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\begin{document}

\title{Engineering robust yeast for neutralizing-agent-free production of
lactic acid from sugarcane bagasse hydrolysate}



\author[1]{Radityo Pangestu}%
\author[1]{Prihardi Kahar}%
\author[2]{Lutfi Kholida}%
\author[2]{Urip Perwitasari}%
\author[2]{Ahmad Thontowi}%
\author[2]{Fahrurrozi Fahrurrozi}%
\author[2]{Puspita Lisdiyanti}%
\author[2]{Yopi Yopi}%
\author[1]{Chiaki Ogino}%
\author[3]{Bambang Prasetya}%
\author[4]{Akihiko Kondo}%
\affil[1]{Kobe University Graduate School of Engineering Faculty of Engineering}%
\affil[2]{National Research and Innovation Agency Republic of Indonesia}%
\affil[3]{National Standardization Agency of Indonesia}%
\affil[4]{Kobe University}%


\vspace{-1em}



  
  \date{May 18, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
From a sustainability standpoint, utilizing lignocellulose-based
material for lactic acid production is highly advantageous. By-products
generated by pre-treatment of biomass, however, tend to exhibit an
inhibitory effect on microbial hosts. Supplementation of neutralizing
agents also produces harmful waste for the environment. Here, we report
a rapid and non-neutralized fermentation by an original strain,
Saccharomyces cerevisiae BTCC3. This robust host was metabolically
engineered by the double disruption of PDC1/5 and the introduction of
L-LDH. Within only 15 h, 51\% of glucose from sugarcane bagasse (SCB)
hydrolysate was converted to lactic acid (productivity at 1.69
g\selectlanguage{ngerman}·L-1·h-1) without neutralizing agent supplementation at any stage of
fermentation. Cultivation using YPD medium under similar conditions
achieved productivity of 3.68 g·L-1·h-1 and the strain could still
generate lactic acid after several glucose-feeding treatment without
neutralization. This study is the first report of lactic acid production
from SCB using a genetically modified host in a process that requires
neither detoxification nor neutralization. Overall, this method offers
an alternative to tolerance engineering that involves extensive genetic
manipulations.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Engineering robust yeast for neutralizing-agent-free production
of lactic acid from sugarcane bagasse hydrolysate}

Radityo Pangestu\textsuperscript{1,2+}, Prihardi
Kahar\textsuperscript{1+}, Lutfi Nia Kholida\textsuperscript{2}, Urip
Perwitasari\textsuperscript{2}, Ahmad Thontowi\textsuperscript{2},
Fahrurrozi\textsuperscript{2}, Puspita Lisdiyanti\textsuperscript{2},
Yopi\textsuperscript{2,3}, Chiaki Ogino\textsuperscript{1,*}, Bambang
Prasetya\textsuperscript{3}, Akihiko Kondo\textsuperscript{1,4}

\textsuperscript{1}Graduate School of Engineering, Kobe University, 1-1
Rokkodaicho, Nada-ku, Kobe 657-8501, Japan

\textsuperscript{2}Research Center for Biotechnology, National Research
and Innovation Agency, Jl. Raya Bogor Km 46, Cibinong, 16911 Bogor, West
Java, Indonesia

\textsuperscript{3}Research Center and HRD, National Standardization
Agency of Indonesia (BSN), Building 430 PUSPIPTEK, South Tangerang
15314, Indonesia

\textsuperscript{4}Graduate School of Science, Technology and Innovation
(STIN), Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan

\textsuperscript{+}These authors have contributed equally to this work
and share the main authorship

\textbf{* Corresponding author:}

Chiaki Ogino (Email: ochiaki@port.kobe-u.ac.jp; Phone: +81-78-803-6193)

Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho,
Nada-ku, Kobe 657-8501, Japan

\textbf{Running Title:}

Neutralizing-agent-free lactic acid fermentation

ABSTRACT

From a sustainability standpoint, utilizing lignocellulose-based
material for lactic acid production is highly advantageous. By-products
generated by pre-treatment of biomass, however, tend to exhibit an
inhibitory effect on microbial hosts. Supplementation of neutralizing
agents also produces harmful waste for the environment. Here, we report
a rapid and non-neutralized fermentation by an original
strain,\emph{Saccharomyces cerevisiae} BTCC3. This robust host was
metabolically engineered by the double disruption of \emph{PDC1/5} and
the introduction of L-\emph{LDH} . Within only 15 h, 51\% of glucose
from sugarcane bagasse (SCB) hydrolysate was converted to lactic acid
(productivity at 1.69 g\selectlanguage{ngerman}·L\textsuperscript{-1}·h\textsuperscript{-1})
without neutralizing agent supplementation at any stage of fermentation.
Cultivation using YPD medium under similar conditions achieved
productivity of 3.68 g·L\textsuperscript{-1}·h\textsuperscript{-1} and
the strain could still generate lactic acid after several
glucose-feeding treatment without neutralization. This study is the
first report of lactic acid production from SCB using a genetically
modified host in a process that requires neither detoxification nor
neutralization. Overall, this method offers an alternative to tolerance
engineering that involves extensive genetic manipulations.

\textbf{Keywords:}

Lactic acid; lignocellulose; neutralizing agent; acid stress tolerance;

\emph{Saccharomyces cerevisiae}

INTRODUCTION

Lactic acid is currently one of the essential chemical commodities due
to widespread commercial applications in the pharmaceutical, cosmetics,
chemical, and food industries (Martinez et al., 2013). With a worldwide
production of 400,000 tons per year, lactic acid is considered a
top-value platform chemical (Becker et al., 2015; Choi et al., 2015).
Lactic acid is the precursor for poly-lactic acid--a popular
biodegradable plastic with physicochemical, thermal, and mechanical
properties that are competitive with traditional petroleum-based
polymers, such as polypropylene (PP) and low-density polypropylene
(LDPE) (Ajala et al., 2020; Nduko \& Taguchi, 2019). In recent years,
about 90\% of industrial lactic acid has been manufactured via
fermentation rather than chemical synthesis (Dusselier et al., 2013).
The former strategy is more environmentally friendly, less
energy-intensive, and yields an optically pure product.

Production of lactic acid is even more lucrative when low-cost feedstock
is employed in the process. Due to its abundant availability,
lignocellulosic biomass has been a prominent material for producing of
various bio-based chemicals (Chen et al., 2020; Kim et al., 2019; Tian
et al., 2020). Moreover, sugarcane bagasse (SCB) generated by the sugar
and alcohol industry is ideal for this objective. Data show that more
than 1.8 billion tons of sugarcane were produced worldwide in 2017 (de
Matos et al., 2020). About 31.8\% composition of sugarcane is bagasse--a
residual fraction obtained after the milling process (Cortez et al.,
2020). Considering its availability, the utility of SCB has become the
subject of numerous studies related to establishing a circular
bioeconomy and sustainable industries.

Despite the compelling benefits, the utilization of lignocellulose as a
feedstock for bioprocessing has several bottlenecks. The generation of
various by-products during the pre-treatment process is one of the
challenging issues. These by-products, which include furan derivatives
(furfural, 5-HMF, etc.), weak organic acids (formic acid, acetic acid,
etc.), and phenolic compounds (vanillin, syringaldehyde, ferulic acid,
etc.) (Ling et al., 2014), inhibit microbial metabolism, which renders
fermentation and diminishes productivity. Biological, physical, and
chemical methods have been explored in a quest to detoxify this process
(Palmqvist \& Hahn-Hägerdal, 2000). However, these methods necessitate
additional equipment, which drives up cost. These processes also
decrease the quantity of fermentable sugar (Hahn-Hägerdal et al., 2007).
Therefore, employing a stress-tolerant microorganism in the fermentation
step would undoubtedly be more desirable than modifying the operational
process.

During lactic acid bioproduction, the product itself may cause
additional stress for a microbial host. Many microorganisms,
particularly bacteria, suffer growth-rate inhibition under highly acidic
conditions. Commonly, neutralizing agent, such as calcium carbonate
(Yang et al., 2015), is added to maintain the pH of the medium. In
addition to increasing cost, neutralizing agents are often toxic for
microorganisms. In addition, calcium salts generally have low solubility
in water. Therefore, they can easily mix with biomass and complicate the
subsequent downstream process. Calcium salts also react with lactic acid
to form calcium lactate. An acidification step using sulfuric acid
followed by subsequent purification is needed to obtain the free form of
lactic acid. This acidification process generates a harmful by-product
known as gypsum, which must be disposed of in landfills (Komesu et al.,
2017) and could cause additional environmental issues. Approximately one
ton of gypsum is formed per ton of lactic acid production (Dusselier et
al., 2013). Hence, from both industrial and environmental perspectives,
eliminating the use of neutralizing agents would be advantageous. It can
be achieved by employing a robust microbial host during the fermentation
process.

Various approaches to obtain a robust microorganism have been proposed.
Tolerance engineering by genetic modification is an example of common
tools to enhance strain robustness. For instance, the co-expression of
both \emph{TAL1} and \emph{ADH1} in \emph{Saccharomyces
cerevisiae}enhances ethanol production in a medium containing furfural
(Hasunuma et al., 2014). Co-overexpression of both \emph{HAA1} and
\emph{PRS3} and disruption of \emph{FPS1} also improve acetic acid
tolerance (Cunha et al., 2018; Zhang et al., 2011). Nevertheless, due to
the complexity of biomass chemical composition, the successive
introduction of a large number of tolerance-related genes could be
cumbersome.

On the contrary, rather than performing extensive genetic engineering,
the strategy proposed here focuses on increasing the lactic acid
production of an originally robust microorganism via a handful of
genetic modifications. In the present study, we selected newly isolated
yeast, identified as \emph{S. cerevisiae} BTCC3, obtained from the
screening of Ascomycota yeasts deposited in Indonesian Culture
Collection (InaCC). This strain has the ability to survive at relatively
low pH (up to 2.5), as well as the presence of lignocellulose-derived
inhibitors such as furfural, formic acid, acetic acid, etc. However,
akin to other yeasts, this strain lacks the metabolic pathway for lactic
acid production. Therefore, we introduced an exogenous L-\emph{LDH} gene
to enable lactic acid fermentation from glucose as an example. This
experiment intended to construct a microbial strain with phenotypes
suitable for utilizing lignocellulosic biomass as a carbon feedstock,
such as SCB. Also, we considered the potential of this recombinant
strain to ferment glucose to lactic acid without the use of a
neutralizing agent.

MATERIALS AND METHODS

\textbf{Strains and medium}

All strains used in this study are compiled in \textbf{Table 1}
.\emph{S. cerevisiae} BTCC3 (haploid) was obtained from Indonesian
Culture Collection (InaCC, Cibinong Science Center, BRIN, Indonesia).
This strain and its derivatives were cultivated using a
yeast/peptone/dextrose (YPD) medium (20 g\selectlanguage{english}[?]L\textsuperscript{-1} Bacto
peptone {[}BD, San Jose, CA{]}, 10 g\selectlanguage{english}[?]L\textsuperscript{-1} Bacto yeast
extract {[}BD{]}, and 50 g\selectlanguage{english}[?]L\textsuperscript{-1} glucose
{[}BD{]}).\emph{Escherichia coli} JM109 (Takara Bio, Shiga, Japan) was
used for cloning. Luria-Bertani medium (10 g\selectlanguage{english}[?]L\textsuperscript{-1}
tryptone {[}Nacalai, Japan{]}, 5 g\selectlanguage{english}[?]L\textsuperscript{-1} yeast extract
{[}BD{]}, and 10 g\selectlanguage{english}[?]L\textsuperscript{-1} sodium chloride {[}Nacalai
tesque Inc., Japan{]}) was used for cultivation.

\textbf{Plasmid design and construction}

All plasmids used in this study are compiled in \textbf{Table 1} and
illustrated in \textbf{Figure 1} , whereas all primers are listed
in\textbf{Supplementary Table S1} . Codon-optimized L-\emph{LDH}
from\emph{Lactobacillus casei} (GenBank accession number
MF582630.1),\emph{LcLLDH} , was selected as an \emph{LDH} gene source.
DNA fragments were assembled via the NEBuilder HiFi DNA Assembly method
(NEB, County Road Ipswich, MA) and transformed into \emph{E. coli} JM109
(\emph{recA1} ,~\emph{endA1} ,~\emph{gyrA96} ,~\emph{thi} -\emph{1}
,\emph{hsdR17} (\emph{r\textsubscript{K}}
\textsuperscript{-}~\emph{m\textsubscript{K}}
\textsuperscript{+}),\emph{e14} \textsuperscript{-}~(\emph{mcrA}
\textsuperscript{-}),~\emph{supE44} ,~\emph{relA1} , D(\emph{lac-proAB}
)/F' {[}\emph{traD36} ,~\emph{proAB} \textsuperscript{+},~\emph{lac
I\textsuperscript{q}} ,~\emph{lacZ} D M15{]}). Plasmid-harboring
transformants were cultivated overnight (37 degC, 190 rpm) using LB medium
supplemented with 0.1 g\selectlanguage{english}[?]L\textsuperscript{-1} of ampicillin
(Sigma-Aldrich, USA) as a selection marker. Plasmids were extracted
using a LaboPassTM Plasmid Mini kit (Cosmo Genetech, Seoul, Korea).

Introduction of the \emph{LDH} gene into \emph{S. cerevisiae} genome
using a genome-integrated plasmid yields a higher accumulation of lactic
acid than using an episomal type plasmid (Ishida et al., 2005).
Therefore, the former plasmid type was employed to construct all
engineered strains in this experiment. For the simultaneous expression
of the \emph{LDH} gene and disruption of \emph{PDC} genes, partial
coding sequences of \emph{PDC1} /\emph{PDC5} genes were cloned into
plasmids containing an \emph{LDH} expression cassette. The sequence
of\emph{LDH} was fused with the \emph{TDH3} promoter and terminator
sequences at both its upstream and downstream regions, respectively, to
construct constitutively expressed \emph{LDH} systems:
pAUR101-TDH3pro-LcLLDH-dPDC1 and pAUR101-TDH3pro-LcLLDH-dPDC5. After the
integration of the respective gene-expressing cassettes into the genome,
there can be a possibility that both promoters control the expression of
the \emph{LDH} gene. The orientation of\emph{P\textsubscript{TDH3}}
-\emph{LcLLDH} -\emph{T\textsubscript{TDH3}}was flipped to avoid that
effect, as shown in \textbf{Figure 1(B)} . Moreover,
pAUR101-BTCC3PDC1-LcLLDH has a sequence of \emph{LDH} gene integrated
between the promoter and terminator of \emph{PDC1} gene cloned from the
genome, and we intended to utilize this native glucose-dependent
promoter. In contrast, pP01-BTCC3PDC5KO contains only the partial coding
sequence of \emph{PDC5} and \emph{KanMx} , a marker gene, to distort the
expression of the corresponding gene without the introduction of the
\emph{LDH} gene.

\textbf{Breeding yeast}

All plasmids were digested by the restriction enzymes (NEB) shown
in\textbf{Figure 1} to yield the five derived strains mentioned
in\textbf{Table 1} . Digested plasmids were transformed to a yeast
strain by following a LiAc/single-stranded carrier DNA/PEG method (Gietz
\& Woods, 2002). Each transformant was selected on a YPD agar containing
the respective selection marker(s). Sequencing analysis using Applied
Biosystems 3500 Genetic Analyzer was performed to confirm that the
fragment(s) of interest were successfully inserted.

\textbf{Fermentation}

A yeast strain was initially pre-cultured (30 degC, 150 rpm) using YPD
medium until the optical density (OD\textsubscript{600nm}) reached
around 50. Cells were cultivated (30 degC, 90 rpm) in a 100-mL Erlenmeyer
flask containing 12 mL of YPD medium supplemented with 50
g\selectlanguage{english}[?]L\textsuperscript{-1} sterilized calcium carbonate (Sigma-Aldrich).
For fermentation without a neutralizing agent, however, the medium was
not supplemented with calcium carbonate.

Furthermore, recombinant yeast was also cultivated using
sterile-filtered sugarcane bagasse hydrolysate obtained from a
pre-treatment process that mixed liquefied C6 and C5 fractions obtained
from sugarcane bagasse following treatment with hot water. This medium
(pH 4.5) contained 50 g*L\textsuperscript{-1} glucose and 25
g*L\textsuperscript{-1} xylose with inhibitory chemical compounds (ICC)
of furfural (4.5 mM), 5-HMF (3.4 mM), acetic acid (66 mM), formic acid
(7.2 mM), and lactic acid (7 mM). After pre-culturing (30 degC, 150 rpm),
cells were cultivated (30 degC, 90 rpm) in a 100-mL Erlenmeyer flask
containing 12 mL of sterile hydrolysate without the addition of calcium
carbonate.

\textbf{Analysis of fermentation products}

The medium broth was periodically sampled. After centrifugation at10,000
rpm for 5 min, the supernatant was filtered through a 0.45-\selectlanguage{greek}μ\selectlanguage{english}M
syringeless PTFE filter. Concentrations of glucose and other
fermentation products were determined via High Performance Liquid
Chromatography (HPLC) (LC-20AB, Shimadzu, Japan) using a Coregel-87H3
column (7.8 mm, I.D. x 300 mm Transgenomic) at 80 \selectlanguage{ngerman}°C for 40 min using 5
mM of sulfuric acid as eluent (flow rate at 0.6
mL\selectlanguage{english}[?]min\textsuperscript{-1}). A refractive index detector was used.

\textbf{Statistical analysis}

The values shown represent the mean of results from three biological
replicates \emph{+} SD (standard deviation). A significant difference
was statistically analyzed using a two-tailed Student's t-test
at\emph{P} \emph{\textless{}} 0.05.

RESULTS

\textbf{Effect of pathway manipulation on the metabolism}

As previously described, BTCC3 (wild type) strain did not produce lactic
acid at a detectable level due to the lack of a metabolic pathway for
lactic acid production from sugar. Here, we inserted an
exogenous\emph{LDH} gene and disrupted several \emph{PDC} genes.
\textbf{Figure 2 s} hows how this pathway adjustment affected the
product accumulation. As expected, the insertion of LDH genes enhanced
the production of lactic acid in all mutant strains. LX1 strain
(\emph{PDC1} \textsuperscript{--},\emph{LDH} \textsuperscript{+})
generated a 20-fold higher amount of lactic acid compared with the
results using LX5 strain (\emph{PDC5} \textsuperscript{--}, \emph{LDH}
\textsuperscript{+}). Our results also revealed that the generation of
lactic acid elevated the accumulation of ethanol and glycerol owing to
the fact that both mutant strains exhibited higher levels of production
compared with the levels from wild-type strains.

Moreover, two \emph{PDC} genes were knocked out to generate LA1 strain
(\emph{PDC1} \textsuperscript{--}, \emph{PDC5}
\textsuperscript{--},\emph{LDH} \textsuperscript{+}). This strain
exhibited a 1.9-fold increase in lactic acid generation and a 1.8-fold
decrease in ethanol accumulation compared with LX1 (\emph{PDC1}
\textsuperscript{--},\emph{LDH} \textsuperscript{+}) without a
noticeable drop-in glucose uptake rate (\textbf{Supplementary Figure S1}
). Surprisingly, the concentration of lactic acid produced from two
copies of the \emph{LDH}gene-harboring strain, namely LA15 (\emph{PDC1}
\textsuperscript{--},\emph{LDH} \textsuperscript{+}, \emph{PDC5}
\textsuperscript{--},\emph{LDH} \textsuperscript{+}) and LA1
(\emph{PDC1} \textsuperscript{--},\emph{LDH} \textsuperscript{+},
\emph{PDC5} \textsuperscript{--}), showed no significant differences
(\emph{P} \emph{\textless{}} 0.05).

The effect of promoter strength on the production of lactic acid in this
strain was also examined. In addition to the LA1 strain (\emph{PDC1}
\textsuperscript{--},
\emph{PDC5\textsuperscript{-},P\textsubscript{TDH3}} -\emph{LDH}
\textsuperscript{+}) that possesses \emph{LDH} gene integrated to a
constitutive promoter, LA2 strain (\emph{PDC1} \textsuperscript{--},
\emph{PDC5\textsuperscript{-},P\textsubscript{PDC1}} -\emph{LDH}
\textsuperscript{+}) containing a\emph{LDH} gene with glucose-dependent
promoter was also constructed. Interestingly, our result revealed that
the LA2 strain produced 43.23 g*L\textsuperscript{-1} of lactic acid
(approximately two times higher than LA1). This result indicated that
the use of a constitutive promoter might not be suitable for our strain,
although this type of promoter is commonly used in numerous experiments.

\textbf{Strain performance without a neutralizing agent}

Considering the promising result of the previous experiment, the LA2
strain was selected for further observation. \textbf{Figure 3} shows
that with and without the addition of calcium carbonate, the LA2 strain
produced lactic acid in concentrations of 43.2 and 33.2
g*L\textsuperscript{-1}, respectively. Even though the accumulation of
lactic acid declined, the removal of calcium carbonate did not cause a
significant decrease in the rate of glucose uptake because the glucose
was completely consumed within only 9 h under both conditions.
Nonetheless, as shown in \textbf{Table 2} , these results were
relatively competitive by comparison with those of the microbial hosts
described in other studies (Ilmen et al., 2013; Ishida et al., 2005;
Mitsui et al., 2020; Park et al., 2018). Despite its lower yield, the
LA2 strain maintained the ability to utilize glucose rapidly and
exhibited an even more stable level of productivity when the
neutralizing agent was removed. With the addition of a neutralizing
agent, the productivity was 4.80
g*L\textsuperscript{-1}*h\textsuperscript{-1}. Meanwhile, under a
non-neutralized condition, the productivity remained high (3.68
g*L\textsuperscript{-1}*h\textsuperscript{-1}), although the pH of the
medium dropped to 3.00 after 9 h of cultivation (\selectlanguage{english}[?]pH = \emph{+} 1.5,
compared with a neutralized condition). Other studies included
in\textbf{Table 2} have exhibited a drop in productivity by as much as
50\%, or more, when no neutralizing agent was supplemented into the
medium.

Based on the result shown in \textbf{Figure 3} , lactic acid generation
was terminated because the glucose was completely consumed within 9 h.
However, as depicted in \textbf{Figure 4} , the LA2 strain could still
accumulate lactic acid when glucose was fed to the medium even without
supplementation with a neutralizing agent. After being fed with
concentrated glucose three times at 6 h, 10 h and 14 h, the productivity
of engineered strain stood at 3.45
g*L\textsuperscript{-1}*h\textsuperscript{-1}(\emph{C}
\textsubscript{LA} = 62.0 g*L\textsuperscript{-1}) and 2.35
g*L\textsuperscript{-1}*h\textsuperscript{-1}(\emph{C}
\textsubscript{LA} = 42.2 g*L\textsuperscript{-1}) under the neutralized
and non-neutralized condition, respectively. Akin to the results from
batch cultivation, this strain exhibited high productivity under both
conditions, with a drop in value less than 35\% when the neutralizing
agent was removed from the medium.

\textbf{Fermentation using bagasse hydrolysate}

Previous results show that LA2 exhibited a strong ability to tolerate
acidic conditions and produce lactic acid without a neutralizing agent.
Subsequently, the robustness of this strain against
lignocellulose-derived inhibitors was examined. The hydrolysate obtained
from hot water pre-treatment was directly used as a medium without prior
treatment of enzymatic hydrolysis, detoxification or neutralization. In
addition to glucose, xylose was also present in the hydrolysate.
However, our yeast strain possesses no active genes that connect the
lactic acid generation and xylose metabolism pathways. Therefore, the
concentrations of xylose were not included in any of the calculations.

As can be seen in \textbf{Figure 5} , LA2 strain generated lactic acid
in a concentration of 25.34 \emph{+} 3.25 g*L\textsuperscript{-1} at 15
h from an initial glucose concentration of 49.57 \emph{+} 0.49
g*L\textsuperscript{-1} (yield = 0.51 g*g\textsuperscript{-1} glucose;
productivity = 1.69 g*L\textsuperscript{-1}*h\textsuperscript{-1}). The
productivity of our strain was slightly lower than that of other reports
(shown in \textbf{Table 3} ). In those reports, however, sodium
hydroxide had to be added to maintain the pH of the medium during
fermentation. Meanwhile, in this experiment, cultivation was performed
without the neutralizing treatment at any stage of fermentation.
Interestingly, despite a decline in the rate of glucose uptake and
productivity, LA2 strain converted about 51\% of the available glucose
when SCB hydrolysate was used as a medium, whereas cultivation using the
YPD medium only converted 33\% of the glucose (non-neutralized
condition). Also, the ratio of lactic acid to ethanol was remarkably
improved (2.67 at 15 h) using SCB hydrolysate compared with cultivation
using a YPD medium (0.91 at 9 h). This result indicates that the stress
of chemical inhibitors up to particular concentration levels could
instead provide a positive impact by shifting the metabolism and
accumulating lactic acid in higher concentration.

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 \emph{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 \emph{PDC1} , \emph{PDC} 5
and\emph{PDC6} genes inhibited cell growth because these genes are
essential for NAD\textsuperscript{+} production (van Maris et al.,
2004). However, a study has revealed that the \emph{PDC6} gene shows the
lowest expression from among the three, and a mere double deletion
of\emph{PDC1} and \emph{PDC5} genes would be sufficient to significantly
diminish the specific activity of pyruvate decarboxylase (Hohmann,
1991). In addition, another gene candidate for disruption is
the\emph{ADH1} 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 \emph{PDC1} and
\emph{PDC5}genes 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\textsuperscript{+}, 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 \emph{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. \emph{P\textsubscript{TDH3}} , 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 \emph{LDH} genes.

Unexpectedly, an engineered strain containing the \emph{LDH} gene under
the control of the \emph{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 \emph{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 \emph{P\textsubscript{PDC1}} is
a native promoter. In addition, although the \emph{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 \& Gorgens, 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~\emph{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\textsuperscript{-1}*h\textsuperscript{-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\textsuperscript{-1}*h\textsuperscript{-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.

\emph{CONCLUSIONS}

Sustainable and greener lactic acid production is possible using
lignocellulose biomass as a raw material. In this work, we demonstrated
the rapid fermentation of lactic acid from hydrolysate of sugarcane
bagasse without the use of a neutralizing agent. This process was
catalyzed by an originally robust microorganism engineered through only
three metabolic modifications---introduction of exogenous
L-\emph{LDH}and double disruptions of \emph{PDC} s---with retaining high
tolerance to acid and inhibitor stresses. Compared with other approaches
that have attempted to construct a recombinant host with extensive
genetic interruptions, this strategy is efficient, more practical and
expected to provide a more stable system for industrial lactic acid
bioprocess that utilizes lignocellulose biomass as its feedstock.

CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.

ACKNOWLEDGEMENTS

This work was financially supported by the International Joint Program,
Science and Technology Research Partnership for Sustainable Development
(SATREPS) (Innovative Bioproduction Indonesia; iBioI) from the Japan
Science and Technology Agency and the Japan International Cooperation
Agency (JST and JICA).

AUTHOR CONTRIBUTIONS

\textbf{Radityo Pangestu:} Investigation, Methodology, Data curation,
Formal analysis, Visualization, Writing -- original draft, Writing --
review \& editing. \textbf{Prihardi Kahar:} Conceptualization,
Methodology, Resources, Data curation, Writing -- original draft,
Writing -- review \& editing. \textbf{Lutfi Nia Kholid \& Urip
Perwitasari:} Investigation, Data curation. \textbf{Ahmad
Thontowi,Fahrurrozi \&} \textbf{Puspita Lisdiyanti:}
Resource.\textbf{Yopi:} Resource, Funding acquisition. \textbf{Chiaki
Ogino:}Funding acquisition, Supervision, Conceptualization, Resources,
Project administration, Writing -- review \& editing. \textbf{Bambang
Prasetya \& Akihiko Kondo:} Funding acquisition.

FIGURE LEGENDS

\textbf{Figure 1.} Maps of the plasmid vectors used for the introduction
of \emph{LDH} and disruption of \emph{PDC1/5} . (a)
pAUR101-BTCC3PDC1-LcLLDH for disruption of \emph{PDC1} and introduction
of \emph{LDH} with expression under the control of an inducible
promoter; (b) pAUR101-TDH3pro-LcLLDH-dPDC1 for disruption of
\emph{PDC1}and introduction of \emph{LDH} with expression under the
control of a constitutive promoter; (c) pPC01-BTCC3PDC5KO for disruption
of\emph{PDC5} without introducing \emph{LDH} ; (d)
pAUR101-TDH3pro-LcLLDH-dPDC5 for disruption of \emph{PDC5} and
introduction of \emph{LDH} with expression under the control of a
constitutive promoter. Restriction enzymes indicate the locations for
integration to a genome via homologous recombination.

\textbf{Figure 2.} Comparison of major products produced
by\emph{Saccharomyces cerevisiae} BTCC3 and all its derived strains.
Values represent the average measurement of three biological replicates.
Error bars represent the standard deviation of measurements. Symbols (+)
and (-) indicate introduction and disruption,
respectively.\emph{P\textsubscript{TDH3}} and
\emph{P\textsubscript{PDC1}} represent constitutive and inducible
promoters, respectively.

\textbf{Figure 3.} Fermentation profile of \emph{Saccharomyces
cerevisiae} BTCC3 LA2 using YPD\textsubscript{100} medium with and
without the addition of neutralizing agent. Concentrations of (a)
glucose; (b) lactic acid; (c) ethanol; (d) glycerol; and (e) pH change
have been indicated.

\textbf{Figure 4.} Fed-batch fermentation profile of \emph{Saccharomyces
cerevisiae} BTCC3 LA2 using YPD\textsubscript{100} medium with and
without the addition of neutralizing agent. Concentrations of (a)
glucose; (b) lactic acid; and (c) ethanol have been indicated.

\textbf{Figure 5.} Fermentation profile of \emph{Saccharomyces
cerevisiae} BTCC3 LA2 using hydrolysate from the pre-treatment of
sugarcane bagasse as a medium without the addition of a neutralizing
agent. Concentrations of (a) glucose; (b) lactic acid; (c) ethanol; and,
(d) pH change have been indicated.

APPENDICES

\textbf{Supplementary} \textbf{Table S1.} List of primers used in this
study

\textbf{Supplementary Figure S1.} Comparison of major products produced
by \emph{Saccharomyces cerevisiae} BTCC3 and all its derived strains
(extended data from Figure 2)

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