Plasmid mini kit

Genomic DNA was isolated by the CTAB method (Wilson 2001 (link)). Plasmid isolation was performed with the Plasmid Midi AX or the Plasmid Mini kits (A&A Biotechnology) while DNA purification was conducted with the Clean-up Concentrator kit (A&A Biotechnology) according to manufacturer instructions. The genome of Acidovorax sp. A1169 was sequenced using an Illumina MiSeq apparatus (Illumina Inc., USA). The Illumina paired-end sequencing library construction was performed with 1 μg of post-nebulized DNA extract and the KAPA Library Preparation Kit reagents (KAPA Biosystems, USA), according to the manufacturer’s instructions. The library was pooled and sequenced on a MiSeq platform using the 600-cycle MiSeq reagent Kit v.3 (Illumina, USA). Sequence quality metrics were assessed using FASTQC (Andrews 2010 ).

Primary strains used in this study were Y. lipolytica A101 [61 (link)] and AJD with overexpression of YALI0E32769g—diacylglycerol acyltransferase Dga1p [40 (link)]. Both strains belong to the Department of Biotechnology and Food Microbiology at Wroclaw University of Environmental and Life Sciences, Poland. Escherichia coli DH5α was primarily used for molecular cloning. Vector pAD [62 (link)], carrying the UAS1_B16-TEF promoter, was the basis for developing new plasmids with the native XR, XDH or XK gene fragment. All plasmids and strains used in this study are listed in Additional file 1: Table S1 in the supplemental material. The all XYL genes fragments were amplified from the Y. lipolytica A101 genomic DNA. The list of primers used is shown in Additional file 1: Table S2. The PCR amplified genes were cloned into the pAD vector using SgsI and NheI/Pml1 sites, T4 DNA Ligase (Thermo Fisher Scientific) and used for transformation of E. coli. The obtained plasmids were isolated using the Plasmid Mini Kit (A&A Biotechnology, Poland), sequenced (Genomed, Poland) and digested with MssI. Linear expression cassettes were used to transform yeast according to the lithium acetate method [63 (link)]. The restriction enzymes were acquired from FastDigest Thermo Scientific.

Isolation of plasmid DNA was carried out according to the protocol of the Plasmid Mini kit (A&A Biotechnology). DNA fragments were isolated from agarose gels following the standard procedure of the DNA Gel-Out kit (A&A Biotechnology). DNA purification after enzyme treatment was performed according to the instructions in the DNA Clean-up kit (A&A Biotechnology). DNA digestion with restriction enzymes was carried out according to the enzyme supplier’s instructions. DNA fragments were ligated, and E. coli cells were prepared and transformed according to the standard methods (Sambrook et al. 1989 ).

All restriction enzymes were purchased from FastDigest Thermo Scientific and all of the digestions were performed according to standard protocols. The PCR reactions were set up using recommended conditions and Phusion high-fidelity DNA polymerase (Thermo Scientific). The ligation reactions were performed for 10 min at room temperature using T4 DNA Ligase (Thermo Scientific). The gel extractions were performed using the Gel Out extraction kit purchased from A&A Biotechnology (Poland). The E. coli minipreps were performed using the Plasmid Mini Kit (A&A Biotechnology). Transformation of E. coli strains was performed using standard chemical protocols¹⁷ .
For transformation of Yarrowia lipolytica only strains with auxotrophy for uracil were used.
Transformation was performed according to the lithium acetate method^{24 (link)} and transformants were plated out on selective media without uracil. They were analyzed for proper integration by gDNA extraction and PCR amplification with two primer pairs. Genomic DNA (gDNA) was extracted from Y. lipolytica using the Genomic Mini AX Yeast Spin kit (A&A Biotechnology, Poland).

Plasmids and primers used in this study are presented in Tables 2 and 3 [72 (link), 73 (link)]. Synthesis of primers was performed by Genomed (Warsaw, Poland).
Yeast genomic DNA library in the pFL44L plasmid was used (Lacroute, Gif-sur-Yvette).
Plasmid preparation, E. coli transformation and agarose gel electrophoresis were carried out as described in Sambrook and Russel [74 ].
Isolation of plasmid DNA was performed using standard alkaline lysis method or with Plasmid Mini Kit (A&A Biotechnology), according to the product manual.

The recombinant pUC19c23oB61 plasmid carrying mutated c23o gene was isolated from DH5C23OB61 clone with Plasmid Mini Kit (A&A Biotechnology, Poland) and digested with EcoRI and HindIII endonucleases (Fermentas) by standard procedures [24 ]. After gel purification, the DNA carrying the mutagenized gene was ligated into the EcoRI and HindIII endonuclease sites of pET-22(b) with T4 DNA ligase (Fermentas). The ligation mixture was then transformed into competent E. coli BL21 cells and plated on LB medium supplemented with ampicillin (100 μg/mL). Transformation of E.coli with plasmid DNA was performed by the RbCl procedure [25 (link)]. From transformants plasmids were isolated and those containing the correct insert were identified by restriction enzyme analysis. The presence of mutations in c23oB61 gene was then confirmed by DNA sequencing.

Yeast strains were transformed using the LiAc/ssDNA/PEG method [104 (link)]. Total yeast DNA was isolated using the Genomic Mini AX Yeast Spin kit (A&A Biotechnology, Gdansk, POLAND). E. coli cells were transformed as previously described [105 ] and bacterial plasmids were isolated using the Plasmid mini kit (A&A Biotechnology, Gdansk, POLAND).