Trichoderma reesei was cultivated in chemostat cultures on lactose-containing medium. The cultures were characterized for growth, consumption of the carbon source and protein production. Secreted proteins were produced most efficiently at low specific growth rates, 0·022–0·033 h−1, the highest specific rate of total protein production being 4·1 mg g−1 h−1 at the specific growth rate 0·031 h−1. At low specific growth rates, up to 29 % of the proteins produced were extracellular, in comparison to only 6–8 % at high specific growth rates, 0·045–0·066 h−1. To analyse protein synthesis and secretion in more detail, metabolic labelling of proteins was applied to analyse production of the major secreted protein, cellobiohydrolase I (CBHI, Cel7A). Intracellular and extracellular labelled CBHI was quantified and analysed for pI isoforms in two-dimensional gels, and the synthesis and secretion rates of the molecule were determined. Both the specific rates of CBHI synthesis and secretion were highest at low specific growth rates, the optimum being at 0·031 h−1. However, at low specific growth rates the secretion rate/synthesis rate ratio was significantly lower than that at high specific growth rates, indicating that at low growth rates the capacity of cells to transport the protein becomes limiting. In accordance with the high level of protein production and limitation in the secretory capacity, the transcript levels of the unfolded protein response (UPR) target genes pdi1 and bip1 as well as the gene encoding the UPR transcription factor hac1 were induced.
Comparative analysis of early divergent land plants and construction of DNA tools for hyper-expression in Marchantia chloroplasts
