Genomic integration of unclonable gene expression cassettes in Saccharomyces cerevisiae using rapid cloning‐free workflows

Abstract Most DNA assembly methods require bacterial amplification steps, which restrict its application to genes that can be cloned in the bacterial host without significant toxic effects. However, genes that cannot be cloned in bacteria do not necessarily exert toxic effects on the final host. In order to tackle this issue, we adapted two DNA assembly workflows for rapid, cloning‐free construction and genomic integration of expression cassettes in Saccharomyces cerevisiae. One method is based on a modified Gibson assembly, while the other relies on a direct assembly and integration of linear PCR products by yeast homologous recombination. The methods require few simple experimental steps, and their performance was evaluated for the assembly and integration of unclonable zeaxanthin epoxidase expression cassettes in yeast. Results showed that up to 95% integration efficiency can be reached with minimal experimental effort. The presented workflows can be employed as rapid gene integration tools for yeast, especially tailored for integrating unclonable genes.

Particularly, the latter has been widely adopted in the community because of its simplicity for joining multiple DNA parts in a single isothermal reaction. As in OE-PCR, Gibson assembly employs overlapping PCR products, but in this case, a T5 exonuclease is used to hydrolyze 5′ ends, thereby generating complementary overhangs for specific annealing. Lastly, the DNA polymerase and Taq ligase sequentially repair the double strand yielding a covalently joined seamless product.
The PCR products are then directly transformed in yeast where the circular vector is assembled by HR. Using this approach, an assembly of up to nine fragments in a 21-kb vector was carried out using 60-bp overlap regions (Kuijpers et al., 2013). Furthermore, so far this method has been shown to be much more effective for dealing with large DNA fragments than traditional cloning in Escherichia coli (Kouprina & Larionov, 2016). For instance, due to instability issues of large DNA constructs in E. coli, in vivo DNA assembly in yeast was critical for assembling the first synthetic bacterial genomes (Gibson, Benders, Andrews-Pfannkoch, et al., 2008;Gibson, Benders, Axelrod, et al., 2008;Gibson et al., 2010).
Besides its use as DNA assembly tool and owed to its well-studied and highly tunable genetics, yeast is currently considered a model organism for biotechnological applications (Lian, Mishra, & Zhao, 2018). Shuttle vectors are plasmids commonly used for gene expression in S. cerevisiae (Gnügge & Rudolf, 2017). These plasmids have genetic sequences that enable their maintenance in both E. coli and S. cerevisiae. This feature enables the construction (usually using in vitro assembly methods), analysis, and amplification of the plasmid in E. coli for subsequent yeast transformation. However, some genes are unclonable in E. coli even in the absence of a bacterial promoter, pointing to DNA toxicity and/or genetic instability (Kimelman et al., 2012). Examples of known genes that cannot be cloned in E. coli include Vssc1 sodium (Lee & Soderlund, 2009) and Cch1 calcium (Vu, Bautos, Hong, & Gelli, 2013) channels, to name a few. In yeast, extrachromosomal expression vectors can be directly assembled by in vivo recombination, thereby bypassing bacterial transformation.
However, this approach has been less explored for direct assembly of integrative constructs. Interestingly, Shao, Zhao, & Zhao (2009) evaluated and demonstrated a high capacity of yeast for assembling and integrating functional expression constructs from PCR-amplified fragments in a single transformation event. These results motivated us to develop simpler assembly and integration workflows for yeast that avoid bacterial transformation altogether.
In this work, we report two simple and rapid workflows for direct assembly and site-specific integration of gene expression cassettes in S. cerevisiae without bacterial cloning steps. One method is based on a modification of Gibson assembly -here termed full in vitro Gibson assembly-whereas the other is based on a direct assembly of PCR-amplified fragments by yeast HR. The methods were validated for the construction and high efficiency integration of expression cassettes for Haematococcus lacustris and Solanum lycopersicum zeaxanthin epoxidase (ZEP) genes. These genes cannot be cloned in E. coli due to toxic effects. The presented workflows provide simple, rapid, and efficient gene assembly and integration alternatives for yeast, especially suitable but not limited to unclonable genes.

| Strains, growth conditions, and DNA templates
Codon-optimized ZEP genes from H. lacustris (HlZEP) and S. lycopersicum (SlZEP) were synthesized by Genscript. Full gene sequences can be found in Table A1. The XI-5 integrative vector with bidirectional PGK1/TEF1 promoters was constructed using the plasmid set described by Mikkelsen et al. (2012). Saccharomyces cerevisiae BY4742 strain was used in all transformations. Cultures were grown in complete YPD medium (20 g/L of peptone, 20 g/L of glucose, and 10 g/L of yeast extract) at 30°C. Yeast transformants were incubated in synthetic medium plates containing: 1.8 g/L of yeast nitrogen base, 5 g/L of ammonium sulfate, 0.8 g/L of CSM-Ura mixture (Sunrise Science Products), 20 g/L of glucose, and 20 g/L of agar.

| DNA construction and assembly
Direct assembly of expression cassettes by HR was performed using three PCR-amplified fragments (F1, F2, and F3, Figure 1). To generate each set of fragments, six primers were designed: UP-F, UP-R, DOWN-F, DOWN-R, ZEP-F, and ZEP-R. Overlapping regions between fragments were included in the 5′ sequence of the primers (exemplified in Figure 2). To evaluate the effect of the overlap length on the assembly efficiency, three sets of primers were designed for each HlZEP and SlZEP expression cassette with overlap lengths of 40, 60, and 100 bp.
Fragments for Gibson assembly were generated with the same primers used for the 40 bp homologous recombination cassettes: DOWN-F/ UP-R and ZEP-F/ZEP-R. Primer sequences are listed in Table A2. DNA fragments for both assembly methods were amplified by PCR using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific).
PCR reactions were carried out in 100 µl containing 0.5 pmol/μl of each primer, HF buffer 5×, and 0.02 U/μl of Phusion DNA polymerase. The PCR protocol consisted of an initial denaturation at 98°C for 2 min, then 35 cycles of amplification (98°C for 10 s, 60°C for 30 s, and 72°C for 3 to 6 min), followed by a final extension of 72°C for 10 min. PCR products were purified by gel extraction using Wizard SV Gel and PCR Clean-Up kit (Promega), according to the manufacturer's instructions.
Assembly was achieved by mixing 2.5 µl containing 100 ng of equimolar fragments with 7.5 µl of master mix and incubated at 50°C for 2 hr.
Finally, 4 μl of the reaction products was used as PCR templates where the UP-F and DOWN-R primers were employed for the amplification of the UP-DOWN cassettes. These PCR products were digested with 5 U of DpnI for 1 hr to eliminate the residual parental vector.

| Yeast transformation
Transformations were performed by LiAc/SS carrier DNA/PEG method (Gietz & Schiestl, 2007) with a slight modification. In order to increase the volume of DNA fragments, a more diluted transformation mix was employed (0.09 M of lithium acetate). In the case of assembly by HR, transformations were performed using 3 pmol of each fragment mixed to a 100-μl final volume. In full in vitro Gibson assemblies, all the resulting PCR product (~1.2 pmol in 100 μl) was used for the transformation. The transformed cells were plated on SC-Ura agar and incubated for two to three days at 30°C. The XI-5 empty vector linearized by SwaI digestion was used as transformation control.

| Evaluation of integration efficiency
In each transformation, ten colonies were individually picked and cultured for 16 hr at 30°C in liquid YPD media. Genomic DNAs were then extracted using Wizard Genomic DNA Purification Kit (Promega). Confirmation of correct chromosomal integration of the F I G U R E 1 Schematic overview of cloning-free methods for assembly and integration of expression cassettes exemplified for ZEP expression constructs. Full in vitro Gibson assembly bypasses bacterial amplification using the reaction product as a template for PCR amplification of the desired integration cassette. Direct assembly by HR is based on the transformation of linear overlapping PCR products which are assembled and integrated into the genome in a single transformation event. The primers used in each method are indicated next to the arrows. F1, F2, and F3 refer to fragments 1, 2, and 3 assembled expression cassettes was carried out by PCR amplification of the previously extracted DNA. Four PCR rounds were performed on each strain for efficiency analysis: one that amplified the entire assembled cassette from the UP to DOWN region and three that amplified between the recombination regions of each fragment.
PCR reactions were carried out with Phusion High-Fidelity DNA Polymerase using the same cycling parameter described in section 2.2. The list of used primers can be found in Table A2.

| RE SULTS AND D ISCUSS I ON
We could not clone HlZEP and SlZEP genes in the yeast integrative vector by traditional Gibson assembly regardless of the E. coli strain evaluated (TOP10, DH5α, and K12). This result suggests high toxicity or instability of the ZEP expression cassettes in E. coli. As previously reported, many gene products, either noncoding RNA or proteins, can be toxic in E. coli (Kimelman et al., 2012). Although in this study ZEP genes were cloned under the control of yeast PGK1 promoter, some eukaryotic promoters can still drive gene expression in E. coli (Antonucci, Wen, & Rutter, 1989;Gognies, Bahkali, Moslem, & Belarbi, 2012). Thus, ZEP genes may have been expressed in the transformed cells causing toxicity. Another plausible cause is related to the toxicity of the DNA itself (Kouprina & Larionov, 2016).
Unclonable noncoding DNA regions have been suggested to exert such effect, but the underpinning molecular mechanisms have not been fully elucidated. Some cloned sequences can seemingly cause toxicity due to their high capacity to recruit and titrate essential DNA binding proteins such as replicator initiator DnaA (Kimelman et al., 2012) or RNA polymerase (Lamberte et al., 2017).
To tackle the above limitations, we developed two strategies that enable assembly and chromosomal integration of expression constructs in yeast without the need of bacterial transformation.
As a proof of concept, the ZEP genes were used in this study. Our approaches employ the set of plasmids designed by Mikkelsen et al. (2012) as transcriptional backbones. Briefly, these vectors enable the integration of one or two genes controlled by a bidirectional promoter in specific chromosomal sites. The vector set uses URA3 as a selectable marker, which is flanked by a direct repeat to enable marker recycling and more transformation rounds. As illustrated in Figure 1, we assembled the ZEP genes into expression cassettes using the vector XI-5 as a backbone (i.e., integration in site 5 of chromosome XI) by two different strategies: full in vitro Gibson assembly and direct assembly by HR. Both methods are PCR-based and do not need bacterial transformation nor plasmid isolation steps.

F I G U R E 2
Illustration of primer design for generation of overlapping fragments. The homology region between fragments is included in the 5′ nonpriming sequences of the primers

| Full in vitro Gibson assembly
Gibson assembly requires one reaction to join DNA fragments into a vector. Typically, the reaction product is transformed and amplified in E. coli. For shuttle integrative vectors in yeast, like as XI-5 (Figure 1), additional digestion with SwaI and gel purification are necessary steps for isolating the desired integrating DNA fragment and discarding bacterial elements (Ori and Amp). Here, we propose a simple modification of this protocol, where the Gibson assembly product is used as DNA template in a PCR reaction that amplifies only the segment that will be integrated into the yeast genome (Figure 1). This PCR reaction can be transformed directly after digestion with Dpn1 (to eliminate the parental vector), without subsequent clean-up steps. Thus, both the assembly and amplification of the Gibson assembly product occur in vitro, as opposed to the conventional Gibson method where the assembly takes place in vitro and the amplification occurs in E. coli. This method was applied to integrate HlZEP and SlZEP expression cassettes in yeast. Resulting colonies were screened for successful integration by genomic PCR with a set of primers that amplified three segments and the whole integrated construct ( Figure 3). As shown in Table 1, a 95% chromosomal integration efficiency was achieved for HlZEP and SlZEP expression cassettes, slightly less than that for SwaI digestion of the empty vector (100%).
Notably, this method is likely limited by the length of the integrating fragment that can be amplified by high-fidelity polymerases. However, for integrative cassettes of one or two genes (5-8 kb), one PCR reaction using high-fidelity polymerases can easily render the required DNA amount (1 pmol) for efficient yeast transformation.

| Direct assembly by homologous recombination
Based on the recombination scheme proposed by Shao et al. (2009), we developed a direct in vivo DNA assembly and site-specific integration method from linear PCR products. Three overlapping fragments were generated by PCR using the backbone vector and the gene of interest as templates (Figure 1). Similar to Gibson assembly, primers were designed with a nonpriming sequence at the 5′ end that is homologous to the 5′ end of the fragment to join (exemplified in Figure 2). All fragments were cotransformed in yeast, which assembled and ultimately integrated the construct by HR. To evaluate the effect of the overlap length on the assembly, we transformed each HlZEP and SlZEP construct with a set of fragments with 40, 60, and 100 bp of homology. Correct assembly and integration were verified by genomic PCR analysis of transformants using primers that annealed specifically in the overlap regions ( Figure 3). Examples of the analysis of more colonies can be found in Figure A1. The overlap length had a strong positive effect on the assembly and integration efficiency, reaching up to 85% efficiency when a 100 bp overlap segment was employed (Table 1). Notably, direct assembly by HR requires only one PCR round (full in vitro Gibson assembly requires two), and thus can be readily performed in a day. F I G U R E 3 Assembly and integration of ZEP expression cassettes. (a) Scheme of assembled ZEP expression cassette with the corresponding verification primers. Genomic PCR analysis of the integrated HlZEP and SlZEP constructs for fullin-vitro Gibson assembly (b) and direct assembly by HR (c). S: 1 kb DNA ladder. F1, F2, and F3 refer to Fragments 1, 2, and 3, and U/D represents the UP/DOWN region. Empty refers to the backbone vector linearized by SwaI digestion. Expected PCR products ( (Shao et al., 2009). The proposed method avoids multiple integrations events that usually occur in δ sites (Sakai, Shimizu, & Hishinuma, 1990;Wang, Wang, & Da Silva, 1996), allowing finer control of the gene copy number. Finally, instead of single homology arm integration, we proposed a double crossing-over configuration, which avoids direct repeats sequences and increases the genomic stability of the construct (Gnügge & Rudolf, 2017;Taxis & Knop, 2006). the plasmid can be assembled extrachromosomally in yeast and converted to an integrative vector in bacteria, which can be then used to transform the yeast again. This time-consuming cloning strategy requires several transformations and plasmid isolation steps. In contrast, the direct assembly by HR proposed here simplifies the assembly and integration of expression cassettes to only few simple steps.

| CON CLUS ION
In this work, we presented two simple, rapid, and effective workflows for cloning-free assembly and integration of gene expression cassettes in S. cerevisiae. While both approaches are inspired on reported assembly strategies, the introduced adaptations enabled substantial reductions in experimental efforts while maintaining high integration efficiencies. The first method-termed full in vitro Gibson assemblyshowed the best integration efficiency (95%), while the second-direct assembly by HR-was faster (it can be performed in a day) with a reasonably high efficiency (85%). Importantly, both techniques can be readily employed to join more than three fragments, for example, construction of bidirectional expression cassettes by a four-fragment assembly. Although the tools presented here are particularly tailored for genes that are unclonable in E. coli, they can also be used as generalpurpose, rapid, and efficient gene integration alternatives methods.

ACK N OWLED G M ENTS
This work was funded by FONDECYT grant number 1170745 from CONICYT. Vicente F. Cataldo was supported by a PhD fellowship from CONICYT.

CO N FLI C T O F I NTE R E S T
None declared. Direct assembly by HR-60 bp overlap 50

AUTH O R CO NTR I B UTI O N S
Direct assembly by HR-100 bp overlap 85