A short-chain dehydrogenase plays a key role in cellulosic D-lactic acid fermentability of Pediococcus acidilactici

Article abstract of DOI:10.1016/j.biortech.2019.122473

Phenolic aldehydes from lignocellulose pretreatment are strong inhibitors of cell growth and metabolism of cellulosic lactic acid bacteria. Their low solubility and recalcitrance highly reduce the removal efficiency of various detoxification methods. This study shows a simultaneous conversion of phenolic aldehydes and fermentation of D-lactic acid by Pediococcus acidilactici using corn stover feedstock. Vanillin was found to be the strongest phenolic aldehyde inhibitor to P. acidilactici. The overexpression of a short-chain dehydrogenase encoded by the gene CGS9114_RS09725 from Corynebacterium glutamicum was identified to play a key role in D-lactic acid fermentability of P. acidilactici. The engineered P. acidilactici with the genome integration of CGS9114_RS09725 showed the accelerated vanillin reduction and improved cellulosic D-lactic acid production. This study reveals that vanillin conversion is crucial for D-lactic acid fermentation, and the direct expression of a specific vanillin reduction gene in lactic acid bacterium efficiently improves cellulosic D-lactic acid production.

Full text of DOI:10.1016/j.biortech.2019.122473

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A short-chain dehydrogenase plays a key role in cellulosic D-lactic acid fer-
mentability of Pediococcus acidilactici
Zhongyang Qiu, Chun Fang, Qiuqiang Gao, Jie Bao
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Please cite this article as: Qiu, Z., Fang, C., Gao, Q., Bao, J., A short-chain dehydrogenase plays a key role in
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Original Research Article manuscript submitted to Bioresource Technology
A short-chain dehydrogenase plays a key role in cellulosic D-lactic acid fermentability of
Pediococcus acidilactici
Zhongyang Qiu1, 2, #, Chun Fang1, #, Qiuqiang Gao1, Jie Bao1, *
1 State Key Laboratory of Bioreactor Engineering, East China University of Science and
Technology, 130 Meilong Road, Shanghai 200237, China
2 Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, Huaiyin
Normal University, 111 West Changjiang Road, Huaian 223300, Jiangsu, China
# These authors are equally contributed to this work.
* Corresponding author: Jie Bao, Tel/fax: +86 21 64251799, email: jbao@ecust.edu.cn
1

ABSTRACT
Phenolic aldehydes from lignocellulose pretreatment are strong inhibitors of cell growth
and metabolism of cellulosic lactic acid bacteria. Their low solubility and recalcitrance highly
reduce the removal efficiency of various detoxification methods. This study shows a
simultaneous conversion of phenolic aldehydes and fermentation of D-lactic acid by
Pediococcus acidilactici using corn stover feedstock. Vanillin was found to be the strongest
phenolic aldehyde inhibitor to P. acidilactici. The overexpression of a short-chain
dehydrogenase encoded by the gene CGS9114_RS09725 from Corynebacterium glutamicum
was identified to play a key role in D-lactic acid fermentability of P. acidilactici. The
engineered P. acidilactici with the genome integration of CGS9114_RS09725 showed the
accelerated vanillin reduction and improved cellulosic D-lactic acid production. This study
reveals that vanillin conversion is crucial for D-lactic acid fermentation, and the direct
expression of a specific vanillin reduction gene in lactic acid bacterium efficiently improves
cellulosic D-lactic acid production.
Keywords: Lignin derived inhibitors; biodetoxification; Pediococcus acidilactici; D-lactic
acid; lignocellulose
2

1. Introduction
Pretreatment is the crucial step of the lignocellulose biorefinery chain (Chandel et al.,
2018; Galbe and Zacchi, 2012; Wyman et al., 2005). One of the outcomes of the harsh
pretreatment operation is the generation of various small molecular weight compounds such
as furan aldehydes (furfural and 5-hydroxymethylfurfural (HMF)), weak acids (acetic acid
and formic acid) and phenolic aldehydes (4-hydroxybenaldehyde, vanillin and
syringaldehyde) (Jonsson et al., 2013; Klinke et al., 2004; Larsson et al., 1999; Parawira and
Tekere, 2011). These compounds severely inhibit cellulase enzyme activity in the enzymatic
hydrolysis step and reduce the viability of microbes in the fermentation step (Palmqvist and
Hahn-Hägerdal, 2000; Zhang et al., 2010b). Therefore, the complete removal of inhibitors is
essential for efficient production of biofuels and bio-based products from lignocellulose
feedstock.
Phenolic aldehydes are lignin derivatives and the representatives include
4-hydroxybenaldehyde, vanillin and syringaldehyde by functional classification of the
p-hydroxyphenyl group (H), guaiacyl group (G) and syringyl group (S), respectively.
Complete removal of these phenolic aldehyde compounds by conventional detoxification
methods such as water-washing and over-liming is difficult because of their low water
solubility and hydrophobicity (Gu et al., 2015; Thomsen et al., 2009). Biological
detoxification can completely degrade furfural, HMF, acetic acid and phenolic aldehydes but
a long period is required (>72 hours), resulting in the consumption of 30%–40% of the total
xylose in pretreated lignocellulose biomass and a considerable loss of the final target product
yield during the fermentation step (Gao et al., 2018; He et al., 2016; Wang et al., 2015; Yi et
al., 2019; Zhang et al., 2010b). Ideally, a short biodetoxification period should be used to
minimize xylose consumption and enable the complete degradation of furfural, HMF and
acetic acid. However, a short biodetoxification period only partially degrades the phenolic
3

aldehydes and the phenolic aldehyde residues in the lignocellulose feedstock negatively affect
the fermentability of the fermentation strains (He et al., 2016; Yi et al., 2016). Thus, the
complete removal of phenolic aldehydes leads to a considerable loss of xylose, whereas
retaining the desired xylose is at the expense of incomplete removal of the toxic phenolic
aldehydes. We propose to combine biodetoxification and fermentation into one simultaneous
process to solve this issue. In the first step, the shortened biodetoxification is conducted to
completely remove furfural, HMF, acetic acid, and partially remove phenolic aldehydes
within a standard period (36–48 h), while xylose in the pretreated lignocellulose biomass is
preserved. In the second step, the residual phenolic aldehydes and the sugars (glucose and
xylose) in the biodetoxified lignocellulose are simultaneously converted into less toxic or
non-toxic derivatives and the target products by microbial fermenting strains.
D-lactic acid is an important monomer chemical of the biodegradable poly-lactic acid
(PLA) plastic (Abdel-Rahman et al., 2011; Farah et al., 2016; Nampoothiri et al., 2010). In
our previous studies, a D-lactic acid producing strain P. acidilactici ZY15 was developed for
efficient co-utilization of glucose and xylose from lignocellulose feedstock (corn stover and
wheat straw) (Qiu et al., 2017; Yi et al., 2016). In this study, vanillin was identified to be the
strongest phenolic aldehyde inhibitor of P. acidilactici. A heterologous short-chain
dehydrogenase gene was discovered to be a key gene for increasing vanillin reduction
conversion and accelerating D-lactic acid fermentability using corn stover feedstock. This
study proved that directly expressing a heterologous vanillin reduction gene in the D-lactic
acid bacterium strain improved the D-lactic acid fermentability from lignocellulose feedstock.
2. Materials and methods
2.1 Strains, media and growth conditions
The strains used in this study are shown in Supplementary materials. D-lactic acid
producing strain P. acidilactici ZY15 with efficient xylose-assimilation ability was
4

constructed previously (CGMCC 13612, Chinese General Microorganisms collection center,
Beijing, China) (Qiu et al., 2017). Escherichia coli XLI-blue for construction of recombinant
plasmids was stored in our laboratory. Biodetoxification strain Amorphotheca resinae ZN1
(CGMCC 7452) was isolated in previous work (Zhang et al., 2010b). Zymomonas mobilis
ZM4 (ATCC 31821, American Type Culture Collection, Manassas, VA, USA), Pseudomonas
putida KT2440 (ATCC 47054) and Corynebacterium glutamicum S9114 (SIIM B460,
Shanghai Industrial Institute of Microorganism, Shanghai, China) were used for heterogenous
oxidoreductase gene amplification.
P. acidilactici strains were grown at 42 °C in the simplified Man-Rogosa-Sharp (MRS)
medium containing 20 g/L glucose, 10 g/L peptone, 10 g/L yeast extract, 5 g/L sodium
acetate, 2 g/L ammonium citrate dibasic, 0.58 g/L MgSO4·7H2O, 2 g/L K2HPO4 and 0.25 g/L
MnSO4·H2O (Yi et al., 2016; Qiu et al., 2017). E. coli strains were cultured in Luria-Bertani
(LB) medium at 37 °C and 400 μg/mL of erythromycin was added to the medium for the
construction of the recombinant plasmids. A. resinae ZN1 was maintained at 28 °C on a
potato dextrose agar (PDA) slant (Zhang et al., 2010b).
2.2 Enzymes and reagents
Commercial cellulase enzyme Cellic CTec 2.0 was purchased from Novozymes (Beijing,
China). The filter paper activity, cellobiase activity and protein concentration of Cellic CTec
2.0 were 203.2 FPU/mL, 4,900 CBU/mL and 87.3 mg/mL, respectively, determined
according to the methods described by Adney and Baker (1996), Ghose (1987) and Bradford
(1976). DNA polymerase and the ligation kit were purchased from Takara (Otsu, Japan). The
restriction endonucleases were from Thermo Scientific, Wilmington, DE, USA. The genomic
DNA and plasmid extraction kits were from Generay Biotech, Shanghai, China. Erythromycin
was from Biosharp Biotech, Beijing, China. Yeast extract and peptone were from Oxoid,
Hampshire, UK. Phenolic aldehydes and their derivatives were purchased from local
5

suppliers.
2.3 Construction of P. acidilactici recombinants
The plasmids and primers used are listed in Supplementary materials. The genomic DNA
of P. acidilactici ZY15, Z. mobilis ZM4, P. putida KT2440 and C. glutamicum S9114 were
extracted separately using the genome extraction kit. Promoter PldhD was amplified from the
300 bp upstream of the start codon of the D-lactate dehydrogenase gene ldhD of the P.
acidilactici ZY15 genome. The ZMO1885 gene was amplified from Z. mobilis ZM4.
PP_3151, PP_5120 and PP_5258 were amplified from P. putida KT2440.
CGS9114_RS10340 and CGS9114_RS09725 were amplified from C. glutamicum S9114.
The original promoter P32 in pMG36e was replaced with PldhD at EcoR I and Xba I to
obtain the plasmid pZY36e for gene expression in P. acidilactici ZY15. Genes ZMO1885,
PP_5120, CGS9114_RS10340 and CGS9114_RS09725 were then inserted into pZY36e at
Xba I and Sal I to obtain plasmids pZY36e-ZMO1885, pZY36e-PP_5120,
pZY36e-CGS9114_RS10340 and pZY36e-CGS9114_RS09725, respectively. Genes PP_3151
and PP_5258 were inserted into pZY36e at Xba I and Pst I to generate plasmids
pZY36e-PP_3152 and pZY36e-PP_5258, respectively. The above seven plasmids were
transformed separately into P. acidilactici ZY15 to obtain the seven recombinants.
The pSET4E-ΔackA2 for deletion of the acetate kinase gene ackA2 was constructed in
Qiu et al. (2018). Expression cassette PldhD_CGS9114_RS09725 was amplified from plasmid
pZY36e-CGS9114_RS09725, and inserted into pSET4E-ΔackA2 at Xba I and Pst I to generate
the integration plasmid pSET4E-ΔackA2::CGS9114_RS09725 for substitution of ackA2 with
the above expression cassette. The integration plasmid was transformed into P. acidilactici
ZY15. The substitution of ackA2 with PldhD_CGS9114_RS09725 was conducted using the
method described in Qiu et al. (2017) and Yi et al. (2015).
6

2.4 Dry acid pretreatment and biodetoxification
Corn stover was collected in the spring of 2018 from Nanyang, Henan, China. The
cellulose and hemicellulose content in the raw corn stover were 34.4% (w/w) and 23.6%
(w/w), respectively, which were determined using a cellulose analyzer (Cellulose Analyzer
220, Ankom Technology, Macedon, NY, USA).
Dry acid pretreatment of corn stover was conducted according to Zhang et al. (2011) and
He et al. (2014). The pretreated corn stover contained 32.2 mg glucose and 144.3 mg xylose,
as well as 5.8 mg furfural, 3.5 mg HMF, 23.0 mg acetic acid, 0.4 mg 4-hydroxybenzaldehyde,
3.3 mg vanillin and 2.2 mg syringaldehyde per gram of dry corn stover (dry matter, DM).
The pretreated corn stover was biodetoxified using A. resinae ZN1 to remove the
inhibitors (He et al., 2016; Zhang et al., 2010b). Furfural, HMF and acetic acid were removed
completely. The phenolic aldehydes were partially removed and the residual phenolic
aldehydes in the pretreated and biodetoxified corn stover included 0.1 mg
4-hydroxybenzaldehyde, 0.6 mg vanillin and 1.5 mg syringaldehyde per gram DM. The
cellulose and hemicellulose contents were essentially constant. The monosaccharides included
18.3 mg glucose and 124.3 mg xylose per gram DM.
2.5 Simultaneous saccharification and D-lactic acid co-fermentation (SSCF)
SSCF was carried out in a 5 L helical agitated bioreactor using the pretreated and
biodetoxified corn stover at 30% (w/w) solids loading with addition of 10 mg cellulase
protein/g cellulose (Qiu et al., 2017; Zhang et al., 2010a). After 6 h prehydrolysis at 50 °C and
150 rpm, SSCF was initiated by adding P. acidilactici seeds with 10% (v/v) inoculation at
42 °C, pH 5.5 and 150 rpm. The nutrients including 10 g/L peptone, 10 g/L yeast extract, 2
g/L ammonium citrate dibasic and 0.25 g/L MgSO4 were added to the SSCF operation. The
pH was maintained at 5.5 by adding a 25% (w/w) Ca(OH)2 slurry.
The D-lactic acid yield in SSCF was defined according to Qiu et al. (2017).
7

2.6 Analytical methods
Cell growth was determined periodically by measuring the optical density at 600 nm
(OD600) using a Biomate 3S spectrophotometer (Thermo Scientific, Massachusetts, USA).
Glucose, xylose and D-lactic acid were determined using an HPLC (LC-20AD,
refractive index detector RID-10A, Shimadzu, Kyoto, Japan) equipped with a Bio-Rad
Aminex HPX-87H column. The mobile phase was a 5 mM sulfuric acid solution and operated
at 65 °C with flow rate of 0.6 mL/min.
The phenolic compounds were determined by HPLC (UV/Vis detector SPD-20A,
Shimadzu, Kyoto, Japan) with a YMC-Pack ODS-A column (YMC Co., Kyoto, Japan),
according to the method described by Gu et al. (2015) and Khoddami et al. (2013).
3. Results and discussion
3.1 Tolerance evaluation of the D-lactic acid producing P. acidilactici to phenolic aldehydes
We tested the tolerance of the D-lactic acid producing strain P. acidilactici ZY15 to the
three typical phenolic aldehydes (4-hydroxybenaldehyde, vanillin and syringaldehyde), as
well as the corresponding alcohol and acid derivatives (Fig. 1). The concentrations of
phenolic aldehydes were selected according to that found in the cellulosic lactic acid
fermentation system (0.3 mM 4-hydroxybenzaldehyde, 1.5 mM vanillin and 3.1 mM
syringaldehyde). Vanillin strongly inhibited P. acidilactici with an LC50 of 1.3 mM (LC50,
lethal concentration of 50% decrease in cell growth) (Fig. 1a), which is similar to the vanillin
concentration found in cellulosic lactic acid fermentation (~1.5 mM). Syringaldehyde was
also a strong inhibitor but its LC50 (4.4 mM) was about 3-fold greater than that of vanillin
(~1.3 mM) and 1.4-fold greater than that found in cellulosic lactic acid fermentation (~3.1
mM). 4-Hydroxybenzaldehyde was a negligible inhibitor with 24.6 mM of
4-hydroxybenzaldehyde showing slight inhibition on P. acidilactici, which is approximately
19-fold greater than that of vanillin (~1.3 mM) and 80-fold greater than that found in
8

cellulosic lactic acid fermentation (~0.3 mM). The corresponding alcohol derivatives
(4-hydroxybenzyl alcohol, vanillyl alcohol and syringic alcohol) and acid derivatives
(4-hydroxybenzoic acid, vanillic acid and syringic acid) showed only slight inhibition on P.
acidilactici even at high concentrations (Figs. 1b-1c). The conversion of phenolic aldehydes
by P. acidilactici ZY15 was also evaluated. Fig. 2 shows that P. acidilactici was able to
convert 4-hydroxybenzaldehyde into its alcohol and acid forms, vanillin was converted into
its alcohol form (vanillyl alcohol) but not into its acid form, and syringaldehyde was stable
and not converted into its alcohol or acid form.
The results showed that vanillin was the strongest phenolic aldehyde inhibitor of P.
acidilactici, and P. acidilactici was able to convert vanillin into its less toxic alcohol form
(vanillyl alcohol). Strengthening the conversion of vanillin into its less toxic alcohol or acid
derivatives should potentially improve D-lactic acid fermentation by P. acidilactici.
3.2 Improving vanillin reduction conversion by integrating the CGS9114_RS09725 gene
Biodetoxification of the pretreated corn stover feedstock was conducted to completely
remove the highly toxic and highly concentrated inhibitors such as furfural, HMF and acetic
acid, but phenolic aldehydes were only partially removed. The residual phenolic aldehydes
in the cellulosic fermentation broth still inhibited D-lactic acid production by P. acidilactici
ZY15 from corn stover feedstock (Fig. 6). Acceleration of the conversion of toxic phenolic
aldehydes into less toxic phenolic alcohol or acid derivatives is an effective strategy to
improve the phenolic aldehyde tolerance in fermenting strains (Wang et al., 2016; Yi et al.,
2015). Here, we overexpressed six oxidoreductase genes in P. acidilactici ZY15 for
improving the conversion of phenolic aldehydes, especially vanillin. The genes were
ZMO1885 encoding NADH: flavin oxidoreductase/NADH oxidase from Z. mobilis ZM4 (Yi
et al., 2015), CGS9114_RS10340 encoding inositol 2-dehydrogenase, CGS9114_RS09725
encoding short-chain dehydrogenase from C. glutamicum S9114 (Zhou et al., 2019), PP_3151
9

encoding NAD+-dependent succinate semialdehyde dehydrogenase, PP_5120 encoding
coniferyl aldehyde dehydrogenase and PP_5258 encoding L-piperidine-6-carboxylate
dehydrogenase from P. putida KT2440 (Simon et al., 2014). Among these genes, ZMO1885,
CGS9114_RS10340 and CGS9114_RS09725 showed a strong response to phenolic aldehydes
reduction, whereas PP_3151, PP_5120 and PP_5258 showed a significant response to
vanillin oxidation.
The genes were inserted into the plasmid pZY36e under the control of promoter PldhD
and then introduced into P. acidilactici to generate six recombinants. A modified MRS
medium containing 1.3 mM vanillin, 3.3 mM syringaldehyde and 24.6 mM
4-hydroxybenaldehyde was designed to evaluate the D-lactic acid fermentability of the
recombinants, which was based on their individual minimum inhibition concentrations of P.
acidilactici. CGS9114_RS09725 from C. glutamicum S9114 was the only gene that promoted
D-lactic acid fermentation under phenolic aldehydes stress (Fig. 3).
To obtain a stable recombinant, the CGS9114_RS09725 gene was integrated into the
gene ackA2 locus of the P. acidilactici ZY15 genome by thermo-sensitive homologous
recombination with no antibiotic resistance markers remaining in the genome. The engineered
strain P. acidilactici ZY15-ΔackA2::CGS9114_RS09725 was examined in MRS medium
containing gradient concentrations of vanillin (Fig. 4), and a significant improvement of
D-lactic acid fermentation was observed when vanillin approached its LC50 concentration
(1.3–1.6 mM). The time course of vanillin conversion at the LC50 of vanillin (i.e., 1.6 mM;
Fig. 5) showed that about 30% of the original vanillin (0.46 mM) was converted into vanillyl
alcohol (0.27 mM) within 24 h, which is more than double that of the parental strain (reduced
0.25 mM vanillin and produced 0.13 mM vanillyl alcohol). The imbalance between vanillin
reduction and vanillyl alcohol production was observed in both strains. Several reasons may
cause this imbalance: (1) conversion of vanillin to other vanillin derivatives other than
10

vanillyl alcohol; (2) volatilization of vanillin at high culture temperature (42 °C for 24 h).
Besides, no vanillic acid was detected, indicating that CGS9114_RS09725 expression only
improved the reduction of vanillin with no improvement in the oxidation of vanillin to vanillic
acid.
We failed to observe any improvements for the conversion of syringaldehyde (data not
shown) and syringaldehyde tolerance (see Supplementary materials) by genome expression of
CGS9114_RS09725, indicating that CGS9114_RS09725 expression specifically accelerated
the reduction of vanillin and consequently enhanced vanillin tolerance of P. acidilactici.
3.3 Cellulosic D-lactic acid fermentation by the engineered P. acidilactici strain
The engineered strain P. acidilactici ZY15-ΔackA2::CGS9114_RS09725 was applied for
D-lactic acid fermentation using corn stover feedstock for evaluating the improvement in
fermentability. The corn stover feedstock was dry acid pretreated and then biodetoxified to
completely remove furfural, HMF, acetic acid and partially remove the phenolic aldehydes
with residuals of 0.1, 0.6 and 1.5 mg/g DM of 4-hydroxybenaldehyde, vanillin and
syringaldehyde, respectively. The pretreated and biodetoxified corn stover was then
simultaneously saccharified and fermented into D-lactic acid under 30% (w/w) solids loading
(Fig. 6). The engineered P. acidilactici with improved vanillin tolerance significantly
accelerated glucose consumption and increased D-lactic acid production by 13.9% in D-lactic
acid titer (from 101 g/L to 115 g/L), 14.3% in productivity (from 1.4 g/L/h to 1.6 g/L/h) and
18.4% in overall yield (from 51.6% to 61.1%). We attempted to compare the vanillin
reduction and vanillyl alcohol production between the engineered and parental strains, but it
failed. This is probably because phenolic aldehydes usually have the low solubility or even
insolubility (Palmqvist and Hahn-Hägerdal, 2000). Unlike the hydrolysate fermentation, high
solid particles were contained in the SSCF. These slightly water-soluble phenolic aldehydes
easily precipitated on solid particles and resulted in the inaccurate detection of vanillin and
11

vanillyl alcohol (Thomsen et al., 2009; Gu et al., 2014).
Complete removal of phenolic aldehydes does not work by conventional water-washing
and over-liming methods because of their low water solubility and hydrophobicity (Gu et al.,
2015; Thomsen et al., 2009). Biodetoxification method is able to remove furfural, HMF and
acetic acid completely, but the complete removal of phenolic aldehydes requires a very long
period, which leads to the considerable loss of xylose (Gao et al., 2018; He et al., 2016;Wang
et al., 2015; Yi et al., 2019; Zhang et al., 2010b). Thus, phenolic aldehydes are inevitably
remained in the pretreated and biodetoxified lignocellulose feedstock to ensure that the
fermentable xylose sugar is not lost and consequently the lactic acid fermentability was
negatively affected. This study demonstrated a practical solution by applying the D-lactic acid
fermenting strain to finalize the conversion of the residual phenolic aldehyde (vanillin) into its
less toxic alcohol during the fermentation period. We accelerated the bioconversion of
vanillin into vanillyl alcohol by the genome expression of a specific gene CGS9114_RS09725
encoding a short-chain dehydrogenase from C. glutamicum, and the tolerance of P.
acidilactici to vanillin was significantly improved. Consequently, the D-lactic acid
fermentability from the dry acid pretreated and biodetoxified corn stover with vanillin
contained was also improved.
Syringaldehyde is also a strong toxic phenolic aldehyde inhibitor, but the present P.
acidilactici strain in this study was unable to convert syringaldehyde into its less toxic alcohol
or acid derivative as in the case of vanillin (Fig. 2). This is probably because vanillin has one
methoxyl group and syringaldehyde has two methoxyl groups, and the space shelling
generated by two methoxyl groups may inhibit specific enzymes binding with syringaldehyde,
which blocks the bioconversion of syringaldehyde(Klinke et al., 2004; Yi et al., 2015). This
study failed to construct bioconversion pathways of syringaldehyde by overexpression of
oxidoreductase genes, and failed to enhance the tolerance of P. acidilactici to syringaldehyde.
12

Future efforts such as adaptive evolution should be made to enhance the tolerance of P.
acidilactici to syringaldehyde for the construction of a robust cellulosic D-lactic acid
bacterium.
4. Conclusion
Vanillin was found to be the strongest phenolic aldehyde inhibitor on P. acidilactici
ZY15. Genome integration of an exogenous gene CGS9114_RS09725 encoding short-chain
dehydrogenase into P. acidilactici significantly enhanced vanillin reduction conversion,
resulting in improved vanillin tolerance. The obtained strain P. acidilactici
ZY15-ΔackA2::CGS9114_RS09725 showed accelerated D-lactic acid fermentation from the
dry acid pretreated and biodetoxified corn stover with phenolic aldehydes contained.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This research was supported by the National Natural Science Foundation of China
(31961133006, 21978083) and the Natural Science Foundation of the Jiangsu Higher
Education Institutions of China (19KJB180012).
Appendix A. Supplementary data
E-supplementary data of this work can be found in online version of the paper.
References
[1] Abdel-Rahman, M., Tashiro, Y., Sonomoto, K., 2011. Lactic acid production from
lignocellulose-derived sugars using lactic acid bacteria: Overview and limits. J.
Biotechnol. 156, 286-301.
[2] Adney, B., Baker, J., 1996. Measurement of cellulase activities. Laboratory analytical
procedure. Technical Report NREL/TP510-42628. National Renewable Energy
13

Laboratory (NREL), Golden CO.
[3] Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,
248-254.
[4] Chandel, A.K., Garlapati, V.K., Singh, A.K., Antunes, F.A.F., da Silva, S.S., 2018. The
path forward for lignocellulose biorefineries: Bottlenecks, solutions, and perspective on
commercialization. Bioresour. Technol. 264, 370-381.
[5] Farah, S., Anderson, D.G., Langer, R., 2016. Physical and mechanical properties of PLA,
and their functions in widespread applications– a comprehensive review. Adv. Drug
Deliver. Rev. 107, 367-392.
[6] Galbe, M., Zacchi, G., 2012. Pretreatment: the key to efficient utilization of
lignocellulosic materials. Biomass Bioenergy 46, 70-78.
[7] Gao, X., Gao, Q.Q., Bao J., 2018. Tolerance response and metabolism of acetic acid by
biodetoxification fungus Amorphotheca resinae ZN1. J. Biotechnol. 275, 31-39.
[8] Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257-268.
[9] Gu, H.Q., Zhang, J., Bao, J., 2014. Inhibitor analysis and adaptive evolution of
Saccharomyces cerevisiae for simultaneous saccharification and ethanol fermentation
from industrial waste corncob residues. Bioresour. Technol. 157, 6-13.
[10]Gu, H.Q., Zhang, J., Bao, J., 2015. High tolerance and physiological mechanism of
Zymomonas mobilis to phenolic inhibitors in ethanol fermentation of corncob residue.
Biotechnol. Bioeng. 112(9), 1770-1782.
[11]He, Y.Q., Zhang, L.P., Zhang, J., Bao, J., 2014. Helically agitated mixing in dry dilute
acid pretreatment enhances the bioconversion of corn stover into ethanol. Biotechnol.
Biofuels 7, 1.
[12]He, Y.Q., Zhang, J., Bao, J., 2016. Acceleration of biodetoxification on dilute acid
14

pretreated lignocellulose feedstock by aeration and the consequent ethanol fermentation
evaluation. Biotechnol. Biofuels 9, 19.
[13]Jonsson, L.J., Alriksson, B., Nilvebrant, N.O., 2013. Bioconversion of lignocellulose:
inhibitors and detoxification. Biotechnol. Biofuels 6(1), 16.
[14]Khoddami, A., Wilkes, M., Roberts, T., 2013. Techniques for analysis of plant phenolic
compounds. Molecules 18(2), 2328-2375.
[15]Klinke, H.B., Thomsen, A.B., Ahring, B.K., 2004. Inhibition of ethanol-producing yeast
and bacteria by degradation products produced during pre-treatment of biomass. Appl.
Microbiol. Biotechnol. 66, 10-26.
[16]Larsson, S., Palmqvist, E., Hahn-Hagerdal, B., Tengborg, C., Stenberg, K., Zacchi, G.,
Nilvebrant, N.O., 1999. The generation of fermentation inhibitors during dilute acid
hydrolysis of softwood. Enzyme Microb. Technol. 24(3-4), 151-159.
[17]Nampoothiri, N.K., Nair, N.R., John, R.P., 2010. An overview of the recent
developments in polylactide (PLA) research. Bioresour. Technol. 101(22), 8493-8501.
[18]Palmqvist, E., Hahn-Hägerdal, B., 2000. Fermentation of lignocellulosic hydrolysates. I:
inhibition and detoxification. Bioresour. Technol. 74(1), 17-24.
[19]Parawira, W., Tekere, M., 2011. Biotechnological strategies to overcome inhibitors in
lignocellulose hydrolysates for ethanol production: review. Crit. Rev. Biotechnol. 31(1),
20-31.
[20]Qiu, Z.Y., Gao, Q.Q., Bao, J., 2017. Constructing xylose-assimilating pathways in
Pediococcus acidilactici for high titer d-lactic acid fermentation from corn stover
feedstock. Bioresour. Technol. 245, 1369-1376.
[21]Qiu, Z.Y., Gao, Q.Q., Bao, J., 2018. Engineering Pediococcus acidilactici with xylose
assimilation pathway for high titer cellulosic L-lactic acid fermentation. Bioresour.
Technol. 249, 9-15.
15

[22]Simon, O., Klaiber, I., Huber, A., Pfannstiel, J., 2014. Comprehensive proteome analysis
of the response of Pseudomonas putida KT2440 to the flavor compound vanillin. J.
Proteomics 109, 212-27.
[23]Thomsen, M.H., Thygesen, A., Thomsen, A.B., 2009. Identification and characterization
of fermentation inhibitors formed during hydrothermal treatment and following SSF of
wheat straw. Appl. Microbiol. Biotechnol. 83(3), 447-455.
[24]van de Guchte, M., van der Vossen, J.M., Kok, J., Venema, G., 1989. Construction of a
lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus
lactis subsp. lactis. Appl. Environ. Microbiol. 55(1), 224-228.
[25]Wang, X., Gao, Q.Q., Bao, J., 2015. Transcriptional analysis of Amorphotheca resinae
ZN1 on biological degradation of furfural and 5-hydroxymethylfurfural derived from
lignocellulose pretreatment. Biotechnol. Biofuels 8, 136.
[26]Wang, X., Liang, Z., Hou, J., Bao, X., Shen, Y., 2016. Identification and functional
evaluation of the reductases and dehydrogenases from Saccharomyces cerevisiae
involved in vanillin resistance. BMC Biotechnol. 16, 31.
[27]Wyman, C.E., Dale, B.E., Elander, R.T., Holtzapple, M., Ladisch, M.R., Lee, Y.Y., 2005.
Coordinated development of leading biomass pretreatment technologies. Bioresour.
Technol. 96, 1959-1966.
[28]Yi, X., Gao, Q.Q., Zhang, L., Wang, X., He, Y.Q., Hu, F.X., Zhang, J., Zou, G., Yang,
S.H., Zhou, Z.H., Bao, J., 2019. Heterozygous diploid structure of Amorphotheca resinae
ZN1 contributes efficient biodetoxification on solid pretreated corn stover. Biotechnol.
Biofuels 12, 126.
[29]Yi, X., Gu, H., Gao, Q.Q., Liu, Z.L., Bao, J., 2015. Transcriptome analysis of
Zymomonas mobilis ZM4 reveals mechanisms of tolerance and detoxification of phenolic
aldehyde inhibitors from lignocellulose pretreatment. Biotechnol. Biofuels 8, 153.
16

[30]Yi, X., Zhang, P., Sun, J.E., Tu, Y., Gao, Q.Q., Zhang, J., Bao, J., 2016. Engineering
wild-type robust Pediococcus acidilactici strain for high titer l- and d-lactic acid
production from corn stover feedstock. J. Biotechnol. 217, 112-121.
[31]Zhang, J., Wang, X.S., Chu, D.Q., He, Y.Q., Bao, J., 2011. Dry pretreatment of
lignocellulose with extremely low steam and water usage for bioethanol production.
Bioresour. Technol. 102, 4480-4488.
[32]Zhang, J., Chu, D., Huang, J., Yu, Z., Dai, G., Bao, J., 2010a. Simultaneous
saccharification and ethanol fermentation at high corn stover solids loading in a helical
stirring bioreactor. Biotechnol. Bioeng. 105(4), 718-728.
[33]Zhang, J., Zhu, Z.N., Wang, X.F., Wang, N., Wang, W., Bao, J., 2010b. Biodetoxification
of toxins generated from lignocellulose pretreatment using a newly isolated fungus
Amorphotheca resinae ZN1 and the consequent ethanol fermentation. Biotechnol.
Biofuels 3, 26.
[34]Zhou, P.P., Khushk, I., Gao, Q.Q., Bao, J., 2019. Tolerance and transcriptional analysis
of Corynebacterium glutamicum on biotransformation of toxic furaldehyde and
benzaldehyde inhibitory compounds. J. Ind. Microbiol. Biotechnol. 46(7), 951-963.
17

Figure captions
Fig. 1 Inhibition of phenolic compounds on the cell growth (OD600) and D-lactic acid
production of P. acidilactici ZY15. (a) Phenolic aldehydes inhibition on P. acidilactici
ZY15; (b) Phenolic alcohols inhibition on P. acidilactici ZY15; (c) Phenolic acids
inhibition on P. acidilactici ZY15. The relative cell growth (or relative D-lactic acid
production) was defined as the percentage of the cell growth (or D-lactic acid production)
under inhibitor stress to that without inhibitor stress in 12 h. The fermentation conditions:
50 mL of MRS medium with gradient concentration of phenolic compounds addition in
250 mL shaking flasks, 10% (v/v) inoculate size, 42 °C, 150 rpm. Cells were collected
for measurement of optical density (OD) at 600 nm without pH control. D-lactic acid was
measured with the pH value controlled by adding 0.6 g of CaCO3 per g of glucose.
Fig. 2 4-hydroxybenzaldehyde, vanillin and syringaldehyde conversion evaluations of
P. acidilactici ZY15. (a) 4-hydroxybenzaldehyde conversion; (b) vanillin conversion; (c)
syringaldehyde conversion. The fermentation conditions: 50 mL of MRS medium with
4-hydroxybenzaldehyde, vanillin or syringaldehyde addition in the 250 mL shaking
flasks, 10% (v/v) inoculate size, 42 °C, 150 rpm. The data were calculated by deducting
the self-volatilization of phenolic aldehydes during the 24 h fermentation conducted
without strains inoculation.
Fig. 3 Fermentation evaluation of P. acidilactici ZY15 recombinants expressing
oxidoreductase genes under mixed phenolic aldehydes stress. (a) Glucose
consumption; (b) D-lactic acid production. The fermentation conditions: 24.6 mM of
4-hydroxybenaldehyde, 1.3 mM of vanillin and 3.3 mM of syringaldehyde added; 50 mL
of MRS medium in 250 mL shaking flasks with 5 μg/mL of erythromycin addition.
Fig. 4 D-lactic acid fermentability of the engineered strain P. acidilactici
ZY15-ΔackA2::CGS9114_RS09725 under stress of gradient concentrations of
18

vanillin. (a) Cell growth (OD600); (b) D-lactic acid production. The fermentation
conditions: 50 mL of MRS medium in 250 mL shaking flasks, 10% (v/v) inoculate size,
42 °C, 150 rpm for 12 h. The parental strain P. acidilactici ZY15 was used as control.
Fig. 5 Vanillin conversion evaluation of the engineered strain P. acidilactici
ZY15-ΔackA2::CGS9114_RS09725 in vanillin contained MRS medium. The
fermentation conditions: 1.6 mM of vanillin added; 50 mL of MRS medium in 250 mL
shaking flasks, 42 °C, 150 rpm, 10% (v/v) inoculate size. The parental strain P.
acidilactici ZY15 was used as control. No vanillic acid was detected during the
fermentation.
Fig. 6 Simultaneous saccharification and co-fermentation (SSCF) of D-lactic acid by
the engineered strain P. acidilactici ZY15-ΔackA2::CGS9114_RS09725 using
phenolic aldehydes contained corn stover feedstock. The SSCF was conducted using
the 30% (w/w) solids loading of dry acid pretreated and biodetoxified corn stover, 10 mg
cellulase protein per gram cellulose. The initial concentration of 4-hydroxybenaldehyde,
vanillin and syringaldehyde in the SSCF system were 0.3, 1.5 and 3.1 mM, respectively.
pH was maintained by automatic feeding of a 25% (w/w) Ca(OH)2 slurry. The parental
strain P. acidilactici ZY15 was used as control.
19

(a) Phenolic aldehydes inhibtion on P. acidilactici ZY15
Relative cell growth Relative D-lactic acid production
120
100
80
60
40
20
0
0.0
4-hydroxybenzaldehyde (mM)
(b) Phenolic alcohols inhibtion on P. acidilactici ZY15
Relative cell growth Relative D-lactic acid production
6.1
12.3
24.6
0.0
0.7
1.3
1.6
0.0
0.0
0.0
2.2
3.3
4.4
Vanillin (mM)
Syringaldehyde (mM)
120
100
80
60
40
20
0
0.0
4-hydroxybenzyl alcohol (mM)
(c) Phenolic acids inhibtion on P. acidilactici ZY15
Relative cell growth Relative D-lactic acid production
6.4
12.9
25.8
0.0
3.9
7.8
15.6
3.3
6.5
13.0
Vanillyl alcohol (mM)
Syringic alcohol (mM)
120
100
80
60
40
20
0
0.0
5.8
11.6
23.2
0.0
3.6
7.1
14.3
3.0
6.1
12.1
Vanillic acid (mM)
Syringic acid (mM)
4-hydroxybenzoic acid (mM)
Fig. 1 Inhibition of phenolic compounds on the cell growth (OD600) and D-lactic acid
production of P. acidilactici ZY15. The relative cell growth or relative D-lactic acid
production was defined as the percentage of the cell growth or D-lactic acid production under
inhibitor stress to that without inhibitor stress in 12 h. The fermentation conditions: 50 mL of
MRS medium with gradient concentration of phenolic compounds addition in 250 mL
20

shaking flasks, 10% (v/v) inoculate size, 42 °C, 150 rpm. Cells were collected for
measurement of optical density (OD) at 600 nm without pH control. D-lactic acid was
measured with the pH value controlled by adding 0.6 g of CaCO3 per g of glucose.
21

(a) 4-hydroxybenzaldehyde conversion
4-hydroxybenzaldehyde
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.74
4-hydroxybenzyl alcohol
4-hyroxybenzoic acid
0.55
0.36
0.31
0.17
12
0.07
0.00
0
0.02
0.00
24
Time (h)
(b) Vanillin conversion
Vanillin
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Vanillyl alcohol
Vanillic acid
0.60
0.43
0.28
0.22
0.14
12
0.00
0.00
0.00
0.00
24
0
Time (h)
(c) Syringaldehyde conversion
Syringaldehyde
Syringic alcohol
Syringic acid
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.51
0.49
0.48
0.00
12
0.00
0
0.00
0.00
0.00
0.00
24
Time (h)
Fig. 2 4-hydroxybenzaldehyde, vanillin and syringaldehyde conversion evaluations of
P. acidilactici ZY15. The fermentation conditions: 50 mL of MRS medium with
4-hydroxybenzaldehyde, vanillin or syringaldehyde addition in the 250 mL shaking flasks,
22

10% (v/v) inoculate size, 42 °C , 150 rpm. The data were calculated by deducting the
self-volatilization of phenolic aldehydes during the 24 h fermentation conducted without
strains inoculation.
23

P. acidilactici ZY15(pZY36e) (Control)
P. acidilactici ZY15(pZY36e-PP_5258)
P. acidilactici ZY15(pZY36e-PP_3151)
P. acidilactici ZY15(pZY36e-PP_5120)
P. acidilactici ZY15(pZY36e-ZMO1885)
P. acidilactici ZY15(pZY36e-CGS9114_RS10340)
P. acidilactici ZY15(pZY36e-CGS9114_RS09275)
(a) Glucose consumption
40
35
30
25
20
15
10
5
0
0
6
12
18
24
30
36
42
48
Time (h)
(b) D-lactic acid production
40
35
30
25
20
15
10
5
0
0
6
12
18
24
30
36
42
48
Time (h)
Fig. 3 Fermentation evaluation of P. acidilactici ZY15 recombinants expressing
oxidoreductase genes under mixed phenolic aldehydes stress. The fermentation conditions:
24.6 mM of 4-hydroxybenaldehyde, 1.3 mM of vanillin and 3.3 mM of syringaldehyde added;
50 mL of MRS medium in 250 mL shaking flasks with 5 μg/mL of erythromycin addition.
24

Parental strain (Control)
(a) Cell growth
Engineered strain
5
4
3
2
1
0
0.0
0.7
1.3
1.6
Vanillin concentration (mM)
(b) D-lactic acid production
20
15
10
5
0
0.0
0.7
1.3
1.6
Vanillin concentration (mM)
Fig. 4 D-lactic acid fermentability of the engineered strain P. acidilactici
ZY15-ΔackA2::CGS9114_RS09725 under stress of gradient concentrations of vanillin.
(a) Cell growth (OD600); (b) D-lactic acid production. The fermentation conditions: 50 mL of
MRS medium in 250 mL shaking flasks, 10% (v/v) inoculate size, 42 °C, 150 rpm for 12 h.
The parental strain P. acidilactici ZY15 was used as control.
25

Parental strain(Control)
Engineered strain
1.8
1.6
1.4
1.2
1.0
0.5
0.4
0.3
0.2
0.1
0.0
0
6
12
18
24
Time (h)
Fig. 5 Vanillin conversion evaluation of the engineered strain P. acidilactici
ZY15-ΔackA2::CGS9114_RS09725 in vanillin contained MRS medium. The fermentation
conditions: 1.6 mM of vanillin added; 50 mL of MRS medium in 250 mL shaking flasks,
42 °C, 150 rpm, 10% (v/v) inoculate size. The parental strain P. acidilactici ZY15 was used
as control. No vanillic acid was detected during the fermentation.
26

Parental strain(Control)
Engineered strain
120
100
80
60
40
20
0
0
12
24
36
48
60
72
84
Time (h)
Fig. 6 Simultaneous saccharification and co-fermentation (SSCF) of D-lactic acid by
the engineered strain P. acidilactici ZY15-ΔackA2::CGS9114_RS09725 using phenolic
aldehydes contained corn stover feedstock. The SSCF was conducted using the 30% (w/w)
solids loading of dry acid pretreated and biodetoxified corn stover, 10 mg cellulase protein
per gram cellulose. 0.1, 0.6 and 1.5 mg/g DM of 4-hydroxybenaldehyde, vanillin and
syringaldehyde were remained in the pretreated and biodetoxified corn stover, respectively.
pH was maintained by automatic feeding of a 25% (w/w) Ca(OH)2 slurry. The parental strain
P. acidilactici ZY15 was used as control.
Author Contributions Section
JB conceived of the study. ZYQ conducted the metabolic engineering experiments. ZYQ and
CF conducted the assay and the fermentation experiments. QQG partially participated the
27

metabolic engineering experiment and helped the analysis. ZYQ and JB wrote the manuscript.
CF and QQF partially participated the writing of the manuscript. All authors approved the
manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Highlights



Genome integration of CGS9114_RS09725 accelerated vanillin reduction.
Simultaneous vanillin conversion and D-lactic acid fermentation was conducted.
High titer D-lactic acid production from corn stover was achieved by SSCF.
Graphical Abstract
28

Corn stover
Dry acid pretreatme
Biodetoxification
CHO
Feedstock con
OCH3
OH
Vanillin
High solids loa
Pediococcus
acidilactici
CH2OH
Genome integration
of the short-chain
dehydrogenase gene
CGS9114_RS09725
 Simultaneous vanillin
chiral lactic acid ferm
 Enhanced chiral lactic
OCH3
OH
Vanillyl alcohol
29