Data for simultaneous fermentation of galacturonic acid and five-carbon sugars by engineered Saccharomyces cerevisiae

Saccharomyces cerevisiae expressing heterologous pathways for xylose, arabinose, and galacturonic acid metabolism has been constructed by a Cas9-based genome editing technology [1]. The fermentation performance of the final strain (YE9) was tested under various substrate conditions, and the fermentation parameters were calculated. The dataset can be used for designing bioprocesses for pectin-rich biomass.

are presented. In Table 1, the fermentation profiles of the YE9 strain with xylose, arabinose, and galacturonic acid in comparison to its wild type strain (D452-2). In Fig. 2, the YE9 strain was tested for xylose and galacturonic acid consumption rates in a mixture of 40 g/L xylose and various galactornic acid concentrations. In Table 2, the fermentation parameters of the YE9 strain with a mixture of galacturonic acid and co-substrates.

Strain construction by Cas9-based genome editing
To construct the YE9 strain, four consecutive transformations were performed as summarized in Fig. 3 using strains listed in Table 3. Briefly, the strain construction includes three parts: 1) guide RNA (gRNA) plasmid construction, 2) donor DNA preparation, and 3) yeast transformation. 1) Guide RNA (gRNA) plasmid construction gRNA sequences are designed to be target cut site-specific and 20-bp long, as listed in Table 4. The plasmids expressing each gRNA sequence were constructed by the fast cloning method [2], which is a PCR-based protocol for plasmid mutagenesis. To construct the pRS42H-ALD6.1 plasmid, for example, the pRS42H-GND1.1 plasmid (a template plasmid) [3] was amplified with the primers Kim044/Kim045 ( Table 5). The PCR products were treated with DpnI and used to transform E. coli TOP10 (Invitrogen, Carlsbad, CA, USA). The transformants were selected on an LBA (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, and 100 mg/mL ampicillin) agar plate. The gRNA sequence of the resulting plasmid was confirmed by Sanger sequencing using a universal primer for the T3 promoter. All other gRNA plasmids were constructed using the same procedure but different primers, as listed in Table 5.
Specifications Table   Subject Applied Microbiology and Biotechnology  Specific subject area  Yeast metabolic engineering  Type of data  Tables and Figures  How data were acquired  The fermentation data were obtained by HPLC (Agilent Technologies 1260  Value of the Data The dataset contains the construction strategy and fermentation data for the engineered strain simultaneously fermenting representative three carbon sources (xylose, arabinose, galacturonic acid) in pectin-rich biomass.
The fermentation data of the YE9 strain expressing the three pathways can be useful for process design utilizing pectinrich biomass consisting mainly of galacturonic acid and arabinose. Based on the fermentation data of the YE9 strain, feasible options for strain engineering can be broadened for industrial bioprocesses.
2) Donor DNA preparation Donor DNA fragments were prepared by PCR using the primers listed in Table 6. Each of the fragments was flanked by 40e50 bp to allow in vivo assembly and genome integration through homologous recombination. Each assembly was an expression cassette of a heterologous gene as described in Fig. 3A. The donor DNAs for the xylose expression cassettes were designed to achieve complete removal of a target gene when genome integrated. On the other hand, the expression cassettes of the arabinose pathway and galacturonic acid pathway were integrated into an intergenic region without interfering neighboring genes.

3) Yeast transformation
For yeast transformation, a gRNA plasmid (4 mg) and donor DNA fragments (4 mg each) were used to transform a designated strain harboring pRS41N-Cas9 [3]. The resulting transformants were selected on a YPD agar plate supplemented with 100 mg/mL nourseothricin sulfate (Gold Biotechnology, St. Louis, MO, USA) and 300 mg/mL hygromycin B (Invitrogen, Carlsbad, CA, USA). Selected transformants were serially sub-cultured in YPD medium supplemented with 100 mg/mL nourseothricin sulfate to only remove the existing gRNA plasmids. Correct assembly and integration was then confirmed by yeast colony PCR with the primers listed in Table 7. Through four consecutive transformations, as described in Fig. 3, the YE9 strain was finally constructed.  Table 1 Fermentation profiles of the native S. cerevisiae strain (D452-2) and engineered strain (YE9) expressing heterologous pathways for metabolizing D-xylose, L-arabinose, and D-galacturonic acid (galUA).

Strain
Substrate Substrate consumed (g/L)