DISCUSSIONS
Establishing a robust microbial host is crucial for sustainable and greener bio-based chemical production. Rather than employing extensive genetic modification to enhance strain tolerance, we attempted to establish a lactic acid bioproduction process facilitated by a robust microbial platform with high tolerance to acid and numerous lignocellulose-derived inhibitors. For that reason, we used S. cerevisiae BTCC3 as a robust yeast platform and enabled its lactic acid production by performing a modest genetic modification. Pathway engineering was conducted to reduce the metabolic flux to ethanol, a major product of fermentation by yeast, by disrupting the pyruvate decarboxylation genes. Triple deletion of PDC1 , PDC 5 andPDC6 genes inhibited cell growth because these genes are essential for NAD+ production (van Maris et al., 2004). However, a study has revealed that the PDC6 gene shows the lowest expression from among the three, and a mere double deletion ofPDC1 and PDC5 genes would be sufficient to significantly diminish the specific activity of pyruvate decarboxylase (Hohmann, 1991). In addition, another gene candidate for disruption is theADH1 gene. However, the deletion of this gene is known to slow the cell growth rate due to the accumulation of toxic acetaldehyde (Tokuhiro et al., 2009). Therefore, only PDC1 and PDC5genes were knocked out in this study.
Although our strain has a high tolerance to various stresses, several negative impacts appeared as the genetic modifications became more extensive. For instance, in all mutants, the accumulations of by-products, such as ethanol and glycerol, were higher than in wild-type strain, which could have been because of the response of the microbial host to cope with acid stress. In fact, the accumulation of ethanol and glycerol induces the generation of NAD+, which has an essential role in countering the negative impact of various stressors in cells (Kato & Lin, 2014; Massudi et al., 2012). Also, our results revealed that inserting an additional copy of the LDH gene into the same locus does not necessarily improve lactic acid production. However, this could be the consequence of employing an identical promoter, i.e. PTDH3 , in two different plasmids, pAUR101-TDH3pro-LcLLDH-dPDC1 and pAUR101-TDH3pro-LcLLDH-dPDC5. Promoter rivalry may have resulted in a conflict in the use of transcription factors during the expression of the two LDH genes.
Unexpectedly, an engineered strain containing the LDH gene under the control of the TDH3 promoter yielded low lactic acid generation, although this constitutive promoter is commonly utilized in consideration of its high expression (Baek et al., 2016, 2017; Saitoh et al., 2005). Meanwhile, the LA2 strain harboring the LDH gene under the control of a glucose-dependent promoter showed the highest lactic acid production. There are several plausible rationalizations for this result. It could simply be because PPDC1 is a native promoter. In addition, although the TDH3 promoter is categorized as a constitutive promoter, its expression declines in the presence of ethanol (Peng et al., 2015)–one of the major products generated by all strains in this type of experiment. Also, it is worth noting that the employment of constitutive promoters could increase the metabolic load (Balbas & Lorence, 2004), affecting the cell growth rate and metabolic flux of microbial hosts.
Since our strain displayed a high tolerance in acidic condition, we performed the fermentation without adding a neutralizing agent. This strategy is important for the fact that one of the bottlenecks in developing the industrial lactic acid process is the inability to generate a free form of lactic acid that requires no subsequent acidification. In fact, according to the life-cycle assessment and techno-economic analysis of SCB valorization to lactic acid, the removal of neutralizing agents by employing an acid-tolerant host lowers the environmental burden caused by gypsum and reduce total capital investment because the process would no longer require an acidification reactor unit (Daful & Görgens, 2017). Based on our best knowledge, to date, the utilization of SCB for second-generation lactic acid bioproduction still requisites the neutralizing step, either at the beginning or during the fermentation (Baral et al., 2020; de Oliveira et al., 2019; Unrean, 2018; van der Pol et al., 2016).
Our results show that our engineered S. cerevisiae  BTCC3 LA2 strain could facilitate a neutralizing-agent-free lactic acid fermentation using the hydrolysate of SCB. This strain could demonstrate a rapid generation of lactic acid at a productivity of 1.69 g·L-1·h-1, which is competitive to other studies in which the neutralizing treatment is still included at the beginning or during fermentation. Moreover, non-neutralized fermentation using YPD medium could achieve productivity as high as 3.68 g·L-1·h-1. All fermentable glucose in the YPD medium was consumed within only 9 h and higher accumulation of lactic acid could still be accomplished after three times glucose feeding treatment even without any neutralizing agent supplemented.
Based on our best knowledge, no previous reports discuss the metabolic engineering approach to developing a lactic acid bioproduction using SCB as raw material without neutralizing or detoxifying treatment. The genetic modification was performed at a modest level to maintain this strain’s robustness, with merely two genes disrupted and one exogenous gene inserted. In contrast, many other studies carried out more complex genetic manipulations in which the number of genes knocked in and out concurrently could reach ten or even more (Baek et al., 2017; Tsuge et al., 2019; Zhong et al., 2019, 2020). Therefore, this strategy is more efficient and potentially generates a more stable host for lactic acid production.
For upcoming experiments, disrupting other gene candidates without severely jeopardizing the rate of cell growth could be accomplished by constructing a switchable metabolic tool (Zhao et al., 2018). Adaptation strategy could also be carried out to attain higher product accumulation when performing fed-batch strategy with more feeding treatments. In addition, the content of xylose in lignocellulosic biomass is the second largest composition after glucose, so the use of xylose as an additional carbon source is essential for efficient production of lactic acid. Therefore, introducing a xylose-assimilating pathway to the microbial host is a good objective for upcoming experiments. In addition, a combination of the utilization of genetically engineered feedstock and the employment of our recombinant strain should lead to the development of a fourth-generation lactic acid.