Differential proteomic analysis highlights metabolic strategies associated with balhimycin production in Amycolatopsis balhimycina chemostat cultivations

Gallo, Giuseppe; Alduina, Rosa; Renzone, Giovanni; Thykaer, Jette; Bianco, Linda; Eliasson-Lantz, Anna; Scaloni, Andrea; Puglia, Anna Maria
January 2010
Microbial Cell Factories;2010, Vol. 9, p95
Academic Journal
Background: Proteomics was recently used to reveal enzymes whose expression is associated with the production of the glycopeptide antibiotic balhimycin in Amycolatopsis balhimycina batch cultivations. Combining chemostat fermentation technology, where cells proliferate with constant parameters in a highly reproducible steady-state, and differential proteomics, the relationships between physiological status and metabolic pathways during antibiotic producing and non-producing conditions could be highlighted. Results: Two minimal defined media, one with low Pi (0.6 mM; LP) and proficient glucose (12 g/l) concentrations and the other one with high Pi (1.8 mM) and limiting (6 g/l; LG) glucose concentrations, were developed to promote and repress antibiotic production, respectively, in A. balhimycina chemostat cultivations. Applying the same dilution rate (0.03 h-1), both LG and LP chemostat cultivations showed a stable steady-state where biomass production yield coefficients, calculated on glucose consumption, were 0.38 ± 0.02 and 0.33 ± 0.02 g/g (biomass dry weight/glucose), respectively. Notably, balhimycin was detected only in LP, where quantitative RT-PCR revealed upregulation of selected bal genes, devoted to balhimycin biosynthesis, and of phoP, phoR, pstS and phoD, known to be associated to Pi limitation stress response. 2D-Differential Gel Electrophoresis (DIGE) and protein identification, performed by mass spectrometry and computer-assisted 2 D reference-map http://www.unipa.it/ ampuglia/Abal-proteome-maps matching, demonstrated a differential expression for proteins involved in many metabolic pathways or cellular processes, including central carbon and phosphate metabolism. Interestingly, proteins playing a key role in generation of primary metabolism intermediates and cofactors required for balhimycin biosynthesis were upregulated in LP. Finally, a bioinformatic approach showed PHO box-like regulatory elements in the upstream regions of nine differentially expressed genes, among which two were tested by electrophoresis mobility shift assays (EMSA). Conclusion: In the two chemostat conditions, used to generate biomass for proteomic analysis, mycelia grew with the same rate and with similar glucose-biomass conversion efficiencies. Global gene expression analysis revealed a differential metabolic adaptation, highlighting strategies for energetic supply and biosynthesis of metabolic intermediates required for biomass production and, in LP, for balhimycin biosynthesis. These data, confirming a relationship between primary metabolism and antibiotic production, could be used to increase antibiotic yield both by rational genetic engineering and fermentation processes improvement.


Related Articles

  • Developments in the use of Bacillus species for industrial production. Schallmey, Marcus; Singh, Ajay; Ward, Owen P. // Canadian Journal of Microbiology;Jan2004, Vol. 50 Issue 1, p1 

    Bacillus species continue to be dominant bacterial workhorses in microbial fermentations. Bacillus subtilis (natto) is the key microbial participant in the ongoing production of the soya-based traditional natto fermentation, and some Bacillus species are on the Food and Drug Administration's...

  • Evidence from proteomics that some of the enzymes of actinorhodin biosynthesis have more than one form and may occupy distinctive cellular locations. Hesketh, Andrew; Chater, Keith F. // Journal of Industrial Microbiology & Biotechnology;Aug2003, Vol. 30 Issue 8, p523 

    An important attribute of proteome analysis carried out with the aid of two-dimensional gel electro-phoresis is that post-translational modifications of proteins can often be revealed. Large-scale proteomic analysis of Streptomyces coelicolor A3(2) has been made possible with the availability of...

  • Role of extracellular protease in nitrogen substrate management during antibiotic fermentation: a process model and experimental validation. Bapat, Prashant M.; Sinha, Avinash; Wangikar, Pramod P. // Applied Microbiology & Biotechnology;Aug2011, Vol. 91 Issue 4, p1019 

    Kinetics of extracellular protease (ECP) production has typically been studied for processes that involve protease as a product. We argue that ECP is equally important in fermentations where protease is not a product of interest. Industrial fermentations typically use complex nitrogen...

  • Lipase production by recombinant strains ofAspergillus nigerexpressing a lipase-encoding gene fromThermomyces lanuginosus. Prathumpai, Wai; Flitter, Simon J.; McIntyre, Mhairi; Nielsen, Jens // Applied Microbiology & Biotechnology;Oct2004, Vol. 65 Issue 6, p714 

    Two recombinant strains ofAspergillus niger(NW 297-14 and NW297-24) producing a heterologous lipase fromThermomyces lanuginosuswere constructed. The heterologous lipase was expressed using the TAKA amylase promoter fromAspergillus oryzae. The production kinetics of the two strains on different...

  • A physiological and enzymatic study of Debaryomyces hansenii growth on xylose- and oxygen-limited chemostats. Nobre, A.; Duarte, L. C.; Roseiro, J. C.; Gírio, F. M. // Applied Microbiology & Biotechnology;Aug2002, Vol. 59 Issue 4/5, p509 

    The effect of changing growth rate and oxygen transfer rate (OTR) on Debaryomyces hansenii physiology was studied using xylose-limited and oxygen-limited chemostat cultures, respectively, and complemented with enzymatic assays. Under xylose-limited chemostat (oxygen-excess), neither ethanol nor...

  • Optimization of Physical and Nutritional Parameters for Hyaluronidase Production by Streptococcus mitis. SAHOO, S.; PANDA, P. K.; MISHRA, S. R.; NAYAK, A.; DASH, S. K.; ELLAIAH, P. // Indian Journal of Pharmaceutical Sciences;2008, Vol. 70 Issue 5, p661 

    The effect of some physical and nutritional parameters were studied for the optimum production of extracellular enzyme hyaluronidase employing Streptococcus mitis MTCC*2695 by submerged fermentation. The effects of initial pH, incubation temperature and time, inoculum level and age of inoculum...

  • Katı Faz Fermentasyon Yöntemi ile Enzim Ãœretimi. Konak, Ülgen İlknur; Karakaş, Barçın; Certel, Muharrem; Erem, Fundagül // Academic Food Journal / Akademik GIDA;Jul2012, Vol. 10 Issue 3, p84 

    As an alternative method to Submerged Fermentation (SmF) for specific applications, Solid State Fermentation (SSF) has been applied in many fields of science and industry due to the interest in SSF from researchers and manufacturers recently. In general, SSF is a well-adapted and cheaper process...

  • Proteomic Analysis and Label-Free Quantification of the Large Clostridium difficile Toxins. Moura, Hercules; Terilli, Rebecca R.; Woolfitt, Adrian R.; Williamson, Yulanda M.; Wagner, Glauber; Blake, Thomas A.; Solano, Maria I.; Barr, John R. // International Journal of Proteomics;2013, p1 

    Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitals worldwide, due to hypervirulent epidemic strains with the ability to produce increased quantities of the large toxins TcdA and TcdB. Unfortunately, accurate quantification of TcdA and TcdB from different...

  • The effect of decreasing oxygen feed rates on growth and metabolism ofTorulaspora delbrueckii. Hanl, Lars; Sommer, Peter; Arneborg, Nils // Applied Microbiology & Biotechnology;Apr2005, Vol. 67 Issue 1, p113 

    The effect of decreasing oxygen feed rates on the growth and metabolism ofTorulaspora delbrueckiiandSaccharomyces cerevisiaein chemostat cultures was investigated. The biosynthetic oxygen requirement, i.e. the minimum specific oxygen consumption rate required for steady-state growth at a...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics