Document Type: Final File

Authors

1 1Department of Plant protection, Faculty of Sciences, University of Sari Agricultural Sciences and Natural Resources, Sari, Islamic Republic of Iran

2 2Genetic and Agricultural Biotechnology Institute of Tabarestan (GABIT), University of Sari Agricultural Sciences and Natural Resources, Sari, Islamic Republic of Iran

Abstract

In this study, after identification of genes involved in biosynthesis of penicillin, we evaluated the expression of pcbAB and pcbC genes in P.  Chrysogenum.  A quantitative PCR (qPCR) approach was used to determine how these genes were expressed in different time courses. In addition, the produced penicillin content was measured using HPLC. qPCR analysis of mRNAs extracted from P. chrysogenum indicated that the expression  levels  of  pcbAB,  pcbC increased in seven days after inoculation compared  to  the  expressed levels with others time courses, but this difference was not significant statistically. The analysis of chromatograms from injected showed that the highest content of penicillin in media observed in 7 days after cultivation, except in P. chrysogenum PTCC 5033.  In addition, the results of comparative analysis among the tested strains showed that the penicillin content in 5, 7 and 11 days after cultivation was highest in  P. chrysogenum PTCC 5037, P. chrysogenum PTCC 5031 and P. chrysogenum PTCC 5033, respectively. However, the results showed an evident relationship between the expression levels of penicillin biosynthesis genes and yielded penicillin.

Keywords

Main Subjects

1. Kozlovskii A.G., Antipova T.V., Zhelifonova V.P., Baskunov B.P., Kochkina G.A. and Ozerskaya S.M. Exometabolites of the fungal isolates (Genus Penicillium, Section Chrysogena) from low-temperature ecotopes. Microbiol. 85: 2. 157-164 (2016).

2. Veiga T., Gombert A.K., Landes N., Verhoeven M.D., Kiel J.A., Krikken A.M., Nijland J.G., Touw H., Luttik M.A., Van der Toorn J.C. and Driessen A.J. Metabolic engineering of β-oxidation in Penicillium chrysogenum for improved semi-synthetic cephalosporin biosynthesis. Metab. eng. 14: 4. 437-448 ( 2012).

3.  Abastabar M., Mirhendi H., Hedayati M.T., Shokohi T., Rezaei-Matehkolaei A., Mohammadi R., Badali H., Moazeni M., Haghani I., Ghojoghi A. and Akhtari J. Genetic and Morphological Diversity of the Genus Penicillium From Mazandaran and Tehran Provinces, Iran. Jundishapur J. Microbiol. 9: 1 (2016).

4.  Visagie C. M., Houbraken J., Dijksterhuis J., Seifert K. A., Jacobs K. and Samson R. A. A taxonomic review of Penicillium species producing conidiophores with solitary phialides, classified in section Torulomyces. Persoonia. 36: 134-155 (2016).

5.  Hassan M. E. production, immobilization and industrial uses of penicillin G acylase. J. Cur. Res. Rev. 8: 15. 11-22 (2016).

6.  Machida M., Asai K., Sano M., Tanaka T., Kumagai T., Terai G., Kusumoto K.-I., Arima T., Akita O. and Kashiwagi Y. Genome sequencing and analysis of Aspergillus oryzae, Nature. 438:  1157-1161 (2005).

7. Wortman J.R., Gilsenan J.M., Joardar V., Deegan J., Clutterbuck J., Andersen M.R., Archer D., Bencina M., Braus G. and Coutinho P. The 2008 update of the Aspergillus nidulans genome annotation: A community effort, Fungal. Genet. Biol. 46:  S2-S13 (2009).

8.  van den Berg M.A., Albang R., Albermann K., Badger J.H., Daran J.-M., Driessen A.J., Garcia-Estrada C., Fedorova N.D., Harris D.M. and Heijne W.H. Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum, Nature biotech. 26: 1161-1168 (2008).

9. Zhang S., Chen Z., Wen Q., Yang L., Wang W., and Zheng J. Effectiveness of bulking agents for co-composting penicillin mycelial dreg (PMD) and sewage sludge in pilot-scale system. Env. Sci. Poll. Res. 23: 2. 1362-1370 (2016).

10. Wu C., Zhao Y., Chen R., Liu D., Liu M., Proksch P. and Lin W. Phenolic metabolites from mangrove-associated Penicillium pinophilum fungus with lipid-lowering effects. RSC Adv. 6: 26. 21969-21978 (2016).

11. Golabgir A. and Herwig C. Combining Mechanistic Modeling and Raman Spectroscopy for Real-Time Monitoring of Fed-Batch Penicillin Production. Chem. Ing. Tech. 88: 6. 764-776 (2016).

12. Martin J.F., Ullan R.V. and García‐Estrada C.  Regulation and compartmentalization of β‐lactam biosynthesis. Microbial. Biotech. 3: 285-299 (2010).

13. Shwab E.K., Keller N.P. Regulation of secondary metabolite production in filamentous ascomycetes, Mycological research. 112: 225-230 (2008).

14. Keller N.P., Turner G. and Bennett J.W. Fungal secondary metabolism-from biochemistry to genomics, Nature Reviews Microbiol. 3:  937-947 (2005).

15. Demain A.L., Adrio J.L. Contributions of microorganisms to industrial biology. Molecul. biotech. 38: 41-55 (2008).

16. Manzoni M., Rollini M. Biosynthesis and biotechnological production of statins by filamentous fungi and application of these cholesterol-lowering drugs, App. Microb. Biotech. 58: 555-564 (2002).

17. Banani H., Marcet-Houben M., Ballester A. R., Abbruscato P., Gonzalez-Candelas L., Gabaldon T. and Spadaro D. Genome sequencing and secondary metabolism of the postharvest pathogen Penicillium griseofulvum. BMC gen. 17: 1. 1. (2016).

18. Song F., He H., Ma R., Xiao X., Wei Q., Wang Q. and Capon R. J. Structure revision of the Penicillium alkaloids haenamindole and citreoindole. Tetra. Lett. 57: 34. 3851-3852 (2016).

19. Frere J. M., Sauvage E. and Kerff F. From “An Enzyme Able to Destroy Penicillin” to Carbapenemases: 70 Years of Beta-lactamase Misbehaviour. Cur. Drg. tar. 17: 9. 974-982 (2016).

20. Fierro F., García-Estrada C., Castillo N.I., Rodriguez R., Velasco-Conde T. and Martin J.F. Transcriptional and bioinformatic analysis of the 56.8 kb DNA region amplified in tandem repeats containing the penicillin gene cluster in Penicillium chrysogenum, Fungal. Genet. Biol. 43:  618-629 (2006).

21.van den Berg M.A., Westerlaken I., Leeflang C., Kerkman R. and Bovenberg R.A. Functional characterization of the penicillin biosynthetic gene cluster of Penicillium chrysogenum Wisconsin54-1255. Fungal. Genet. Biol. 44: 830-844 (2007).

22. Scherlach K., Hertweck C. Triggering cryptic natural product biosynthesis in microorganisms. Org. Biomol. Chem. 7: 1753-1760 (2009).

23. Martin J.F. Molecular control of expression of penicillin biosynthesis genes in fungi: regulatory proteins interact with a bidirectional promoter region, J. bacteriol. 182: 2355-2362 (2000).

24. Garcia-Estrada C. and Martin J.F. Biosynthetic gene clusters for relevant secondary metabolites produced by Penicillium roqueforti in blue cheeses. App. Microb. Biotech. 100: 19. 8303-8313 (2016).

25. Hamza L.F., Kamal S.A. and Hameed I.H. Determination of metabolites products by Penicillium expansum and evaluating antimicobial activity. J. Pharma. Phyto. 7: 194-220 (2015).

26. Praube M. T., Schauble S., Guthke R. and Schuster S. Computing the various pathways of penicillin synthesis and their molar yields. Biotech. bioengin. 113: 1. 173-181 (2016).

27. Van Gulik W., De Laat W., Vinke J. and Heijnen J. Application of metabolic flux analysis for the identification of metabolic bottlenecks in the biosynthesis of penicillin‐G. Biotech. Bioengin. 68: 602-618 (2000).

28. VanGulik W., Antoniewicz M., DeLaat W., Vinke J. and Heijnen J. Energetics of growth and penicillin production in a high‐producing strain of Penicillium chrysogenum. Biotech. Bioengin. 72: 185-193 (2001).

29. Henriksen C.M., Holm S., Schipper D., Jorgensen H., Nielsen J. and Villadsen J. Kinetic studies on the carboxylation of 6-amino-penicillanic acid to 8-hydroxy-penillic acid. Pro. Biochem. 32: 85-91 (1997).

30. Barredo J.L., Diez B., Alvarez E. and Martin J.F. Large amplification of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high penicillin producing strains of Penicillium chrysogenum. Curr. genet. 16: 453-459 (1989).

31. Theilgaard H.A., van den Berg M.A., Mulder C.A., Bovenberg R.A. and Nielsen J. Quantitative analysis of Penicillium chrysogenum Wis54‐1255 transformants overexpressing the penicillin biosynthetic genes. Biotechnol. Bioengine. 72: 379-388 (2001).

 

32. Newbert R., Barton B., Greaves P., Harper J. and Turner G. Analysis of a commercially improved Penicillium chrysogenum strain series: involvement of recombinogenic regions in amplification and deletion of the penicillin biosynthesis gene cluster. J. Industrial. Microbiol. Biotech. 19: 18-27 (1997).

33. Kiel J.A., Van den Berg M.A., Fusetti F., Poolman B., Bovenberg R.A., Veenhuis M. and Van der Klei I.J. Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells. Funct. Integr. Genomics. 9: 167-184 (2009).

34. Jami M.-S., Barreiro C., Garcia-Estrada C. and Martin J.-F. Proteome Analysis of the Penicillin Producer Penicillium chrysogenum characterization of protein changes during the industrial strain improvement. Mol. Cell. Proteom. 9: 1182-1198 (2010).

35. Renno D.V., Saunders G., Bull A.T. and Holt G. Transcript analysis of penicillin genes from Penicillium chrysogenum. Curr. genet. 21: 49-54 (1992).

36. Petit P., Lucas E.M., Abreu L.M., Pfenning L.H. and Takahashi J.A. Novel antimicrobial secondary metabolites from a Penicillium sp. isolated from Brazilian cerrado soil. Electronic. J. Biotech. 12: 8-9 (2009).

37. Veerapagu M., Jeya K. and Ponmurugan K. Mutational effect of Penicillium chrysogenum on Antibiotic Production. Adv. Biotech. 3: 16-19 (2008).

38. Wang Y., Angelatos A.S., Dunstan D.E. and Caruso F. Infiltration of macromolecules into nanoporous silica particles. Macromol. 40: 7594-7600 (2007).

39. Agweyu A., Gathara D., Oliwa J., Muinga N., Edwards T., Allen E., Maleche-Obimbo E., English M., Aweyo F., Awuonda B. and Chabi M. Oral amoxicillin versus benzyl penicillin for severe pneumonia among Kenyan children: a pragmatic randomized controlled noninferiority trial. Clinic. Infect. Diseas. 60: 1216-1224 (2015).

40. Sibirny A.A. Yeast peroxisomes: structure, functions and biotechnological opportunities. FEMS yeast research. 16: 38-45 (2016).

41. Dembitsky Valery M. Naturally occurring bioactive Cyclobutane-containing (CBC) alkaloids in fungi, fungal endophytes, and plants. Phytomedic. 21: 1559-1581 (2014).

42. Vederas John C. Explorations of fungal biosynthesis of reduced polyketides–a personal viewpoint. Nature. Pro. rep. 31: 1253-1259 (2014).