A Straightforward Approach to Synthesize 7-Aminocephalosporanic Acid In Vivo in the Cephalosporin C Producer Acremonium chrysogenum
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains, Plasmids, and Culture Conditions
2.2. Tools for Codon Adaptation, Gene Synthesis, and Protein Analysis
2.3. Construction of Fungal Gene Expression Vectors Using Codon-Adapted Bacterial Acylase Genes
2.4. Protein Extraction and Western Blot Analysis
2.5. Fluorescence Microscopy
2.6. Southern Blots
2.7. HPLC Analysis of Acylase Substrates and Products from Mycelia and Supernatants
2.8. Protein LC-MS/MS Analysis of Fragmented Peptides from CCAA and CCAB
3. Results
3.1. Gene Synthesis and Vector Construction for Introducing Three Codon-Optimized Cca Genes into A. chrysogenum
3.2. Bacterial Acylase Expression in A. chrysogenum
3.3. Evidence for Alpha and Beta CCA Subunits by MS Analysis
3.4. Time-Dependent Processing of the CCA Precursor and Detection in Mycelia and Supernatants
3.5. Detection of Substrates and Products of CCAs in Fungal Mycelia and Culture Supernatants
3.6. Exploration of the Optimal Settings for Active Acylases
3.7. Comparative Investigation of Transformants under Optimal Acylase Incubation Conditions
4. Discussion
4.1. The Heterologous Genes Encoding Cephalosporin C Acylase Are Efficiently Expressed in a Fungal Host
4.2. Bacterial Cephalosporin C Acylase Is Efficiently Processed in the Fungal Cell
4.3. Conversion of CPC into 7-ACA in the Culture Supernatant Has Applied Relevance
4.4. The Heterologous Cephalosporin C Acylase Shows Enzymatic Activity
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Newton, G.G.; Abraham, E.P. Cephalosporin C, a new antibiotic containing sulphur and D-alpha-aminoadipic acid. Nature 1955, 175, 548. [Google Scholar] [CrossRef] [PubMed]
- Abraham, E.P.; Newton, G.G. The structure of cephalosporin C. Biochem. J. 1961, 79, 377–393. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Harrison, C.J.; Bratcher, D. Cephalosporins: A review. Pediatr. Rev. 2008, 29, 264–267. [Google Scholar] [CrossRef] [PubMed]
- Tartaglione, T.A.; Polk, R.E. Review of the new second-generation cephalosporins: Cefonicid, ceforanide, and cefuroxime. Drug Intell. Clin. Pharm. 1985, 19, 188–198. [Google Scholar] [CrossRef]
- Pichichero, M.E.; Casey, J.R. Safe use of selected cephalosporins in penicillin-allergic patients: A meta-analysis. Otolaryngol. Head Neck Surg. 2007, 136, 340–347. [Google Scholar] [CrossRef]
- Garau, J.; Wilson, W.; Wood, M.; Carlet, J. Fourth-generation cephalosporins: A review of in vitro activity, pharmacokinetics, pharmacodynamics and clinical utility. Clin. Microbiol. Infect. 1997, 3, S87–S101. [Google Scholar] [CrossRef] [Green Version]
- Wilson, W.R. The role of fourth-generation cephalosporins in the treatment of serious infectious diseases in hospitalized patients. Diagn Microbiol Infect. Dis. 1998, 31, 473–477. [Google Scholar] [CrossRef]
- Bui, T.; Preuss, C.V. Cephalosporins; StatPearls Publishing: Treasure Island, FL, USA, 2022. [Google Scholar]
- Zhanel, G.G.; Sniezek, G.; Schweizer, F.; Zelenitsky, S.; Lagace-Wiens, P.R.; Rubinstein, E.; Gin, A.S.; Hoban, D.J.; Karlowsky, J.A. Ceftaroline: A novel broad-spectrum cephalosporin with activity against methicillin-resistant Staphylococcus aureus. Drugs 2009, 69, 809–831. [Google Scholar] [CrossRef]
- Huber, F.M.; Chauvette, R.R.; Jackson, B.G. Preparative methods for 7-aminocephalosporanic acid and 6-aminopenicillanic acid. In Cephalosporins and Penicillins: Chemistry and Biology; Flynn, E.H., Ed.; Academic Press: New York, NY, USA, 1972; pp. 27–73. [Google Scholar]
- Cabri, W. Industrial synthesis design with low environmental impact in the pharma industry. In New Methodologies and Techniques for a Sustainable Organic Chemistry; Mordini, A., Faigl, F., Eds.; Springer Science & Business Media: Berlin/Heidelberg, Germany, 2008; Volume 246, pp. 119–129. [Google Scholar]
- Morin, R.B.; Roeske, R.W.; Flynn, E.H.; Jackson, B.G. Chemistry of cephalosporin antibiotics.1. 7-Aminocephalosporanic acid from cephalosporin C. J. Am. Chem Soc. 1962, 84, 3400–3401. [Google Scholar] [CrossRef]
- Riethorst, W.; Reichert, A. An industrial view on enzymes for the cleavage of cephalosporin C. Chimia 1999, 53, 600–607. [Google Scholar]
- Wohlgemuth, R. Large-scale applications of hydrolases in biocatalytic asymmetric synthesis, 2nd ed. In Asymmetric Catalysis on Industrial Scale; Wiley-VCH: Hoboken, NJ, USA, 2010; pp. 249–264. [Google Scholar]
- Groeger, H.; Pieper, M.; Koenig, B.; Bayer, T.; Schleich, H. Industrial landmarks in the development of sustainable production processes for the beta-lactam antibiotic key intermediate 7-aminocephalosporanic acid (7-ACA). Sustain. Chem. Pharm. 2017, 5, 72–79. [Google Scholar] [CrossRef]
- Aramori, I.; Fukagawa, M.; Tsumura, M.; Iwami, M.; Isogai, T.; Ono, H.; Ishitani, Y.; Kojo, H.; Kohsaka, M.; Ueda, Y. Cloning and nucleotide sequencing of new glutaryl 7-ACA and cephalosporin C acylase genes from Pseudomonas strains. J. Ferment. Bioeng. 1991, 72, 232–243. [Google Scholar] [CrossRef]
- Pollegioni, L.; Rosini, E.; Molla, G. Cephalosporin C acylase: Dream and(/or) reality. Appl. Microbiol. Biotechnol. 2013, 97, 2341–2355. [Google Scholar] [CrossRef] [PubMed]
- Oh, B.; Kim, M.; Yoon, J.; Chung, K.; Shin, Y.; Lee, D.; Kim, Y. Deacylation activity of cephalosporin acylase to cephalosporin C is improved by changing the side-chain conformations of active-site residues. Biochem. Biophys. Res. Commun. 2003, 310, 19–27. [Google Scholar] [CrossRef]
- Kim, Y.; Yoon, K.; Khang, Y.; Turley, S.; Hol, W.G. The 2.0 A crystal structure of cephalosporin acylase. Structure 2000, 8, 1059–1068. [Google Scholar] [CrossRef]
- Kim, Y.; Hol, W.G. Structure of cephalosporin acylase in complex with glutaryl-7-aminocephalosporanic acid and glutarate: Insight into the basis of its substrate specificity. Chem. Biol. 2001, 8, 1253–1264. [Google Scholar] [CrossRef] [Green Version]
- Otten, L.G.; Sio, C.F.; Van Der Sloot, A.M.; Cool, R.H.; Quax, W.J. Mutational analysis of a key residue in the substrate specificity of a cephalosporin acylase. ChemBioChem 2004, 5, 820–825. [Google Scholar] [CrossRef]
- Shin, Y.C.; Jeon, J.Y.; Jung, K.H.; Park, M.R.; Kim, Y.; Sandoz, A.G. Cephalosporin C Acylase Mutant and Method for Preparing 7-ACA Using Same. US7592168B2, 2009. [Google Scholar]
- Zhu, X.; Luo, H.; Chang, Y.; Su, H.; Li, Q.; Yu, H.; Shen, Z. Characteristic of immobilized cephalosporin C acylase and its application in one-step enzymatic conversion of cephalosporin C to 7-aminocephalosporanic acid. World J. Microbiol. Biotechnol. 2011, 27, 823–829. [Google Scholar] [CrossRef]
- Bloemendal, S.; Loper, D.; Terfehr, D.; Kopke, K.; Kluge, J.; Teichert, I.; Kück, U. Tools for advanced and targeted genetic manipulation of the beta-lactam antibiotic producer Acremonium chrysogenum. J. Biotechnol. 2014, 169, 51–62. [Google Scholar] [CrossRef]
- Hu, Y.; Zhu, B. Study on genetic engineering of Acremonium chrysogenum, the cephalosporin C producer. Synth. Syst. Biotechnol. 2016, 1, 143–149. [Google Scholar] [CrossRef] [Green Version]
- Sambrook, J.; Russell, D.W. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: New York, NY, USA, 2001; Volume 3. [Google Scholar]
- Engler, C.; Kandzia, R.; Marillonnet, S. A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 2008, 3, e3647. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Bullock, W.O.; Fernandez, J.M.; Short, J.M. Xl1-Blue-a high-efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. Biotechniques 1987, 5, 376–379. [Google Scholar]
- Walz, M.; Kück, U. Targeted integration into the Acremonium chrysogenum genome-disruption of the pcbC gene. Curr. Genet. 1993, 24, 421–427. [Google Scholar] [CrossRef] [PubMed]
- Gsaller, F.; Blatzer, M.; Abt, B.; Schrettl, M.; Lindner, H.; Haas, H. The first promoter for conditional gene expression in Acremonium chrysogenum: Iron starvation-inducible mir1(P). J. Biotechnol. 2013, 163, 77–80. [Google Scholar] [CrossRef]
- Radzio, R.; Kück, U. Efficient synthesis of the blood-coagulation inhibitor hirudin in the filamentous fungus Acremonium chrysogenum. Appl. Microbiol. Biotechnol. 1997, 48, 58–65. [Google Scholar] [CrossRef]
- Dreyer, J.; Eichhorn, H.; Friedlin, E.; Kürnsteiner, H.; Kück, U. A homologue of the Aspergillus velvet gene regulates both cephalosporin C biosynthesis and hyphal fragmentation in Acremonium chrysogenum. Appl. Environ. Microbiol. 2007, 73, 3412–3422. [Google Scholar] [CrossRef] [Green Version]
- Kück, U.; Hoff, B. Application of the nourseothricin acetyltransferase gene (nat1) as dominant marker for the transformation of filamentous fungi. Fungal Genet. Newsl. 2006, 53, 9. [Google Scholar] [CrossRef] [Green Version]
- Mahmoudjanlou, Y.; Hoff, B.; Kück, U. Construction of a codon-adapted Nourseothricin-resistance marker gene for efficient targeted gene deletion in the mycophenolic acid producer Penicillium brevicompactum. J. Fungi 2019, 5, 96. [Google Scholar] [CrossRef] [Green Version]
- Schmidt, S.; Märker, R.; Ramsak, B.; Beier-Rosberger, A.M.; Teichert, I.; Kück, U. Crosstalk between pheromone signaling and NADPH oxidase complexes coordinates fungal developmental processes. Front. Microbiol 2020, 11, 1722. [Google Scholar] [CrossRef]
- Schmidt, S. Interaction of Conserved Signaling Pathways during Cellular Development in Sordaria macrospora. Ph.D. Thesis, Ruhr University Bochum, Bochum, Germany, 2020. [Google Scholar] [CrossRef]
- Dahlmann, T.A.; Terfehr, D.; Becker, K.; Teichert, I. Golden Gate vectors for efficient gene fusion and gene deletion in diverse filamentous fungi. Curr Genet. 2021, 67, 317–330. [Google Scholar] [CrossRef]
- Bayram, O.; Krappmann, S.; Ni, M.; Bok, J.W.; Helmstaedt, K.; Valerius, O.; Braus-Stromeyer, S.; Kwon, N.J.; Keller, N.P.; Yu, J.H.; et al. VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism. Science 2008, 320, 1504–1506. [Google Scholar] [CrossRef] [PubMed]
- Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem 1976, 72, 248–254. [Google Scholar] [CrossRef]
- Stein, V.; Blank-Landeshammer, B.; Müntjes, K.; Märker, R.; Teichert, I.; Feldbrügge, M.; Sickmann, A.; Kück, U. The STRIPAK signaling complex regulates dephosphorylation of GUL1, an RNA-binding protein that shuttles on endosomes. PLOS Genet. 2020, 16, e1008819. [Google Scholar] [CrossRef] [PubMed]
- Hoff, B.; Schmitt, E.K.; Kück, U. CPCR1, but not its interacting transcription factor AcFKH1, controls fungal arthrospore formation in Acremonium chrysogenum. Mol. Microbiol 2005, 56, 1220–1233. [Google Scholar] [CrossRef]
- Schneider, C.A.; Rasband, W.S.; Eliceiri, K.W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 2012, 9, 671–675. [Google Scholar] [CrossRef] [PubMed]
- Kraus, A.; Weskamp, M.; Zierles, J.; Balzer, M.; Busch, R.; Eisfeld, J.; Lambertz, J.; Nowaczyk, M.M.; Narberhaus, F.; Becker, A. Arginine-rich small proteins with a domain of unknown function, DUF1127, play a role in phosphate and carbon metabolism of Agrobacterium tumefaciens. J. Bacteriol 2020, 202, e00309–e00320. [Google Scholar] [CrossRef]
- Cormann, K.U.; Möller, M.; Nowaczyk, M.M. Critical assessment of protein cross-linking and molecular docking: An updated model for the interaction between photosystem II and Psb27. Front. Plant. Sci 2016, 7. [Google Scholar] [CrossRef]
- Terfehr, D.; Dahlmann, T.A.; Specht, T.; Zadra, I.; Kürnsteiner, H.; Kück, U. Genome sequence and annotation of Acremonium chrysogenum, producer of the beta-lactam antibiotic cephalosporin C. Genome Announc 2014, 2. [Google Scholar] [CrossRef] [Green Version]
- Lee, Y.-s.; Han-chul, Y.; Sung-soo, P. Cloning and characterization of GL-7-ACA acylase gene from Pseudomonas sp. GK16. J. Microbiol Biotechnol 1996, 6, 375–380. [Google Scholar]
- Matsuda, A.; Komatsu, K.-I. Molecular cloning and structure of the gene for 7 beta-(4-carboxybutanamido) cephalosporanic acid acylase from a Pseudomonas strain. J. Bacteriol 1985, 163, 1222–1228. [Google Scholar] [CrossRef] [Green Version]
- Matsuda, A.; Matsuyama, K.; Yamamoto, K.; Ichikawa, S.; Komatsu, K. Cloning and characterization of the genes for two distinct cephalosporin acylases from a Pseudomonas strain. J. Bacteriol 1987, 169, 5815–5820. [Google Scholar] [CrossRef] [Green Version]
- Tan, Q.; Qiu, J.; Luo, X.; Zhang, Y.; Liu, Y.; Chen, Y.; Yuan, J.; Liao, W. Progress in one-pot bioconversion of cephalosporin C to 7-aminocephalosporanic acid. Curr. Pharm. Biotechnol. 2018, 19, 30–42. [Google Scholar] [CrossRef] [PubMed]
- Shin, Y.C.; Park, C.; Wang, E.S.; Jung, K.H. Mutated Enzyme for Producing Cephalosporin Antibiotics Raw Material (7-ACA). CN103937764A, 2014. Available online: https://patents.google.com/patent/CN103937764A/en (accessed on 15 March 2022).
- König, B. A novel mutant acylase gene encoding an enzyme to convert cephalosporin C into 7-ACA. manuscript in preparation.
- Kopke, K.; Hoff, B.; Kück, U. Application of the Saccharomyces cerevisiae FLP/FRT recombination system in filamentous fungi for marker recycling and construction of knockout strains devoid of heterologous genes. Appl. Environ. Microbiol. 2010, 76, 4664–4674. [Google Scholar] [CrossRef] [Green Version]
- Kluge, J.; Terfehr, D.; Kück, U. Inducible promoters and functional genomic approaches for the genetic engineering of filamentous fungi. Appl Microbiol Biotechnol 2018, 102, 6357–6372. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Menne, S.; Walz, M.; Kück, U. Expression studies with the bidirectional pcbAB-pcbC promoter region from Acremonium chrysogenum using reporter gene fusions. Appl. Microbiol. Biotechnol. 1994, 42, 57–66. [Google Scholar] [CrossRef] [PubMed]
- Yin, J.; Deng, Z.X.; Zhao, G.P.; Huang, X. The N-terminal nucleophile serine of cephalosporin acylase executes the second autoproteolytic cleavage and acylpeptide hydrolysis. J. Biol. Chem. 2011, 286, 24476–24486. [Google Scholar] [CrossRef] [Green Version]
- Lee, Y.S.; Park, S.S. Two-step autocatalytic processing of the glutaryl 7-aminocephalosporanic acid acylase from Pseudomonas sp. strain GK16. J. Bacteriol. 1998, 180, 4576–4582. [Google Scholar] [CrossRef] [Green Version]
- Kim, S.; Kim, Y. Active site residues of cephalosporin acylase are critical not only for enzymatic catalysis but also for post-translational modification. J. Biol Chem 2001, 276, 48376–48381. [Google Scholar] [CrossRef] [Green Version]
- Pollegioni, L.; Lorenzi, S.; Rosini, E.; Marcone, G.L.; Molla, G.; Verga, R.; Cabri, W.; Pilone, M.S. Evolution of an acylase active on cephalosporin C. Protein Sci. 2005, 14, 3064–3076. [Google Scholar] [CrossRef] [Green Version]
- Crawford, L.; Stepan, A.M.; McAda, P.C.; Rambosek, J.A.; Conder, M.J.; Vinci, V.A.; Reeves, C.D. Production of cephalosporin intermediates by feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity. Biotechnol. (N. Y.) 1995, 13, 58–62. [Google Scholar] [CrossRef] [PubMed]
- Robin, J.; Jakobsen, M.; Beyer, M.; Noorman, H.; Nielsen, J. Physiological characterisation of Penicillium chrysogenum strains expressing the expandase gene from Streptomyces clavuligerus during batch cultivations. Growth and adipoyl-7-aminodeacetoxycephalosporanic acid production. Appl. Microbiol. Biotechnol. 2001, 57, 357–362. [Google Scholar] [CrossRef] [PubMed]
- Robin, J.; Bonneau, S.; Schipper, D.; Noorman, H.; Nielsen, J. Influence of the adipate and dissolved oxygen concentrations on the beta-lactam production during continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus. Metab. Eng. 2003, 5, 42–48. [Google Scholar] [CrossRef]
- Velasco, J.; Luis Adrio, J.; Ángel Moreno, M.; Díez, B.; Soler, G.; Luis Barredo, J. Environmentally safe production of 7-aminodeacetoxycephalosporanic acid (7-ADCA) using recombinant strains of Acremonium chrysogenum. Nat. Biotechnol. 2000, 18, 857–861. [Google Scholar] [CrossRef] [PubMed]
- Gutiérrez, S.; Díez, B.; Alvarez, E.; Barredo, J.L.; Martín, J.F. Expression of the penDE gene of Penicillium chrysogenum encoding isopenicillin N acyltransferase in Cephalosporium acremonium: Production of benzylpenicillin by the transformants. Mol. Genet. Genom. 1991, 225, 56–64. [Google Scholar] [CrossRef]
- Morita, S.; Kuriyama, M.; Nakatsu, M.; Kitano, K. High level expression of Fusarium alkaline protease gene in Acremonium chrysogenum. Biosci. Biotechnol. Biochem. 1994, 58, 627–630. [Google Scholar] [CrossRef]
- Zhgun, A.; Dumina, M.; Valiakhmetov, A.; Eldarov, M. The critical role of plasma membrane H+-ATPase activity in cephalosporin C biosynthesis of Acremonium chrysogenum. PLoS ONE 2020, 15, e0238452. [Google Scholar] [CrossRef]
- DeModena, J.A.; Gutierrez, S.; Velasco, J.; Fernandez, F.J.; Fachini, R.A.; Galazzo, J.L.; Hughes, D.E.; Martin, J.F. The production of cephalosporin C by Acremonium chrysogenum is improved by the intracellular expression of a bacterial hemoglobin. Biotechnol. (N. Y.) 1993, 11, 926–929. [Google Scholar] [CrossRef]
- Souza, P.M.d.; Bittencourt, M.L.d.A.; Caprara, C.C.; Freitas, M.d.; Almeida, R.P.C.d.; Silveira, D.; Fonseca, Y.M.; Ferreira, E.X.; Pessoa, A.; Magalhães, P.O. A biotechnology perspective of fungal proteases. Braz. J. Microbiol. 2015, 46, 337–346. [Google Scholar] [CrossRef] [Green Version]
- Honda, G.; Matsuda, A.; Zushi, M.; Yamamoto, S.; Komatsu, K.-i. Heterologous protein poroduction in Acremonium chrysogenum: Expression of bacterial cephalosporin C acylase and human thrombomodulin genes. Biosci. Biotechnol. Biochem. 1997, 61, 948–955. [Google Scholar] [CrossRef]
- Serrano-Amatriain, C.; Ledesma-Amaro, R.; López-Nicolás, R.; Ros, G.; Jiménez, A.; Revuelta, J.L. Folic acid production by engineered Ashbya gossypii. Metab. Eng. 2016, 38, 473–482. [Google Scholar] [CrossRef]
- Stahmann, K.-P.; Revuelta, J.; Seulberger, H. Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production. Appl. Microbiol. Biotechnol. 2000, 53, 509–516. [Google Scholar] [CrossRef]
- Förster, C. Biochemische und Molekularbiologische Charakterisierung des Riboflavintransports in Ashbya gossypii. Ph.D. Thesis, Forschungszentrum Jülich, Jülich, Germany, 1999. [Google Scholar]
- Förster, C.; Santos, M.A.; Ruffert, S.; Krämer, R.; Revuelta, J.L. Physiological consequence of disruption of the VMA1 gene in the riboflavin overproducer Ashbya gossypii. J. Biol. Chem. 1999, 274, 9442–9448. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Isogai, T.; Fukagawa, M.; Aramori, I.; Iwami, M.; Kojo, H.; Ono, T.; Ueda, Y.; Kohsaka, M.; Imanaka, H. Construction of a 7-aminocephalosporanic acid (7ACA) biosynthetic operon and direct production of 7-ACA in Acremonium chrysogenum. Nat. Biotechnol. 1991, 9, 188–191. [Google Scholar] [CrossRef] [PubMed]
- Liu, Y.; Gong, G.; Zhu, C.; Zhu, B.; Hu, Y. Environmentally safe production of 7-ACA by recombinant Acremonium chrysogenum. Curr. Microbiol. 2010, 61, 609–614. [Google Scholar] [CrossRef] [PubMed]
Strains | Genotypes | Source |
---|---|---|
A3/2 | Producer strain, nats | [29] |
XUL-4.1, -4.2, -4.3, -4.4, -4.5, -4.6, -4.7, -4.19, | A3/2, pXUL-4 (ccaA), natr | This work |
XUL-22.1, -22.2, -22.3, -22.4, -22.5, -22.6, -22.7 | A3/2, pXUL-22 (ccaB), natr | This work |
XUL-2.1, -2.2, -2.3, -2.4, -2.5, | A3/2, pXUL-2 (ccaC), natr | This work |
XUL-pAB-nat | A3/2, pAB-nat, natr | This work |
Plasmids | Genotypes | Source |
---|---|---|
pEX K248 >CCAA | HA::ccaA::HA, kanr, amps | This work (1) |
pEX-K248->CCAB | His::ccaB-HA, kanr, amps | This work (1) |
pEX-K168->CCAC | HA::ccaC::HA, kanr, amps | This work (1) |
pGG-C-EGFP | PgpdA::egfp::TtrpC, natr, kans, ampr | [35] |
pAB-nat | PgpdA::TtrpC, natr, kans, ampr | Modified from pGG-C-EGFP [36] |
pXUL-2 | PgpdA::HA::ccaC::HA::TtrpC, natr, kans, ampr | This work |
pXUL-4 | PgpdA::His::ccaA-HA::TtrpC, natr, kans, ampr | This work |
pXUL-10 | PgpdA::His::ccaA::HA::egfp::TtrpC, natr, kans, ampr | This work |
pXUL-22 | PgpdA::His::ccaB::HA::TtrpC, natr, kans, ampr | This work |
Name | Class | Source Strain | Predicted Molecular Weight (kDa) | Subunit Structure α + β (kDa) | Spacer (aa) | References |
---|---|---|---|---|---|---|
CCAA | I | Pseudomonas sp. GK16 | 79 | 19 + 60 | 10 | [51] |
CCAB | I | Pseudomonas sp. GK16 | 79 | 19 + 60 | 10 | Patent 2014, CN103937764B, (Amicogen Inc., Jinju, South Korea) |
CCAC | III | Pseudomonas sp. SE 83 (AcyII) | 84 | 24 + 60 | 10 | Patent 2009, US7592168B2, (Sandoz AG, Basel, Switzerland) |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Lin, X.; Lambertz, J.; Dahlmann, T.A.; Nowaczyk, M.M.; König, B.; Kück, U. A Straightforward Approach to Synthesize 7-Aminocephalosporanic Acid In Vivo in the Cephalosporin C Producer Acremonium chrysogenum. J. Fungi 2022, 8, 450. https://doi.org/10.3390/jof8050450
Lin X, Lambertz J, Dahlmann TA, Nowaczyk MM, König B, Kück U. A Straightforward Approach to Synthesize 7-Aminocephalosporanic Acid In Vivo in the Cephalosporin C Producer Acremonium chrysogenum. Journal of Fungi. 2022; 8(5):450. https://doi.org/10.3390/jof8050450
Chicago/Turabian StyleLin, Xuemei, Jan Lambertz, Tim A. Dahlmann, Marc M. Nowaczyk, Burghard König, and Ulrich Kück. 2022. "A Straightforward Approach to Synthesize 7-Aminocephalosporanic Acid In Vivo in the Cephalosporin C Producer Acremonium chrysogenum" Journal of Fungi 8, no. 5: 450. https://doi.org/10.3390/jof8050450
APA StyleLin, X., Lambertz, J., Dahlmann, T. A., Nowaczyk, M. M., König, B., & Kück, U. (2022). A Straightforward Approach to Synthesize 7-Aminocephalosporanic Acid In Vivo in the Cephalosporin C Producer Acremonium chrysogenum. Journal of Fungi, 8(5), 450. https://doi.org/10.3390/jof8050450