Improved Microbial Production of Lysine by Developing a New Auxotrophic Mutant of Corneybacterium glutamicum (original) (raw)
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PAKISTAN JOURNAL OF …, 2007
Homoserine auxotrophic mutants were developed from a local strain of Corynebacterium glutamicum (PCSIR-BI4) By using N-methyl-N-nitro-N nitrosoguanidine (NTG) as a mutagen. The developed mutants were screened for the production of lysine in cane molasses based fermentation medium. The lysine over producing mutant was designated as CGH-15. Different initial sugar concentrations of cane molasses were employed to obtain better lysine production in fermentation medium by the selected homoserine auxotroph. The optimal sugar level was found to be 60 gl -1 after 120 hours of incubation at 30°C, which enhanced lysine production from 4.00 to 25.05 g -1 . All the kinetic parameters including yield coefficients and volumetric rates revealed the overproduction of lysine by the mutant using black strap molasses as a sole source of carbon.
Recent Advances in engineering Corynebacterium glutamicum for production of Lysine
Research Journal of Biotechnology, 2022
Lysine is an essential amino acid utilised in diverse industrial areas, mostly as animal feed and food. It is synthesized by various organisms but the novel Corynebacterium glutamicum (gram positive, non sporulating bacteria) is selected anticipating to the enormous research. This research has paved a way towards enhancing the lysine productivity. Different strains of Corynebacterium glutamicum utilised many technologies for the enhanced production of L-Lysine. This literature study examines the current status of Llysine industrial production with a focus on technology used for development irrespective of the strain and fermentation technologies through different approach of understanding of provisions (NADPH, Media source, PTS, exploit of RAM, Vector systems and promoters) with use of different methodologies. The technologies were divided into metabolic engineering, conventional gene mutations, optimization of fermentation parameters (RSM, ANN, GA), gene overexpression, gene switching and exploit of CRISPR, electric current. This review presents the methodologies which are superior at manufacturing enhanced amounts of lysine and report the techniques which assist in producing the best yield in collation with the earlier. This also focuses on the genes related to lysine export and provides conviction of utilizing lysine as an animal feed.
L-lysine (C6H14N2O2), one of the essential and commercially important amino acids, is found in naturally-occurring proteins of all living organisms. One of the most commonly used approaches for improving the production of L-Lysine in Corynebacterium glutamicum was classical mutagenesis which involves the repeated mutation and selection of the desired mutant. Single and / or combined cloning and expression of the genes or disruption of certain genes in Corynebacterium glutamicum enabled the analysis of carbon flux control in response to elevation or removal of the respective enzyme activity. Based on these analyses, new strategies for the manipulation of this industrially important amino acid producer become possible. A quantitative description of how a pathway flux is controlled by individual pathway reactions and how this control changes in response to environmental and genetic changes will provide a rational basis for genetic manipulation. The key aspect of this approach is to enable a production strain to make full use of its intrinsic ability through eliminating all undesirable mutations accumulated in its genome. This review focuses on the approaches in the last 30 years in the field of industrial production of L-Lysine in Corynebacterium glutamicum from conventional methods like classical mutagenesis, metabolic flux analysis to the recent advancements like DNA microarray, genome based strain breeding and genome sequencing and functional genomics.
3 Biotech, 2015
Establishing a cost and time efficient approach for bioprocess optimization is desired but is challenging. In the present work, we have addressed the effectiveness of using immobilized cells for aerobic processes, behaviour of immobilized cells, optimization and upstream bioprocess analysis for the production of lysine by immobilized cells of Corynebacterium glutamicum ATCC 13032 and MH 20-22 B in stirred tank bioreactor. Optimized operational conditions for maximal yield and productivity were determined with six parameters i.e., pH, temperature, fermentation time, airflow rate, glucose concentration and aeration rate. With the obtained results, it was evident that the optimum values for the upstream parameters viz., fermentation time, pH, temperature, glucose concentration, air flow rate and agitation rate are 96 h, 7.5, 30°C, 90 g/l, 1.0 vvm and 200 rpm for both immobilized cells of C. glutamicum ATCC 13032 and MH 20-22 B. Immobilized cells of C. glutamicum MH 20-22 B, which is a leucine auxotroph has yielded more L-lysine compare to the immobilized cells of wild type strain C. glutamicum ATCC 13032.
Folia Microbiologica, 1998
Activity and regulation of key enzymes of the lysine biosynthetic pathway were investigated in Brevibacterium linens, a natural excretor of lysine, its lysine-overproducing homoserine auxotroph (Horn-1) and its auxotrophic and multianalogue-resistant high-yielding mutant (AEC NV 20r50). The activity of aspartate kinase (AK) and aspartaldehydate dehydrogenase (AD) was maximum during the mid-exponential phase of growth and decreased thereafter. The mutants showed 10 and 20 % more activity of AK and AD than the wild-type lysine excretor. B. linens (natural excretor) has a single AK and AD repressed and inhibited bivalently by lysine and threonine. Lysine slightly repressed and inhibited dihydrodipicolinate synthase (DS) and diaminopimelate decarboxylase (DD) of the wild type and of the mutant Horn -I. The mutant AEC NV 20r50 showed DS and DD to be insensitive to lysine inhibition and repression. Persistence of a major part of the maximal activity of these enzymes during the late stationary phase of growth allowed prolonged synthesis and excretion of lysine. Stepwise addition of resistance to the different analogues of lysine in the mutant AEC Nv20r50 resulted in an increase of enzyme activity and reduced repressibilities of enzymes that contributed to the high yield of lysine.
Metabolic Engineering of Lysine Producing Corynebacterium glutamicum Strains
Cytology and Genetics, 2020
The review is devoted to the analysis of the current achievements of Corynebacterium glutamicum metabolic engineering for the production of lysine. Key genes of lysine biosynthesis in C. glutamicum and ways of creating new genetically modified strains are considered. The role of different plasmids, vector cassettes, and promoter types for the regulation of gene expression in C. glutamicum is described. Information is provided on the use of carbon-containing substrates (hexose, pentose, lactic acid, mannitol) for the production of lysine. Possibilities of using CRISPR technology in genetic engineering of C. glutamicum are considered. Genetic changes in C. glutamicum allowed the use of alternative substrates and contributed to the increase of lysine accumulation in the culture fluid. The data that may be used for the creation of new lysine overproduction strains are summarized.
Engineering Corynebacterium glutamicum for fast production of l-lysine and l-pipecolic acid
Applied Microbiology and Biotechnology, 2016
The Gram-positive Corynebacterium glutamicum is widely used for fermentative production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose uptake and phosphorylation by C. glutamicum mainly occur by the phosphotransferase system (PTS) and to lesser extent by inositol permeases and glucokinases. Heterologous expression of the genes for the highaffinity glucose permease from Streptomyces coelicolor and Bacillus subtilis glucokinase fully compensated for the absence of the PTS in Δhpr strains. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and glucokinase from B. subtilis were overproduced with balanced translation initiation rates using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid or with pVWEx1-IolTBest, 18 to 20 % higher volumetric productivities and 70 to 72 % higher specific productivities as compared to the parental strain resulted. The non-proteinogenic amino acid L-pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics, or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the constructed L-lysine-producing strain, the L-lysine 6-dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5carboxylate reductase gene proC were overexpressed as synthetic operon. This enabled C. glutamicum to produce L-PA with a yield of 0.09 ± 0.01 g g −1 and a volumetric productivity of 0.04 ± 0.01 g L −1 h −1 .To the best of our knowledge, this is the first fermentative process for the production of L-PA from glucose.