D-Lactic Acid Production from Sugarcane Bagasse by Genetically Engineered Saccharomyces cerevisiae

Abstract

Lactic acid (LA) is a promising bio-based chemical that has broad applications in food, nutraceutical, and bioplastic industries. However, production of the D-form of LA (D-LA) from fermentative organisms is lacking. In this study, Saccharomyces cerevisiae harboring the D-lactate dehydrogenase (DLDH) gene from Leuconostoc mesenteroides was constructed (CEN.PK2_DLDH). To increase D-LA production, the CRISPR/Cas12a system was used for the deletion of gpd1, gpd2, and adh1 to minimize glycerol and ethanol production. Although an improved D-LA titer was observed for both CEN.PK2_DLDHΔgpd and CEN.PK2_DLDHΔgpdΔadh1, growth impairment was observed. To enhance the D-LA productivity, CEN.PK2_DLDHΔgpd was crossed with the weak acid-tolerant S. cerevisiae BCC39850. The isolated hybrid2 showed a maximum D-LA concentration of 23.41 ± 1.65 g/L, equivalent to the improvement in productivity and yield by 2.2 and 1.5 folds, respectively. The simultaneous saccharification and fermentation using alkaline pretreated sugarcane bagasse by the hybrid2 led to an improved D-LA conversion yield on both the washed solid and whole slurry (0.33 and 0.24 g/g glucan). Our findings show the exploitation of natural yeast diversity and the potential strategy of gene editing combined with conventional breeding on improving the performance of S. cerevisiae for the production of industrially potent products.

Keywords: D-lactic acid; Saccharomyces cerevisiae; adh deletion; glycerol-3-phosphate dehydrogenase genes; gpd deletion; hybrid yeast.

Figure 1

Metabolic and growth profiles of engineered strains. (A) Production of D-lactic acid, (B) ethanol, (C) glycerol, and (D) growth profile of the engineered strains. The cultures were grown at 30 °C and 250 rpm in an orbital shaking incubator. Samples were taken after 3 days, and the supernatants were analyzed on HPLC to quantify the content of each metabolite (N.D. indicates that the metabolite was not detected). Data represent mean values of biological triplicates and error bars indicate standard deviations.

Figure 2

Construction of S. cerevisiae strains from yeast mating. (A) PCR amplification of Lm.ldhA gene (1kb) from the genomic DNA of the strain CEN.PK2_DLDHΔgpd (lane 1), hybrid2 (lane 2), hybrid35 (lane 3), hybrid36 (lane 4), wild-type laboratory strain CEN.PK2-1C (lane 5), and wild isolate BCC39850 (lane 6). (B) PCR amplification of gpd genes (gpd1 and gpd2; indicated above the lanes) from the genomic DNA of the strain CEN.PK2_DLDHΔgpd (lane 1), hybrid2 (lane 2), hybrid35 (lane 3), hybrid36 (lane 4), wild-type laboratory strain CEN.PK2-1C (lane 5), and wild isolate BCC39850 (lane 6). The products from PCR amplification were separated by agarose gel electrophoresis and stained with ethidium bromide. Sizes of PCR amplicons were estimated by comparison with DNA markers (lanes marked M), in which fragments of known length are indicated on the left.

Figure 3

Lactic acid tolerance of S. cerevisiae BCC39850 (wild-type parent), CEN.PK2_DLDHΔgpd (transgenic parental strain), and progeny hybrid2, hybrid35, and hybrid36. Strains were grown in liquid culture to an initial yeast inoculum OD₆₀₀ of 0.5 before spotting on plates. (A) Growth at day 1 after spotting on a YPD plate containing 0, 30, 40, 50, and 60 g/L DL-lactic acid and incubation at 30 °C. (B). Growth at day 3 after spotting on a YPD plate containing 0, 30, 40, 50, and 60 g/L DL-lactic acid and incubation at 30 °C.

Figure 4

Time courses of glucose conversion to D-lactic acid and ethanol under aerobic conditions. D-lactic acid production in YPD medium containing 100 g/L glucose without synthetic hydrolysate toxins (A); ethanol production (B) and consumed glucose (C). D-lactic acid production in YPD medium containing 100 g/L glucose with synthetic hydrolysate toxins (0.36 g/L formic acid, 1.65 g/L acetic acid, 0.025 g/L levulinic acid, 0.05 g/L vanillin, and 0.018 g/L syringaldehyde) (D); ethanol production (E) and consumed glucose (F). Data represent mean values of biological triplicates and error bars indicate standard deviations.

Figure 5

Simultaneous saccharification and fermentation (SSF) of D-lactic acid and ethanol by CEN.PK2_DLDHΔgpd and hybrid2: (A) SSF of washed solid fraction of alkaline-pretreated sugarcane bagasse supplemented with Cellic^® Ctec2 at 30 FPU/g biomass. (B) SSF of whole slurry of alkaline-pretreated biomass (6% solid loading) supplemented with Cellic^® Ctec2 at 30 FPU/g biomass. Glucose concentration in SSF medium is shown by the solid blue line; parental strain CEN.PK2_DLDHΔgpd (open circle), hybrid2 (open triangle). Production of D-lactic acid is shown by the solid black line; parental strain CEN.PK2_DLDHΔgpd (open circle), hybrid2 (open triangle). Production of ethanol is shown by the solid black line; CEN.PK2_DLDHΔgpd (closed circle), hybrid2 (closed triangle). All SSF experiments were performed in triplicate at the temperature of 30 °C with shaking at 200 rpm.