Effect of inhibitory chemical complexes on metabolic dynamics
At a minimum, the wild-type BTCC3 and F118 strains have demonstrated to be able to tolerate toxic byproducts produced during the pretreatment of lignocellulosic feedstock, up to a 20% concentration level (Kahar et al., 2022; Pangestu et al., 2022). We hypothesized that flocculation-induced cell coagulation might provide increased protection to the cells against exposure to toxic chemicals present in the fermentation medium. Figure 2a confirms that the F118L strain was still able to accumulate lactic acid in the presence of 20% ICC, although depletions were observed to 0.16 ± 0.01 g·g glucose-1 at low cell density and 0.11 ± 0.01 g·g glucose-1 at high cell density, which were 0.07 g·g glucose-1 and 0.17 g·g glucose-1 lower than the yield obtained in the absence of ICC, respectively. In contrast, the BTCC3L strain exhibited nearly negligible lactic acid production (less than 0.01 g·g glucose-1) in both low- and high-density cell cultures. In terms of glucose consumption, a reduction in the consumption rate caused by 20% ICC was observed when both strains were cultured at high cell density (Figure 2b, red and blue lines). At low cell density, however, the presence of 20% ICC appeared to slightly decelerate glucose consumption in the F118L strain (straight vs. dashed green lines), while the BTCC3L strain did not seem to be affected (straight vs. dashed yellow lines). Higher levels of ethanol accumulation were observed when both strains were cultivated in the presence of 20% ICC, with this trend being most pronounced in the BTCC3L strain at high cell density (Figure 2a). These observations indicate that the effects of 20% ICC on the metabolism of both strains may exhibit distinct characteristics.
Furthermore, we conducted a comparison of the relative expression levels of genes in the absence and presence of 20% ICC at both low and high cell densities (L vs. IL and H vs. IH ). In general, supplementation with 20% ICC yielded only a few DEGs in both strains, likely due to their strong robustness, as previously reported (Kahar et al., 2022; Pangestu et al., 2022). In the F118L strain, as depicted in Figure 5a-b, only the expression of one gene, SLT2 (also known asMPK1 ), which encodes a protein kinase involved in the cell wall integrity pathway and flocculation, exhibited significant upregulation (LFC = 6.88, -log10 adjusted p -value = 3.15) in low-density cell culture. Interestingly, in high-density cell culture, the expression levels of all the examined genes did not show significant changes, indicating that performing fermentation at higher cell density might reduce the vulnerability of the flocculating cells to chemical stress. It is important to note that there were still several genes that displayed substantial differences in expression under chemical inhibitor stress, yet they were not referred to as DEGs since their -log10 adjusted p-values were low due to the broad variances among biological replicates. Therefore, we also examined non-DEGs with LFC ≥ 2 and adjusted -log10 adjustedp -value < 1.30 (Figure 5b) and performed functional enrichment analysis to elucidate their ontologies (Figure S4c-d). After including these genes, we observed that ICC affected monosaccharide binding (PFK2 and FLO5 ), which might correlate with decreased glucose consumption rates, as well as flocculation (SLT2 and FLO5 ) in low-density cell culture (Figure S4d). At high cell density, on the other hand, the presence of toxic compounds led to increased expression of the heat shock protein genes,HSP26 and HSP32 .
In the BTCC3L strain, a higher number of DEGs were observed in high-cell density culture compared to low-density cell culture (Figure 5a). This result could be attributed to the increased metabolic burden of responding to chemical stress present in the medium and the accumulation of higher levels of fermentation products, resulting in multiple activations of cellular coping mechanisms. In high-cell density culture (Figure 5b), the gene ADH4 was significantly upregulated (LFC = 2.37, -log10 adjusted p -value = 1.58), which rationalizes the observed increase in ethanol accumulation as a response to chemical stress, consistent with findings from previous studies. Additionally, two significantly downregulated genes, SPI1 andTDH1 , were associated with glycolysis. When non-DEGs with high LFC were included, the list expanded to include ZWF1 ,TKL2 , HXK1 , PGM2 , and GPM2 . The expression levels of the glucose transporters HXT5 and HXT7 , as well as the exogenous lactate dehydrogenase gene LDH , were also reduced, providing an explanation for the observed reductions in the glucose consumption rate and lactic acid accumulation. Interestingly, the expression of other glucose transporters, GAL1 andHXT1 , was enhanced, suggesting the preference of specific glucose transporters under different conditions. At low cell density, nearly all DEGs were associated with gluconeogenesis and PPP, specificallyPGM1 , FBP1 , and SOL4 .
We also conducted a comparison of gene expression levels between low-density cell culture containing 20% ICC and high-density cell culture containing 20% ICC (IL vs. IH ). The combined effect of increasing cell density and 20% ICC supplementation resulted in the significant upregulation of 14 genes in the BTCC3L strain (Figure 5c). Compared to the genes shown in Figure 3c, 23 DEGs disappeared, 5 new DEGs were added, and 9 DEGs remained after 20% ICC supplementation (displayed in detail in Figure S5a, c). Most of these genes were associated with glycolysis-gluconeogenesis, PPP, and ethanol metabolism. As depicted in Figure 5d, increasing cell density in the presence of 20% ICC resulted in the reduced expression of HXK1 , PFK1 , and FBA1 , while TPI1 , TDH1 , TDH2 ,TDH3 , PGK1 , GPM1 , and ENO1 remained significantly upregulated (LFC ≥ 2, -log10 adjustedp -value ≥ 1.30), albeit also showing reductions compared to their LFC values in the absence of ICC. The former group (HXK1 ,PFK1 , and FBA1 ) represents glycolytic genes involved in the energy investment phase, where ATP molecules are required for the reactions, while the latter represents glycolytic genes in the energy payoff phase, where energy in the form of ATP and NADH molecules is formed. Hence, we concluded that chemical stress impacts the ATP supply to glycolysis. Additionally, our study revealed that genes related to PPP were no longer significantly upregulated when 20% ICC was present in the medium, despite previous studies highlighting the role of NADPH generated from this pathway in countering oxidative stress (Grant, 2008; Kwolek-Mirek et al., 2019; Mullarky & Cantley, 2015; Slekar et al., 1996). This finding suggests that in the BTCC3L strain, which inherently exhibits strong robustness against various chemical stresses, the induction of PPP might be more correlated to lactic acid tolerance as opposed to ICC resistance (at least at a level of 20%). Additionally, in the presence of 20% ICC, more genes related to ethanol metabolism were significantly upregulated, rationalizing the observed increase in ethanol accumulation. In the absence of ICC, bothHSP26 and HSP150 were significantly upregulated, whereas in the presence of ICC, only HSP26 showed significant upregulation (Figure S4), likely due to lower lactic acid accumulation.
In the F118L strain, as shown in Figure 5c, the combination of increasing cell density and ICC supplementation resulted in significant upregulation of ACT1 and downregulation of PDR8 andSLT2 (|LFC| ≥ 2, -log10adjusted p -value ≥ 1.30), whereas no DEGs were observed in the absence of ICC (Figure 3a). As previously discussed, the SLT2gene is essential for inducing flocculation as a response to chemical stress. When fermentation was conducted with a high cell density, more flocs were formed since the beginning of cultivation, thus providing higher protection to the cells located in the inner layer against exposure to ICC. Therefore, at higher cell densities, a high expression level of SLT2 may not be necessary. The downregulation ofSLT2 results in reduced activity of the SLT2/MAPK pathway, consequently diminishing the phosphorylation and inactivation of Msn2/4 by SLT2 . Dephosphorylated Msn2/4 transcription factors can then translocate into the nucleus, promoting stress-inducible dual specificity phosphatase 1 (SDP1 ), which acts as a negative regulator of the PKA pathway (Hahn & Thiele, 2002), leading to an upregulation of PKA, which consequently upregulates theACT1 gene. Additionally, PDR8 , encoding an ATP-binding cassette (ABC) transporter that plays a crucial role in multidrug resistance (Hikkel et al., 2003), was significantly upregulated in the low-density cell culture. In the high-density cell culture, where many cells are protected within the coagulant structure, high expression of this gene may not be essential, leading to its downregulation.
In general, these observations imply that the BTCC3L strain responds to chemical stresses by adjusting its central carbon metabolism, thereby significantly influencing the quantities of its fermentation products. On the other hand, in the flocculant F118L strain, genes associated with the cell wall and stress response were more actively involved in cellular adaptation during chemical stresses. The activities of these genes still require ATP, which may impact cell growth and several intracellular processes. However, the fluctuations in their expression levels have a less direct effect on key pathways, such as glycolysis and energy metabolism.