Lipases or triacylglycerol hydrolases are widely spread in nature and are particularly common in the microbial world. The filamentous fungus Mucor circinelloides is a potential lipase producer, as it grows well in triacylglycerol-contained culture media. So far only one lipase from M. circinelloides has been characterized, while the majority of lipases remain unknown in this fungus. In the present study, 47 potential lipase genes in M. circinelloides WJ11 and 30 potential lipase genes in M. circinelloides CBS 277.49 were identified by extensive bioinformatics analysis. An overview of these lipases is presented, including several characteristics, sub-cellular location, phylogenetic analysis and expression profiling of the lipase genes during growth and lipid accumulation. All of these proteins contained the consensus sequence for a classical lipase (GXSXG motif) and were divided into four types including α/β-hydrolase_1, α/β-hydrolase_3, class_3 and GDSL lipase (GDSL) based on gene annotations. Phylogenetic analyses revealed that class_3 family and α/β-hydrolase_3 family were the conserved lipase family in M. circinelloides. Additionally, some lipases also contained a typical acyltransferase motif of H-(X) 4-D, and these lipases may play a dual role in lipid metabolism, catalyzing both lipid hydrolysis and transacylation reactions. The differential expression of all lipase genes were confirmed by quantitative real-time PCR, and the expression profiling were analyzed to predict the possible biological roles of these lipase genes in lipid metabolism in M. circinelloides. We preliminarily hypothesized that lipases may be involved in triacylglycerol degradation, phospholipid synthesis and beta-oxidation. Moreover, the results of sub-cellular localization, the presence of signal peptide and transcriptional analyses of lipase genes indicated that four lipase in WJ11 most likely belong to extracellular lipases with a signal peptide. These findings provide a platform for the selection of candidate lipase genes for further detailed functional study.
The filamentous fungus Mucor circinelloides has been widely used as a model organism to investigate the mechanisms of lipid accumulation. Although a lot of work has been done to analyze and explore many of the essential enzymes/genes related to lipid accumulation in M. circinelloides, the function of lipase in this fungus has not been studied at all. In this study, we report some important characteristics of all 30 lipases, and especially 4 lipases Lip6, Lip10, Lip19 and Lip24 with functional analyses in Mucor circinelloides CBS 277.49 based on the genome sequences. Transcriptional analyses revealed that the Lip6 or Lip10 gene expression increased significantly during 24 h to 72 h of lipid accumulation while Lip19 and Lip24 genes were down-regulated along with the increase in lipid accumulation. Over-expression of either lipase Lip6 or Lip10 led to a slight increase in cell dry weight, but no significant effect on lipid accumulation.More interestingly over-expression of Lip6 resulted in a 17% increase in C18:3 content of the lipids.However, over-expression of lipase Lip19 and Lip24 decreased the cell dry weight by 9.08-18.8% and lipid content by 37.86-41.42%, respectively. The fatty acid profiles of strains with Lip19 and Lip24 overexpression were also significantly changed as compared to control. Analysis of lipase genes revealed that the sequences of Lip6 and Lip10 not only contained the typical lipase motif (G/A) XSXG, but also the consensus sequence motif HXXXXD (a typical of acyltransferase motif). These results suggest that Lip6 or Lip10 may control acyltransferase activity in Mucor circienlloides and play a role in fatty acid reconstruction of TAG, while Lip19 or Lip24 may work as a TAG lipase involved in TAG metabolism/ degradation. † Electronic supplementary information (ESI) available: Table S1 sequence of primers used in quantitative RT-PCR. Table S2 sequence of primers used in gene over-expression of Lip6 (161426), Lip10 (115761), Lip19 (143450) and Lip24 (107413). Table S3 the sub-cellular location, and the presence and location of signal peptide of lipases from M. circinelloides CBS 277.49. See
The xylose isomerase (XI) pathway, which converts xylose in lignocellulosic materials into intermediate metabolites, is characterized for the first time in M. circinelloides.
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