Transcriptomic insights into the physiology of Aspergillus niger approaching a specific growth rate of zero

Abstract

The physiology of filamentous fungi at growth rates approaching zero has been subject to limited study and exploitation. With the aim of uncoupling product formation from growth, we have revisited and improved the retentostat cultivation method for Aspergillus niger. A new retention device was designed allowing reliable and nearly complete cell retention even at high flow rates. Transcriptomic analysis was used to explore the potential for product formation at very low specific growth rates. The carbon- and energy-limited retentostat cultures were highly reproducible. While the specific growth rate approached zero (<0.005 h(-1)), the growth yield stabilized at a minimum (0.20 g of dry weight per g of maltose). The severe limitation led to asexual differentiation, and the supplied substrate was used for spore formation and secondary metabolism. Three physiologically distinct phases of the retentostat cultures were subjected to genome-wide transcriptomic analysis. The severe substrate limitation and sporulation were clearly reflected in the transcriptome. The transition from vegetative to reproductive growth was characterized by downregulation of genes encoding secreted substrate hydrolases and cell cycle genes and upregulation of many genes encoding secreted small cysteine-rich proteins and secondary metabolism genes. Transcription of known secretory pathway genes suggests that A. niger becomes adapted to secretion of small cysteine-rich proteins. The perspective is that A. niger cultures as they approach a zero growth rate can be used as a cell factory for production of secondary metabolites and cysteine-rich proteins. We propose that the improved retentostat method can be used in fundamental studies of differentiation and is applicable to filamentous fungi in general.

FIG. 1.

Cell retention device for in situ filtration of mycelial culture broth. (A) Scaled picture of filter components: housing (h), mesh (m), frame nut (f), septum (s), air inlet (a), and drain (d) for continuous removal of filtrate (effluent). (B) Schematic illustration of separation principle: a, adjustable cell retention device; b, antifouling mechanism regulated by a computer controlled solenoid valve. (C) Comparison of pore size (51 μm) and organism.

FIG. 2.

Morphological differentiation of A. niger N402 in maltose-limited retentostat cultures. (A) Mycelium from exponentially growing culture. Scale bar, 50 μm. (B) Mycelium after 1 day of substrate-limited growth. Scale bar, 50 μm. (C) SEM of conidiospore-forming mycelium after 8 days of substrate-limited growth. Scale bar, 10 μm. (D) TEM showing compartmentalization within a mycelium after 8 days. Arrow, septum plugged with Woronin body. (E) TEM of conidiogenous cell and newly formed conidium (arrow).

FIG. 3.

Physiological and molecular differentiation of A. niger N402 in maltose-limited retentostat cultures. (A) Biomass accumulation in three replicate cultures (□, Odense, Denmark; ○ and ▵, Leiden, Netherlands) and the specific growth rate (μ; solid line). The dashed line indicates maximum theoretical maintained cell density calculated from a maintenance requirement of 0.026 g g_DW⁻¹ h⁻¹; the three arrows mark time points used in the transcriptomic analysis. (B) Accumulation of cell protein (□), total RNA (○), and melanin (▴) in a retentostat culture (per kg of culture); the dashed line marks a transition in biomass composition. (C) Venn diagram showing the number of genes differentially expressed (4,251 genes in total) in transcriptomic comparisons of 0, 2, and 8 days of retentostat cultivation. (D) Northern blot analyses of the central regulator of conidiation, brlA (An01g10540), and the most highly transcribed reproductive and vegetative genes, respectively, a hydrophobin gene (An08g09880) and the glucoamylase gene (glaA [An03g06550]). Exposure time was 5 h for all three genes, 1 h for the first 18S rRNA panel (brlA comparison), and overnight for the second 18S rRNA panel.