Something old, something new: challenges and developments in Aspergillus niger biotechnology

Abstract

The filamentous ascomycete fungus Aspergillus niger is a prolific secretor of organic acids, proteins, enzymes and secondary metabolites. Throughout the last century, biotechnologists have developed A. niger into a multipurpose cell factory with a product portfolio worth billions of dollars each year. Recent technological advances, from genome editing to other molecular and omics tools, promise to revolutionize our understanding of A. niger biology, ultimately to increase efficiency of existing industrial applications or even to make entirely new products. However, various challenges to this biotechnological vision, many several decades old, still limit applications of this fungus. These include an inability to tightly control A. niger growth for optimal productivity, and a lack of high-throughput cultivation conditions for mutant screening. In this mini-review, we summarize the current state-of-the-art for A. niger biotechnology with special focus on organic acids (citric acid, malic acid, gluconic acid and itaconic acid), secreted proteins and secondary metabolites, and discuss how new technological developments can be applied to comprehensively address a variety of old and persistent challenges.

Keywords: Aspergillus niger; citric acid; glucoamylase; macromorphology; micro-computer tomography; secondary metabolite.

Figure 1. Technological and biological highlights from 100 years of A. niger biotechnology

Figure 2. Current and future A. niger strain engineering cycles

The cycle begins with a candidate gene list(s) for functional analysis derived from previous datasets/experiments. Numbers in parentheses denote either the total amount of time for each step (months per scientist, M) or an exemplar number of genes remaining in each step (e.g., n = 50 candidate genes). Refinement of functional predictions is now possible by publicly available coexpression resources for A. niger. Mutants can be generated in-house using kusA mutants/genome editing technology, or in future from acquiring isolates from a mutant library resource (dotted arrow). Next, preliminary fermentation can be conducted, currently in shake flask, but in future via MTP cultivation. Putative hyperproducers (which in this example has generated 1-5 isolates) can then be tested via extensive design of experiment (DoE) approaches or, in future, by functional analysis in pre-generated chassis strains with a defined macromorphology. Pilot production strains can then be further analysed for another iteration of strain engineering, or taken for scale up during trial industrial production. This engineering cycle is similar for other industrially used filamentous fungi, both with regards to the main steps, time-frames, and major bottle-necks.