Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains.

Assembly of DNA parts into DNA constructs is a foundational technology in the emerging field of synthetic biology. An efficient DNA assembly method is particularly important for high-throughput, automated DNA assembly in biofabrication facilities and therefore we investigated one-step, scarless DNA assembly via ligase cycling reaction (LCR). LCR assembly uses single-stranded bridging oligos complementary to the ends of neighboring DNA parts, a thermostable ligase to join DNA backbones, and multiple denaturation-annealing-ligation temperature cycles to assemble complex DNA constructs. The efficiency of LCR assembly was improved ca. 4-fold using designed optimization experiments and response surface methodology. Under these optimized conditions, LCR enabled one-step assembly of up to 20 DNA parts and up to 20 kb DNA constructs with very few single-nucleotide polymorphisms (<1 per 25 kb) and insertions/deletions (<1 per 50 kb). Experimental comparison of various sequence-independent DNA assembly methods showed that circular polymerase extension cloning (CPEC) and Gibson isothermal assembly did not enable assembly of more than four DNA parts with more than 50% of clones being correct. Yeast homologous recombination and LCR both enabled reliable assembly of up to 12 DNA parts with 60-100% of individual clones being correct, but LCR assembly provides a much faster and easier workflow than yeast homologous recombination. LCR combines reliable assembly of many DNA parts via a cheap, rapid, and convenient workflow and thereby outperforms existing DNA assembly methods. LCR assembly is expected to become the method of choice for both manual and automated high-throughput assembly of DNA parts into DNA constructs.

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Free‐energy (ATP) conservation during product formation is crucial for the maximum product yield that can be obtained, but often overlooked in metabolic engineering strategies. Product pathways that do not yield ATP or even demand input of free energy (ATP) require an additional pathway to supply the ATP needed for product formation, cellular maintenance, and/or growth. On the other hand, product pathways with a high ATP yield may result in excess biomass formation at the expense of the product yield. This mini‐review discusses the importance of the ATP yield for product formation and presents several opportunities for engineering free‐energy (ATP) conservation, with a focus on sugar‐based product formation by Saccharomyces cerevisiae. These engineering opportunities are not limited to the metabolic flexibility within S. cerevisiae itself, but also expression of heterologous reactions will be taken into account. As such, the diversity in microbial sugar uptake and phosphorylation mechanisms, carboxylation reactions, product export, and the flexibility of oxidative phosphorylation via the respiratory chain and H+‐ATP synthase can be used to increase or decrease free‐energy (ATP) conservation. For product pathways with a negative, zero or too high ATP yield, analysis and metabolic engineering of the ATP yield of product formation will provide a promising strategy to increase the product yield and simplify process conditions.

Mixed culture fermentations are of interest for the low-cost production of organic acids from complex agricultural waste streams. Models are developed for these processes in order to predict the product spectrum as a function of the environmental process conditions. An important assumption in many existing models for anaerobic mixed culture fermentations is that the NADH/NAD(+) ratio is directly coupled to the dissolved hydrogen partial pressure (pH2, liquid). In this study, this assumption was tested experimentally with mixed culture chemostats operated at dilution rates of 0.05 and 0.125 h(-1) for a wide range of calculated dissolved hydrogen partial pressures (0.04-6.8 atm). No correlation was found between pH2, liquid and the NADH/NAD(+) ratio. This result, together with thermodynamic calculations, suggests that additional electron carriers such as ferredoxin and formate should be included in models predicting product formation by mixed cultures.

Laboratory evolution is a powerful approach in applied and fundamental yeast research, but complete elucidation of the molecular basis of evolved phenotypes remains a challenge. In this study, DNA microarray-based transcriptome analysis and whole-genome resequencing were used to investigate evolution of novel lactate transporters in Saccharomyces cerevisiae that can replace Jen1p, the only documented S. cerevisiae lactate transporter. To this end, a jen1Δ mutant was evolved for growth on lactate in serial batch cultures. Two independent evolution experiments yielded growth on lactate as sole carbon source (0.14 and 0.18 h(-1) , respectively). Transcriptome analysis did not provide leads, but whole-genome resequencing showed different single-nucleotide changes (C755G/Leu219Val and C655G/Ala252Gly) in the acetate transporter gene ADY2. Introduction of these ADY2 alleles in a jen1Δ ady2Δ strain enabled growth on lactate (0.14 h(-1) for Ady2p(Leu219Val) and 0.12 h(-1) for Ady2p(Ala252Gly) ), demonstrating that these alleles of ADY2 encode efficient lactate transporters. Depth of coverage of DNA sequencing, combined with karyotyping, gene deletions and diagnostic PCR, showed that an isochromosome III (c. 475 kb) with two additional copies of ADY2(C755G) had been formed via crossover between retrotransposons YCLWΔ15 and YCRCΔ6. The isochromosome formation shows how even short periods of selective pressure can cause substantial karyotype changes.

Laboratory evolution is a powerful approach in applied and fundamental yeast research, but complete elucidation of the molecular basis of evolved phenotypes remains a challenge. In this study, DNA microarray‐based transcriptome analysis and whole‐genome resequencing were used to investigate evolution of novel lactate transporters in Saccharomyces cerevisiae that can replace Jen1p, the only documented S. cerevisiae lactate transporter. To this end, a jen1Δ mutant was evolved for growth on lactate in serial batch cultures. Two independent evolution experiments yielded growth on lactate as sole carbon source (0.14 and 0.18 h−1, respectively). Transcriptome analysis did not provide leads, but whole‐genome resequencing showed different single‐nucleotide changes (C755G/Leu219Val and C655G/Ala252Gly) in the acetate transporter gene ADY2. Introduction of these ADY2 alleles in a jen1Δ ady2Δ strain enabled growth on lactate (0.14 h−1 for Ady2pLeu219Val and 0.12 h−1 for Ady2pAla252Gly), demonstrating that these alleles of ADY2 encode efficient lactate transporters. Depth of coverage of DNA sequencing, combined with karyotyping, gene deletions and diagnostic PCR, showed that an isochromosome III (c. 475 kb) with two additional copies of ADY2C755G had been formed via crossover between retrotransposons YCLWΔ15 and YCRCΔ6. The isochromosome formation shows how even short periods of selective pressure can cause substantial karyotype changes.