Parallel laboratory evolution and rational debugging reveal genomic plasticity to S. cerevisiae synthetic chromosome XIV defects.

Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic Saccharomyces cerevisiae genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of S. cerevisiae chromosome XIV as part of the Synthetic Yeast Genome project. In parallel to the use of rational engineering approaches to restore wild-type fitness, we used adaptive laboratory evolution to generate a general growth-defect-suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the synthetic chromosome recombination and modification by loxPsym-mediated evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications.

Author Correction: Building a global alliance of biofoundries.

The original version of this Comment contained errors in the legend of Figure 2, in which the locations of the fifteenth and sixteenth GBA members were incorrectly given as '(15) Australian Genome Foundry, Macquarie University; (16) Australian Foundry for Advanced Biomanufacturing, University of Queensland.'. The correct version replaces this with '(15) Australian Foundry for Advanced Biomanufacturing (AusFAB), University of Queensland and (16) Australian Genome Foundry, Macquarie University'. This has been corrected in both the PDF and HTML versions of the Comment.

Isolation, characterization and expression of the hex1 gene from Trichoderma reesei.

Polymers of the HEX1 protein produce Woronin bodies in filamentous fungi. We have isolated and sequenced the hex1 gene and flanking regions from the industrially exploited fungus Trichoderma reesei. Multiple transcription start sites (TSS) and the 5' untranslated region (UTR) were identified by 5'RACE PCR. There are three hex1 transcript types, two of which originate from two TSSs at approximately -320 and -1335 from the start codon, which are separated by a 500-bp intron within the 5'UTR. The third transcript type results from alternative splicing of the intron within the coding sequence at the 3' end, which results in the inclusion or exclusion of an unconserved histidine-rich coding region. The three transcripts code for two forms of HEX1 protein. N-terminal sequencing of HEX1 separated by 2D gel electrophoresis confirms that there are two forms of HEX1 protein which are modified further by alternative cleavage of the N-terminus. The dominant form of HEX1 is coded by a cDNA with TSS at position -1335. Expression of hex1 on cellulase-inducing medium peaks strongly within 24 h of growth but the protein is expressed at a lower and more consistent level in medium containing glucose. This is the first investigation of expression of the hex1 gene encoding a protein unique to filamentous fungi.

Prospecting for novel lipase genes using PCR.

A PCR method suitable for the isolation of lipase genes directly from environmental DNA is described. The problems associated with the low levels of similarity between lipase genes were overcome by extensive analysis of conserved regions and careful primer design. Using this method, a lipase gene (oli-lipase) was isolated directly from environmental DNA. This lipase showed less than 20% similarity with other known lipases at the amino acid level. The study also revealed that distantly related members of the alpha/beta hydrolase superfamily share similar conserved motifs with the lipases, thus making these genes targets for gene prospecting by PCR.