The metabolic switch can be activated in a recombinant strain of Streptomyces lividans by a low oxygen transfer rate in shake flasks

Abstract

Background: In Streptomyces, understanding the switch from primary to secondary metabolism is important for maximizing the production of secondary metabolites such as antibiotics, as well as for optimizing recombinant glycoprotein production. Differences in Streptomyces lividans bacterial aggregation as well as recombinant glycoprotein production and O-mannosylation have been reported due to modifications in the shake flask design. We hypothetized that such differences are related to the metabolic switch that occurs under oxygen-limiting conditions in the cultures.

Results: Shake flask design was found to affect undecylprodigiosin (RED, a marker of secondary metabolism) production; the RED yield was 12 and 385 times greater in conventional normal Erlenmeyer flasks (NF) than in baffled flasks (BF) and coiled flasks (CF), respectively. In addition, oxygen transfer rates (OTR) and carbon dioxide transfer rates were almost 15 times greater in cultures in CF and BF as compared with those in NF. Based on these data, we obtained respiration quotients (RQ) consistent with aerobic metabolism for CF and BF, but an RQ suggestive of anaerobic metabolism for NF.

Conclusion: Although the metabolic switch is usually related to limitations in phosphate and nitrogen in Streptomyces sp., our results reveal that it can also be activated by low OTR, dramatically affecting recombinant glycoprotein production and O-mannosylation and increasing RED synthesis in the process.

Keywords: Metabolic switch; Orbital shaking; Oxygen transfer rate; Recombinant glycoproteins; Shaken bioreactors; Streptomyces lividans; Undecylprodigiosin.

Fig. 1

Kinetics of recombinant S. lividans producing rAPA from M. tuberculosis in conventional normal (NF, squares), coiled (CF, circles), and baffled (BF, triangles) shake flask cultures. a Biomass dry weight growth; the inset presents the growth by a logarithmic axis. b Characteristic dissolved oxygen tension (DOT) trends in NF (continuous lane), BF (dotted lane), and CF (dashed line). c Undecylprodigiosin (RED) production. d Oxygen transfer rate (OTR) trends. e Carbon transfer rate (CTR) trends. f Respiration quotient (RQ). All cultures were carried out at 30 °C, 150 rpm, and 2.5 cm orbital-shaking diameter, in 250-mL shake flasks with 50-mL filling volume. Symbols represent the median and the standard deviation of at least three independents experiments

Fig. 2

ATR-FTIR structural analysis of undecylprodigiosin. a Standard prodigiosin spectrum (C₂₀H₂₅N_3O M.W. 323.44 g/mol, Merck-Sigma-Aldrich, Darmstadt, Germany). b ATR-FTIR spectrum from NF, BF, and CF cultures; names above each peak indicate the functional group, CF (dashed line), BF (dotted line), and NF (continuous line). c HPLC: Elution profile of undecylprodigiosin, CF in dashed line, BF in dotted line, and NF in continuous line. (Inset: Standard prodigiosin, Merck-Sigma-Aldrich, Darmstadt, Germany)

Fig. 3

Metabolic pathway proposed when no oxygen limitation (solid line) or oxygen limitation (dotted line) occurs in recombinant S. lividans cultures