Catabolite Repression of Aox inPichia pastorisIs Dependent on Hexose Transporter PpHxt1 and Pexophagy

Abstract

In this work, the identification and characterization of two hexose transporter homologs in the methylotrophic yeast Pichia pastoris, P. pastoris Hxt1 (PpHxt1) and PpHxt2, are described. When expressed in a Saccharomyces cerevisiae hxt-null mutant strain that is unable to take up monosaccharides, either protein restored growth on glucose or fructose. Both PpHXT genes are transcriptionally regulated by glucose. Transcript levels of PpHXT1 are induced by high levels of glucose, whereas transcript levels of PpHXT2 are relatively lower and are fully induced by low levels of glucose. In addition, PpHxt2 plays an important role in glycolysis-dependent fermentative growth, since PpHxt2 is essential for growth on glucose or fructose when respiration is inhibited. Notably, we firstly found that the deletion of PpHXT1, but not PpHXT2, leads to the induced expression of the alcohol oxidase I gene (AOX1) in response to glucose or fructose. We also elucidated that a sharp dropping of the sugar-induced expression level of Aox at a later growth phase is caused mainly by pexophagy, a degradation pathway in methylotrophic yeast. The sugar-inducible AOX1 promoter in an ⌬hxt1 strain may be promising as a host for the expression of heterologous proteins. The functional analysis of these two hexose transporters is the first step in elucidating the mechanisms of sugar metabolism and catabolite repression in P. pastoris.Pichia pastoris, a methylotrophic yeast, has been developed as a successful expression platform for heterologous proteins. The increasing popularity of this particular expression system can be attributed to several reasons (3, 6): (i) the easy techniques needed for the molecular genetic manipulation and the simple growth medium or culture conditions required for growth compared with those for mammalian cells; (ii) high levels of protein expression at the intra-or extracellular level; (iii) the ability to perform higher-eukaryotic protein modifications, such as glycosylation, disulfide bond formation, and proteolytic processing; and (iv) the availability of the expression system as a commercially available kit.In the P. pastoris system, the expression of foreign genes is usually driven by the outstanding promoter of the alcohol oxidase I gene (AOX1), which encodes the first enzyme in the methanol utilization pathway. The expression of Aox can be regulated with both repression and derepression mechanisms responding to different carbon sources. The AOX1 promoter (P AOX1 ) is induced only in response to methanol and repressed by other carbon sources, such as glucose or ethanol (3, 5, 6). However, the P AOX1 -based platform is not free of drawbacks (12, 23). The strength of induction of P AOX1 is strictly dependent on methanol as the carbon source. Methanol is derived mainly from petrochemical sources, which is unsuitable for use in the production of certain food products and additives. Methanol metabolism in methylotrophic yeast produces a toxic byproduct, hydrogen peroxide (H 2 O 2 ), which causes oxidative stress and eli...

Identification and characterization of novel promoters for recombinant protein production in yeast Pichia pastoris

“…Genomic DNA was removed and cDNA was synthesized using ReverTra Ace quantitative PCR RT Kit (Toyobo). Quantitative PCR was carried out as described previously using primers (Table S1) designed by Beacon designer 7.9 (Zhang et al, ).…”

“…After cultured at 30°C for 4 h, cell pellets were harvested and subsequently used to isolate ReverTra Ace quantitative PCR RT Kit (Toyobo). Quantitative PCR was carried out as described previously using primers (Table S1) designed by Beacon designer 7.9 (Zhang et al, 2010).…”

Kinase Screening in Pichia pastoris Identified Promising Targets Involved in Cell Growth and Alcohol Oxidase 1 Promoter (PAOX1) Regulation

“…So far several protein factors have been reported to participate in P AOX1 regulation. Hexose sensor Gss1 [4] and transporter Hxt1 [5] were reported to function at the first stage of P AOX1 repression pathways when P . pastoris are cultured in glucose.…”

Control of specific growth rate for the enhanced production of human interferon α2b in glycoengineered Pichia pastoris: process analytical technology guided approach

“…Growth phase is characterized by the utilization of glycerol, which shows repressing effect on the AOX transcription elements. As a result it leads to the channelling of major carbon flux towards biomass formation with relatively lesser maintenance requirement . This can be observed from the higher biomass yields ( achieved at the end of glycerol batch phase as shown in Table .…”

“…As a result it leads to the channelling of major carbon flux towards biomass formation with relatively lesser maintenance requirement. 43 This can be observed from the higher biomass yields (Y X ∕ gly ) achieved at the end of glycerol batch phase as shown in Table 1. During the batch growth phases of pulsed feed strategy (glycerol as sole carbon source), significant biomass production was achieved (Table 1).…”