A protease-deficient strain of Aspergillus niger has been used as a host for the production of human tissue plasminogen activator (t-PA). In defined medium, up to 0.07 mg t-PA (g biomass)(-1) was produced in batch and fed-batch cultures and production was increased two- to threefold in two-phase batch cultures in which additional glucose was provided as a single pulse at the 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 medium. The rate of t-PA production in batch cultures supplemented with soy peptone (0.2 to 0.6 mg t-PA L(-1) h(-1)) was comparable to rates observed previously in high-producing mammalian or insect cell cultures. In glucose-limited 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 increased expression of genes (bipA, pdiA, and cypB) involved in the unfolded protein response (UPR). However, when cypB was overexpressed in a t-PA-producing 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, suggesting that the two-chain t-PA may have been incorrectly processed or that initial proteolytic cleavage occurred within the proteolytic domain of the protein. Total t-PA (detected by enzyme-linked immunoassay) also eventually disappeared from culture supernatants, confirming significant extracellular proteolytic activity, even though the host strain was protease-deficient.

The life cycle of Streptomyces coelicolor during development on solid medium has been studied from a physiological perspective. A biphasic growth pattern was demonstrated, evidenced by a continuous transition from an initial exponential growth period into a slower phase of biomass accretion. The switch between the two phases coincided with the exhaustion of nitrate from the medium. The depletion of nitrate from the medium coincided with the initiation of aerial mycelium formation within the cultures and the development of hydrophobic surface properties. During secondary growth, cultures remained metabolically active, continuing to accumulate DNA, despite a cessation in the levels of RNA and cell protein accretion. In addition, the accumulation of glycogen and lipid contributed to the observed accretion of biomass in this phase. The depletion of nitrate also marked an increase in the production of a-ketoglutarate by the culture and a coincident decrease in medium pH. Latter stages of the secondary growth phase saw the development of spores within the culture, this in turn was associated with a decrease in cellular glycogen. This supported previous observations that glycogen degradation and spore maturation were intimately associated.

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