Abstract
Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence diversity in the long terminal repeats (LTR) of Ty retrotransposons, we developed a new set of integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a range of expression levels were obtained, as assessed with the green fluorescent protein (GFP) reporter system. The EasyCloneMulti vector set was applied to balance the expression of the rate-controlling step in the β-alanine pathway for biosynthesis of 3-hydroxypropionic acid (3HP). The best 3HP producing clone, with 5.45 g.L-1 of 3HP, produced 11 times more 3HP than the lowest producing clone, which demonstrates the capability of EasyCloneMulti vectors to impact metabolic pathway enzyme activity.
Highlights
Budding yeast Saccharomyces cerevisiae is used in the biotech industry for the production of a wide range of chemicals, ranging from biofuels and bulk chemicals to nutraceuticals and pharmaceuticals [1,2,3,4]
long terminal repeats (LTR) of Ty retrotransposons are scattered in multiple copies throughout the entire genome of S. cerevisiae and are present on all chromosomes
These sequences should preferably share a high degree of identity with a maximum number of LTRs belonging to the same family while being highly dissimilar to sequences belonging to other families
Summary
Budding yeast Saccharomyces cerevisiae is used in the biotech industry for the production of a wide range of chemicals, ranging from biofuels and bulk chemicals to nutraceuticals and pharmaceuticals [1,2,3,4]. S. cerevisiae is an excellent host for heterologous production of metabolites and fine chemicals at industrial scale, since it has a GRAS (Generally Recognized As Safe) status and is tolerant to low pH. In order to produce a chemical of interest, yeast strains are usually metabolically engineered by inserting and optimizing the required heterologous pathways as well as by improving the host properties, such as precursor and co-factor supply, tolerance to the product, or by-products formation [12]. Using high copy plasmids such as 2μ-based vectors is not the preferred option, since these plasmids are not mitotically stable and thereby lead to heterogeneous gene expression in a population of cells [15,16]. Integrating genes in multiple copies into the genome to maintain stable production of the desired molecule for many generations is of high interest
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