L-lysine Production Research Articles

The pyruvate dehydrogenase complex of Corynebacterium glutamicum: An attractive target for metabolic engineering

The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative thiamine pyrophosphate-dependent decarboxylation of pyruvate to acetyl-CoA and CO2. Since pyruvate is a key metabolite of the central metabolism and also the precursor for several relevant biotechnological products, metabolic engineering of this multienzyme complex is a promising strategy to improve microbial production processes. This review summarizes the current knowledge and achievements on metabolic engineering approaches to tailor the PDHC of Corynebacterium glutamicum for the bio-based production of l-valine, 2-ketosiovalerate, pyruvate, succinate and isobutanol and to improve l-lysine production.

HETEROLOGOUS EXPRESSION OF Escherichia coli FRUCTOSE-1,6-BISPHOSPHATASE IN Corynebacterium glutamicum AND EVALUATING THE EFFECT ON CELL GROWTH AND L-LYSINE PRODUCTION

Fructose-1,6-bisphosphatase (FBPase), which is mainly used to supply NADPH, has an important role in increasing L-lysine production by Corynebacterium glutamicum. However, C. glutamicum FBPase is negatively regulated at the metabolic level. Strains that overexpressed Escherichia coli fructose-1,6-bisphosphatase in C. glutamicum were constructed, and the effects of heterologous FBPase on cell growth and L-lysine production during growth on glucose, fructose, and sucrose were evaluated. The heterologous fructose-1,6-bisphosphatase is insensitive to fructose 1-phosphate and fructose 2,6-bisphosphate, whereas the homologous fructose-1,6-bisphosphatase is inhibited by fructose 1-phosphate and fructose 2,6-bisphosphate. The relative enzyme activity of heterologous fructose-1,6-bisphosphatase is 90.8% and 89.1% during supplement with 3 mM fructose 1-phosphate and fructose 2,6-bisphosphate, respectively. Phosphoenolpyruvate is an activator of heterologous fructose-1,6-bisphosphatase, whereas the homologous fructose-1,6-bisphosphatase is very sensitive to phosphoenolpyruvate. Overexpression of the heterologous fbp in wild-type C. glutamicum has no effect on L-lysine production, but fructose-1,6-bisphosphatase activities are increased 9- to 13-fold. Overexpression of the heterologous fructose-1,6-bisphosphatase increases L-lysine production in C. glutamicum lysC T311I by 57.3% on fructose, 48.7% on sucrose, and 43% on glucose. The dry cell weight (DCW) and maximal specific growth rate (μ) are increased by overexpression of heterologous fbp. A “funnel-cask” diagram is first proposed to explain the synergy between precursors supply and NADPH supply. These results lay a definite theoretical foundation for breeding high L-lysine producers via molecular target.

Thick Juice-Based Production of Amino Acihttp://www.omicsonline.org/open-access/thick-juice-based-production-of-amino-acids-2155-952X-4-167.php?aid=34446ds and Putrescine by Corynebacterium glutamicum

Thick juice (also regarded as syrup) is an intermediate product of sugar processing. It is cheaper than processed sugar and is mainly composed of sucrose. Sucrose is a preferred carbon source of Corynebacterium glutamicum, a workhorse of biotechnology used for million-ton-scale amino acid production. Here, it is shown for C. glutamicum that sugar beet thick juice led to higher growth rates and faster carbon source consumption than pure sucrose. Comparative DNA microarray analysis revealed differential expression of genes for butyrate and citrate catabolism and of NAD and biotin biosynthesis suggesting provision of these compounds by the thick juice. Thick juice was also shown to be superior to sucrose in production of the amino acids L-lysine, L-glutamate and L-arginine as well as of the diamine putrescine since higher volumetric productivities than with pure sucrose could be achieved. Taken together, sugar beet thick juice was shown to be a carbon source for growth and amino acid and diamine production of C. glutamicum superior to pure sucrose.

Improvement of L-lysine production combines with minimization of by-products synthesis inCorynebacterium glutamicum

BACKGROUND Corynebacterium glutamicum was engineered for improvement of L-lysine production and minimization of by-products synthesis by genetically engineering. RESULTS The most promising recombinant strain C. glutamicum Lys9 produced 62.1 mmol L-1 L-lysine with substrate-specific yield (YP/S) of 0.28 mol per mol of glucose in shake flasks fermentation, whereas parental strain showed more than four times lower L-lysine production and more than 10 times lower biomass-specific yield (YP/X) than that of C. glutamicum Lys9. L-lysine production and cell growth were drastically decreased by isocitrate dehydrogenase (ICD) attenuation in aceE deletion strains, indicating that down-regulation of ICD activity in aceE deletion strains adversely affects L-lysine production. In fed-batch fermentation, C. glutamicum Lys9 produced 526 mmol L-1 L-lysine, i.e. 96.8 g L-1 L-lysine-HCl with high yield of 0.422 mol per mol of glucose and productivity of 2.69 g L-1 h-1. Corynebacterium glutamicum Lys9 was devoid of any detectable L-alanine and L-lactate synthesis. CONCLUSION Superior to classical producers to some degree, C. glutamicum Lys9 is more adaptable for industrial L-lysine production. In addition to L-lysine, pyruvate, oxaloacetate (OAA) and L-valine were produced by C. glutamicum Lys9, suggesting further optimization to improve L-lysine production by engineering the L-lysine and/or NADPH biosynthetic pathway. © 2013 Society of Chemical Industry

Improvement of cell growth and l-lysine production by genetically modified Corynebacterium glutamicum during growth on molasses

Fructose-1,6-bisphosphatase (FBPase) and fructokinase (ScrK) have important roles in regenerating glucose-6-phosphate in the pentose phosphate pathway (PPP), and thus increasing L-lysine production. This article focuses on the development of L-lysine high-producing strains by heterologous expression of FBPase gene fbp and ScrK gene scrK in C. glutamicum lysC (fbr) with molasses as the sole carbon source. Heterologous expression of fbp and scrK lead to a decrease of residual sugar in fermentation broth, and heterologous expression of scrK prevents the fructose efflux. Heterologous expression of fbp and scrK not only increases significantly the activity of corresponding enzymes but also improves cell growth during growth on molasses. FBPase activities are increased tenfold by heterologous expression of fbp, whereas the FBPase activity is only increase fourfold during co-expression of scrK and fbp. Compared with glucose, the DCW of heterologous expression strains are higher on molasses except co-expression of fbp and scrK strain. In addition, heterologous expression of fbp and scrK can strongly increase the L-lysine production with molasses as the sole carbon source. The highest increase (88.4%) was observed for C. glutamicum lysC (fbr) pDXW-8-fbp-scrK, but the increase was also significant for C. glutamicum lysC (fbr) pDXW-8-fbp (47.2%) and C. glutamicum lysC (fbr) pDXW-8-scrK (36.8%). By-products, such as glycerol and dihydroxyacetone, are decreased by heterologous expression of fbp and scrK, whereas trehalose is only slightly increased. The strategy for enhancing L-lysine production by regeneration of glucose-6-phosphate in PPP may provide a reference to enhance the production of other amino acids during growth on molasses or starch.

Reduction of hydrogen peroxide stress derived from fatty acid beta-oxidation improves fatty acid utilization in Escherichia coli.

Fatty acids are a promising raw material for substance production because of their highly reduced and anhydrous nature, which can provide higher fermentation yields than sugars. However, they are insoluble in water and are poorly utilized by microbes in industrial fermentation production. We used fatty acids as raw materials for l-lysine fermentation by emulsification and improved the limited fatty acid-utilization ability of Escherichia coli. We obtained a fatty acid-utilizing mutant strain by laboratory evolution and demonstrated that it expressed lower levels of an oxidative-stress marker than wild type. The intracellular hydrogen peroxide (H2O2) concentration of a fatty acid-utilizing wild-type E. coli strain was higher than that of a glucose-utilizing wild-type E. coli strain. The novel mutation rpsA D210Y identified in our fatty acid-utilizing mutant strain enabled us to promote cell growth, fatty-acid utilization, and l-lysine production from fatty acid. Introduction of this rpsA D210Y mutation into a wild-type strain resulted in lower H2O2 concentrations. The overexpression of superoxide dismutase (sodA) increased intracellular H2O2 concentrations and inhibited E. coli fatty-acid utilization, whereas overexpression of an oxidative-stress regulator (oxyS) decreased intracellular H2O2 concentrations and promoted E. coli fatty acid utilization and l-lysine production. Addition of the reactive oxygen species (ROS) scavenger thiourea promoted l-lysine production from fatty acids and decreased intracellular H2O2 concentrations. Among the ROS generated by fatty-acid β-oxidation, H2O2 critically affected E. coli growth and l-lysine production. This indicates that the regression of ROS stress promotes fatty acid utilization, which is beneficial for fatty acids used as raw materials in industrial production.

Direct l-lysine production from cellobiose by Corynebacterium glutamicum displaying beta-glucosidase on its cell surface

We constructed beta-glucosidase (BGL)-displaying Corynebacterium glutamicum, and direct L-lysine fermentation from cellobiose was demonstrated. After screening active BGLs, Sde1394, which is a BGL from Saccharophagus degradans, was successfully displayed on the C. glutamicum cell surface using porin as an anchor protein, and cellobiose was directly assimilated as a carbon source. The optical density at 600nm of BGL-displaying C. glutamicum grown on cellobiose as a carbon source reached 23.5 after 48h of cultivation, which was almost the same as that of glucose after 24h of cultivation. Finally, Sde1394-displaying C. glutamicum produced 1.08g/l of L-lysine from 20g/l of cellobiose after 4days of cultivation, which was about threefold higher than the amount of produced L-lysine using BGL-secretory C. glutamicum strains (0.38g/l after 5days of cultivation). This is the first report on amino acid production using cellobiose as a carbon source by BGL-expressing C. glutamicum.

Cloning, expression, crystallization and preliminary structural studies of dihydrodipicolinate reductase from Acinetobacter baumannii.

Acinetobacter baumannii is a virulent pathogenic bacterium that is resistant to most currently available antibiotics. Therefore, the design of drugs for the treatment of infections caused by A. baumannii is urgently required. Dihydrodipicolinate reductase (DHDPR) is an important enzyme which is involved in the biosynthetic pathway that leads to the production of L-lysine in bacteria. In order to design potent inhibitors against this enzyme, its detailed three-dimensional structure is required. DHDPR from A. baumannii (AbDHDPR) has been cloned, expressed, purified and crystallized. Here, the preliminary X-ray crystallographic data of AbDHDPR are reported. The crystals were grown using the hanging-drop vapour-diffusion method with PEG 3350 as the precipitating agent The crystals belonged to the orthorhombic space group P222, with unit-cell parameters a = 80.0, b = 100.8, c = 147.6 Å, and contained four molecules in the asymmetric unit. The complete structure determination of AbDHDPR is in progress.

Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation

Recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance. Here, we establish recombineering for Corynebacterium glutamicum using RecT of prophage Rac and combine this with our recently developed nanosensor technology, which enables the detection and isolation of productive mutants at the single-cell level via fluorescence-activated cell sorting (FACS). We call this new technology RecFACS, which we use for genomic site-directed saturation mutagenesis without relying on pre-constructed libraries to directly isolate l-lysine-producing cells. A mixture of 19 different oligonucleotides was used targeting codon 81 in murE of the wild-type, at a locus where one single mutation is known to cause l-lysine production. Using RecFACS, productive mutants were screened and isolated. Sequencing revealed 12 different amino acid exchanges in the targeted murE codon, which caused different l-lysine production titers. Apart from introducing a rapid genome construction technology for C. glutamicum, the present work demonstrates that RecFACS is suitable to simply create producers as well as genetic diversity in one single step, thus establishing a new general concept in synthetic biology.

Optimization of physical parameter for the production of L-lysine by a mutant strain Micrococcus glutamicus AB2oo

Optimization of physical parameter for the production of L-lysine by a mutant strain Micrococcus glutamicus AB2oo

Production of l-lysine on different silage juices using genetically engineered Corynebacterium glutamicum

Corynebacterium glutamicum, the best established industrial producer organism for lysine was genetically modified to allow the production of lysine on grass and corn silages. The resulting strain C. glutamicum lysCfbrdldPsodpycPsodmalEPsodfbpPsodgapXPsod was based on earlier work (Neuner and Heinzle, 2011). That mutant carries a point mutation in the aspartokinase (lysC) regulatory subunit gene as well as overexpression of d-lactate dehydrogenase (dld), pyruvate carboxylase (pyc) and malic enzyme (malE) using the strong Psod promoter. Here, we additionally overexpressed fructose 1,6-bisphosphatase (fbp) and glyceraldehyde 3-phosphate dehydrogenase (gapX) using the same promoter. The resulting strain grew readily on grass and corn silages with a specific growth rate of 0.35h−1 and lysine carbon yields of approximately 90C-mmol (C-mol)−1. Lysine yields were hardly affected by oxygen limitation whereas linear growth was observed under oxygen limiting conditions. Overall, this strain seems very robust with respect to the composition of silage utilizing all quantified low molecular weight substrates, e.g. lactate, glucose, fructose, maltose, quinate, fumarate, glutamate, leucine, isoleucine and alanine.

Determination of auxotropic nature of the mutant Micrococcus glutamicus AB2oo for improved L-lysine production

Determination of auxotropic nature of the mutant Micrococcus glutamicus AB2oo for improved L-lysine production

Exploring the allosteric mechanism of dihydrodipicolinate synthase by reverse engineering of the allosteric inhibitor binding sites and its application for lysine production

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyzes the first committed reaction of L-lysine biosynthesis in bacteria and plants and is allosterically regulated by L-lysine. In previous studies, DHDPSs from different species were proved to have different sensitivity to L-lysine inhibition. In this study, we investigated the key determinants of feedback regulation between two industrially important DHDPSs, the L-lysine-sensitive DHDPS from Escherichia coli and L-lysine-insensitive DHDPS from Corynebacterium glutamicum, by sequence and structure comparisons and site-directed mutation. Feedback inhibition of E. coli DHDPS was successfully alleviated after substitution of the residues around the inhibitor's binding sites with those of C. glutamicum DHDPS. Interestingly, mutagenesis of the lysine binding sites of C. glutamicum DHDPS according to E. coli DHDPS did not recover the expected feedback inhibition but an activation of DHDPS by L-lysine, probably due to differences in the allosteic signal transduction in the DHDPS of these two organisms. Overexpression of L-lysine-insensitive E. coli DHDPS mutants in E. coli MG1655 resulted in an improvement of L-lysine production yield by 46 %.

Production of l-lysine from l-lysine monohydrochloride by bipolar membrane electrodialysis

Bipolar membrane electrodialysis consisting of one bipolar membrane, one anion-exchange membrane and one cation-exchange membrane was performed to achieve the production of l-lysine from l-lysine (l-Lys) monohydrochloride. Several experimental parameters including the operation voltage, the initial l-Lys·HCl concentration and operation temperature were compared and discussed. The Cl- removal ratio, the electric conductivity, the pH in various compartments, the current efficiency (CE) and the energy consumption (EC) were presented and analyzed respectively. When the initial concentration of l-Lys·HCl is 0.6 mol/L and the constant voltage is 40 V, the removal ratio of Cl- reached 86.6%, the CE 24% and the EC 28.2 kW h/kg. Elevating the operation temperature can promote the electrodialysis process slightly.

A disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell level

In the continuously growing field of industrial biotechnology the scale-up from lab to industrial scale is still a major hurdle to develop competitive bioprocesses. During scale-up the productivity of single cells might be affected by bioreactor inhomogeneity and population heterogeneity. Currently, these complex interactions are difficult to investigate. In this report, design, fabrication and operation of a disposable picolitre cultivation system is described, in which environmental conditions can be well controlled on a short time scale and bacterial microcolony growth experiments can be observed by time-lapse microscopy. Three exemplary investigations will be discussed emphasizing the applicability and versatility of the device. Growth and analysis of industrially relevant bacteria with single cell resolution (in particular Escherichia coli and Corynebacterium glutamicum) starting from one single mother cell to densely packed cultures is demonstrated. Applying the picolitre bioreactor, 1.5-fold increased growth rates of C. glutamicum wild type cells were observed compared to typical 1 litre lab-scale batch cultivation. Moreover, the device was used to analyse and quantify the morphological changes of an industrially relevant l-lysine producer C. glutamicum after artificially inducing starvation conditions. Instead of a one week lab-scale experiment, only 1 h was sufficient to reveal the same information. Furthermore, time lapse microscopy during 24 h picolitre cultivation of an arginine producing strain containing a genetically encoded fluorescence sensor disclosed time dependent single cell productivity and growth, which was not possible with conventional methods.

Cleaner Design of Single-Product Biotechnological Facilities through the Integration of Process Simulation, Multiobjective Optimization, Life Cycle Assessment, and Principal Component Analysis

Bioprocesses have been typically optimized according to their economic performances. In this work we present a novel framework for their optimal design that allows for the simultaneous consideration of economic and environmental concerns. Our approach relies on the combined use ofsimulation packages, multiobjective optimization (MOO), life cycle assessment (LCA), and principal component analysis (PCA). The capabilities of the proposed methodology are illustrated through its application to the production of the amino acid L-lysine.

Biotin protein ligase from Corynebacterium glutamicum: role for growth and l-lysine production

Corynebacterium glutamicum is a biotin auxotrophic Gram-positive bacterium that is used for large-scale production of amino acids, especially of L-glutamate and L-lysine. It is known that biotin limitation triggers L-glutamate production and that L-lysine production can be increased by enhancing the activity of pyruvate carboxylase, one of two biotin-dependent proteins of C. glutamicum. The gene cg0814 (accession number YP_225000) has been annotated to code for putative biotin protein ligase BirA, but the protein has not yet been characterized. A discontinuous enzyme assay of biotin protein ligase activity was established using a 105aa peptide corresponding to the carboxyterminus of the biotin carboxylase/biotin carboxyl carrier protein subunit AccBC of the acetyl CoA carboxylase from C. glutamicum as acceptor substrate. Biotinylation of this biotin acceptor peptide was revealed with crude extracts of a strain overexpressing the birA gene and was shown to be ATP dependent. Thus, birA from C. glutamicum codes for a functional biotin protein ligase (EC 6.3.4.15). The gene birA from C. glutamicum was overexpressed and the transcriptome was compared with the control strain revealing no significant gene expression changes of the bio-genes. However, biotin protein ligase overproduction increased the level of the biotin-containing protein pyruvate carboxylase and entailed a significant growth advantage in glucose minimal medium. Moreover, birA overexpression resulted in a twofold higher L-lysine yield on glucose as compared with the control strain.

Impact of a new glucose utilization pathway in amino acid-producing Corynebacterium glutamicum

Corynebacterium glutamicum imports and phosphorylates glucose, fructose and sucrose by the phosphoenolpyruvate-dependent phosphotransferase carbohydrate uptake system (PTS). Recently, we have discovered how glucose can be utilized by C. glutamicum in a PTS-independent manner. PTS-independent glucose uptake is mediated by one of two inositol permeases (IolT1 or IolT2) and the second function of PTS, substrate phosphorylation, is catalyzed by one of two glucokinases (Glk or PpgK). PTS-deficient C. glutamicum strains exclusively utilizing glucose via this system grew comparably well on glucose minimal media as the parental strain. Furthermore, PTS-deficient L-lysine producing C. glutamicum strains overexpressing genes for inositol permease and glucokinase showed increased L-lysine production and reduced formation of by-products derived from pyruvate. Here, we discuss the impact of our findings on engineering strategies of C. glutamicum strains used in various biotechnological production processes.

Hybrid simulation-optimization based approach for the optimal design of single-product biotechnological processes

In this work, we present a systematic method for the optimal development of bioprocesses that relies on the combined use of simulation packages and optimization tools. One of the main advantages of our method is that it allows for the simultaneous optimization of all the individual components of a bioprocess, including the main upstream and downstream units. The design task is mathematically formulated as a mixed-integer dynamic optimization (MIDO) problem, which is solved by a decomposition method that iterates between primal and master sub-problems. The primal dynamic optimization problem optimizes the operating conditions, bioreactor kinetics and equipment sizes, whereas the master levels entails the solution of a tailored mixed-integer linear programming (MILP) model that decides on the values of the integer variables (i.e., number of equipments in parallel and topological decisions). The dynamic optimization primal sub-problems are solved via a sequential approach that integrates the process simulator SuperPro Designer ® with an external NLP solver implemented in Matlab ®. The capabilities of the proposed methodology are illustrated through its application to a typical fermentation process and to the production of the amino acid L-lysine.

Phosphotransferase system-independent glucose utilization in corynebacterium glutamicum by inositol permeases and glucokinases.

Phosphoenolpyruvate-dependent glucose phosphorylation via the phosphotransferase system (PTS) is the major path of glucose uptake in Corynebacterium glutamicum, but some growth from glucose is retained in the absence of the PTS. The growth defect of a deletion mutant lacking the general PTS component HPr in glucose medium could be overcome by suppressor mutations leading to the high expression of inositol utilization genes or by the addition of inositol to the growth medium if a glucokinase is overproduced simultaneously. PTS-independent glucose uptake was shown to require at least one of the inositol transporters IolT1 and IolT2 as a mutant lacking IolT1, IolT2, and the PTS component HPr could not grow with glucose as the sole carbon source. Efficient glucose utilization in the absence of the PTS necessitated the overexpression of a glucokinase gene in addition to either iolT1 or iolT2. IolT1 and IolT2 are low-affinity glucose permeases with K(s) values of 2.8 and 1.9 mM, respectively. As glucose uptake and phosphorylation via the PTS differs from glucose uptake via IolT1 or IolT2 and phosphorylation via glucokinase by the requirement for phosphoenolpyruvate, the roles of the two pathways for l-lysine production were tested. The l-lysine yield by C. glutamicum DM1729, a rationally engineered l-lysine-producing strain, was lower than that by its PTS-deficient derivate DM1729Δhpr, which, however, showed low production rates. The combined overexpression of iolT1 or iolT2 with ppgK, the gene for PolyP/ATP-dependent glucokinase, in DM1729Δhpr enabled l-lysine production as fast as that by the parent strain DM1729 but with 10 to 20% higher l-lysine yield.