YeastFab:the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae

It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biological parts, which can be used to assemble transcriptional units in a single-tube reaction. In addition, standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the production through combinatorial assembly of a pathway using hundreds of regulatory biological parts

Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome

INTRODUCTION Although much effort has been devoted to studying yeast in the past few decades, our understanding of this model organism is still limited. Rapidly developing DNA synthesis techniques have made a “build-to-understand” approach feasible to reengineer on the genome scale. Here, we report on the completion of a 770-kilobase synthetic yeast chromosome II (synII). SynII was characterized using extensive Trans-Omics tests. Despite considerable sequence alterations, synII is virtually indistinguishable from wild type. However, an up-regulation of translational machinery was observed and can be reversed by restoring the transfer RNA (tRNA) gene copy number. RATIONALE Following the “design-build-test-debug” working loop, synII was successfully designed and constructed in vivo. Extensive Trans-Omics tests were conducted, including phenomics, transcriptomics, proteomics, metabolomics, chromosome segregation, and replication analyses. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP -mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium. RESULTS To efficiently construct megabase-long chromosomes, we developed an I- Sce I–mediated strategy, which enables parallel integration of synthetic chromosome arms and reduced the overall integration time by 50% for synII. An I- Sce I site is introduced for generating a double-strand break to promote targeted homologous recombination during mitotic growth. Despite hundreds of modifications introduced, there are still regions sharing substantial sequence similarity that might lead to undesirable meiotic recombinations when intercrossing the two semisynthetic chromosome arm strains. Induction of the I- Sce I–mediated double-strand break is otherwise lethal and thus introduced a strong selective pressure for targeted homologous recombination. Since our strategy is designed to generate a markerless synII and leave the URA3 marker on the wild-type chromosome, we observed a tenfold increase in URA3 -deficient colonies upon I- Sce I induction, meaning that our strategy can greatly bias the crossover events toward the designated regions. By incorporating comprehensive phenotyping approaches at multiple levels, we demonstrated that synII was capable of powering the growth of yeast indistinguishably from wild-type cells (see the figure), showing highly consistent biological processes comparable to the native strain. Meanwhile, we also noticed modest but potentially significant up-regulation of the translational machinery. The main alteration underlying this change in expression is the deletion of 13 tRNA genes. A growth defect was observed in one very specific condition—high temperature (37°C) in medium with glycerol as a carbon source—where colony size was reduced significantly. We targeted and debugged this defect by two distinct approaches. The first approach involved phenotype screening of all intermediate strains followed by a complementation assay with wild-type sequences in the synthetic strain. By doing so, we identified a modification resulting from PCRTag recoding in TSC10 , which is involved in regulation of the yeast high-osmolarity glycerol (HOG) response pathway. After replacement with wild-type TSC10 , the defect was greatly mitigated. The other approach, debugging by SCRaMbLE, showed rearrangements in regions containing HOG regulation genes. Both approaches indicated that the defect is related to HOG response dysregulation. Thus, the phenotypic defect can be pinpointed and debugged through multiple alternative routes in the complex cellular interactome network. CONCLUSION We have demonstrated that synII segregates, replicates, and functions in a highly similar fashion compared with its wild-type counterpart. Furthermore, we believe that the iterative “design-build-test-debug” cycle methodology, established here, will facilitate progression of the Sc2.0 project in the face of the increasing synthetic genome complexity. SynII characterization. ( A ) Cell cycle comparison between synII and BY4741 revealed by the percentage of cells with separated CEN2-GFP dots, metaphase spindles, and anaphase spindles. ( B ) Replication profiling of synII (red) and BY4741 (black) expressed as relative copy number by deep sequencing. ( C ) RNA sequencing analysis revealed that the significant up-regulation of translational machinery in synII is induced by the deletion of tRNA genes in synII. </jats:sec

Design, Construction, and Functional Characterization of a tRNA Neochromosome in Yeast

Here we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs

Design, construction, and functional characterization of a tRNA neochromosome in yeast

Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs

Design, construction, and functional characterization of a tRNA neochromosome in yeast

Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs

Genetic variations in the flanking regions of miR-101-2 are associated with increased risk of breast cancer.

Genetic variants in human microRNA (miRNA) genes may alter mature miRNA processing and/or target selection, and likely contribute to cancer susceptibility and disease progression. Previous studies have suggested that miR-101 may play important roles in the development of cancer by regulating key tumor-associated genes. However, the role of single nucleotide polymorphisms (SNPs) of miR-101 in breast cancer susceptibility remains unclear. In this study, we genotyped 11 SNPs of the miR-101 genes (including miR-101-1 and miR-101-2) in a case-control study of 1064 breast cancer cases and 1073 cancer-free controls. The results revealed that rs462480 and rs1053872 in the flank regions of pre-miR-101-2 were significantly associated with increased risk of breast cancer (rs462480 AC/CC vs AA: adjusted OR = 1.182, 95% CI: 1.030-1.357, P = 0.017; rs1053872 CG/GG vs CC: adjusted OR = 1.179, 95% CI: 1.040-1.337, P = 0.010). However, the remaining 9 SNPs were not significantly associated with risk of breast cancer. Additionally, combined analysis of the two high-risk SNPs revealed that subjects carrying the variant genotypes of rs462480 and rs1053872 had increased risk of breast cancer in a dose-response manner (P(trend) = 0.002). Compared with individuals with "0-1" risk allele, those carrying "2-4" risk alleles had 1.29-fold risk of breast cancer. In conclusion, these findings suggested that the SNPs rs462480 and rs1053872 residing in miR-101-2 gene may have a solid impact on genetic susceptibility to breast cancer, which may improve our understanding of the potential contribution of miRNA SNPs to cancer pathogenesis

Responses of Soil Respiration and Organic Carbon to Straw Mulching and Ridge Tillage in Maize Field of a Triple Cropping System in the Hilly Region of Southwest China

Soil disturbance by tillage practices promotes soil respiration which is a main source of carbon dioxide emission into the atmosphere. The present study was conducted to investigate the effect of different tillage practices on soil respiration and the carbon source/sink characteristics of maize farmland ecosystems in the wheat&#8722;maize&#8722;soybean cropping system. Six tillage treatments, namely, traditional tillage (T), ridge tillage (R), traditional tillage + straw mulching (TS), ridge tillage + straw mulching (RS), traditional tillage + straw mulching + decomposing inoculants (TSD), and ridge tillage + straw mulching + decomposing inoculants (RSD), were used to measure the soil respiration and its hydrothermal factors. The results showed that the intensity of soil respiration increased initially and decreased afterwards throughout the growth period of maize ranging from 1.011 to 5.575 &#956;mol (m2&#183;s)&#8722;1. The soil respiration rate under different treatments varied remarkably presenting a trend of RSD &gt; TSD &gt; TS &gt; RS &gt; T &gt; R. Ridge tillage reduced the soil respiration rate of maize farmland while straw mulching improved it. Meanwhile, ridge tillage and straw mulching increased the soil temperature sensitivity index of soil respiration, but the addition of decomposing inoculants reduced this trend. The soil moisture response threshold under ridge tillage was lower, while the straw mulching was found to increase it, compared with the control. Moreover, there was a positive correlation between trapped soil fauna and soil respiration. Compared with the control, ridge tillage and straw mulching were beneficial to the carbon sink of the farmland ecosystem as shown by the maize field for the entire growing season

Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond

INTRODUCTION Total synthesis of designer chromosomes and genomes is a new paradigm for the study of genetics and biological systems. The Sc2.0 project is building a designer yeast genome from scratch to test and extend the limits of our biological knowledge. Here we describe the design, rapid assembly, and characterization of synthetic chromosome VI (synVI). Further, we investigate the phenotypic, transcriptomic, and proteomic consequences associated with consolidation of three synthetic chromosomes–synVI, synIII, and synIXR—into a single poly-synthetic strain. RATIONALE A host of Sc2.0 chromosomes, including synVI, have now been constructed in discrete strains. With debugging steps, where the number of bugs scales with chromosome length, all individual synthetic chromosomes have been shown to power yeast cells to near wild-type (WT) fitness. Testing the effects of Sc2.0 chromosome consolidation to uncover possible synthetic genetic interactions and/or perturbations of native cellular networks as the number of designer changes increases is the next major step for the Sc2.0 project. RESULTS SynVI was rapidly assembled using nine sequential steps of SwAP-In (switching auxotrophies progressively by integration), yielding a ~240-kb synthetic chromosome designed to Sc2.0 specifications. We observed partial silencing of the left- and rightmost genes on synVI, each newly positioned subtelomerically relative to their locations on native VI. This result suggests that consensus core X elements of Sc2.0 universal telomere caps are insufficient to fully buffer telomere position effects. The synVI strain displayed a growth defect characterized by an increased frequency of glycerol-negative colonies. The defect mapped to a synVI design feature in the essential PRE4 gene ( YFR050C ), encoding the β7 subunit of the 20 S proteasome. Recoding 10 codons near the 3′ end of the PRE4 open reading frame (ORF) caused a ~twofold reduction in Pre4 protein level without affecting RNA abundance. Reverting the codons to the WT sequence corrected both the Pre4 protein level and the phenotype. We hypothesize that the formation of a stem loop involving recoded codons underlies reduced Pre4 protein level. Sc2.0 chromosomes (synI to synXVI) are constructed individually in discrete strains and consolidated into poly-synthetic (poly-syn) strains by “endoreduplication intercross.” Consolidation of synVI with synthetic chromosomes III (synIII) and IXR (synIXR) yields a triple-synthetic (triple-syn) strain that is ~6% synthetic overall—with almost 70 kb deleted, including 20 tRNAs, and more than 12 kb recoded. Genome sequencing of double-synthetic (synIII synVI, synIII synIXR, synVI synIXR) and triple-syn (synIII synVI synIXR) cells indicates that suppressor mutations are not required to enable coexistence of Sc2.0 chromosomes. Phenotypic analysis revealed a slightly slower growth rate for the triple-syn strain only; the combined effect of tRNA deletions on different chromosomes might underlie this result. Transcriptome and proteome analyses indicate that cellular networks are largely unperturbed by the existence of multiple synthetic chromosomes in a single cell. However, a second bug on synVI was discovered through proteomic analysis and is associated with alteration of the HIS2 transcription start as a consequence of tRNA deletion and loxPsym site insertion. Despite extensive genetic alterations across 6% of the genome, no major global changes were detected in the poly-syn strain “omics” analyses. CONCLUSION Analyses of phenotypes, transcriptomics, and proteomics of synVI and poly-syn strains reveal, in general, WT cell properties and the existence of rare bugs resulting from genome editing. Deletion of subtelomeres can lead to gene silencing, recoding deep within an ORF can yield a translational defect, and deletion of elements such as tRNA genes can lead to a complex transcriptional output. These results underscore the complementarity of transcriptomics and proteomics to identify bugs, the consequences of designer changes in Sc2.0 chromosomes. The consolidation of Sc2.0 designer chromosomes into a single strain appears to be exceptionally well tolerated by yeast. A predictable exception to this is the deletion of tRNAs, which will be restored on a separate neochromosome to avoid synthetic lethal genetic interactions between deleted tRNA genes as additional synthetic chromosomes are introduced. Debugging synVI and characterization of poly-synthetic yeast cells. ( A ) The second Sc2.0 chromosome to be constructed, synVI, encodes a “bug” that causes a variable colony size, dubbed a “glycerol-negative growth-suppression defect.” ( B ) Synonymous changes in the essential PRE4 ORF lead to a reduced protein level, which underlies the growth defect. ( C ) The poly-synthetic strain synIII synVI synIXR directs growth of yeast cells to near WT fitness levels. </jats:sec