Project description:Tremella fuciformis is a popular edible fungus with fruiting bodies that can be produced in large quantities at low costs, while it is easy to transform and cultivate as yeast. This makes it an attractive potential bioreactor. Enhanced heterologous gene expression through codon optimization would be useful, but until now codon usage preferences in T. fuciformis remain unknown. The only available genome of a species in the genus Tremella was that of Tremella mesenterica. To precisely determine the preferred codon usage of T. fuciformis we sequenced the genome of strain Tr26 resulting in a 24.5 Mb draft genome with 10,040 predicted genes. 3288 of the derived predicted proteins matched the UniProtKB/Swiss-Prot databases with 40% or more similarity. Corresponding gene models of this subset were subsequently optimized trough repetitive comparison of alternative start codons and selection of best length matching gene models. For experimental confirmation of gene models, 96 random clones from an existing T. fuciformis cDNA library were sequenced, generating 80 complete CDSs. Calculated optimal codons (RSCU and RFCU values) for the 3288 predicted and the 80 cloned CDSs were highly similar, indicating sufficient accuracy of predicted gene models for codon usage analysis. T. fuciformis showed a strong preference for C and then G (C+G; 66.4 %) at the third base pair position of used codons, while average GC content of predicted genes was slightly higher (58.5 %) than he total genome sequence average (55.9%). Most frequently used codons (optimal codons) all ended in C or G except for one (Ter; AGU), and an increased frequency of C ending codons was observed in genes with higher expression levels. Surprisingly, the preferred codon usage in T. fuciformis strongly differed from T. mesenterica (same genus) and C. neoformans (same family). Instead, optimal codon usage was similar to more distant related species such as Ustilago maydis and Neurospora crassa. Despite much higher overall sequence homology between T. fuciformis and T. mesenterica only 7 out of 21 optimal codons were equal, whereas T. fuciformis shared up to 20 out 21 optimal codons with other species. Clearly, codon usage in Tremella can differ largely and should be estimated for individual species. The precise identification of optimal and high expression related codons is therefore an important step in the development of T. fuciformis as a bioreactor system.

Project description:Tremella fuciformis is a popular edible fungus with fruiting bodies that can be produced in large quantities at low costs, while it is easy to transform and cultivate as yeast. This makes it an attractive potential bioreactor. Enhanced heterologous gene expression through codon optimization would be useful, but until now codon usage preferences in T. fuciformis remain unknown. The only available genome of a species in the genus Tremella was that of Tremella mesenterica. To precisely determine the preferred codon usage of T. fuciformis we sequenced the genome of strain Tr26 resulting in a 24.5 Mb draft genome with 10,040 predicted genes. 3288 of the derived predicted proteins matched the UniProtKB/Swiss-Prot databases with 40% or more similarity. Corresponding gene models of this subset were subsequently optimized trough repetitive comparison of alternative start codons and selection of best length matching gene models. For experimental confirmation of gene models, 96 random clones from an existing T. fuciformis cDNA library were sequenced, generating 80 complete CDSs. Calculated optimal codons (RSCU and RFCU values) for the 3288 predicted and the 80 cloned CDSs were highly similar, indicating sufficient accuracy of predicted gene models for codon usage analysis. T. fuciformis showed a strong preference for C and then G (C+G; 66.4 %) at the third base pair position of used codons, while average GC content of predicted genes was slightly higher (58.5 %) than he total genome sequence average (55.9%). Most frequently used codons (optimal codons) all ended in C or G except for one (Ter; AGU), and an increased frequency of C ending codons was observed in genes with higher expression levels. Surprisingly, the preferred codon usage in T. fuciformis strongly differed from T. mesenterica (same genus) and C. neoformans (same family). Instead, optimal codon usage was similar to more distant related species such as Ustilago maydis and Neurospora crassa. Despite much higher overall sequence homology between T. fuciformis and T. mesenterica only 7 out of 21 optimal codons were equal, whereas T. fuciformis shared up to 20 out 21 optimal codons with other species. Clearly, codon usage in Tremella can differ largely and should be estimated for individual species. The precise identification of optimal and high expression related codons is therefore an important step in the development of T. fuciformis as a bioreactor system. Using the Total RNA was extracted from yeast-like cells of T. fuciformis Tr21 in their exponential growth stage, and double-stranded cDNA was synthesized for tag library construction and digital gene expression tag (DGE) sequencing at BGI Tech Solutions Co., Ltd (Shenzhen, China). Image analysis, base calling, extraction of tags, and tag counting were conducted using the Illumina pipeline. Clean tags were mapped to predicted gene models of the draft genome with a mismatch tolerance of 1bp. The number of tags for each CDS was calculated and then normalized to TPM (number of transcripts per million clean tags) digital gene expression (DGE)

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