Zhongyi Yuan

Effects of different glycerol feeding strategies on S-adenosyl-l-methionine biosynthesis by PGAP-driven Pichia pastoris overexpressing methionine adenosyltransferase

Methionine adenosyltransferase (MAT) was overexpressed within Pichia pastoris employing the promoter of glyceraldehyde-3-phosphate dehydrogenase gene (P(GAP)), to biosynthesize S-adenosyl-l-methionine (SAM). Effects of five glycerol feeding tactics on MAT activity were first investigated. Strategies A-C were based on limited feeding correlated with dissolved oxygen (DO) at 50.0%, 25.0% and 0.0%, respectively. For strategies D and E, unlimited supplementation was executed by pulsed feeding mode. Gradual decline (2-0%) (w:v) of the residual glycerol level was shown between any two pulses in strategy D, while a nearly stable content (2%) throughout fed-batch cultivation with strategy E. With shifting strategies A-E in alphabetical order, gradual improvements of MAT activities were achieved, with the maximum of 9.05Ug(-1) dried biomass for strategy E, since the specific glycerol consumption rate (F(G)) ascended due to the elevated specific oxygen uptake rate (qO(2)). The success was ascribed to the enhancement of oxygen transfer rate (OTR), because 2% glycerol improved oxygen saturation content in broth (C*) and volumetric oxygen transfer coefficient (k(L)a). Strategy E also led to the highest values of ATP and biomass besides MAT. Consequently, the highest SAM yield and volumetric level were obtained at 0.058gg(-1) and 9.26gl(-1), respectively.

An alkaline pH control strategy for methionine adenosyltransferase production in Pichia pastoris fermentation

Pichia pastoris is a successful system for expressing heterologous proteins and its fermentation pH is always maintained below 7.0. However, particular proteins are unstable under acidic conditions, such as methionine adenosyltransferase (MAT), and thus fermentation under acidic pH conditions is unsuitable because protein activity is lost owing to denaturation. Here, a strategy employing alkaline pH in the late fermentation period was developed to improve MAT production. Initially, P. pastoris KM71 was transformed with the mat gene to overexpress MAT. After 72 h of in vitro incubation at different pH values, the expressed MAT displayed highest stability at pH 8.0; however, pH 8.0 inhibited cell growth and induced cell rupture, thus affecting protein production. To balance MAT stability and Pichia cell viability, different pH control strategies were compared. In strategy A (reference), the induction pH was maintained at 6.0, whereas in strategy B, it was gradually elevated to 8.0 through a 25 h transition period (80 ∼ 105 h). MAT activity was 0.86 U/mg (twofold higher than the control). However, MAT content was reduced by 50% when compared with strategy A, because of proteases released upon cell lysis. To improve cell viability under alkaline conditions, glycerol was added in addition to methanol (strategy C). When compared with strategy B, the MAT-specific activity remained nearly constant, whereas the expression level increased to 1.27 g/L. The alkaline pH control strategy presented herein for MAT production represents an excellent alternative for expressing proteins that are stable only under alkaline conditions.