Direct production of commodity chemicals from lignocellulose using Myceliophthora thermophila
Introduction
Plant biomass has become the major feasible stock for production of renewable fuels and chemicals because of its abundance, sustainability and environmental friendliness (Lynd et al., 2002). However, the plant cell wall is structurally complex and recalcitrant to depolymerization, making it necessary to employ chemical and enzymatic pretreatment to degrade lignocellulose into fermentable sugars for processing by microbes (Himmel et al., 2007; Lynd, 2017; Stephanopoulos, 2007). For the economically efficient conversion of lignocellulose into commodity chemicals, several major processing steps in the use of microbial technologies must be improved, including the pretreatment, saccharification, and fermentation steps. In particular, the currently used pretreatment and saccharification steps are complex and require the addition of large amounts of extra cellulase (Klein-Marcuschamer et al., 2012). Although considerable efforts have been made toward making a bioeconomy a reality, the capital cost of pretreatment and cellulase enzymes remain the major cost hurdles to overcome.
The strategy of consolidated bioprocessing (CBP), which combines saccharification (and even pretreatment) and fermentation processes in a one-pot reaction with a single microorganism (Wargacki et al., 2012) or microbial consortium (Mintya et al., 2013; Scholz et al., 2018) has been widely recognized as the ultimate configuration for cellulosic biomass conversion (Chung et al., 2014; Olson et al., 2012). There are two main strategies to construct microorganisms suitable for a CBP platform: the heterologous strategy, and the native strategy (Xu et al., 2009). The heterologous strategy uses microbes that ferment substrates efficiently, such as yeast and Escherichia coli, and equips them with heterologous cellulases (Peralta-Yahya et al., 2012; Zhang et al., 2011). The native strategy focuses on engineering native lignocellulolytic organisms to enhance chemical production. Most investigations using the native strategy have focused on bacteria such as Clostridia (Lynd et al., 2002; Olson et al., 2012). Cellulolytic fungi have largely been overlooked as CBP hosts, although fungi as a group are the most efficient producers of cellulases. Genera such as Trichoderma and Penicillium have been shown to produce abundant cellulases (Gusakov, 2011; Peterson and Nevalainen, 2012).
Myceliophthora thermophila is a highly efficient cellulose and hemicellulose degrader and capable of efficiently using various monosaccharides from plant biomass hydrolysis. The genome of this thermophilic mold encodes a large number of lignocellulolytic enzymes (>200) covering most of the recognized families required for efficient degradation of polysaccharides. There are about 20 polysaccharide monooxygenases belonging to family AA9 in M. thermophila, significantly accelerating the degradation of polysaccharides into oligosaccharides relative to Trichoderma and Aspergillus (Berka et al., 2011; Singh, 2014). The cellulase product from M. thermophila achieved “Generally Recognized As Safe” (GRAS) status (Visser et al., 2011). Additionally, the optimal growth temperature (45–50 °C) matches that for cellulase activity (around 50 °C for fungal hydrolases) (Bhat and Maheshwari, 1987; Liu et al., 2017; Visser et al., 2011). Traditionally this fungus was studied as a cellulase producer or reservoir of thermophilic enzymes. There are some works focusing on the intricate regulatory mechanisms of cellulase secretion (Liu et al., 2019; Visser et al., 2011; Wang et al., 2015b; Yang et al., 2015). Recently, with the development of tools for genetic manipulation, especially CRISPR-Cas9 based genome editing techniques in this fungus (Liu et al., 2017; Xu et al., 2015), workers have tried to engineer it into a cell factory to produce fumaric acid from glucose (Gu et al., 2018). Herein, M. thermophila was used as a promising candidate for application of CBP technology to produce biochemicals. Four-carbon dicarboxylic acids (C4-diacids; malic and succinic acid), which are important building blocks for both commodity biopolymers and specialty chemicals with a potential addressable market of $10 billion per annum, were chosen as the target chemicals for production using our fungal CBP platform.
Section snippets
Strains and growth conditions
Myceliophthora thermophila ATCC 42464 was obtained from the American Type Culture Collection. The wild-type strain and its derivates were grown on Vogel's minimal medium (VM medium) supplemented with 2% glucose or xylose at 35 °C for 15 days to obtain mature conidia. PEG-mediated protoplast transformation was employed for heterologous expression of interesting genes in M. thermophila. Antibiotics were added when needed for transformant screening. E. coli DH5α was used for construction and
Metabolic engineering for C4-diacid production in M. thermophila
In microbes, pyruvate carboxylase (encoded by pyc) catalyzes the conversion of pyruvic acid to oxaloacetic acid with the fixation of CO2; the oxaloacetic acid is then converted into malic acid by malate dehydrogenase (encoded by mdh), and then to fumaric acid and succinic acid through further steps in the reductive tricarboxylic acid (rTCA) pathway; 25% of the carbon in C4 organic acids comes from CO2 (Fig. 1A). Theoretically, 2 mol of malic acid can be synthesized from 1 mol of glucose
Discussion
Plant cell walls represent a sustainable and renewable resource for biosynthesis of biofuels and commodity chemicals. However, plant cellulosic biomass is structurally complex and recalcitrant to degradation to soluble fermentable sugars, and the cost of pretreatment and cellulase enzymes remain major cost hurdles to overcome (Himmel et al., 2007). CBP strategy has been discussed extensively, and is thought to be a promising approach to economical biorefineries (Olson et al., 2012). Because of
Author contributions
C.G.T. and J.G.L. designed the project. J.G.L, L.C.L., T.S., J.X., J.X.J and Q.L. performed the study. J.G.L. and C.G.T. analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.
Competing financial interests
All authors are inventors on patents applied for by Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, which cover organic acid production by this fungus.
Acknowledgments
We thank Zhidan Zhang for assistance with intracellular metabolite analysis. This work was supported by funding from the Chinese Academy of Sciences (XDA21060900 and ZDRW-ZS-2016-3K) and the National Natural Science Foundation of China (Grant Nos.: 31670042, 31601013, 31640049, and 31771386).
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2023, Metabolic EngineeringCitation Excerpt :In addition, the U.S. Department of Energy (DOE) has proposed L-MA as one of the top twelve building block chemicals that have the potential to be produced from renewable resources, and its market demand was estimated at over 200,000 metric tons per year (Dai et al., 2018b; Sauer et al., 2008). In the past decades, bacteria (Li et al., 2018; Zhang et al., 2011), yeasts (Kang et al., 2021; Zelle et al., 2008) and filamentous fungi (Li et al., 2020; Liu et al., 2017; Shigeo et al., 1962) have been engineered to produce enantiomerically pure L-MA (Table 1). However, only filamentous fungi were successfully adopted for large-scale production.