Modular toolkit structure - The toolkit consists of seven modules for quick and easy assembly of integrative constructs and Cas9-helper plasmids. Lvl0 Module: single parts in entry vectors. Exp Module: assembly of overexpression constructs. Pro Module: assembly and screening of new promoters. Del Module: assembly of disruption constructs. Int Module: changing integration loci by homology arms exchange. MEx Module: assembly of marker-free constructs by selectable marker excision. Cas Module: redirection of Cas9-helper to new genome loci. Five of the modules - Pro, Del, Cas, Int, and MEx Modules - represent a new methodology that functionally extends previously used GG assembly systems. Assembly of Pro, Del and Int Modules are based on single GG reactions, while Cas and MEx Modules involve homologous and site-specific recombination taking place in the special E. coli strains. The arrows between modules indicate different orders in which they can be applied to enable variable genome engineering techniques as shown in the top panel, Yeast. Credit: Nature Communications Biology, doi: https://doi.org/10.1038/s42003-023-05202-5

Promoter library screening using CRISPR/Cas9. In the Pro Module an amplified promoter can be easily assembled in a single GG step using RFP dropout selection. Resultant Pro-series plasmids can be used for both TU assembly in the Exp Module and for promoter assay after integration into the yeast genome. Single-copy integration into the standard locus is facilitated by a co-transformed Cas9-helper plasmid. Transformants are first selected by Nat-resistance encoded by the episomal helper, followed by Ura⁺ phenotype verification confirming integration of the construct. The 96-well plate pictured demonstrates fluorescence of the Y. lipolytica transformant library with 93 native promoters grown in YPD medium (Section 8.1 of Supplementary material). Two bottom panels contain bar charts with 93 native and 43 hybrid promoters that were assayed using plate reader and flow cytometer respectively. Data were blanked using the parent strain S234 (0%) and normalized by TEF1 promoter activity (100%). For each promoter the GFP florescence was assayed in minimal (blue) and rich (red) media with either 2% glucose (YNBD, YPD) or 2% glycerol (YNBG and YPG) as the carbon source. Credit: Nature Communications Biology, doi: https://doi.org/10.1038/s42003-023-05202-5

Validation of the toolkit for metabolic engineering. a) Schematic representation of the HGA producing pathway engineered in Y. lipolytica. Green arrows, overexpressed steps. Red arrows, inactivated reactions. Purple arrows, enzymatic reactions thought to be encoded by two other ORFs in the Y. lipolytica HGA degradation cluster. Genes encoding corresponding enzymatic steps are shown next to the reactions. Metabolites are shortened as follows: PEP phosphoenolpyruvate, E4P erythrose-4-phosphate, DAHP 3-deoxy-D-arabino-heptulosonate 7-phosphate, DHQ 3-dehydroquinate, DHS 3-dehydro-shikimate, SHIK shikimate, SHP shikimate-3-phosphate, EP3P 5-enolpyruvyl-shikimate-3-phosphate, CHA chorismate, PPA prephenate, HPP para-hydroxy-phenylpyruvate, PPY phenylpyruvate, MAA 4-maleyl-acetoacetate, FAA 4-fumaryl-acetoacetate, FUM fumarate, AAС acetoacetate, Tyr L-tyrosine, Phe L-phenylalanine. b) Summary of modifications introduced in HGA producing strains. The symbols of delta and up arrow are used to indicate deletion and overexpression respectively of corresponding genes. c) Accumulation of HGA by engineered strains after 14-day cultivation in YNB medium with 9% glucose. Two biological replicates are shown. d) Visible accumulation of pyomelanin by engineered strains in the cultural broth after seven-day cultivation in the YNB medium with 2% citrate. Credit: Nature Communications Biology, doi: https://doi.org/10.1038/s42003-023-05202-5

The principle of accelerated metabolic engineering using the CRISPR/Cas9 toolkit. The toolkit allows frequent switching between marker-based and marker-free integration combining the advantages of both technologies. More robust marker-based integration requires at least 11 days, since it includes the marker recovery procedure. Application of the marker-free integration with CRISPR/Cas9 enables a single integration round in five days (image, top). Any single GG assembly reaction could be used for production of vectors with and without marker. At the same time, using the HA exchange system, any assembled integration cassette for overexpression, regardless of its complexity, can be redirected to alternative loci of the genome in two days. Moreover, both gRNA and donor can be redirected to alternative loci just in two days (image, bottom left). Credit: Nature Communications Biology, doi: https://doi.org/10.1038/s42003-023-05202-5