EasyClone: method for iterative chromosomal integration of multiple genes in <em>Saccharomyces cerevisiae</em>

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 Log(10) 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

Biosynthesis of insect sex pheromone precursors via engineered β-oxidation in yeast

Mating disruption with insect sex pheromones is an attractive and environmentally friendly technique for pest management. Several Lepidoptera sex pheromones have been produced in yeast, where biosynthesis could be accomplished by the expression of fatty acyl-CoA desaturases and fatty acyl-CoA reductases. In this study, we aimed to develop yeast Yarrowia lipolytica cell factories for producing Lepidoptera pheromones which biosynthesis additionally requires β-oxidation, such as (Z)-7-dodecenol (Z7-12:OH), (Z)-9-dodecenol (Z9-12:OH), and (Z)-7-tetradecenol (Z7-14:OH). We expressed fatty acyl-CoA desaturases from Drosophila melanogaster (Dmd9) or Lobesia botrana (Lbo_PPTQ) and fatty acyl-CoA reductase from Helicoverpa armigera (HarFAR) in combinations with 11 peroxisomal oxidases of different origins. Yeast cultivations were performed with supplementation of methyl myristate (14:Me). The oxidase Lbo_31670 from L. botrana provided the highest titers of (Z)-7-dodecenoate, (Z)-9-dodecenoate, and (Z)-7-tetradecenoate. However, no chain-shortened fatty alcohols were produced. The mutation of fatty acid synthase (Fas2p(I1220F)) to increase myristate production did not lead to targeted fatty alcohol production. The problem was solved by directing the reductase into peroxisomes, where the strain with Dmd9 produced 0.10 ± 0.02 mg/l of Z7-12:OH and 0.48 ± 0.03 mg/l of Z7-14:OH, while the strain with Lbo_PPTQ produced 0.21 ± 0.03 mg/l of Z9-12:OH and 0.40 ± 0.07 mg/l of Z7-14:OH. In summary, the engineering of β-oxidation in Y. lipolytica allowed expanding the portfolio of microbially produced insect sex pheromones

Glucose-Dependent Promoters for Dynamic Regulation of Metabolic Pathways

For an industrial fermentation process, it can be advantageous to decouple cell growth from product formation. This decoupling would allow for the rapid accumulation of biomass without inhibition from product formation, after which the fermentation can be switched to a mode where cells would grow minimally and primarily act as catalysts to convert substrate into desired product. The switch in fermentation mode should preferably be accomplished without the addition of expensive inducers. A common cell factory Saccharomyces cerevisiae is a Crabtree-positive yeast and is typically fermented at industrial scale under glucose-limited conditions to avoid the formation of ethanol. In this work, we aimed to identify and characterize promoters that depend on glucose concentration for use as dynamic control elements. Through analysis of mRNA data of S. cerevisiae grown in chemostats under glucose excess or limitation, we identified 34 candidate promoters that strongly responded to glucose presence or absence. These promoters were characterized in small-scale batch and fed-batch cultivations using a quickly maturing rapidly degrading green fluorescent protein yEGFP3-Cln2PEST as a reporter. Expressing 3-hydroxypropionic acid (3HP) pathway from a set of selected regulated promoters allowed for suppression of 3HP production during glucose-excess phase of a batch cultivation with subsequent activation in glucose-limiting conditions. Regulating the 3HP pathway by the ICL1 promoter resulted in 70% improvement of 3HP titer in comparison to PGK1 promoter

Presentation_1_Glucose-Dependent Promoters for Dynamic Regulation of Metabolic Pathways.PDF

<p>For an industrial fermentation process, it can be advantageous to decouple cell growth from product formation. This decoupling would allow for the rapid accumulation of biomass without inhibition from product formation, after which the fermentation can be switched to a mode where cells would grow minimally and primarily act as catalysts to convert substrate into desired product. The switch in fermentation mode should preferably be accomplished without the addition of expensive inducers. A common cell factory Saccharomyces cerevisiae is a Crabtree-positive yeast and is typically fermented at industrial scale under glucose-limited conditions to avoid the formation of ethanol. In this work, we aimed to identify and characterize promoters that depend on glucose concentration for use as dynamic control elements. Through analysis of mRNA data of S. cerevisiae grown in chemostats under glucose excess or limitation, we identified 34 candidate promoters that strongly responded to glucose presence or absence. These promoters were characterized in small-scale batch and fed-batch cultivations using a quickly maturing rapidly degrading green fluorescent protein yEGFP3-Cln2_PEST as a reporter. Expressing 3-hydroxypropionic acid (3HP) pathway from a set of selected regulated promoters allowed for suppression of 3HP production during glucose-excess phase of a batch cultivation with subsequent activation in glucose-limiting conditions. Regulating the 3HP pathway by the ICL1 promoter resulted in 70% improvement of 3HP titer in comparison to PGK1 promoter.</p

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast.

Whole-cell biocatalysts have proven a tractable path toward sustainable production of bulk and fine chemicals. Yet the screening of libraries of cellular designs to identify best-performing biocatalysts is most often a low-throughput endeavor. For this reason, the development of biosensors enabling real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily of LysR-type transcriptional regulators (LTTRs). We identified a design supporting LTTR-dependent activation of reporter gene expression in the presence of cognate small-molecule inducers. As proof of principle, we applied the biosensors for in vivo screening of cells producing naringenin or cis,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications

Chromosomal distribution of Ty sequences on <i>S</i>. <i>cerevisiae</i> S288C genome.

<p>Ty sequences and their location were downloaded from <a href="http://www.yeastgenome.org" target="_blank">www.yeastgenome.org</a>. Green bars: Ty sequences, red circles: centromeres, black squares: telomeres.</p

Copy numbers and number of integration loci estimated from next generation sequencing data.

<p>Copy numbers and number of integration loci estimated from next generation sequencing data.</p

Phylogenetic tree based on alignments of LTRs from Ty elements of <i>S</i>. <i>cerevisiae</i> S288C.

<p>LTRs longer than 200 nucleotides were retrieved and a multiple alignment was performed at <a href="http://www.ebi.ac.uk/Tools/msa/clustalw2" target="_blank">www.ebi.ac.uk/Tools/msa/clustalw2</a>. A) A phylogenetic tree was created using the maximum likelihood method for tree building provided by the software MEGA6. The main cluster for each Ty family is highlighted blue (Ty1), green (Ty2), light pink (Ty3) or dark pink (Ty4). Five consensus sequences—Ty1Cons1, Ty1Cons2, Ty2Cons, Ty3Cons, and Ty4Cons—are specified in bold letters. B) Per Ty consensus sequence, the number of genome sequences sharing >0.95, >0.97 or >0.98 level of identity is reported. C) Pair-wise comparison of identity levels between consensus sequences.</p