Strain modification and response to exogenous lactate dehydrogenase
In this study, the nonflocculant S. cerevisiae BTCC3 strain, a budding yeast isolated from cocoa beans, was selected due to its rapid cell growth and high acid tolerance during lactic acid fermentation (Pangestu et al., 2022). On the other hand, the flocculant S. cerevisiae F118 strain was chosen for its high sugar consumption rate and cell coagulation propensity under chemical stress conditions (Kahar et al., 2022). Both strains are haploid yeasts and displayed stronger robustness against various lignocellulose-derived byproducts compared to mainstreamS. cerevisiae strains, such as BY4741 and S228C, rendering them well suited for the objectives of this study. Additionally, similar to most budding yeasts, these two strains do not naturally accumulate lactic acid at a detectable level. Therefore, pathway adjustment was conducted by introducing a copy of codon-optimized L-lactate dehydrogenase, LDH , from Lactobacillus casei under the control of the TDH3 promoter (depicted in Figure S1a), as similarly carried out in our previous work (Pangestu et al., 2022). However, in this study, we also intended to analyze how the studied parameters affect metabolic flux to ethanol. Therefore, the exogenous gene was integrated into the CYB2 gene, an L-lactate cytochrome-c oxidoreductase localized in the mitochondrial intermembrane space that converts lactate back to pyruvate, while all PDC genes remained undisrupted.
After transforming the gene to generate the lactic-acid-producing strains, eight colonies from the recombinant yeasts, namely, the nonflocculant BTCC3L and flocculant F118L strains, were randomly selected, checked, and cultured without pH controlling treatment for early screening (Figure S1b). Figure 1a shows that only one copy of the exogenous LDH gene was already sufficient to significantly induce lactic acid production in the F118L strain from 0 g·L-1 to 28.2 g·L-1 (yield = 0.32 g·g glucose-1, in the highest accumulating colony) despite high variance among colonies. To the best of our knowledge, this was the highest yield of lactic acid obtained from glucose in S. cerevisiae without any alterations to the ethanol pathway and stress tolerance compared to other reports (Branduardi et al., 2006; Dequin & Barre, 1994; Pacheco et al., 2012; Porro et al., 1995; Sugiyama et al., 2016; Turner et al., 2015). Meanwhile, in the BTCC3L strain, the accumulation of lactic acid was still very low ([Lactic acid]highest = 0.5 g·L-1). The flocculation trait of the F118L strain was much stronger than that of the BTCC3L strain (Figure 1b-c, Figure S1c), indicating the easiness of the former to instantly form cell coagulation. Additionally, our previous study revealed that the wild-type F118 strain demonstrated a gradual increase in cell wall hydrophobicity in response to elevated concentrations of ICC (Kahar et al., 2022). However, our current results, as shown in Figure 1c, indicate that the cell wall hydrophobicity of the LDH -incorporating F118L strain remained high (over 90%) even in the absence of ICC. This observation suggests that lactic acid accumulated by the yeast itself could induce cell coagulation in the flocculant strain, contributing to the high cell wall hydrophobicity regardless of the presence of ICC.
Colonies with the highest lactic acid accumulation for each strain were selected for further investigation. The effects of increasing cell density and the presence of ICC were evaluated by performing flask-scale fermentation with no pH control treatment at different initial cell concentrations (i.e., OD600nm = 1 and 50) and concentrations of ICC (i.e., no inhibitor and 20% ICC). For higher lactate production, cultivation was conducted in low-agitation mode (90 rpm). Our results showed that nonflocculant and flocculant budding yeast strains exhibited distinctive profiles in terms of fermentation product accumulation and glucose uptake rate (Figure 2a-b). These differences were further investigated by profiling the expression levels of 94 genes related to glycolysis-gluconeogenesis, the pentose phosphate pathway (PPP), the electron transfer chain (ETC), ethanol and lactate metabolism, sugar and lactate transport, energy metabolism, cytokinetic processes, stress responses, cell‒cell adhesion, and other relevant processes under various cultivation conditions (Figure 2c-d).