Host cell properties and external pH affect proinsulin production by Saccharomyces yeast

Abstract

The expression of a hybrid gene encoding an alpha-factor prepro leader peptide-miniproinsulin (MPI) fusion [MPI is the same as the LysArg human insulin precursor described by Thim et al. (1986)] was tested in a series of isogenic yeast strains to investigate the influence of some genetic and physiological factors on heterologous production in yeast. We found that: (i) an MF alpha 1 gene disruption in haploid cells, as well as MF alpha 1 gene product expression in diploid cells, do not affect the MPI secretion level; (ii) under conditions of exogenous leucine availability, MPI production is hindered by leucine auxotrophy (a leu2 mutation); (iii) rho- mutations increase the per-cell MPI yield approximately three-fold; (iv) the MPI yield is apparently dependent on the pH of the culture medium: the higher the external pH, the larger the per-cell MPI yield.

Comparison of three expression systems for heterologous xylanase production byS. cerevisiaein defined medium

“…The growth of the auxotrophic strain in the chemically defined medium was supported by adding the amino acids, histidine (165 mg/l), leucine (870 mg/l) and tryptophan (664 mg/l), resulting in a total amino acid concentration of 10.8 m M in the defined medium. The histidine and tryptophan amino acids were added to compensate for the presence of his3 and trp1 auxotrophic markers in the transformant, and leucine because of high consumption levels in defined medium previously reported (Albers, 2000; Kozlov et al , 1995; L. Gustafsson, Department of Molecular Biotechnology, Chalmers University of Technology, Gothenburg, Sweden). The concentrations of amino acids were selected by considering requirements for biomass formation (Greasham and Herber, 1997).…”

“…The presence of auxotrophic markers in transformant strains represents an aspect of host strain genetics that may have strong deleterious effects on heterologous protein production levels (Pronk, 2002). Excessive auxotrophic markers in transformed S. cerevisiae strains require the presence of the necessary metabolite in the cultivation medium, often resulting in overconsumption of the required metabolite and difficulties in growth, protein production and genetic stability (Mendoza‐Vega et al , 1994; VanDusen et al , 1997; Pronk, 2002; Chopra et al , 1999; Beretta et al , 1991; Korogodin et al , 1991; Çakar et al , 1999; Kozlov et al , 1995). Auxotrophic strains grown in nutrient supplemented medium are thus not necessarily physiologically equivalent to the complemented transformants, as mentioned in previous reports (Chopra et al , 1999; Pronk, 2002).…”

High-Level Production of Human Parathyroid Hormone (hPTH) by Induced Expression inSaccharomyces cerevisiae

“…Environmental parameters can influence the secretion capacity and the stability of a heterologous protein (13,14). To test such factors, the cells were grown at different conditions.…”

Inulase‐secreting strain of Saccharomyces cerevisiae produces fructose

“…7). It is not inconceivable that this exponential relationship was due to inhibition of inulase activity by fructose as we have observed the 10-fold reduction in (Kozlov et al, 1995) in which the EcoRI/BclI-region with the yeast 2 m replicon (ori) was deleted and the XbaI site was changed for the XhoI site using the XhoI linker. To create pINT-INU, this derivative was ligated with the BglII/XhoI fragment of the cloned INU1 Km gene (2 kb) via the PstI/BamHI connecting fragment (junction) encoding C-terminal region of SP extending to the potential cleavage site for the S. cerevisiae signal peptidase.…”

“…The rich media used were as follows: YEP medium (1% yeast extract and 2% bactopeptone) supplemented with 2% glucose (YEPD) or 2% inulin (YEPI) or 2% ethanol plus 3% glycerol plus inulin in varied amounts (2, 4, 6, 8, or 10%) (YEPEGI). The selective media were based on the synthetic minimal medium (Kozlov et al, 1995) supplemented with 2% glucose or 2% fructose. Cells were incubated at 30°C.…”