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.
Highlights
An essential challenge for next-generation cellulosic biofuels is overcoming the biomass recalcitrance and converting the biomassderived sugars to biofuels [1,2]
Microbial conversion of biomass to biofuels is an attractive route for biofuel development, because some microorganisms, including the model fungus Mucor circinelloides, are able to accumulate lipids by growing on the sugars released from the pretreatment of biomass; the lipids can be used as feedstock for biodiesel production [3,4], catalysed by chemicals or lipases
In the M. circinelloides genome, we found four GH5, two GH8, four GH9, and three GH45 enzymes; whereas glycoside hydrolase (GH) families 6, 7, and 12 are unrepresented (Table 3)
Summary
An essential challenge for next-generation cellulosic biofuels is overcoming the biomass recalcitrance and converting the biomassderived sugars to biofuels [1,2]. Microbial conversion of biomass to biofuels is an attractive route for biofuel development, because some microorganisms, including the model fungus Mucor circinelloides, are able to accumulate lipids by growing on the sugars released from the pretreatment of biomass; the lipids can be used as feedstock for biodiesel production [3,4], catalysed by chemicals or lipases. Lipase from M. circinelloides has been commercialized, and its immobilization and utilization in fine chemistry and oil and fats modification have been reviewed recently [5], adding to the relevance of studying this species for developing biodiesel. The current state-of-the-art and prospects on microbial biodiesel production have been discussed by several recent reviews of the literature [6,7,8,9,10]. Its genome sequence is available, making possible genomic as well as secretomic approaches
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