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 PII Signal Transduction Protein GlnK Alleviates Feedback Inhibition of N-Acetyl-L-Glutamate Kinase by L-Arginine in Corynebacterium glutamicum

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Meijuan Xu ◽  
Mi Tang ◽  
Jiamin Chen ◽  
Taowei Yang ◽  
Xian Zhang ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Corynebacterium Glutamicum ◽  
Feedback Inhibition ◽  
Amino Acid Biosynthesis ◽  
Arginine Biosynthesis ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Pii Protein

ABSTRACT PII signal transduction proteins are ubiquitous and highly conserved in bacteria, archaea, and plants and play key roles in controlling nitrogen metabolism. However, research on biological functions and regulatory targets of PII proteins remains limited. Here, we illustrated experimentally that the PII protein Corynebacterium glutamicum GlnK (CgGlnK) increased l-arginine yield when glnK was overexpressed in Corynebacterium glutamicum. Data showed that CgGlnK regulated l-arginine biosynthesis by upregulating the expression of genes of the l-arginine metabolic pathway and interacting with N-acetyl-l-glutamate kinase (CgNAGK), the rate-limiting enzyme in l-arginine biosynthesis. Further assays indicated that CgGlnK contributed to alleviation of the feedback inhibition of CgNAGK caused by l-arginine. In silico analysis of the binding interface of CgGlnK-CgNAGK suggested that the B and T loops of CgGlnK mainly interacted with C and N domains of CgNAGK. Moreover, F11, R47, and K85 of CgGlnK were identified as crucial binding sites that interact with CgNAGK via hydrophobic interaction and H bonds, and these interactions probably had a positive effect on maintaining the stability of the complex. Collectively, this study reveals PII-NAGK interaction in nonphotosynthetic microorganisms and further provides insights into the regulatory mechanism of PII on amino acid biosynthesis in corynebacteria. IMPORTANCE Corynebacteria are safe industrial producers of diverse amino acids, including l-glutamic acid and l-arginine. In this study, we showed that PII protein GlnK played an important role in l-glutamic acid and l-arginine biosynthesis in C. glutamicum. Through clarifying the molecular mechanism of CgGlnK in l-arginine biosynthesis, the novel interaction between CgGlnK and CgNAGK was revealed. The alleviation of l-arginine inhibition of CgNAGK reached approximately 48.21% by CgGlnK addition, and the semi-inhibition constant of CgNAGK increased 1.4-fold. Furthermore, overexpression of glnK in a high-yield l-arginine-producing strain and fermentation of the recombinant strain in a 5-liter bioreactor led to a remarkably increased production of l-arginine, 49.978 g/liter, which was about 22.61% higher than that of the initial strain. In conclusion, this study provides a new strategy for modifying amino acid biosynthesis in C. glutamicum.

scholarly journals Production ofN-Acetyl-d-Neuraminic Acid by Use of an Efficient Spore Surface Display System

2011 ◽  
Vol 77 (10) ◽  
pp. 3197-3201 ◽  
Author(s):  
Xiaoman Xu ◽  
Chao Gao ◽  
Xifeng Zhang ◽  
Bin Che ◽  
Cuiqing Ma ◽  
...  
Keyword(s):  
Surface Display ◽  
Value Added ◽  
Neuraminic Acid ◽  
High Yield ◽  
Permeability Barrier ◽  
Chemical Production ◽  
High Concentration ◽  
Suitable Alternative ◽  
Content Type ◽  
Whole Cells

ABSTRACTProduction ofN-acetyl-d-neuraminic acid (Neu5Ac) via biocatalysis is traditionally conducted using isolated enzymes or whole cells. The use of isolated enzymes is restricted by the time-consuming purification process, whereas the application of whole cells is limited by the permeability barrier presented by the microbial cell membrane. In this study, a novel type of biocatalyst, Neu5Ac aldolase presented on the surface ofBacillus subtilisspores, was used for the production of Neu5Ac. Under optimal conditions, Neu5Ac at a high concentration (54.7 g liter−1) and a high yield (90.2%) was obtained under a 5-fold excess of pyruvate overN-acetyl-d-mannosamine. The novel biocatalyst system, which is able to express and immobilize the target enzyme simultaneously on the surface ofB. subtilisspores, represents a suitable alternative for value-added chemical production.

Structure and performance of the Italian alpine “core”: a counterfactual analysis

2020 ◽  
Vol 12 (4) ◽  
pp. 387-407
Author(s):  
Alberto Bramanti ◽  
Sofia Ricci
Keyword(s):  
Inner Core ◽  
Statistical Significance ◽  
Balance Sheet ◽  
Value Added ◽  
Counterfactual Analysis ◽  
Content Type ◽  
The Core ◽  
The Future ◽  
Future Challenges ◽  
Matching Techniques

Purpose The competitiveness of the Alpine regions is strongly influenced by environmental constraints and its relationship with the urban network in the valley floor, which cannot be one of pure dependence. This study aims to analyse the health of the Italian Alpine economy through the performance of its capital companies, defined as those operating in the strictly mountainous are-as within the territories covered by the Alpine Convention. The authors compare the performance (2012-2018) of the “inner core” firms with a counterfactual sample of companies from neighbouring territories to delineate the strengths and weaknesses of the Alpine enterprises. The paper addresses policymakers and practitioners who will design the future policies for the high lands, exploiting a vast collaborative planning network. Design/methodology/approach The study analyses two broad strands of literature on territorial competitiveness. It uses the coarsened exact matching techniques for the selection of a counterfactual sample at the enterprise level. The study follows a policy-oriented design, offering answers to future challenges. Findings The Alpine region has several different local production systems, with a significant level of heterogeneity among firms that differentiate the top 25% from the rest. The counterfactual analysis carried out does not provide clear evidence of significant differences. Instead, it con-firms strong similarities between the Alpine core and the peri-Alpine belt. It is only in terms of employment growth that the core grows less (with a high statistical significance). Finally, the authors introduce the analysis of sustainable value added (SVA) in the core area and use the “tourism chain” to compare different models. The focus here is on two keywords – rarefied and uncontaminated – that enable the transformation of some typical weaknesses of the “minor (or marginal) mountain” into assets for development, provided that place-based and network policies are activated. Research limitations/implications The study focusses on the Italian Alps and could be extended in the future to the other countries participating in the Alpine Convention. It may also be enriched by qualitative analyses of partnerships and sole proprietorships that are not identified by the balance sheet analysis. Practical implications The study follows a policy-oriented design, offering possible solutions to future challenges. Social implications The study offers some suggestions on the post-COVID-19 phase. The bottom-up, reluctant and community dimension are possible strengths to face the challenges that are opening up. Originality/value The study is one of the very few to carry out a counterfactual analysis of Alpine enterprises. It offers evidence on the strengths and weaknesses of the productive fabric of the high lands and updates the assessment of the health status of Alpine enterprises to accompany future fact-based policies after the COVID pandemic.

scholarly journals Identification and Characterization of γ-Aminobutyric Acid Uptake System GabPCg(NCgl0464) in Corynebacterium glutamicum

2012 ◽  
Vol 78 (8) ◽  
pp. 2596-2601 ◽  
Author(s):  
Zhi Zhao ◽  
Jiu-Yuan Ding ◽  
Wen-hua Ma ◽  
Ning-Yi Zhou ◽  
Shuang-Jiang Liu
Keyword(s):  
Amino Acid ◽  
Corynebacterium Glutamicum ◽  
Dry Weight ◽  
Aminobutyric Acid ◽  
Gaba Uptake ◽  
Acid Uptake ◽  
Uptake System ◽  
Content Type ◽  
Gaba Transporters ◽  
New Type

ABSTRACTCorynebacterium glutamicumis widely used for industrial production of various amino acids and vitamins, and there is growing interest in engineering this bacterium for more commercial bioproducts such as γ-aminobutyric acid (GABA). In this study, aC. glutamicumGABA-specific transporter (GabPCg) encoded byncgl0464was identified and characterized. GabPCgplays a major role in GABA uptake and is essential toC. glutamicumgrowing on GABA. GABA uptake by GabPCgwas weakly competed byl-Asn andl-Gln and stimulated by sodium ion (Na+). TheKmandVmaxvalues were determined to be 41.1 ± 4.5 μM and 36.8 ± 2.6 nmol min−1(mg dry weight [DW])−1, respectively, at pH 6.5 and 34.2 ± 1.1 μM and 67.3 ± 1.0 nmol min−1(mg DW)−1, respectively, at pH 7.5. GabPCghas 29% amino acid sequence identity to a previously and functionally identified aromatic amino acid transporter (TyrP) ofEscherichia colibut low identities to the currently known GABA transporters (17% and 15% toE. coliGabP andBacillus subtilisGabP, respectively). The mutant RES167 Δncgl0464/pGXKZ9 with the GabPCgdeletion showed 12.5% higher productivity of GABA than RES167/pGXKZ9. It is concluded that GabPCgrepresents a new type of GABA transporter and is potentially important for engineering GABA-producingC. glutamicumstrains.

scholarly journals Complete and Draft Genome Sequences of Amino Acid-Producing Corynebacterium glutamicum Strains ATCC 21799 and ATCC 31831 and Their Genomic Islands

2020 ◽  
Vol 9 (32) ◽  
Author(s):  
Hideo Kawaguchi ◽  
Takashi Sazuka ◽  
Akihiko Kondo
Keyword(s):  
Amino Acid ◽  
Corynebacterium Glutamicum ◽  
Reference Strain ◽  
Draft Genome ◽  
Genomic Islands ◽  
Strain Atcc ◽  
Genome Sequences ◽  
Coding Sequences ◽  
Content Type

ABSTRACT We determined the complete and draft genome sequences of two strains of Corynebacterium glutamicum and revealed their genomic islands (GEIs). The two strains, ATCC 21799 and ATCC 31831, were found to have 3,079 and 3,109 coding sequences, respectively, with 13 GEIs each not present in the reference strain, ATCC 13032.

scholarly journals Engineering of Corynebacterium glutamicum for High-Yield l-Valine Production under Oxygen Deprivation Conditions

2012 ◽  
Vol 79 (4) ◽  
pp. 1250-1257 ◽  
Author(s):  
Satoshi Hasegawa ◽  
Masako Suda ◽  
Kimio Uematsu ◽  
Yumi Natsuma ◽  
Kazumi Hiraga ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Glucose Consumption ◽  
Resistant Mutant ◽  
Oxygen Deprivation ◽  
High Yield ◽  
Acetohydroxy Acid Synthase ◽  
Succinate Production ◽  
Content Type ◽  
High Productivity ◽  
By Products

ABSTRACTWe previously demonstrated efficientl-valine production by metabolically engineeredCorynebacterium glutamicumunder oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis ofl-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase fromLysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase geneldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress thel-valine yield. Eliminating these by-products therefore was deemed key to improving thel-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase geneppc, succinate production was effectively suppressed, but both glucose consumption andl-valine production dropped considerably due to the severely elevated intracellular NADH/NAD+ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, includinggapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higherl-valine yield and productivity. Deleting the alanine transaminase geneavtAsuppressed alanine production. The resultant strain produced 1,280 mMl-valine at a yield of 88% mol mol of glucose−1after 24 h under oxygen deprivation, a vastly improved yield over our previous best.

scholarly journals Degradation Pathways of 2- and 4-Nitrobenzoates in Cupriavidus sp. Strain ST-14 and Construction of a Recombinant Strain, ST-14::3NBA, Capable of Degrading 3-Nitrobenzoate

2016 ◽  
Vol 82 (14) ◽  
pp. 4253-4263 ◽  
Author(s):  
Soumik Basu ◽  
Piyali Pal Chowdhury ◽  
Satamita Deb ◽  
Tapan K. Dutta
Keyword(s):  
Recombinant Strain ◽  
Gene Knockout ◽  
Gene Segment ◽  
Value Added ◽  
Gene Clusters ◽  
Industrial Wastes ◽  
Cloning And Expression ◽  
Bacterial Degradation ◽  
Content Type ◽  
Catabolic Genes

ABSTRACTStrain ST-14, characterized as a member of the genusCupriavidus, was capable of utilizing 2- and 4-nitrobenzoates individually as sole sources of carbon and energy. Biochemical studies revealed the assimilation of 2- and 4-nitrobenzoates via 3-hydroxyanthranilate and protocatechuate, respectively. Screening of a genomic fosmid library of strain ST-14 constructed inEscherichia coliidentified two gene clusters,onbandpob-pca, to be responsible for the complete degradation of 2-nitrobenzoate and protocatechuate, respectively. Additionally, a gene segment (pnb) harboring the genes for the conversion of 4-nitrobenzoate to protocatechuate was unveiled by transposome mutagenesis. Reverse transcription-PCR analysis showed the polycistronic nature of the gene clusters, and their importance in the degradation of 2- and 4-nitrobenzoates was ascertained by gene knockout analysis. Cloning and expression of the relevant pathway genes revealed the transformation of 2-nitrobenzoate to 3-hydroxyanthranilate and of 4-nitrobenzoate to protocatechuate. Finally, incorporation of functional 3-nitrobenzoate dioxygenase into strain ST-14 allowed the recombinant strain to utilize 3-nitrobenzoate via the existing protocatechuate metabolic pathway, thereby allowing the degradation of all three isomers of mononitrobenzoate by a single bacterial strain.IMPORTANCEMononitrobenzoates are toxic chemicals largely used for the production of various value-added products and enter the ecosystem through industrial wastes. Bacteria capable of degrading mononitrobenzoates are relatively limited. Unlike other contaminants, these man-made chemicals have entered the environment since the last century, and it is believed that bacteria in nature evolved not quite efficiently to assimilate these compounds; as a consequence, to date, there are only a few reports on the bacterial degradation of one or more isomers of mononitrobenzoate. In the present study, fortunately, we have been able to isolate aCupriavidussp. strain capable of assimilating both 2- and 4-nitrobenzoates as the sole carbon source. Results of the biochemical and molecular characterization of catabolic genes responsible for the degradation of mononitrobenzoates led us to manipulate a single enzymatic step, allowing the recombinant host organism to expand its catabolic potential to assimilate 3-nitrobenzoate.

scholarly journals Metabolic engineering of Corynebacterium glutamicum for producing branched chain amino acids

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Shengzhu Yu ◽  
Bo Zheng ◽  
Zhenya Chen ◽  
Yi-Xin Huo
Keyword(s):  
Amino Acids ◽  
Metabolic Engineering ◽  
Corynebacterium Glutamicum ◽  
Animal Feed ◽  
Feedback Inhibition ◽  
Value Added ◽  
Branched Chain Amino Acids ◽  
High Yield ◽  
Acetohydroxy Acid Synthase ◽  
Branched Chain

Abstract Background Branched chain amino acids (BCAAs) are widely applied in the food, pharmaceutical, and animal feed industries. Traditional chemical synthetic and enzymatic BCAAs production in vitro has been hampered by expensive raw materials, harsh reaction conditions, and environmental pollution. Microbial metabolic engineering has attracted considerable attention as an alternative method for BCAAs biosynthesis because it is environmentally friendly and delivers high yield. Main text Corynebacterium glutamicum (C. glutamicum) possesses clear genetic background and mature gene manipulation toolbox, and has been utilized as industrial host for producing BCAAs. Acetohydroxy acid synthase (AHAS) is a crucial enzyme in the BCAAs biosynthetic pathway of C. glutamicum, but feedback inhibition is a disadvantage. We therefore reviewed AHAS modifications that relieve feedback inhibition and then investigated the importance of AHAS modifications in regulating production ratios of three BCAAs. We have comprehensively summarized and discussed metabolic engineering strategies to promote BCAAs synthesis in C. glutamicum and offer solutions to the barriers associated with BCAAs biosynthesis. We also considered the future applications of strains that could produce abundant amounts of BCAAs. Conclusions Branched chain amino acids have been synthesized by engineering the metabolism of C. glutamicum. Future investigations should focus on the feedback inhibition and/or transcription attenuation mechanisms of crucial enzymes. Enzymes with substrate specificity should be developed and applied to the production of individual BCAAs. The strategies used to construct strains producing BCAAs provide guidance for the biosynthesis of other high value-added compounds.

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

2013 ◽  
Vol 79 (8) ◽  
pp. 2588-2595 ◽  
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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