Production of tissue plasminogen activator (t-PA) in Aspergillus niger

A protease-deficient strain of Aspergillus niger has been used as a host fo r the production of human tissue plasminogen activator (t-PA). In defined m edium, up to 0.07 mg t-PA (g biomass)(-1) was produced in batch and fed-bat ch cultures and production was increased two- to threefold in two-phase bat ch cultures in which additional glucose was provided as a single pulse at t he end of the first batch growth phase. Production was increased [up to 1.9 mg t-PA (g biomass)(-1)] by the addition of soy peptone to the defined med ium. The rate of t-PA production in batch cultures supplemented with soy pe ptone (0.2 to 0.6 mg t-PA L-1 h(-1)) was comparable to rates observed previ ously in high-producing mammalian or insect cell cultures. In glucose-limit ed chemostat culture supplemented with soy peptone, t-PA was produced at a rate of 0.7 mg t-PA L-1 h(-1). Expression of t-PA in A. niger resulted in i ncreased expression of genes (bipA, pdiA, and cypB) involved in the unfolde d protein response (UPR). However, when cypB was overexpressed in a t-PA-pr oducing strain, t-PA production was not increased. The t-PA produced in A. niger was cleaved into two chains of similar molecular weight to two-chain human melanoma t-PA. The two chains appeared to be stable for at least 16 h in culture supernatant of the host strain. However, in general, <1% of the t-PA produced in A. niger was active, and active t-PA disappeared from the culture supernatant during the stationary phase of batch cultures, suggest ing that the two-chain t-PA may have been incorrectly processed or that ini tial proteolytic cleavage occurred within the proteolytic domain of the pro tein. Total t-PA (detected by enzyme-linked immunoassay) also eventually di sappeared from culture supernatants, confirming significant extracellular p roteolytic activity, even though the host strain was protease-deficient. (C ) 2001 John Wiley & Sons, Inc.