Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log10 mean ± 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out.

The widely used pESC vector series (Stratagene, La Jolla, CA, USA) with the bidirectional GAL1 /GAL10 promoter provides the possibility of simultaneously expressing two different genes from a single vector in Saccharomyces cerevisiae. This system can be induced by galactose and is repressed by glucose. Since S. cerevisiae prefers glucose as a carbon source, and since its growth rate is higher in glucose than in galactose-containing media, we compared and evaluated seven different promoters expressed during growth on glucose (pTEF1, pADH1, pTPI1, pHXT7, pTDH3, pPGK1 and pPYK1 ) with two strong galactose-induced promoters (pGAL1 and pGAL10 ), using lacZ as a reporter gene and measuring LacZ activity in batch and continuous cultivation. TEF1 and PGK1 promoters showed the most constant activity pattern at different glucose concentrations. Based on these results, we designed and constructed two new expression vectors which contain the two constitutive promoters, TEF1 and PGK1, in opposite orientation to each other. These new vectors retain all the features from the pESC-URA plasmid except that gene expression is mediated by constitutive promoters.

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Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharomyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the β-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of β-alanine and its subsequent conversion into 3HP using a novel β-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7±0.3gL(-1) with a 0.14±0.0C-molC-mol(-1) yield on glucose in 80h in controlled fed-batch fermentation in mineral medium at pH 5, and this work therefore lays the basis for developing a process for biological 3HP production.

Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intracellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus, a bacterial-type enzyme from the amoeba Dictyostelium discoideum, and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 M cinnamic acid per unit of optical density at 600 nm [OD 600 ]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p-coumaric acid in Escherichia coli (producing 440 M p-coumaric acid OD 600 unit ؊1 in the medium) and in Lactococcus lactis. The enzymes were also efficient in Saccharomyces cerevisiae, where p-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases. Small organic molecules of biotechnological interest include aromatic structures that are derived from p-coumaric acid (pHCA). pHCA can be formed from phenylalanine either through deamination to cinnamic acid (CA) by phenylalanine ammonialyase (PAL; EC 4.3.1.24) and subsequent hydroxylase activity by trans-cinnamate-4-monooxygenase or through deamination of tyrosine by tyrosine ammonia-lyase (TAL; EC 4.3.1.23) (Fig. 1). Considering that the route from phenylalanine requires activity of a P450 enzyme, which has been shown to be the limiting step in previous engineering strategies (1, 2), deamination of tyrosine for production of pHCA may be preferred in microbial cell factories. Tyrosine ammonia-lyases (TAL) have been employed for the production of plant biochemicals and aromatic compounds, e.g., for pHCA itself (3-5), or in the production of stilbenes, such as resveratrol (6, 7), flavonoids, such as naringenin (8, 9), cinnamoyl anthranilates (10), or plastic precursors, such as p-hydroxystyrene (11, 12), yet the TAL reaction may be the limiting step (10, 13).Due to the significant interest in production of phenolic compounds in biotechnological processes, we aimed at increasing the range of characterized phenylalanine ammonia-lyases (PAL) and tyrosine ammonia-lyases (TAL), and in particular at identifying the optimal TAL for use in microbial cell factories...

The ability to transfer metabolic pathways from the natural producer organisms to the well-characterized cell factory Saccharomyces cerevisiae is well documented. However, as many secondary metabolites are produced by collaborating enzymes assembled in complexes, metabolite production in yeast may be limited by the inability of the heterologous enzymes to collaborate with the native yeast enzymes. This may cause loss of intermediates by diffusion or degradation or due to conversion of the intermediate through competitive pathways. To bypass this problem, we have pursued a strategy in which key enzymes in the pathway are expressed as a physical fusion. As a model system, we have constructed several fusion protein variants in which farnesyl diphosphate synthase (FPPS) of yeast has been coupled to patchoulol synthase (PTS) of plant origin (Pogostemon cablin). Expression of the fusion proteins in S. cerevisiae increased the production of patchoulol, the main sesquiterpene produced by PTS, up to 2-fold. Moreover, we have demonstrated that the fusion strategy can be used in combination with traditional metabolic engineering to further increase the production of patchoulol. This simple test case of synthetic biology demonstrates that engineering the spatial organization of metabolic enzymes around a branch point has great potential for diverting flux toward a desired product.

The yeast Saccharomyces cerevisiae was chosen as a microbial host for heterologous biosynthesis of three different plant sesquiterpenes, namely valencene, cubebol, and patchoulol. The volatility and low solubility of the sesquiterpenes were major practical problems for quantification of the excreted sesquiterpenes. In situ separation of sesquiterpenes in a two-phase fermentation using dodecane as the secondary phase was therefore performed in order to enable quantitative evaluation of different strains. In order to enhance the availability of the precursor for synthesis of sesquiterpenes, farnesyl diphosphate (FPP), the ERG9 gene which is responsible for conversion of FPP to squalene was downregulated by replacing the native ERG9 promoter with the regulatable MET3 promoter combined with addition of 2 mM methionine to the medium. This strategy led to a reduced ergosterol content of the cells and accumulation of FPP derived compounds like target sesquiterpenes and farnesol. Adjustment of the methionine level during fermentations prevented relieving MET3 promoter repression and resulted in further improved sesquiterpene production. Thus, the final titer of patchoulol and farnesol in the ERG9 downregulated strain reached 16.9 and 20.2 mg/L, respectively. The results obtained in this study revealed the great potential of yeast as a cell factory for production of sesquiterpenes.

BackgroundOne of the bottlenecks in production of biochemicals and pharmaceuticals in Saccharomyces cerevisiae is stable and homogeneous expression of pathway genes. Integration of genes into the genome of the production organism is often a preferred option when compared to expression from episomal vectors. Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers.ResultsHere, we have developed a novel method, CrEdit (CRISPR/Cas9 mediated genome Editing), which utilizes targeted double strand breaks caused by CRISPR/Cas9 to significantly increase the efficiency of homologous integration in order to edit and manipulate genomic DNA. Using CrEdit, the efficiency and locus specificity of targeted genome integrations reach close to 100% for single gene integration using short homology arms down to 60 base pairs both with and without selection. This enables direct and cost efficient inclusion of homology arms in PCR primers. As a proof of concept, a non-native β-carotene pathway was reconstructed in S. cerevisiae by simultaneous integration of three pathway genes into individual intergenic genomic sites. Using longer homology arms, we demonstrate highly efficient and locus-specific genome integration even without selection with up to 84% correct clones for simultaneous integration of three gene expression cassettes.ConclusionsThe CrEdit approach enables fast and cost effective genome integration for engineering of S. cerevisiae. Since the choice of the targeting sites is flexible, CrEdit is a powerful tool for diverse genome engineering applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0288-3) contains supplementary material, which is available to authorized users.