Submerged differentiation of Aspergillus niger in carbon-limited cultures approaching zero specific growth rate

This work presents an exploration of submerged differentiation of the ubiquitous saprophyte and industrially important fungus, Aspergillus niger, in response to a limited availability of a sole carbon and energy source, maltose. In aspergilli and other mold fungi, asexual reproduction through formation of elaborate conidiogenic structures normally requires an aerial interface. This requirement is bypassed in submerged culture in response to severe nutrient limitation. Continuous cultures with cell retention (retentostat cultures) were applied to generate a fundamental physiological state, where the specific growth rate approaches zero, as the density of the cell population adapts to the supply of the limiting energy source. Temporal differentiation of mycelium structure and commitment to asexual reproduction were major phenomena, apparent on biochemical, morphological, physiological, and transcriptomic level. The severe substrate limitation had a rapid negative impact on cytoplasmic processes, and promoted endo- and exogenous nutrient mobilization, and hyphal compartmentalisation. The first conidiogenic structures appeared after one day with little additional differentiation until Day 4 to 6, where a transition to full commitment to reproductive growth took place. Submerged conidiation in A. niger involved transcriptional regulation of homologs of the regulatory pathway, centered around the Bristle gene (brlA), and structural genes previously described in other aspergilli. Comparison of transcriptomes, revealed a number of co-regulated gene clusters, which appear to encode secondary metabolite biosynthetic potential. We discuss the concept of maintenance energy in the context of differentiation, a possible physiological trigger for sporulation and the special physiological adaptations of the starved mycelium. We also present a simple and efficient method for in situ retention of filamentous organisms. The dataset consists of 9 Affymetrix arrays derived from defined growth conditions of lab-scale bioreactor cultures (5L). Total RNA was extracted from biomass harvested at three different growth phases: exponential growth phase, 2 and 8 days of retentostat cultivation. For each of the phases, the data is derived from three biological replicates.

本研究针对无处不在的腐生菌、工业重要真菌黑曲霉（Aspergillus niger），在唯一碳源与能源麦芽糖供应受限条件下的浸没培养分化现象展开探索。在曲霉属及其他霉菌中，通过形成复杂产孢结构完成无性生殖通常依赖气生界面。但在浸没培养体系中，这一需求会因严重营养限制而被绕过。采用带细胞滞留的连续培养（retentostat cultures）体系，可构建基础生理状态：当细胞种群密度适配限制性能源供应时，比生长速率趋近于零。菌丝结构的时序分化与无性生殖的启动是核心研究现象，在生化、形态、生理及转录组层面均有显著体现。
严重的底物限制会快速对细胞质过程产生负面影响，并促进内源性与外源性营养物质的动员，以及菌丝分区化。首批产孢结构于培养1天后出现，后续分化进展缓慢，直至第4至6天，此时菌体完全转向生殖生长。黑曲霉的浸没产孢涉及以Bristle基因（brlA）为核心的调控通路同源基因，以及其他曲霉中已报道的结构基因的转录调控。转录组比较分析揭示了多组共调控基因簇，其似乎编码次生代谢物生物合成潜能。
本研究从分化视角探讨了维持能（maintenance energy）概念、孢子形成的潜在生理触发因子，以及饥饿菌丝体的特殊生理适应性。此外，我们提出了一种简便高效的丝状微生物原位滞留方法。本数据集包含9张Affymetrix基因芯片，均来自实验室规模5L生物反应器的特定培养条件。总RNA提取自三个不同生长阶段收获的菌体：指数生长阶段、滞留培养第2天及第8天。每个生长阶段均设置3次生物学重复。