The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium.
Five recombinant S. cerevisiae strains were cultivated under identical conditions to quantify the molecular basis of the metabolic burden of heterologous gene expression, and to evaluate mechanisms for the metabolic burden. Two recombinant S. cerevisiae strains, producing Trichoderma reesei xylanase II under control of either the PGK1 or ADH2 promoters, were compared quantitatively with three references strains, where either the heterologous xylanase II (XYN2) gene, or the heterologous gene and the promoter and terminator were omitted from the recombinant plasmid. Neither the replication of multiple copies of the 2-microm-based YEp352 plasmid nor the replication the foreign XYN2 gene represented a metabolic burden to the cell, as the growth of the host organism was not affected. The inclusion of a glycolytic promoter on the recombinant plasmid, however, reduced the maximum specific growth rate (12% to 15%), biomass yield on glucose (8% to 11%), and specific glucose consumption rate (6% to 10%) of the recombinant strains. The presence of the heterologous XYN2 gene on the recombinant plasmid caused a further reduction in the maximum specific growth rate (11% to 14%), biomass yield (4%), and specific glucose consumption rate (12%) of the host strain during active gene expression, which was dictated by the regulatory characteristics of the promoter utilized. The metabolic effect of foreign gene expression was disproportionally large, with respect to on the amount of heterologous protein produced. This was most likely due to an increased energetic demand for the expression of a foreign gene and/or a competition for limiting amounts of transcription or translation factors, biosynthetic precursors or metabolic energy.
Görgens
J F
JF
Department of Applied Microbiology, Lund University, Box 124, S-221 00 Lund, Sweden.
van Zyl
W H
WH
Knoetze
J H
JH
Hahn-Hägerdal
B
B
D000426:Alcohol Dehydrogenase / Q000235:genetics; D018533:Biomass; D003470:Culture Media; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism*; D010735:Phosphoglycerate Kinase / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000096:biosynthesis* / Q000235:genetics
D003470:Culture Media; D000426:Alcohol Dehydrogenase; D010735:Phosphoglycerate Kinase; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D005947:Glucose