The Journal of Antibiotics (2017) 70, 347–360 & 2017 Japan Antibiotics Research Association All rights reserved 0021-8820/17 www.nature.com/ja REVIEW ARTICLE Production of valuable compounds by molds and yeasts Arnold L Demain1 and Evan Martens2 We are pleased to dedicate this paper to Dr Julian E Davies. Julian is a giant among microbial biochemists. He began his professional career as an organic chemistry PhD student at Nottingham University, moved on to a postdoctoral fellowship at Columbia University, then became a lecturer at the University of Manchester, followed by a fellowship in microbial biochemistry at Harvard Medical School. In 1965, he studied genetics at the Pasteur Institute, and 2 years later joined the University of Wisconsin in the Department of Biochemistry. He later became part of Biogen as Research Director and then President. After Biogen, Julian became Chair of the Department of Microbiology at the University of British Columbia in Vancouver, Canada, where he has contributed in a major way to the reputation of this department for many years. He also served as an Adjunct Professor at the University of Geneva. Among Julian’s areas of study and accomplishment are fungal toxins including α-sarcin, chemical synthesis of triterpenes, mode of action of streptomycin and other aminoglycoside antibiotics, biochemical mechanisms of antibiotic resistance in clinical isolates of bacteria harboring resistance plasmids, their origins and evolution, secondary metabolism of microorganisms, structure and function of bacterial ribosomes, antibiotic resistance mutations in yeast ribosomes, cloning of resistance genes from an antibiotic-producing microbe, gene cloning for industrial purposes, engineering of herbicide resistance in useful crops, bleomycin-resistance gene in clinical isolates of Staphylococcus aureus and many other topics. He has been an excellent teacher, lecturing in both English and French around the world, and has organized international courses. Julian has also served on the NIH study sections, as Editor for several international journals, and was one of the founders of the journal Plasmid. We expect the impact of Julian’s accomplishments to continue into the future. The Journal of Antibiotics (2017) 70, 347–360; doi:10.1038/ja.2016.121; published online 12 October 2016 INTRODUCTION are produced by plants (alkaloids, flavonoids, terpenoids, steroids, Microbes have contributed significantly to improving the health and carbohydrates, etc.) and 5% of these plant products have a microbial well-being of humans. The natural products that they have yielded origin. From all the reported natural products, ∼ 20–25% show have not only helped eradicate disease and alleviate suffering, but also biological activity and, of these, ∼ 10% have been obtained from greatly increased the average life expectancy. The first major contribu- microbes. Microorganisms produce many compounds with biological tion of microbes began back in 1928, when Alexander Fleming activity. From the 22 500 biologically active compounds so far discovered in a Petri dish seeded with Staphylococcus aureus that a obtained from microbes, ∼ 40% are produced by fungi.2,3 The role compound produced by a mold killed the bacterium. The mold, of fungi in the production of antibiotics and other drugs for treatment Penicillium notatum, produced an active agent that was named of noninfective diseases has been dramatic.4 penicillin. Fleming’s discovery began the microbial drug era. By using Biosynthetic genes are present in clusters coding for large, the same method, other naturally occurring substances, like multidomain and multimodular enzymes. Some examples of chloramphenicol and streptomycin, were later isolated from bacterial these enzymes include polyketide synthases, prenyltransferases, fermentations. Naturally occurring antibiotics are produced by nonribosomal peptide synthases and terpene cyclases. Genes adjacent fermentation, an old technique that can be traced back almost 8000 to the biosynthetic gene clusters encode regulatory proteins, years, initially for beer and wine production, and recorded in the oxidases, hydroxylases and transporters. Aspergilli usually contain written history of ancient Egypt and Mesopotamia. During the 30–40 secondary metabolite gene clusters. Strategies to activate silent past 4000 years, Penicillium roqueforti has been utilized for cheese genes have been reviewed by Brakhage and Schroekh.3 production, and for the past 3000 years, soy sauce in Asia and bread in Given that the vast majority of microbes in nature have yet to be Egypt represented examples of traditional fermentations.1 cultured (~99%), there have been major advances in isolating and Natural products from microbes have a broad range of therapeutic growing different microbial species.5 Furthermore, metagenomics— applications and are often produced via primary or secondary that is, the extraction of DNA from soil, plants and marine habitats metabolism. Because of technical improvements in screening pro- and its incorporation into known organisms—is allowing access to a grams and separation and isolation techniques, the number of natural vast untapped reservoir of genetic and metabolic diversity.6,7 The compounds discovered exceeds one million.2 Among them, 50–60% potential for discovery of new secondary metabolites with beneficial 1Research Institute for Scientists Emeriti (RISE), Drew University, Madison, NJ, USA and 2Cempra, Chapel Hill, NC, USA Correspondence: Professor AL Demain, Research Institute for Scientists Emeriti (RISE), Drew University, 36 Madison Avenue, Madison, NJ 07940, USA. E-mail: [email protected] Received 28 June 2016; revised 31 August 2016; accepted 1 September 2016; published online 12 October 2016 Production of valuable compounds by molds and yeasts AL Demain and E Martens 348 use for humans is great. A method to predict secondary metabolite are a family of neutral, highly lipophilic, cyclic undecapeptides gene clusters in filamentous fungi has recently been devised.8 containing some unusual amino acids, synthesized by a nonribosomal Interestingly, microbial production of secondary metabolites is peptide synthetase, cyclosporin synthetase. Discovery of the immuno- limited to a very low level by certain regulatory mechanisms. Despite suppressive activity of this secondary metabolite led to its use in heart, this, the extent of such production is sufficient for the microbe to liver and kidney transplants and to the overwhelming success of the compete with other organisms or maintain a commensal/mutual organ transplant field.16 Cyclosporin was approved for use in 1983. It relationship with other species. The industrial microbiologist, how- is thought to bind to the cytosolic protein cyclophilin (immunophilin) ever, desires a strain that will overproduce the molecule of interest. of immunocompetent lymphocytes, especially T lymphocytes. This Development of higher-producing strains involves mutagenesis and, complex of cyclosporin and cyclophilin inhibits calcineurin that under more recently, recombinant DNA technologies.9 Although some normal circumstances is responsible for activating the transcription of metabolites of interest can be made by plants or animals, or by interleukin-2. It also inhibits lymphokine production and interleukin chemical synthesis, the recombinant microbe is usually the ‘creature of release and therefore leads to a reduced function of effector T cells. ’ choice . Thousand-fold increases in production of small molecules Annual world sales of cyclosporin A are ∼ $2 billion. Cyclosporin A have been obtained by mutagenesis and/or genetic engineering. The also has activity against coronaviruses.17 use of genome mining to discover new fungal natural products has Studies on the mode of action of cyclosporin, and the later 10 been reviewed by Wiemann and Keller. Other important parts of developed immunosuppressants from actinomycetes, such as sirolimus industrial production include creating a proper nutritional environ- (a rapamycin) and FK-506 (tacrolimus), have markedly expanded ment for the organism to grow and produce its product, and the current knowledge of T-cell activation and proliferation. These agents avoidance of negative effects such as inhibition and/or repression by act by interacting with an intracellular protein (an immunophilin), fi carbon, nitrogen and phosphorus sources, metals and the nal thus forming a novel complex that selectively disrupts the signal 4,11 product itself. Avoidance of enzyme decay is also desired. transduction events of lymphocyte activation. Their targets are inhibitors of signal transduction cascades in microbes and humans. BROAD USE OF SECONDARY METABOLITES PRODUCED In humans, the signal transduction pathway is required for activation BY FUNGI of T cells. Fingolimod (FTY720), another immunosuppressant, was Given the diverse array of secondary metabolites that fungi are capable approved by the US Food and Drug Administration (FDA) in 2010, of producing, the pharmaceutical industry began to focus their efforts specifically for relapsing forms of multiple sclerosis. The drug, initially on the screening of compounds for indications other than anti- discovered by Professors Fujita, Yoshitomi and Taito in collaboration, infectives.12,13 As microorganisms are such a prolific source of is a derivative of myriocin isolated from the fungus Isaria sinclairii. structurally diverse bioactive metabolites, the industry extended their The annual world sales of fingolimod are ∼ $2.7 billion. screening