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).