Increasing l-lysine production in Corynebacterium glutamicum by engineering amino acid transporters

AbstractL-Lysine is one of the important amino acid required for humans and animals. It has a high commercial market. Large scale production of this amino acid is essential to meet the commercial demands. Typically, L-lysine is produced by batch fermentation. In the present study, the important process, as well as nutrient parameters such as glucose concentration (g/L), rpm, incubation temperature (°C), pH and incubation time for L-lysine production by Corynebacterium glutamicum ATCC13032, were optimized by a combined approach of response surface methodology (RSM) with artificial neural network (ANN) method. Initially, 32 runs face central composite design was employed. In the first step, the data was analyzed by the RSM and the optimum conditions for L-lysine production were determined. In the second step, the same data was used to train the neural network. A feed-forward neural network with error backpropagation was used. The best network was obtained by optimizing the no of neurons in the hidden layer. From the best network, the optimized weights and predicted responses were used to optimize the conditions of the selected parameters by genetic algorithm (GA). Overall with the combination of RSM-ANN-GA onefold of L-lysine production from Corynebacterium glutamicum ATCC 13032 was improved.

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Phosphotransferase System-Mediated Glucose Uptake Is Repressed in Phosphoglucoisomerase-Deficient Corynebacterium glutamicum Strains

Applied and Environmental Microbiology ◽

10.1128/aem.03231-12 ◽

2013 ◽

Vol 79 (8) ◽

pp. 2588-2595 ◽

Cited By ~ 27

Author(s):

Steffen N. Lindner ◽

Dimitar P. Petrov ◽

Christian T. Hagmann ◽

Alexander Henrich ◽

Reinhard Krämer ◽

...

Keyword(s):

Amino Acid ◽

Glucose Uptake ◽

Corynebacterium Glutamicum ◽

Pentose Phosphate ◽

Phosphotransferase System ◽

Negative Effects ◽

Gene Encoding ◽

Lysine Production ◽

Content Type ◽

Model Strain

ABSTRACTCorynebacterium glutamicumis particularly known for its industrial application in the production of amino acids. Amino acid overproduction comes along with a high NADPH demand, which is covered mainly by the oxidative part of the pentose phosphate pathway (PPP). In previous studies, the complete redirection of the carbon flux toward the PPP by chromosomal inactivation of thepgigene, encoding the phosphoglucoisomerase, has been applied for the improvement ofC. glutamicumamino acid production strains, but this was accompanied by severe negative effects on the growth characteristics. To investigate these effects in a genetically defined background, we deleted thepgigene in the type strainC. glutamicumATCC 13032. The resulting strain,C. glutamicumΔpgi, lacked detectable phosphoglucoisomerase activity and grew poorly with glucose as the sole substrate. Apart from the already reported inhibition of the PPP by NADPH accumulation, we detected a drastic reduction of the phosphotransferase system (PTS)-mediated glucose uptake inC. glutamicumΔpgi. Furthermore, Northern blot analyses revealed that expression ofptsG, which encodes the glucose-specific EII permease of the PTS, was abolished in this mutant. Applying our findings, we optimizedl-lysine production in the model strainC. glutamicumDM1729 by deletion ofpgiand overexpression of plasmid-encodedptsG.l-Lysine yields and productivity withC. glutamicumΔpgi(pBB1-ptsG) were significantly higher than those withC. glutamicumΔpgi(pBB1). These results show thatptsGoverexpression is required to overcome the repressed activity of PTS-mediated glucose uptake inpgi-deficientC. glutamicumstrains, thus enabling efficient as well as fastl-lysine production.

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Expression of the NADP+-Dependent Formate Dehydrogenase Gene from Pseudomonas Increases the Lysine Production in Corynebacterium glutamicum

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-6-21-29 ◽

2019 ◽

Vol 35 (6) ◽

pp. 21-29

Author(s):

T.E. Leonova ◽

T.E. Shustikova ◽

T.V. Gerasimova ◽

Т.А. Ivankova ◽

K.V. Sidorenko Sidorenko ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Formate Dehydrogenase ◽

Ministry Of Education ◽

Lysine Production ◽

Simultaneous Formation ◽

Resource Center ◽

Formate Oxidation ◽

Gene Coding ◽

Dehydrogenase Gene ◽

Lysine Synthesis

Thepsefdh_D221Q gene coding for a mutant formate dehydrogenase (PseFDG_D221Q) from Pseudomonas, which catalyzes the formate oxidation with the simultaneous formation of NADPH, has been expressed in the cells of lysine-producing Corynebacterium glutamicum strains. The psefdh_D221Q gene was introduced into С. glutamicum strains as part of an autonomous plasmid or was integrated into the chromosome with simultaneous inactivation of host formate dehydrogenase genes. It was shown that the С. glutamicum strains with NADP+ -dependent formate dehydrogenase have an increased level of L-lysine synthesis in the presence of formate, if their own formate dehydrogenase is inactivated. L-lysine, formate dehydrogenase, NADPH, Corynebacterium glutamicum The work was carried out using the equipment of the Multipurpose Scientific This work was carried out on the equipment of the Multipurpose Scientific Installation of «All-Russian Collection of Industrial Microorganisms», National Bio-Resource Center, NRC «Kurchatov Institute»- GosNIIgenetika. This work was financially supported by the Ministry of Education and Science of Russia (Unique Project Identifier - RFMEFI61017X0011).

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Amino Acid Transporters on the Guard of Cell Genome and Epigenome

Cancers ◽

10.3390/cancers13010125 ◽

2021 ◽

Vol 13 (1) ◽

pp. 125

Author(s):

Uğur Kahya ◽

Ayşe Sedef Köseer ◽

Anna Dubrovska

Keyword(s):

Amino Acid ◽

Cancer Metabolism ◽

Tumor Imaging ◽

Redox Homeostasis ◽

Amino Acid Uptake ◽

Metabolic Reprogramming ◽

Tumor Evolution ◽

Amino Acid Transporters ◽

Acid Uptake ◽

Growth And Survival

Tumorigenesis is driven by metabolic reprogramming. Oncogenic mutations and epigenetic alterations that cause metabolic rewiring may also upregulate the reactive oxygen species (ROS). Precise regulation of the intracellular ROS levels is critical for tumor cell growth and survival. High ROS production leads to the damage of vital macromolecules, such as DNA, proteins, and lipids, causing genomic instability and further tumor evolution. One of the hallmarks of cancer metabolism is deregulated amino acid uptake. In fast-growing tumors, amino acids are not only the source of energy and building intermediates but also critical regulators of redox homeostasis. Amino acid uptake regulates the intracellular glutathione (GSH) levels, endoplasmic reticulum stress, unfolded protein response signaling, mTOR-mediated antioxidant defense, and epigenetic adaptations of tumor cells to oxidative stress. This review summarizes the role of amino acid transporters as the defender of tumor antioxidant system and genome integrity and discusses them as promising therapeutic targets and tumor imaging tools.

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Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein

Methods and Protocols ◽

10.3390/mps4030051 ◽

2021 ◽

Vol 4 (3) ◽

pp. 51

Author(s):

Satish Kantipudi ◽

Daniel Harder ◽

Sara Bonetti ◽

Dimitrios Fotiadis ◽

Jean-Marc Jeckelmann

Keyword(s):

Amino Acid ◽

Pichia Pastoris ◽

Protein Complexes ◽

Amino Acid Transporter ◽

Amino Acid Transporters ◽

Expression Host ◽

Amino Acids And Derivatives ◽

Heterodimeric Complex ◽

Acid Transporter ◽

And Function

Heterodimeric amino acid transporters (HATs) are protein complexes composed of two subunits, a heavy and a light subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. HATs transport amino acids and derivatives thereof across the plasma membrane. The human HAT 4F2hc-LAT1 is composed of the type-II membrane N-glycoprotein 4F2hc (SLC3A2) and the L-type amino acid transporter LAT1 (SLC7A5). 4F2hc-LAT1 is medically relevant, and its dysfunction and overexpression are associated with autism and tumor progression. Here, we provide a general applicable protocol on how to screen for the best membrane transport protein-expressing clone in terms of protein amount and function using Pichia pastoris as expression host. Furthermore, we describe an overexpression and purification procedure for the production of the HAT 4F2hc-LAT1. The isolated heterodimeric complex is pure, correctly assembled, stable, binds the substrate L-leucine, and is thus properly folded. Therefore, this Pichia pastoris-derived recombinant human 4F2hc-LAT1 sample can be used for downstream biochemical and biophysical characterizations.

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Characterization of the third member of the MCAT family of cationic amino acid transporters. Identification of a domain that determines the transport properties of the MCAT proteins.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)36854-1 ◽

1993 ◽

Vol 268 (28) ◽

pp. 20796-20800

Author(s):

E.I. Closs ◽

C.R. Lyons ◽

C Kelly ◽

J.M. Cunningham

Keyword(s):

Amino Acid ◽

Transport Properties ◽

Amino Acid Transporters ◽

Cationic Amino Acid ◽

The Third

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LAT1 and ASCT2 Related microRNAs as Potential New Therapeutic Agents against Colorectal Cancer Progression

Biomedicines ◽

10.3390/biomedicines9020195 ◽

2021 ◽

Vol 9 (2) ◽

pp. 195

Author(s):

Francisca Dias ◽

Cristina Almeida ◽

Ana Luísa Teixeira ◽

Mariana Morais ◽

Rui Medeiros

Keyword(s):

Colorectal Cancer ◽

Amino Acid ◽

Cancer Progression ◽

Cell Metabolism ◽

Tumor Development ◽

Amino Acid Transporters ◽

Epigenetic Alterations ◽

Therapeutic Approaches ◽

Colorectal Cancer Progression ◽

Made In

The development and progression of colorectal cancer (CRC) have been associated with genetic and epigenetic alterations and more recently with changes in cell metabolism. Amino acid transporters are key players in tumor development, and it is described that tumor cells upregulate some AA transporters in order to support the increased amino acid (AA) intake to sustain the tumor additional needs for tumor growth and proliferation through the activation of several signaling pathways. LAT1 and ASCT2 are two AA transporters involved in the regulation of the mTOR pathway that has been reported as upregulated in CRC. Some attempts have been made in order to develop therapeutic approaches to target these AA transporters, however none have reached the clinical setting so far. MiRNA-based therapies have been gaining increasing attention from pharmaceutical companies and now several miRNA-based drugs are currently in clinical trials with promising results. In this review we combine a bioinformatic approach with a literature review in order to identify a miRNA profile with the potential to target both LAT1 and ASCT2 with potential to be used as a therapeutic approach against CRC.

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