author(s):  
Shunichi Kobayashi ◽  
Shota Atsumi ◽  
Kazunori Ikebukuro ◽  
Koji Sode ◽  
Ryutaro Asano
Keyword(s):  
Production System ◽  
Large Scale ◽  
Expression System ◽  
Green Light ◽  
Value Added ◽  
Acid Decarboxylase ◽  
Light Illumination ◽  
Alcohol Production ◽  
Chemical Inducers ◽  
Pcc 6803

Abstract Background Cyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully produced in engineered cyanobacteria. Chemical inducer-dependent promoters are used to induce the expression of target biosynthetic pathway genes. A chemical inducer is not ideal for large-scale reactions owing to its high cost; therefore, it is important to develop scaling-up methods to avoid their use. In this study, we designed a green light-inducible alcohol production system using the CcaS/CcaR green light gene expression system in the cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803). Results To establish the green light-inducible production of isobutanol and 3-methyl-1-butanol (3MB) in PCC 6803, keto-acid decarboxylase (kdc) and alcohol dehydrogenase (adh) were expressed under the control of the CcaS/CcaR system. Increases in the transcription level were induced by irradiation with red and green light without severe effects on host cell growth. We found that the production of isobutanol and 3MB from carbon dioxide (CO2) was induced under red and green light illumination and was substantially repressed under red light illumination alone. Finally, production titers of isobutanol and 3MB reached 238 mg L−1 and 75 mg L−1, respectively, in 5 days under red and green light illumination, and these values are comparable to those reported in previous studies using chemical inducers. Conclusion A green light-induced alcohol production system was successfully integrated into cyanobacteria to produce value-added chemicals without using expensive chemical inducers. The green light-regulated production of isobutanol and 3MB from CO2 is eco-friendly and cost-effective. This study demonstrates that light regulation is a potential tool for producing chemicals and increases the feasibility of cyanobacterial bioprocesses. Graphical Abstract

A tetracycline inducible expression vector for Corynebacterium glutamicum allowing tightly regulable gene expression

Plasmid ◽  
2012 ◽  
Vol 68 (2) ◽  
pp. 142-147 ◽  
Author(s):  
Frank Lausberg ◽  
Ava Rebecca Chattopadhyay ◽  
Antonia Heyer ◽  
Lothar Eggeling ◽  
Roland Freudl
Keyword(s):  
Gene Expression ◽  
Corynebacterium Glutamicum ◽  
Expression Vector ◽  
Inducible Expression

Production of Recombinant Melittin by Auto-Induction in Escherichia coli

2013 ◽  
Vol 798-799 ◽  
pp. 1007-1012
Author(s):  
Wen He Zhu ◽  
Wei Zhang ◽  
Yan Li ◽  
Jun Jie Xu ◽  
Shi Jie Lv
Keyword(s):  
Fusion Protein ◽  
Large Scale ◽  
Expression Vector ◽  
Human Cancer ◽  
Expression System ◽  
Scale Production ◽  
Recognition Sequence ◽  
Large Scale Production ◽  
N Terminus ◽  
Gst Fusion Protein

Melittin is a novel peptide of biological activity isolated from bee venom. It has potential application value in medicine and agriculture. Here we encoded melittin gene with the EK recognition sequence in the N-terminus into expression vector pGEX-2T.The expressed fusion protein, which is about 29KDa, identified by Western Blot. To facilitate large-scale production of recombinant GST-fusion protein, we optimized different expression conditions to increase the overall production of the fusion protein. The production of the protein had increased about 10-fold when we used an auto-inducing medium. The GST fusion protein showed an equivalent activity with the natural melittin after digested by EK and can inhibited the proliferations of several human cancer lines. The expression system described in this study provides a feasible way for producing melittin in further studies.

scholarly journals A New Strategy for Production of 5-Aminolevulinic Acid in Recombinant Corynebacterium glutamicum with High Yield

2016 ◽  
Vol 82 (9) ◽  
pp. 2709-2717 ◽  
Author(s):  
Peng Yang ◽  
Wenjing Liu ◽  
Xuelian Cheng ◽  
Jing Wang ◽  
Qian Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Corynebacterium Glutamicum ◽  
Recombinant Strain ◽  
Aminolevulinic Acid ◽  
Value Added ◽  
High Yield ◽  
Two Stage ◽  
Content Type ◽  
Nonprotein Amino Acid ◽  
The Future

ABSTRACT5-Aminolevulinic acid (ALA), a nonprotein amino acid involved in tetrapyrrole synthesis, has been widely applied in agriculture, medicine, and food production. Many engineered metabolic pathways have been constructed; however, the production yields are still low. In this study, several 5-aminolevulinic acid synthases (ALASs) from different sources were evaluated and compared with respect to their ALA production capacities in an engineeredCorynebacterium glutamicumCgS1 strain that can accumulate succinyl-coenzyme A (CoA). A codon-optimized ALAS fromRhodobacter capsulatusSB1003 displayed the best potential. Recombinant strain CgS1/pEC-SB produced 7.6 g/liter ALA using a mineral salt medium in a fed-batch fermentation mode. Employing two-stage fermentation, 12.46 g/liter ALA was produced within 17 h, with a productivity of 0.73 g/liter/h, in recombinantC. glutamicum. Through overexpression of the heterologous nonspecific ALA exporter RhtA fromEscherichia coli, the titer was further increased to 14.7 g/liter. This indicated that strain CgS1/pEC-SB-rhtA holds attractive industrial application potential for the future.IMPORTANCEIn this study, a two-stage fermentation strategy was used for production of the value-added nonprotein amino acid 5-aminolevulinic acid from glucose and glycine in a generally recognized as safe (GRAS) host,Corynebacterium glutamicum. The ALA titer represented the highest in the literature, to our knowledge. This high production capacity, combined with the potential easy downstream processes, made the recombinant strain an attractive candidate for industrial use in the future.

scholarly journals Use of a Tetracycline-Inducible System for Conditional Expression in Mycobacterium tuberculosis and Mycobacterium smegmatis

2005 ◽  
Vol 71 (6) ◽  
pp. 3077-3084 ◽  
Author(s):  
Paul Carroll ◽  
D. G. Niranjala Muttucumaru ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Reporter Gene ◽  
Mycobacterium Smegmatis ◽  
Expression System ◽  
Inducible Promoter ◽  
Inducible Expression ◽  
Essential Genes ◽  
In Vivo Studies ◽  
Conditional Expression

ABSTRACT A number of essential genes have been identified in mycobacteria, but methods to study these genes have not been developed, leaving us unable to determine the function or biology of the genes. We investigated the use of a tetracycline-inducible expression system in Mycobacterium tuberculosis and Mycobacterium smegmatis. Using a reporter gene which encodes an unstable variant of GFP, we showed that tetracycline-inducible expression occurred in M. smegmatis and that expression levels were titratable to some extent by varying the concentration of tetracycline. The removal of tetracycline led to cessation of GFP expression, and we showed that this was a controllable on/off switch for fluorescence upon addition and removal of the antibiotic inducer. The system also functioned in M. tuberculosis, giving inducible expression of the reporter gene. We used homologous recombination to construct a strain of M. tuberculosis that expressed the only copy of the tryptophan biosynthetic enzyme, TrpD, from the tetracycline-inducible promoter. This strain was conditionally auxotrophic, showing auxotrophy only in the absence of tetracycline, confirming that trpD was tightly controlled by the foreign promoter. This is the first demonstration of the use of an inducible promoter to generate a conditional auxotroph of M. tuberculosis. The ability to tightly regulate genes now gives us the possibility to define the functions of essential genes by switching them off under defined conditions and paves the way for in vivo studies.

Transient Production of Recombinant Pharmaceutical Proteins in Plants: Evolution and Perspectives

2019 ◽  
Vol 26 (3) ◽  
pp. 365-380 ◽  
Author(s):  
Lilya Kopertekh ◽  
Joachim Schiemann
Keyword(s):  
Transient Expression ◽  
Large Scale ◽  
Ebola Virus ◽  
Expression System ◽  
Expression Vectors ◽  
Scale Production ◽  
Expression Platform ◽  
Large Scale Production ◽  
Pharmaceutical Proteins ◽  
Current Design

During the last two decades, the production of pharmaceutical proteins in plants evolved from proof of concept to established technology adopted by several biotechnological companies. This progress is particularly based on intensive research starting stable genetic transformation and moving to transient expression. Due to its advantages in yield and speed of protein production transient expression platforms became the leading plant-based manufacturing technology. Current transient expression methods rely on Agrobacteriummediated delivery of expression vectors into plant cells. In recent years, great advances have been made in the improvement of expression vectors, host cell engineering as well as in the development of commercial manufacturing processes. Several GMP-certified large-scale production facilities exist around the world to utilize agroinfiltration method. A number of pharmaceutical proteins produced by transient expression are currently in clinical development. The great potential of transient expression platform in respect to rapid response to emerging pandemics was demonstrated by the production of experimental ZMapp antibodies against Ebola virus as well as influenza vaccines. This review is focused on current design, status and future perspectives of plant transient expression system for the production of biopharmaceutical proteins.

A step forward: Compatible and dual-inducible expression vectors for gene co-expression in Corynebacterium glutamicum

Plasmid ◽  
2019 ◽  
Vol 101 ◽  
pp. 20-27 ◽  
Author(s):  
Rahul Gauttam ◽  
Christian Desiderato ◽  
Lisa Jung ◽  
Adnan Shah ◽  
Bernhard J. Eikmanns
Keyword(s):  
Corynebacterium Glutamicum ◽  
Inducible Expression ◽  
Expression Vectors

scholarly journals Effects of inducible and constitutive expression vectors on heterologous expression of LEH mutants BE3 and BG5

2021 ◽  
Author(s):  
Jie Yong ◽  
Binghuan Liu ◽  
Kunlun Wang ◽  
Hui Yang ◽  
Yun Tian ◽  
...  
Keyword(s):  
High Purity ◽  
Large Scale ◽  
Expression System ◽  
Constitutive Expression ◽  
Catalytic Efficiency ◽  
Catalytic Decomposition ◽  
Expression Vectors ◽  
Substrate Affinity ◽  
E Coli ◽  
One Step

Abstract Background Limonene epoxide hydrolase (LEH) is a class of enzymes in Epoxide hydrolases (EHs) that can form chiral products only by one step catalysis. High purity S or R chiral intermediates play an important role in the application of biomedical industry, and can be used to synthesize various drugs such as ibuprofen, linezoline, cilastatin, etc. Therefore, it has application value to find the expression system which can realize the stable and efficient conversion of LEH to produce high purity S and R chiral products. Results We explored the constitutive expression system of LEH for the first time, and tried to realize the expression of LEH in B.subtilis WB800. Firstly, the LEH mutant on the original inducible vector pBAD-Myc-HisA was obtained and connected to the constitutive vector pHY-p43 containing strong promoter p43. E. coli TOP10 and B.subtilis WB800 were used as the host bacteria to realize the non induced and secretory expression of LEH. Conclusions The results showed that the non induced expression of LEH could be achieved successfully by using the constitutive promoter vector pHY-p43, and the substrate affinity and enzyme catalytic efficiency of the mutants have increased. The catalytic decomposition of the substrate and the formation of chiral products by LEH were determined by GC-MS, which also had stable enzyme activity in the system. This study laid a foundation for large-scale fermentation of LEH and catalytic production of chiral products.

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