Catabolite repression of phosphoenolpyruvate carboxykinase by a zinc finger protein under biotin- and pyruvate carboxylase-deficient conditions in Pichia pastoris

Abstract

We have identified a methanol-and biotin-starvation-inducible zinc finger protein named ROP [repressor of phosphoenolpyruvate carboxykinase (PEPCK)] in the methylotrophic yeast Pichia pastoris. When P. pastoris strain GS115 (wild-type, WT) is cultured in biotin-deficient, glucoseammonium (Bio " ) medium, growth is suppressed due to the inhibition of anaplerotic synthesis of oxaloacetate, catalysed by the biotin-dependent enzyme pyruvate carboxylase (PC). Deletion of ROP results in a strain (DROP) that can grow under biotin-deficient conditions due to derepression of a biotin-and PC-independent pathway of anaplerotic synthesis of oxaloacetate. Northern analysis as well as microarray expression profiling of RNA isolated from WT and DROP strains cultured in Bio " medium indicate that expression of the phosphoenolpyruvate carboxykinase gene (PEPCK) is induced in DROP during biotin-or PC-deficiency even under glucose-abundant conditions. There is an excellent correlation between PEPCK expression and growth of DROP in Bio " medium, suggesting that ROP-mediated regulation of PEPCK may have a crucial role in the biotin-and PC-independent growth of the DROP strain. To our knowledge, ROP is the first example of a zinc finger transcription factor involved in the catabolite repression of PEPCK in yeast cells cultured under biotin-or PC-deficient and glucose-abundant conditions.

Computational codon optimization of synthetic gene for protein expression

“…In this sense, we used published microarray data of E. coli [57], L. lactis [58], P. pastoris [59] and S. cerevisiae [60] from various experimental conditions to identify the top 5% of genes with the highest expression value for each microbe. The ICU and CC of these genes were then extracted from their corresponding DNA coding sequences that can be obtained from publicly available genome annotations for E. coli [61], L. lactis [62], P. pastoris [63] and S. cerevisiae [64].…”

Methylotrophy of Pichia pastoris: Current Advances, Applications, and Future Perspectives for Methanol-Based Biomanufacturing

“…Mxr1p has a zinc finger DNA-binding domain, which binds to Mxr1p response elements (MXREs) in the promoters of methanol utilization pathway genes to activate their expression. , Mxr1 mutations make the strain unable to grow on methanol medium . Rop1p is also a zinc finger protein with the same DNA binding specificity as Mxr1p and represses the expression of genes related to the methanol utilization pathway in P. pastoris when cultured in a nutrient-rich medium . Trm1p, a Zn­(II) 2 Cys 6 -type transcription factor, is also a key regulator for controlling gene expression of the methanol utilization pathway in P. pastoris.…”