*2.4. Construction of pTYGS*·*arg*·*pccABE*

The vector pTYGS·*arg* was fully digested using *Asc*I. All codon-optimized *pcc* gene fragments (http://genomes.urv.es/OPTIMIZER/, accessed on: 1 January 2020) flanked with overlaps (by *ca* 30 bp) were synthesized commercially. The plasmids bearing *pccABE* gene fragments were prepared by using double restriction enzymatic digestion (*pccA*-*EcoR*I/*BamH*I, *pccB*-*Xba*I/*BamH*I, *pccE*-*BamH*I/*Xba*I) for the plasmid reassembly in yeast. The competent *Saccharomyces cerevisiae* CEN.PK cells were removed from −80 ◦C storage and placed on ice to thaw. The following components were added to the yeast pellet in order: 240 µL PEG solution (50% (*w*/*v*) polyethylene glycol 3350); 36 µL LiAc (1 M); <sup>50</sup> <sup>µ</sup>L denatured salmon testis DNA (2 mg·mL−<sup>1</sup> in TE buffer) and 34 µL DNA fragments containing the linearized pTYGS·*arg* and three desired inserts in equimolar concentration (the uncut plasmid was used as the positive control and the linearized plasmid was used as the negative control). The mixture was resuspended and incubated for 50 min at 42 ◦C. Cells were pelleted by centrifugation at 11,000× *g* for 15 s and the supernatant was removed. The pellet was resuspended in 500 µL deionized H2O, and 100 µL suspension was spread on selective SM-URA plates, which were incubated for 3 days at 28 ◦C.

Yeast colonies were gathered. Plasmids were extracted from yeast colonies by using the ZymoprepTM Yeast Plasmid Miniprep II kit (Zymo Research, Freiburg Germany). Subsequently, using standard heat-shock protocols, the extracted plasmid mixture was introduced into *<sup>E</sup>*. *coli ccdB* survival 2 T1<sup>R</sup> with the antibiotic carbenicillin (50 mg·mL−<sup>1</sup> ). Large amounts of *E*. *coli* colonies were generated, and then five colonies were picked up and identified by the colony PCR using specific 50 and 30 test primers. Plasmids were then extracted from positive colonies by using the Nucleospin® Extract Kit (Machery-Nagel, Düren, Germany) and identified using the double restriction enzymatic digestion (*Nhe*I+*Swa*I). The construct was checked by gene sequencing for correct construction.
