Genomic, Proteomic, and Biochemical Analyses of Oleaginous Mucor circinelloides: Evaluating Its Capability in Utilizing Cellulolytic Substrates for Lipid Production

Abstract

Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. Our genomic analysis revealed the existence of genes encoding 13 endoglucanases (7 of them secretory), 3 β-D-glucosidases (2 of them secretory) and 243 other glycoside hydrolase (GH) proteins, but not genes for exoglucanases such as cellobiohydrolases (CBH) that are required for breakdown of cellulose to cellobiose. Analysis of the major PAGE gel bands of secretome proteins confirmed expression of two secretory endoglucanases and one β-D-glucosidase, along with a set of accessory cell wall-degrading enzymes and 11 proteins of unknown function. We found that M. circinelloides can grow on CMC (carboxymethyl cellulose) and cellobiose, confirming the enzymatic activities of endoglucanases and β-D-glucosidases, respectively. The data suggested that M. circinelloides could be made usable as a consolidated bioprocessing (CBP) strain by introducing a CBH (e.g. CBHI) into the microorganism. This proposal was validated by our demonstration that M. circinelloides growing on Avicel supplemented with CBHI produced about 33% of the lipid that was generated in glucose medium. Furthermore, fatty acid methyl ester (FAME) analysis showed that when growing on pre-saccharified Avicel substrates, it produced a higher proportion of C14 fatty acids, which has an interesting implication in that shorter fatty acid chains have characteristics that are ideal for use in jet fuel. This substrate-specific shift in FAME profile warrants further investigation.

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

“…Studies evaluating microbial oil production from lignocellulosic substrates are typically limited to the hydrolysates of pretreated biomass and/or enzymatically hydrolyzed biomass (Economou et al, 2011;Gong et al, 2013;Huang et al, 2012;Liang et al, 2012;Ruan et al, 2012Ruan et al, , 2013Tsigie et al, 2011;Yu et al, 2011). Few studies consider oil production from a whole biomass slurry, which includes unhydrolyzed carbohydrate solids without cellulase supplementation (Dey et al, 2011;Hui et al, 2010;Peng and Chen, 2008;Wei et al, 2013). Eliminating the need for a separate enzymatic hydrolysis process step or culture supplementation with cellulolytic enzymes has the potential to reduce costs for biofuels production (Linger and Darzins, 2013).…”

Genome Analysis of a Zygomycete Fungus Choanephora cucurbitarum Elucidates Necrotrophic Features Including Bacterial Genes Related to Plant Colonization

“…The infection caused by this fungus is frequently found in warm and humid climates such as tropical and subtropical regions18. Several dozen Mucorales genomes have been sequenced, including the opportunistic human pathogenic Rhizopus delemar 19, oleaginous Mucor circinelloides 20, human pathogenic Lichtheimia corymbifera 21, and thermophilic Rhizomucor miehei 22. There are some Mucorales fungi reported as plant pathogen including Mucor circinelloides (wound pathogen of noni fruit)17, Rhizopus microsporus (pathogen of maize, sunflower, and rice)4, and Rhizopus stolonifer (pathogen of sweet potato)23, but none of their genomic features have been analyzed and reported in previous studies.…”

Lipid Accumulation by Fungi Grown on Palm Oil Mill Effluent

“…Current practice of biodiesel production is through trans-esterification of vegetable oils or animal fats with short chain alcohols. Feedstock acquires more than 70% of total biofuel production cost, and this limit further expansion of biodiesel (Wei . 2013).…”

“…and sp. accumulate large amounts of lipids greater than 40% per dry cell weight (Wei 2013;Wynn . 2001) under N-limited cultivation conditions.…”