Amino acid supplementation, controlled oxygen limitation and sequential double induction improves heterologous xylanase production by

Abstract

Heterologous endo-beta-1,4-xylanase was produced by Pichia stipitis under control of the hypoxia-inducible PsADH2-promoter in a high-cell-density culture. After promoter induction by a shift to oxygen limitation, different aeration rates (oxygen transfer rates) were applied while maintaining oxygen-limitation. Initially, enzyme production was higher in oxygen-limited cultures with high rates of oxygen transfer, although the maximum xylanase activity was not significantly influenced. Amino acid supplementation increased the production of the heterologous endo-beta-1,4-xylanase significantly in highly aerated oxygen-limited cultures, until glucose was depleted. A slight second induction of the promoter was observed in all cultures after the glucose had been consumed. The second induction was most obvious in amino acid-supplemented cultures with higher oxygen transfer rates during oxygen limitation. When such oxygen-limited cultures were shifted back to fully aerobic conditions, a significant re-induction of heterologous endo-beta-1,4-xylanase production was observed. Re-induction was accompanied by ethanol consumption. A similar protein production pattern was observed when cultures were first grown on ethanol as sole carbon source and subsequently glucose and oxygen limitation were applied. Thus, we present the first expression system in yeast with a sequential double-inducible promoter.

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

“…The use of an inducible promoter, such as AOX1 in P. pastoris, allows for the separation of the growth phase from the induction phase [40]. The general trend regarding recombinant expression by Pichia would appear to suggest that the highest titres of recombinant xylanase were attained following the induction of high cell density cultures [41].…”

Induction by Hypoxia of Heterologous-Protein Production with the Kl PDC1 Promoter in Yeasts

“…A similar system has already been proposed for the production of heterologous proteins with another nonconventional yeast, Pichia stipitis, using the oxygen-regulated ADH2 homologous promoter (19). As with our two-stage induction strategy, in this case the levels of heterologous production benefit from a small amount of air inlet (11). In this paper we demonstrate the possibility of a practical application of hypoxic induction of the KlPDC1 promoter (5) for the production of five different heterologous proteins in K. lactis.…”

Bioethanol a Microbial Biofuel Metabolite; New Insights of Yeasts Metabolic Engineering

“…P. stipitis xylanases allow the direct fermentation of xylan into ethanol, but with very low yields [94,95]. The overexpression of xylanase was achieved through a heterologous endoxylanase expression [95,[173][174][175], or through mutation [176], or by amino acid supplementation [177], which could improve the fermentation rate of xylan. Notably, S. cerevisiae genome does not encode for natural xylanase; however, several scientists have succeeded to engineer S. cerevisiae strains to express heterologously xylanases [175,178,179].…”

“…Notably, the production of xylitol by P. stipites is relatively very low, which accompanies high ethanol yields; however, the xylitol productions increase dramatically when alcohol dehydrogenase (ADH) gene is deleted [177,187]. This suggests that P. stipitis XDH competes with ADH for reductant, apparently NADH.…”