Protein hyperproduction in fungi by design

Applied Microbiology and Biotechnology https://doi.org/10.1007/s00253-018-9265-1 MINI-REVIEW Protein hyperproduction in fungi by design Scott E. Baker 1,2 Received: 1 June 2018 / Revised: 17 July 2018 / Accepted: 19 July 2018 # The Author(s) 2018 Abstract The secretion of enzymes used by fungi to digest their environment has been exploited by humans for centuries for food and beverage production. More than a century after the first biotechnology patent, we know that the enzyme cocktails secreted by these amazing organisms have tremendous use across a number of industrial processes. Secreting the maximum titer of enzymes is critical to the economic feasibility of these processes. Traditional mutagenesis and screening approaches have generated the vast majority of strains used by industry for the production of enzymes. Until the emergence of economical next generation DNA sequencing platforms, the majority of the genes mutated in these screens remained uncharacterized at the sequence level. In addition, mutagenesis comes with a cost to an organism’s fitness, making tractable rational strain design approaches an attractive alternative. As an alternative to traditional mutagenesis and screening, controlled manipulation of multiple genes involved in processes that impact the ability of a fungus to sense its environment, regulate transcription of enzyme-encoding genes, and efficiently secrete these proteins will allow for rational design of improved fungal protein production strains. Keywords Enzyme . Protein . Hyperproduction . Secretion . Biodesign . Fungi . Biotechnology Introduction Fungi are constantly digesting their environment, secreting degradative enzymes, and absorbing the building block nutrients that are released. For centuries, humans have endeavored to harness the secreted enzyme activity, largely for production of food and beverage products such as soy sauce or sake (Abe and Gomi 2008; Baker and Bennett 2007; Machida et al. 2008). However, as the diversity of characterized enzymatic activities grows, so too do the potential uses (reviewed in Østergaard and Olsen 2011). Since the issuance of the first biotechnology patent in 1894 focused on production of starch saccharification enzymes from Aspergillus oryzae (Takamine 1894), fungi have been used to understand the basic biology of enzymes and to develop systems for their industrial production for use in a variety of applications. For example, since World War II, pioneering research and development have been * Scott E. Baker 1 Department of Energy Joint BioEnergy Institute, Emeryville, CA 94608, USA 2 Biosystems Design and Simulation Group, Environmental Molecular Sciences Division, Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA 99352, USA performed in Trichoderma reesei, from basic research that includes the elucidation of the components for the cellulose degradation enzyme system (Reese 1976) to applied research that includes the development of strains such as T. reesei RUTC30 that are the parents of strains used by the industry to produce enzyme cocktails for lignocellulosic biofuel production (Peterson and Nevalainen 2012). In the case of lignocellulosic biofuel and bioproduct production, where an enzyme or enzyme cocktail rather than the enzymatic process is being sold, a key factor for economic viability of enzyme sales is the cost and efficiency of enzyme production (KleinMarcuschamer et al. 2012). Over the last century, traditional forward genetic mutagenesis and screening methods have been utilized to generate strains with increased titer, rates, and yields of desired secreted enzymes. For example, Aspergillus niger strains with improved production of multiple types of enzymes, including glucoamylase (Armbruster 1961; Hu et al. 2017; Nevalainen 1981; Tahoun 1993) and T. reesei strains that produce high titers of cellulase (Mandels et al. 1971; Montenecourt and Eveleigh 1977; Peterson and Nevalainen 2012), have been generated by a variety of mutagenesis and screening regimes. With the continued industrialization and decreasing cost of DNA sequencing, it is now possible to Bresequence^ these mutant strains, identify mutations of interest, and assess mutations in a Bclean^ genetic background for their effect on Appl Microbiol Biotechnol enzyme secretion using reverse genetic methods (Baker 2009; Baker and Bredeweg 2016; Ivanova et al. 2017; Koike et al. 2013; Le Crom et al. 2009; Lichius et al. 2015; Nitta et al. 2012; Vitikainen et al. 2010). In this way, a number of mutations have been characterized that have led to increased enzyme secretion (Nitta et al. 2012; Pei et al. 2015). Derivatives of mutagenized strains continue to be developed and used by the industry for production of enzymes (Schuster et al. 2002; van Dijck et al. 2003). Although mutagenesis is effective at generating strains that secrete significant titers of enzymes, strain improvement often comes with collateral genome damage. For example, in the case of T. reesei, the quest for strains hyperproducing cellulases also led to cellulose-negative strains (Druzhinina et al. 2006; Ivanova et al. 2017; Lichius et al. 2015; Torigoi et al. 1996). Moreover, it is only within the last decade that the sexual cycle of T. reesei was described and the possibility of classical genetic strategies for understanding and improving protein hyper-production explored (Jourdier et al. 2017; Kuck and Bohm 2013; Li et al. 2016; Linke et al. 2015; Seidl and Seiboth 2010; Tisch et al. 2017). Beyond industrial biotechnology enzyme and small molecule production hosts, yeast and filamentous fungi are well studied as model systems for a number of biological processes that include, but are not limited to, protein secretion, cell signaling, cell morphology, and small molecule transport. Approaches from a breadth of biological disciplines, such as genetics, genomics, cell biology, physiology, molecular biology, and biochemistry have been used to understand the biological processes that underlie the fungal lifestyle. Decades of basic and applied fungal research spanning a breadth of methods has generated a knowledgebase that makes it possible to rationally design hypersecreting fungal enzyme production hosts. This mini review focuses on a subset of biological processes involved in ascomycete production of carbohydrate-active enzymes (CAZymes). Enzymatic deconstruction of various plant biomass components is considered a critical step in the production of lignocellulosic biofuels, and there is a vast literature on the genetics, biochemistry, cell biology, and regulation of CAZyme secretion from ascomycetes. In the following sections, I describe three different biological processes that contribute to filamentous fungal enzyme secretion: (1) nutrient sensing; (2) transcriptional regulation, and (3) translation and secretion (Fig. 1). I also overview recent research that uses a rational design strategy for a filamen (...truncated)