Incorporation of alternative amino acids into cyanophycin by different cyanophycin synthetases heterologously expressed in Corynebacterium glutamicum.
Ontology highlight
ABSTRACT:
SUBMITTER: Wordemann R
PROVIDER: S-EPMC8050183 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:UnlabelledElementary flux mode (EFM) analysis is a powerful tool to represent the metabolic network structure and can be further utilized for flux analysis. The method enables characterization and quantification of feasible phenotypes in microbes. EFM analysis was employed to characterize the phenotype of Corynebacterium glutamicum to yield various amino acids. The metabolic network of C. glutamicum yielded 62 elementary modes by incorporating the accumulation of amino acids namely, lysine, alanine, valine, glutamine and glutamate. The analysis also allowed us to compute the maximum theoretical yield for the synthesis of various amino acids. These 62 elementary modes were further used to obtain optimal phenotypic space towards accumulation of biomass and lysine. The study indicated that the optimal solution space from 62 elementary modes forms a super space which incorporates various mutants including lysine producing strain of C. glutamicum. The analysis was also extended to obtain sensitivity of the network to variation in the stoichiometry of NADP in the definition of biomass.Electronic supplementary materialThe online version of this article (doi:10.1007/s11693-011-9073-8) contains supplementary material, which is available to authorized users.
| S-EPMC3065593 | biostudies-literature
Project description:A number of d-amino acids occur in nature, and there is growing interest in their function and metabolism, as well as in their production and use. Here we use the well-established l-amino-acid-producing bacterium Corynebacterium glutamicum to study whether d-amino acid synthesis is possible and whether mechanisms for the export of these amino acids exist. In contrast to Escherichia coli, C. glutamicum tolerates d-amino acids added extracellularly. Expression of argR (encoding the broad-substrate-specific racemase of Pseudomonas taetrolens) with its signal sequence deleted results in cytosolic localization of ArgR in C. glutamicum. The isolated enzyme has the highest activity with lysine (100%) but also exhibits activity with serine (2%). Upon overexpression of argR in an l-arginine, l-ornithine, or l-lysine producer, equimolar mixtures of the d- and l-enantiomers accumulated extracellularly. Unexpectedly, argR overexpression in an l-serine producer resulted in extracellular accumulation of a surplus of d-serine (81 mM d-serine and 37 mM l-serine) at intracellular concentrations of 125 mM d-serine plus 125 mM l-serine. This points to a nonlimiting ArgR activity for intracellular serine racemization and to the existence of a specific export carrier for d-serine. Export of d-lysine relies fully on the presence of lysE, encoding the exporter for l-lysine, which is apparently promiscuous with respect to the chirality of lysine. These data show that d-amino acids can also be produced with C. glutamicum and that in special cases, due to specific carriers, even a preferential extracellular accumulation of this enantiomer is possible.
| S-EPMC3067661 | biostudies-literature
Project description:The hyperphosphorylated guanosine derivatives ppGpp and pppGpp represent global regulators of the bacterial stress response, as they act as central elements of the stringent response system. Although it was assumed that both, (p)ppGpp synthesis and hydrolysis, are catalyzed by one bifunctional RSH-protein in the actinobacterial model organism Corynebacterium glutamicum ATCC 13032, two putative short alarmone synthetases (SASs) were identified by bioinformatic analyses. The predicted sequences of both enzymes, designated as RelP*Cg and RelSCg, exhibit high similarities to the conserved (p)ppGpp synthetase catalytic domain. In the context of sequence analysis, significant differences were found between the RelP variants of different C. glutamicum isolates. In contrast to the bifunctional RelA/SpoT homolog (RSH) protein RelCg, whose gene deletion results in a reduced growth rate, no change in growth characteristics were observed for deletion mutants of the putative SAS proteins under standard growth conditions. The growth deficit of the ?rel strain could be restored by the additional deletion of the gene encoding RelSCg, which clearly indicates a functional relationship between both enzymes. The predicted pyrophosphokinase activity of RelSCg was demonstrated by means of genetic complementation of an Escherichia coli ?relA?spoT strain. For the expression of RelP*Cg , as well as the slightly differing variant RelPCg from C. glutamicum AS1.542, no complementation was observed, concluding that both RelP versions possess no significant pyrophosphokinase activity in vivo. The results were confirmed by in vitro characterization of the corresponding proteins. In the course of this investigation, the additional conversion of GMP to pGpp was determined for the enzyme RelSCg. Since the SAS species analyzed extend both the network of stringent response related enzymes and the number of substances involved, the study of this class of enzymes is an important component in understanding the bacterial stress response. In addition to the comprehension of important biological processes, such as growth rate regulation and the survival of pathogenic species in the host organism, SAS enzymes can be used to produce novel hyperphosphorylated nucleotide species, such as pGpp.
| S-EPMC5566576 | biostudies-literature
Project description:Corynebacterium glutamicum is a non-pathogenic bacterium widely used in industrial amino acid production and metabolic engineering research. Although the genome sequences of some C. glutamicum strains are available, comprehensive comparative genome analyses of these species have not been done. Six wild type C. glutamicum strains were sequenced using next-generation sequencing technology in our study. Together with 20 previously reported strains, we present a comprehensive comparative analysis of C. glutamicum genomes.By average nucleotide identity (ANI) analysis, we show that 10 strains, which were previously classified either in the genus Brevibacterium, or as some other species within the genus Corynebacterium, should be reclassified as members of the species C. glutamicum. C. glutamicum has an open pan-genome with 2359 core genes. An additional NAD+/NADP+ specific glutamate dehydrogenase (GDH) gene (gdh) was identified in the glutamate synthesis pathway of some C. glutamicum strains. For analyzing variations related to amino acid production, we have developed an efficient pipeline that includes three major steps: multi locus sequence typing (MLST), phylogenomic analysis based on single nucleotide polymorphisms (SNPs), and a thorough comparison of all genomic variation amongst ancestral or closely related wild type strains. This combined approach can provide new perspectives on the industrial use of C. glutamicum.This is the first comprehensive comparative analysis of C. glutamicum genomes at the pan-genomic level. Whole genome comparison provides definitive evidence for classifying the members of this species. Identifying an aditional gdh gene in some C. glutamicum strains may accelerate further research on glutamate synthesis. Our proposed pipeline can provide a clear perspective, including the presumed ancestor, the strain breeding trajectory, and the genomic variations necessary to increase amino acid production in C. glutamicum.
| S-EPMC5310272 | biostudies-literature
Project description:Hydroxybenzoic acids are industrially relevant aromatic compounds, which also play key roles in the microbial carbon metabolism, e.g., as precursors for the synthesis of cofactors or metal-chelating molecules. Due to its pronounced resistance to aromatics Corynebacterium glutamicum represents an interesting platform for production of these compounds. Unfortunately, a complex catabolic network for aromatic molecules prevents application of C. glutamicum for microbial production of aromatic compounds other than aromatic amino acids, which cannot be metabolized by this microorganism.We completed the construction of the platform strain C. glutamicum DelAro5, in which the deletion of altogether 27 genes in five gene clusters abolished most of the peripheral and central catabolic pathways for aromatic compounds known in this microorganism. The obtained strain was subsequently applied for the production of 2-hydroxybenzoate (salicylate), 3-hydroxybenzoate, 4-hydroxybenzoate and protocatechuate, which all derive from intermediates of the aromatic amino acid-forming shikimate pathway. For an optimal connection of the designed hydroxybenzoate production pathways to the host metabolism, C. glutamicum was additionally engineered towards increased supply of the shikimate pathway substrates erythrose-4-phosphate and phosphoenolpyruvate by manipulation of the glucose transport and key enzymatic activities of the central carbon metabolism. With an optimized genetic background the constructed strains produced 0.01 g/L (0.07 mM) 2-hydroxybenzoate, 0.3 g/L (2.2 mM) 3-hydroxybenzoate, 2.0 g/L (13.0 mM) protocatechuate and 3.3 g/L (23.9 mM) 4-hydroxybenzoate in shaking flasks.By abolishing its natural catabolic network for aromatic compounds, C. glutamicum was turned into a versatile microbial platform for aromatics production, which could be exemplarily demonstrated by rapidly engineering this platform organism towards producing four biotechnologically interesting hydroxybenzoates. Production of these compounds was optimized following different metabolic engineering strategies leading to increased precursor availability. The constructed C. glutamicum strains are promising hosts for the production of hydroxybenzoates and other aromatic compounds at larger scales.
| S-EPMC5948850 | biostudies-literature
Project description:Two strains of the methylotrophic yeast Pichia pastoris were used to establish cyanophycin (multi-L-arginyl-poly-L-aspartic acid [CGP]) synthesis and to explore the applicability of this industrially widely used microorganism for the production of this polyamide. Therefore, the CGP synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA(6308)) was expressed under the control of the alcohol oxidase 1 promoter, yielding CGP contents of up to 10.4% (wt/wt), with the main fraction consisting of the soluble form of the polymer. To increase the polymer contents and to obtain further insights into the structural or catalytic properties of the enzyme, site-directed mutagenesis was applied to cphA(6308) and the mutated gene products were analyzed after expression in P. pastoris and Escherichia coli, respectively. CphA(6308)Delta1, which was truncated by one amino acid at the C terminus; point mutated CphA(6308)C595S; and the combined double-mutant CphA(6308)Delta1C595S protein were purified. They exhibited up to 2.5-fold higher enzyme activities of 4.95 U/mg, 3.20 U/mg, and 4.17 U/mg, respectively, than wild-type CphA(6308) (2.01 U/mg). On the other hand, CphA proteins truncated by two (CphA(6308)Delta2) or three (CphA(6308)Delta3) amino acids at the C terminus showed similar or reduced CphA enzyme activity in comparison to CphA(6308). In flask experiments, a maximum of 14.3% (wt/wt) CGP was detected after the expression of CphA(6308)Delta1 in P. pastoris. For stabilization of the expression plasmid, the his4 gene from Saccharomyces cerevisiae was cloned into the expression vector used and the constructs were transferred to histidine auxotrophic P. pastoris strain GS115. Parallel fermentations at a one-to-one scale revealed 26 degrees C and 6.0 as the optimal temperature and pH, respectively, for CGP synthesis. After optimization of fermentation parameters, medium composition, and the length of the cultivation period, CGP contents could be increased from 3.2 to 13.0% (wt/wt) in cells of P. pastoris GS115 expressing CphA(6308) and up to even 23.3% (wt/wt) in cells of P. pastoris GS115 expressing CphA(6308)Delta1.
| S-EPMC2820970 | biostudies-literature
Project description:A wide range of noncanonical amino acids (ncAAs) can be incorporated into proteins in living cells by using engineered aminoacyl-tRNA synthetase/tRNA pairs. However, most engineered tRNA synthetases are polyspecific; that is, they can recognize multiple rather than one ncAA. Polyspecificity of engineered tRNA synthetases imposes a limit to the use of genetic code expansion because it prevents specific incorporation of a desired ncAA when multiple ncAAs are present in the growth media. In this study, we employed directed evolution to improve substrate selectivity of polyspecific tRNA synthetases by developing substrate-selective readouts for flow-cytometry-based screening with the simultaneous presence of multiple ncAAs. We applied this method to improve the selectivity of two commonly used tRNA synthetases, p-cyano-l-phenylalanyl aminoacyl-tRNA synthetase ( pCNFRS) and Nε-acetyl-lysyl aminoacyl-tRNA synthetase (AcKRS), with broad specificity. Evolved pCNFRS and AcKRS variants exhibit significantly improved selectivity for ncAAs p-azido-l-phenylalanine ( pAzF) and m-iodo-l-phenylalanine ( mIF), respectively. To demonstrate the utility of our approach, we used the newly evolved tRNA synthetase variant to produce highly pure proteins containing the ncAA mIF, in the presence of multiple ncAAs present in the growth media. In summary, our new approach opens up a new avenue for engineering the next generation of tRNA synthetases with improved selectivity toward a desired ncAA.
| S-EPMC6642615 | biostudies-literature
Project description:BackgroundBio-based aromatic compounds are of great interest to the industry, as commercial production of aromatic compounds depends exclusively on the unsustainable use of fossil resources or extraction from plant resources. γ-amino acid 3-amino-4-hydroxybenzoic acid (3,4-AHBA) serves as a precursor for thermostable bioplastics.ResultsUnder aerobic conditions, a recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI genes derived from Streptomyces griseus produced 3,4-AHBA with large amounts of amino acids as by-products. The specific productivity of 3,4-AHBA increased with decreasing levels of dissolved oxygen (DO) and was eightfold higher under oxygen limitation (DO = 0 ppm) than under aerobic conditions (DO ≥ 2.6 ppm). Metabolic profiles during 3,4-AHBA production were compared at three different DO levels (0, 2.6, and 5.3 ppm) using the DO-stat method. Results of the metabolome analysis revealed metabolic shifts in both the central metabolic pathway and amino acid metabolism at a DO of < 33% saturated oxygen. Based on this metabolome analysis, metabolic pathways were rationally designed for oxygen limitation. An ldh deletion mutant, with the loss of lactate dehydrogenase, exhibited 3.7-fold higher specific productivity of 3,4-AHBA at DO = 0 ppm as compared to the parent strain KT01 and produced 5.6 g/L 3,4-AHBA in a glucose fed-batch culture.ConclusionsOur results revealed changes in the metabolic state in response to DO concentration and provided insights into oxygen supply during fermentation and the rational design of metabolic pathways for improved production of related amino acids and their derivatives.
| S-EPMC8697445 | biostudies-literature
Project description:N-methylated amino acids are found in Nature in various biological compounds. N-methylation of amino acids has been shown to improve pharmacokinetic properties of peptide drugs due to conformational changes, improved proteolytic stability and/or higher lipophilicity. Due to these characteristics N-methylated amino acids received increasing interest by the pharmaceutical industry. Syntheses of N-methylated amino acids by chemical and biocatalytic approaches are known, but often show incomplete stereoselectivity, low yields or expensive co-factor regeneration. So far a one-step fermentative process from sugars has not yet been described. Here, a one-step conversion of sugars and methylamine to the N-methylated amino acid N-methyl-L-alanine was developed. A whole-cell biocatalyst was derived from a pyruvate overproducing C. glutamicum strain by heterologous expression of the N-methyl-L-amino acid dehydrogenase gene from Pseudomonas putida. As proof-of-concept, N-methyl-L-alanine titers of 31.7?g?L-1 with a yield of 0.71?g per g glucose were achieved in fed-batch cultivation. The C. glutamicum strain producing this imine reductase enzyme was engineered further to extend this green chemistry route to production of N-methyl-L-alanine from alternative feed stocks such as starch or the lignocellulosic sugars xylose and arabinose.
| S-EPMC6110843 | biostudies-other
Project description:Methods of genetic code manipulation, such as nonsense codon suppression and genetic code reprogramming, have enabled the incorporation of various nonproteinogenic amino acids into the peptide nascent chain. However, the incorporation efficiency of such amino acids largely varies depending on their structural characteristics. For instance, l-α-amino acids with artificial, bulky side chains are poorer substrates for ribosomal incorporation into the nascent peptide chain, mainly owing to the lower affinity of their aminoacyl-tRNA toward elongation factor-thermo unstable (EF-Tu). Phosphorylated Ser and Tyr are also poorer substrates for the same reason; engineering EF-Tu has turned out to be effective in improving their incorporation efficiencies. On the other hand, exotic amino acids such as d-amino acids and β-amino acids are even poorer substrates owing to their low affinity to EF-Tu and poor compatibility to the ribosome active site. Moreover, their consecutive incorporation is extremely difficult. To solve these problems, the engineering of ribosomes and tRNAs has been executed, leading to successful but limited improvement of their incorporation efficiency. In this review, we comprehensively summarize recent attempts to engineer the translation systems, resulting in a significant improvement of the incorporation of exotic amino acids.
| S-EPMC6386890 | biostudies-literature