Aspergillus Niger Research Timothy C
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Cairns et al. Fungal Biol Biotechnol (2018) 5:13 https://doi.org/10.1186/s40694-018-0054-5 Fungal Biology and Biotechnology REVIEW Open Access How a fungus shapes biotechnology: 100 years of Aspergillus niger research Timothy C. Cairns*, Corrado Nai* and Vera Meyer* Abstract In 1917, a food chemist named James Currie made a promising discovery: any strain of the flamentous mould Aspergillus niger would produce high concentrations of citric acid when grown in sugar medium. This tricarboxylic acid, which we now know is an intermediate of the Krebs cycle, had previously been extracted from citrus fruits for applications in food and beverage production. Two years after Currie’s discovery, industrial-level production using A. niger began, the biochemical fermentation industry started to fourish, and industrial biotechnology was born. A century later, citric acid production using this mould is a multi-billion dollar industry, with A. niger additionally produc- ing a diverse range of proteins, enzymes and secondary metabolites. In this review, we assess main developments in the feld of A. niger biology over the last 100 years and highlight scientifc breakthroughs and discoveries which were infuential for both basic and applied fungal research in and outside the A. niger community. We give special focus to two developments of the last decade: systems biology and genome editing. We also summarize the current interna- tional A. niger research community, and end by speculating on the future of fundamental research on this fascinating fungus and its exploitation in industrial biotechnology. Keywords: Aspergillus niger, Biotechnology, Industrial biology, Systems biology, Genome editing, Citric acid Introduction €4.7 billion, which was expected to reach up to €10 bil- For millennia, humanity has practiced rudimental forms lion within the next decade [2]. In this celebratory his- of biotechnology: by fermenting starch and sugars pre- torical overview, we outline some of the crucial advances sent in grains and fruits, ancient civilizations were able for the flamentous mould Aspergillus niger since the to produce bread, beer, wine, and other alcoholic bever- very frst biotechnological experiments using this fungus ages. Prior to the late nineteenth and early twentieth cen- 100 years ago. tury, these processes were conducted without knowledge of the underlying biological events. Now, brewery and 100 years ago: industrial biotech is born wine making is a well-understood, controlled industrial In contrast to what most people might think, citric acid process. Similarly, in just a century, industrial biotechnol- is not—or not anymore—isolated from citrus fruits, but ogy has changed dramatically and fourished, from initial is industrially produced by the flamentous fungus A. proof-of-principle experimentation in Erlenmeyer fasks, niger. Te process was pioneered by James Currie, a food to a multibillion dollar industry producing megatons of chemist, who 100 years ago published a study describ- useful molecules [1]. Fungal biotechnology is undoubt- ing the superior properties of A. niger for the industrial edly a major contributor and driver of this success. As production of the acid [3]. In particular, Currie showed just one example, the estimated market volume for plant- the necessary growth medium for citric acid biosyn- degrading enzymes from flamentous fungi in 2016 was thesis, and the ability of the fungus to grow at low pH (2.5–3.5), while still being able to produce high amounts *Correspondence: t.cairns@tu‑berlin.de; corrado.nai@tu‑berlin.de; vera. of the metabolite. Moreover, this work demonstrated meyer@tu‑berlin.de the direct correlation between amount of substrate in Department of Applied and Molecular Microbiology, Institute the medium and amount of product, laying the basis for of Biotechnology, Technische Universität Berlin, Gustav‑Meyer‑Allee 25, 13355 Berlin, Germany modern-day industrial fermentation of citric acid [3]. In © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cairns et al. Fungal Biol Biotechnol (2018) 5:13 Page 2 of 14 contrast to other species of fungi that had been reported regarding the history of the A. niger research feld can, to produce citric acid by 1917, every single strain of A. in general terms, be deciphered. We conducted a survey niger that Currie tested could efciently produce this of the A. niger literature since Currie’s seminal study by molecule when grown in sugar solutions. Two years later, interrogating the PubMed database [6] for any publica- the American company Pfzer made a pilot plant for bio- tion containing ‘Aspergillus niger’ in the title. Te result- chemical production of citric acid, and by the mid 1920s, ing articles (> 3000, see Additional fle 1: Table S1) were production using A. niger fermentation far outweighed divided into fve 20-year periods based on their publi- extraction from citrus fruits [4]. cation date, and the 20 most common words from the Te citric acid cycle was comprehensively determined available titles during each time period were visualized over the next several decades, resulting in the award of as word clouds (Fig. 1). Although querying the PubMed the Nobel Prize to Hans Krebs and Fritz Lipmann in database for ‘Aspergillus niger’ in abstract and keywords 1953. Te frst and fnal reaction in the cycle involves the resulted in more returned manuscripts (> 8700 hits), we formation of citrate from oxaloacetate, acetyl-CoA, and decided to limit our word-cloud analysis specifcally to water, by a citrate synthase, ultimately generating chemi- titles. We applied this restriction as searching among cal energy in the form of adenosine triphosphate (ATP). manuscript abstracts returned a majority of hits where Te objective of industrial microbiologists, including researchers used A. niger in simple growth assays to vali- James Currie, was to exploit this cycle, and indeed many date efcacy of putative antifungals. While of interest, other metabolic pathways, to ferment useful molecules. these research eforts (which were extremely common Although there are variations in fermentation tech- from 1977 onwards) are not, specifcally, interested in A. niques, in general, industrial production of citric acid niger biology per se. requires aerobic, submerged growth of A. niger in a Our analysis of 100 years of A. niger publications indi- sugar solution, which is usually derived from inexpensive cated, unsurprisingly, that ‘citric acid’ and ‘fermentation’ sources, such as molasses, corn steep liquor, or hydro- were amongst the most common returned words in titles lysed corn starch, amongst others. After fermentation, from every period (Fig. 1). Clearly, Currie’s discovery of A. niger is physically removed, usually by fltration, and conditions for maximizing citric acid production [3] was citric acid is isolated by precipitation of the fermentation indeed a biotechnological revolution, and one that would mix with calcium hydroxide (lime) to generate calcium constitute a major focus of research over the next cen- citrate salt. Subsequent treatment with sulphuric acid tury. Indeed, A. niger research has rapidly grown over the yields the citric acid product. past 40 years (Fig. 2). Although by no means exhaustive, Te widespread applications of citric acid are shown by distinct historical trends become apparent from our anal- current fgures regarding this metabolite: in 2007, world- ysis (Fig. 1). wide production was estimated at 1.6 million tons, with an estimated value of $2.6 billion in 2014 and predicted The foundations of A. niger research to rise to $3.6 billion by 2020 [1, 5]. As a weak acid, it From the period 1937–1956, A. niger researchers were can be used as an antioxidant, preservative, acidulant, predominantly concerned with the impact of micro/ pH-regulator, or favour in food and beverages, as well as macro nutrients in cultivation media and growth param- comparable applications in the pharmaceutical and cos- eters for optimized citric acid fermentation (Fig. 1, [7, metics industries. Citric acid is currently predominantly 8]). Most studies specifcally utilized submerged culture, produced in China, which accounts for approximately which obviously refects the need to control growth, 60% of global production [1]. However, A. niger industrial morphology, and, ultimately, citric acid production dur- applications are not just limited to the production of cit- ing fermentation (Additional fle 1: Table S1 and [9]). ric acid; as a prolifc secretor, numerous industrially rel- After the award of Hermann Muller’s Nobel Prize in evant enzymes and other molecules are produced by this 1946 for work on mutations via X-ray irradiation, the fungus. Below, we summarize some of the key develop- late 1940s and early 1950s saw the frst mutagenesis stud- ments in the feld over the last century. ies in biological sciences deployed by the A. niger com- munity in order to generate strains with improved citric A historical snapshot of A. niger research acid yields [10]. Te number of UV, X-ray, and chemical Te fundamental and applied scientifc discoveries using mutagenesis eforts increased from 1957 to 1976, with A. niger over the last 100 years are extremely diverse. As most focusing on enhancing citric acid production (e.g. Currie wrote in 1917: ‘Few concise statements can be [11] and Fig. 1). made concerning the metabolism of an organism capa- Notably, the period from 1957 to 1976 saw a paradigm ble of producing such a variety of chemical transforma- shift amongst the A. niger biotechnological research tions as Aspergillus niger’ [3]. Nevertheless, some trends community, with the widespread realization that this Cairns et al.