Engineering the filamentous fungus Neurospora crassa for lipid production from lignocellulosic biomass

Abstract

Microbially produced triacylglycerol (TAG) is a potential feedstock for the production of biodiesel, but its commercialization will require high yields from low-cost renewable feedstocks such as lignocellulose. The present study employs a multi-gene approach to increasing TAG biosynthesis in the filamentous fungus Neurospora crassa. We demonstrate the redirection of carbon flux from glycogen biosynthesis towards fatty acid biosynthesis in a glycogen synthase deletion strain (Δgsy-1). Furthermore, combining Δgsy-1 with an enhanced TAG biosynthetic strain (acyl-Coenzyme A synthase; Δacs-3) of N. crassa yielded a twofold increase in total fatty acid accumulation over the control strain. The cellulose degrading potential of this double deletion strain was improved by deleting of the carbon catabolite regulation transcription factor (Δcre-1) to create the triple deletion strain Δacs-3 Δcre-1; Δgsy-1. This strain exhibited early and increased cellulase expression, as well as fourfold increased total fatty acid accumulation over the control on inhibitor-free model cellulose medium. The Δcre-1 mutation, however, was not beneficial for total fatty acid accumulation from pretreated lignocellulose. Conversion of dilute-acid pretreated Miscanthus to TAG was maximum in the constructed strain Δacs-3; Δgsy-1, which accumulated 2.3-fold more total fatty acid than the wild-type control strain, corresponding to a total fatty acid yield of 37.9 mg/g dry untreated Miscanthus.

A transcriptomic analysis of Neurospora crassa using five major crop residues and the novel role of the sporulation regulator rca-1 in lignocellulase production

“…Investigation of fungal responses to a wide spectrum of biomass substrates is needed not only to gain more knowledge of plant cell wall degradation and utilization but also to rationally engineer filamentous fungi for biomass-based enzyme, ethanol, and chemical production. This information will be especially helpful in using plant biomass direct microbial conversion (DMC), a recently suggested, promising strategy [ 7 , 8 ] whereby filamentous fungi are used to generate valuable products, such as fatty acids produced by Neurospora crassa [ 9 ] and ethanol by Trichoderma reesei and Fusarium oxysporum [ 10 ]. Although transcriptome analysis has been performed in different fungal systems digesting various types of plant biomass (such as Miscanthus giganteus , barley, oat, canola, alfalfa, wheat straw, and bagasse) [ 11 - 16 ], the comparative analysis of genome-wide profiling on a wide spectrum of biomass substrates in a single fungus species can offer outstanding knowledge on specific and common microbe responses to different crop residues, which will be useful for engineering fungal DMC using various sources of plant biomass.…”

“…Studies using this model organism have begun to reveal a more thorough perspective on lignocellulolytic enzyme induction, regulation, and production in filamentous fungi [ 23 ]. These advances can be rationally applied to N. crassa as well as other filamentous fungi to further improve their production of lignocellulolytic enzymes and even target chemicals via DMC [ 9 ].…”

Neurospora crassa: Looking back and looking forward at a model microbe

“…Novel proteins involved in plant cell wall deconstruction have been identified in Neurospora through comparative genomic, transcriptomic, and proteomic studies (Beeson et al, 2012; Phillips et al, 2011), and evidence suggests additional factors for plant biomass degradation are present (Coradetti et al, 2012; Benz et al, 2014). Recent studies have pushed Neurospora research beyond understanding fundamental scientific questions and have used Neurospora for applied research as a consolidated bioprocessing host for lignocellulosic biofuel production and a heterologous protein production host (Dogaris et al, 2013; Dana et al, 2014; Roche et al, 2014). With these studies, Neurospora continues to evolve in its role as a model microbe, now including applied biotechnological research.…”

Transcriptome Profiling-Based Analysis of Carbohydrate-Active Enzymes in Aspergillus terreus Involved in Plant Biomass Degradation

“…Analysis of gene expression in lignocellulolytic fungal species cultivated on different plant biomass substrates as sole carbon source enables identification of conserved and specific mechanisms employed in the microbial degradation of diverse plant cell wall residues ( Roche et al, 2014 ). In this study, we examine differential expression of CAZyme-encoding genes through transcriptome profiling of A. terreus strain BLU24 after growth on sugarcane bagasse and soybean hulls, which are both considered promising lignocellulosic substrates for biorefinery applications, in terms of the high concentration of carbohydrates that can be converted into fermentable sugar residues ( Cheng and Zhu, 2013 ).…”