EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae

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.

Fig 1. Chromosomal distribution of Ty sequences on S. cerevisiae S288C genome.

Ty sequences and their location were downloaded from www.yeastgenome.org. Green bars: Ty sequences, red circles: centromeres, black squares: telomeres.

Fig 2. Phylogenetic tree based on alignments of LTRs from Ty elements of S. cerevisiae S288C.

LTRs longer than 200 nucleotides were retrieved and a multiple alignment was performed at www.ebi.ac.uk/Tools/msa/clustalw2. 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.

Fig 3. Functional map of the EasyCloneMulti vectors.

Two types of EasyCloneMulti were constructed: A) EasyCloneMulti with a USER cassette for cloning purposes and B) EasyCloneMulti with a reporter cassette consisting of P_TEF1-GFP-T_CYC1. All EasyCloneMulti vectors share the following features: 1) upstream and downstream regions for insertion at specific Ty sequences, HR 5’ and HR 3’, flanked by NotI restriction sites for linearization 2) a selective marker, i.e. URA3 from Kluyveromyces lactis, flanked by loxP sites; 3) an in-frame fusion of the marker gene with a degradation signal. HR 5’ and HR 3’ are in direct orientation with respect to one another to trigger gene conversion based integrations. EasyCloneMulti with a USER cassette support the same USER cloning possibilities as EasyClone vectors [16].

Fig 4. Comparison of single cell fluorescence levels of S. cerevisiae strains bearing different types of vectors.

Single cell measurements of fluorescence of strain CEN.PK 113-5D transformed with either of the following vectors: pCfB319 (episomal, 2μ, URA3), pCfB329 (single integrative at locus Chr. X-2, URA3), pCfB321 (multi-integrative, Ty4Cons based, URA3) and pCfB326 (multi-integrative, Ty4Cons, fusion URA3-degradation signal). Low (grey), mid (blue) and high (orange) expression levels were arbitrarily defined and are represented.

Fig 5. Effect of the different EasyCloneMulti vector backbones on fluorescence levels of a GFP reporter gene.

Analysis of specific fluorescence levels of EasyCloneMulti vectors containing the reporter cassette P_TEF1-GFP-T_CYC1 and bearing the indicated consensus sequence for integration at Ty sequences: pCfB1136 (Ty1Cons1), pCfB1137 (Ty1Cons2), pCfB1138 (Ty2Cons), pCfB1139 (Ty3Cons), and pCfB326 (Ty4Cons). Integration of pCfB329 (single integrative, Chr. X-2) is used as reference for GFP expression from a single defined locus. The resulting fluorescence of S. cerevisiae CEN.PK.113-5D bearing either of the vectors was measured on a spectrophotometer. 16 individual clones for each vector type were tested. Average (red bars) and standard deviation (orange bar) are also represented.

Fig 6. 3HP production by S. cerevisiae strains bearing T. castaneum panD on different EasyCloneMulti vectors.

Yeast strain SCE-iL1-155 was transformed with different EasyCloneMulti vectors that are expressing T. castaneum panD: pCfB2099 (Ty1Cons1), pCfB2097 (Ty1Cons2), pCfB2096 (Ty2Cons), or pCfB799 (Ty4cons). Final 3HP titers after cultivation in synthetic fed-batch medium (m2p-Labs) are shown. Average (red bars) and standard deviation (orange bar) are also represented.