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.