Metabolic Engineering 71 (2022) 2–12

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Metabolic Engineering
journal homepage: www.elsevier.com/locate/meteng

Metabolic engineering for the utilization of carbohydrate portions of

lignocellulosic biomass
Jiwon Kim a, b, 1, Sungmin Hwang a, 1, Sun-Mi Lee a, c, d, *
a
Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seoul, 02792, Republic of Korea
b
Department of Biotechnology, Korea University, Seoul, 02841, Republic of Korea
c
Clean Energy and Chemical Engineering, University of Science and Technology, Daejeon, 34113, Republic of Korea
d
Green School (Graduate School of Energy and Environment), Korea University, Seoul, 02841, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: The petrochemical industry has grown to meet the need for massive production of energy and commodities along
Lignocellulosic carbohydrates with an explosive population growth; however, serious side effects such as greenhouse gas emissions and global
Glucose warming have negatively impacted the environment. Lignocellulosic biomass with myriad quantities on Earth is
Xylose
an attractive resource for the production of carbon-neutral fuels and chemicals through environmentally friendly
Metabolic engineering
Yeast
processes of microbial fermentation. This review discusses metabolic engineering efforts to achieve economically
Bacteria feasible industrial production of fuels and chemicals from microbial cell factories using the carbohydrate portion
of lignocellulosic biomass as substrates. The combined knowledge of systems biology and metabolic engineering
has been applied to construct robust platform microorganisms with maximum conversion of monomeric sugars,
such as glucose and xylose, derived from lignocellulosic biomass. By comprehensively revisiting carbon con­
version pathways, we provide a rationale for engineering strategies, as well as their features, feasibility, and
recent representative studies. In addition, we briefly discuss how tools in systems biology can be applied in the
field of metabolic engineering to accelerate the development of microbial cell factories that convert lignocel­
lulosic biomass into carbon-neutral fuels and chemicals with economic feasibility.

1. Introduction originating from the structural complexity make it resistant to microbial

Concerns regarding climate change and global warming driven by
human-induced emissions of greenhouse gases and energy security have
led to the development of renewable energy sources to mitigate the
current excessive use of fossil fuels. Recently, efforts to reduce green­
house gas emissions have gained more attention with the global
commitment to net-zero carbon emissions (Rogelj et al., 2021). Bio­
refinery offers one of the most effective ways to achieve carbon
neutrality especially in the transportation and industry sectors by sup­
plying carbon-neutral fuels and chemicals. With the use of lignocellu­
losic biomass, which exists in large quantities and prevails on Earth,
biorefinery would foster more sustainable and economically feasible
production of fuels and chemicals, supporting the smooth transition to a
post-carbon economy (Kim et al., 2020; Ko and Lee, 2018).
Lignocellulosic biomass is composed of cellulose, hemicellulose, and
lignin. The rigidity and heterogeneity of lignocellulosic biomass
conversion. This requires efficient pretreatment and hydrolysis tech­
nologies, and microbial strains capable of fermenting all carbohydrate
portions of lignocellulosic biomass, which account for 70% of the dry
mass, to improve the economic feasibility of lignocellulosic biorefinery.
In metabolic engineering fields, the development of microbial strains
that effectively convert the carbohydrate portion of lignocellulosic
biomass has been mainly devoted to improving the carbon catabolism of
non-glucose sugars derived from lignocellulosic biomass. While the
cellulose portion provides the most preferable sugar of glucose, the use
of hemicellulose is challenging for microorganisms due to the presence
of secondary sugars including xylose. Xylose comprises up to 40% of
carbohydrates derived from lignocellulosic biomass and serves as the
second most abundant sugar (glucose accounts for 60–70%) in ligno­
cellulosic hydrolysates; thus, the conversion of xylose along with
glucose adds an accessible sugar component in the carbohydrate portion
of lignocellulosic biomass from 60 to over 90% depending on the type of

* Corresponding author. Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seoul, 02792, Republic of Korea.
E-mail address: smlee@kist.re.kr (S.-M. Lee).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.ymben.2021.10.002
Received 2 July 2021; Received in revised form 16 September 2021; Accepted 3 October 2021
Available online 6 October 2021
1096-7176/© 2021 The Authors. Published by Elsevier Inc. on behalf of International Metabolic Engineering Society. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
J. Kim et al.
lignocellulosic biomass (Ko et al., 2020).
Here, we review the metabolic engineering efforts devoted to the
development of microorganisms that efficiently convert the carbohy­
drate portion of lignocellulosic biomass for the sustainable production of
fuels and chemicals. Specifically, the metabolic engineering strategies
adopted in microbial cell factories are discussed with a focus on model
bacteria and yeasts, E. coli and S. cerevisiae, in which carbon catabolite
repression (CCR) and inability to utilize xylose have been the main
bottlenecks in maximizing the conversion of lignocellulosic biomass into
value-added products (Gao et al., 2019; Fox and Prather, 2020). We first
introduce carbon catabolic pathways available to achieve high-yield
conversion of sugars into core intermediates for value-added chem­
icals by detouring central carbon metabolism. Next, we discuss meta­
bolic engineering approaches to improve carbon conversion efficiency
through the carbon catabolic pathways. Then, we discuss recent engi­
neering efforts to balance the energy supply in sugar catabolism, which
better support carbon flux through biosynthetic pathways. Finally, we
highlight the recent progress in lignocellulosic biorefinery, showing the
potential of engineered strains in the bioconversion of lignocellulosic
hydrolysates into value-added products such as fuels and chemicals.
2. Catabolic pathways for the conversion of sugars into core-
intermediates
Once generated through physical, chemical, and biological pre­
treatment processes, the microbially accessible basal units of the
lignocellulosic carbohydrates, monomeric carbon substrates of sugars
with mainly glucose (60–70%) and xylose (30–40%), can be catabolized
through the central carbon metabolism of microbial cell factories. The
conversion of glucose and xylose through central carbon metabolism
generates core intermediates such as glyceraldehyde-3-phosphate, py­
ruvate, and acetyl-CoA, which are further used as building blocks for the
production of fuels and chemicals of polyketides, terpenoids, keto acids,
alcohol, and fatty acid derivatives (Fig. 1).
Fig. 1. Overall catabolic pathways for hexose (glucose) and pentose (xylose) utilization in microorganisms. Orange and red lines indicate the non-phosphorylating
and pentose phosphate pathways, respectively. Black line shows glycolysis pathway. Cofactors, energy produced and carbon loss are shown in blue, green and red
characters, respectively. The energy used for sugar uptake is not counted in the calculation.
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Metabolic Engineering 71 (2022) 2–12
2.1. Conversion of glucose to maximize carbon utilization
Most microorganisms preferably utilize glucose, a major carbon
component of lignocellulosic carbohydrates, via glycolysis. Through this
central carbon metabolism, cells generate energy in the form of ATP
while converting glucose into pyruvate, which is then further converted
to generate more energy through the citric acid cycle or fermentation.
When one mole of glucose is converted through the Embden-Meyerhof-
Parnas (EMP) glycolytic pathway to acetyl-CoA, a core intermediate for
the production of fuels and chemicals via glycolysis, one-third of the
carbon is lost as CO2 decreasing product yields (Fig. 2). When the
biosynthesis of a target product requires NADPH, the EMP pathway can
be modified to comprise NADP+ preferred glyceraldehyde-3-phosphate
dehydrogenase (Martínez et al., 2008). A certain portion of glucose
can also be redirected through the Entner-Doudoroff (ED) glycolytic or
pentose phosphate (PP) pathway from which one or 2 mol of NADPH are
obtained per mole of glucose to generate acetyl-CoA at the expense of
energy or carbon loss, respectively (Flamholz et al., 2013) (Table 1 and
Fig. 2). Glucose conversion through the ED glycolytic pathway prevails
in gram-negative bacteria and archaea, and offers additional benefits of
rapid sugar turnover and low enzyme synthesis cost because of the
relatively simpler biochemical reactions compared to those of the EMP
glycolytic pathway. In E. coli, the substitution of EMP with a heterolo­
gous ED glycolytic pathway adapted from Z. mobilis led to 50–80%
increased isobutanol production in terms of titer, yield, and productivity
(13.67 g/L, 0.5 g/g, and 0.456 g/L/h, respectively) during glucose
fermentation (Liang et al., 2018). Rerouting carbon flux through
non-oxidative glycolysis via the phosphoketolase pathway (PK pathway)
offers an alternative pathway to maximize carbon conversion. In E. coli,
glucose catabolism based on non-oxidative glycolysis results in
near-complete carbon utilization for producing acetyl-CoA, which is
then converted to organic acids (Lin et al., 2018). In S. cerevisiae, glucose
conversion through the PK pathway has improved the conversion yields
of polyhydroxybutyrate (Kocharin et al., 2013), fatty acid ethyl ester (de
Jong et al., 2014), and farnesene (Meadows et al., 2016). Recently, the
J. Kim et al. Metabolic Engineering 71 (2022) 2–12

Fig. 2. Schematic flow diagrams of glucose (green) and xylose (yellow) conversion into metabolites. Flow thickness corresponds to the relative level of metabolites
and the numbers represent the moles generated from 1 mol of the respective sugar. Abbreviations: EMP, Embden-Meyerhof-Parnas; ED, Entner-Doudoroff; PP,
pentose phosphate; PK, phosphoketolase; Iso-PP, isomerase based pentose phosphate; Iso-PK, isomerase based phosphoketolase; OR-PP, oxidoreductase based
pentose phosphate; OR-PK, oxidoreductase based phosphoketolase. The energy used for sugar uptake is not counted in the calculation.

efficiency of carbon conversion through the PK pathway has been
improved by cycling carbon flux with a promiscuous phosphoketolase,
resulting in a 109% increase in the titer of 3-hydroxypropionic acid
(3-HP) compared to the result obtained using the conventional PK
pathway (Hellgren et al., 2020). Similarly, the diversion of carbon flux
from glucose through PK pathways resulted in a 19 and 78% increase in
lipid yield and productivity, respectively, in Y. lipolytica (Kamineni
et al., 2021).
2.2. Conversion of xylose to maximize carbon utilization
In bacteria, xylose is converted into xylulose-5-phosphate via an
isomerase-based pathway, and is then further metabolized through
either the PP or PK pathway (Fig. 3). However, yeasts use the
oxidoreductase-based pathway to convert xylose or arabinose into
xylulose-5-phosphate (Table 1 and Fig. 1). While the isomerase-based
pathway is cofactor neutral, the conversion of pentose through an
oxidoreductase-based pathway causes the diversion of carbon flux to­
ward by-product formation to compensate for cofactor imbalance, thus
lowering the yield of the desired product. The conversion of xylose
through an isomerase-based pathway increases the carbon conversion
yield, reaching a theoretical maximum (Hoang Nguyen Tran et al., 2020;
Yook et al., 2020).
The conversion of xylose through the non-phosphorylating pathway
(Dhams or Weimberg pathway) yields either glycoaldehyde, pyruvate,
or α-ketoglutarate without carbon loss, as well as reducing power in the
form of NADH (Figs. 1 and 3). In E. coli, the production of ethylene
glycol via the Dahms pathway has proven the potential of xylose con­
version through a non-phosphorylating pathway supporting the pro­
duction of ethylene glycol and glycolic acid with a yield reaching the
theoretical maximum (Cabulong et al., 2017, 2021). Redirecting xylose
flux via a non-phosphorylating pathway, which provides a simplified
biosynthetic pathway, resulted in the highest titer of 3-hydrox­
y-γ-butyrolactone production (1.27 g/L) in E. coli (Wang et al., 2017b).
However, this route has potential weaknesses in the accumulation of the
toxic intermediate, xylonic acid, and the induction of CCR when glucose
is present. The first issue could be solved by low-level expression of
heterologous xylose dehydrogenase which reduces the xylonic acid
precursor, xylonolactone. Through this approach, Chae et al. has
improved ethylene glycol productivity up to 2.25 g/L/h (Chae et al.,
2018). The accumulation of xylonic acid has been a common challenge
in taking advantage of these carbon-and energy-saving pathways in
yeasts. The functionality of the complete pathway originated from
Caulobacter crescentus has been limited in S. cerevisiae due to the low
activity of xylonate dehydratase, which results in a significant accu­
mulation of xylonic acid (Salusjarvi et al., 2017; Wasserstrom et al.,
2018). Recently, complete expression of the Weimberg pathway in
S. cerevisiae has been reported through the collaboration of two strate­
gies that the deletion of FRA2 for upregulation of FE-S metabolism,
which may increase D-xylonate dehydrogenase activity, and the

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J. Kim et al. Metabolic Engineering 71 (2022) 2–12

Table 1
Comparison of yield and energy cost in carbon catabolic pathways.
Sugars Catabolic pathway Intermediate NADH yield NADPH yield ATP yield Pathway yield Carbon loss

Glucose EMP-Glycolysis G3P 0 0 − 2 3 0%

EMP-Glycolysis Pyruvate 2 0 2 2 0%
EMP-Glycolysis Acetyl-CoA 4 0 2 2 33%
ED-Glycolysis Pyruvate 1 1 1 2 0%
ED-Glycolysis Acetyl-CoA 3 1 1 2 33%
Glucose PP G3P 0 2 − 2.33 1.67 17%
PP Pyruvate 1.67 2 1 1.67 17%
PP Acetyl-CoA 3.33 2 1 1.67 44%
Glucose PK Acetyl-CoA 2 2 1 2 33%
Xylose Iso-PP G3P 0 0 − 1.67 1.67 0%
Iso-PP Pyruvate 1.67 0 1.67 1.67 0%
Iso-PP Acetyl-CoA 3.33 0 1.67 1.67 33%
Xylose Iso-PK Acetyl-CoA 2 0 1 2 20%
Xylose OR-PP G3P 1 − 1 − 1.67 1.67 0%
OR-PP Pyruvate 2.67 − 1 1.67 1.67 0%
OR-PP Acetyl-CoA 4.33 − 1 1.67 1.67 33%
Xylose OR-PK Acetyl-CoA 3 − 1 1 2 20%
Xylose Dahms Pyruvate + glycoaldehyde 1 0 0 1 0%
Xylose Weimberg α-ketoglutarate 2 0 0 1 0%
Arabinose Iso-PP G3P 0 0 − 1.67 1.67 0%
Iso-PP Pyruvate 1.67 0 1.67 1.67 0%
Iso-PP Acetyl-CoA 3.33 0 1.67 1.67 33%
Arabinose Iso-PK Acetyl-CoA 2 0 1 2 20%
Arabinose OR-PP G3P 2 − 2 − 1.67 1.67 0%
OR-PP Pyruvate 3.67 − 2 1.67 1.67 0%
OR-PP Acetyl-CoA 5.33 − 2 1.67 1.67 33%
Arabinose OR-PK Acetyl-CoA 4 − 2 1 2 20%
Arabinose Dahms Pyruvate + glycoaldehyde 1 0 0 1 0%
Arabinose Weimberg α-ketoglutarate 2 0 0 1 0%

Abbreviations: EMP-Glycolysis, Embden-Meyerhof-Parnas glytolytic pathway; ED-glycolysis, Entner-Doudoroff glycolytic pathway; PP, pentose phosphate pathway;
PK, phosphoketolase pathway; Iso-PP, isomerase based pentose phosphate pathway; Iso-PK, isomerase based phosphoketolase pathway; OR-PP, oxidoreductase based
pentose phosphate pathway; OR-PK, oxidoreductase based phosphoketolase pathway. * The energy used for sugar uptake is not counted in the calculation. ** Pyruvate
to Acetyl-CoA calculation is based on the reaction mediated by pyruvate dehydrogenase under aerobic condition.

Fig. 3. Pentose-specific catabolic

pathways. Cofactors, energy produced
and carbon loss are shown in blue,
green and red characters, respectively.
Abbreviations: XLDH, xylose dehydro­
genase; XLS, xylonolactonase; XDY,
xylonate dehydratase; KDY, 2-keto-3-
deoxyxylonate dehydratase; KSH,
α-KG semialdehyde dehydrogenase;
DAL, 2-keto-3-deoxyxylonate aldolase;
PDH, pyruvate dehydrogenase; XR,
xylose reductase; XDH, xylose dehy­
drogenase; XI, xylose isomerase; XK,
xylose kinase; PK, phosphoketolase;
AcK, acetate kinase.

replacement of α-ketoglutarate semialdehyde dehydrogenase from has been solved, no ATP generation during xylose conversion through
Caulobacter crescentus (xylA) with that from Corynebacterium glutamicum the carbon conserving non-phosphorylating pathway (Fig. 2) could be a
(ksaD) (Borgstrom et al., 2019). Although the issue of toxic intermediate critical challenge especially in industrial settings with limited oxygen

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J. Kim et al. Metabolic Engineering 71 (2022) 2–12

availability. While ATP could be supplied from the oxidation of NADH
released from the pathway under aerobic conditions, the cell would
suffer from lack of energy for growth and maintenance under industrial
settings with limited oxygen availability (Zhou et al., 2009).
3. Improving metabolic flux through a carbon catabolic
pathway
Compared to glucose conversion, xylose conversion efficiency has
been limited in microbial cell factories due to CCR and the absence of
catabolic pathways, which are major challenges in bacterial and yeast
strains, respectively. Through metabolic engineering efforts over the
past few decades, delayed xylose utilization in bacterial strains during
glucose/xylose co-fermentation has been pushed forward by disrupting
cAMP receptor protein and phosphoenolpyruvate transferase system
(Fox and Prather, 2020). In addition, the xylose conversion efficiency in
engineered yeast strains followed up glucose conversion into ethanol
(Ko et al., 2020; Ko and Lee, 2018). Recent evidences show that the
conversion of both glucose and xylose not only improves the overall
product yield per unit biomass in lignocellulosic biorefinery, but also
supports carbon flux and energy through biosynthetic pathways for the
desired target products (Kwak and Jin, 2017; Arora et al., 2019). This
underpins the importance of efficiency and balance in the catabolism of
sugars derived from lignocellulosic biomass through metabolic engi­
neering efforts such as transporter engineering, pathway enzyme engi­
neering, and transcription factor engineering (summarized in Table 2).

Table 2
Engineering strategies for improving carbon conversion efficiency.
Main Strategy Strain Representative Substrates (g/L) Product Titer (g/ Yield Improvements Ref.
Engineering L) (g/g)

Transporter S. cerevisiae Gal2N376F X (0.15–75) N/A N/A N/A 2.4-fold in Km for xylose* (91.4 ± Farwick et al.
engineering 8.9 mM) (2014)
S. cerevisiae Gal2F85S G + X + A (20 Ethanol 10 0.41** 1.4-fold in growth rate on Wang et al.
+ 20+ 20) arabinose (0.035 ± 0.001 h− 1) (2017a)
S. cerevisiae Hxt11/2N361T G + X (80 + 40) Ethanol 49.1* 0.44 1.2-fold in Km for xylose (57.3 ± Shin et al.
5.2 mM) (2017)
1.22-fold in ethanol productivity*
(1.17 ± 0.04 g/L/h)
S. cerevisiae SZ2-H-Gal2, X (20) Ethanol 5.7* 0.29* 1.66-fold in xylose consumption Thomik et al.
SZ1-WH1-XI rate* (0.125 g/L/h) (2017)
2.4-fold in ethanol productivity**
(0.37 g/L/h)
O. polymorpha Hxt1N358A G + X (70 + 30) Ethanol 27.56 0.35 1.73-fold in xylose consumption Vasylyshyn
rate* (0.324 g/L/h) et al. (2020)
1.3-fold in ethanol yield
Z. mobilis EcXylE G + X (50 + 50) Ethanol 39.6 0.46 1.55-fold in xylose consumption Dunn and Rao
rate* (0.82 ± 0.03 g/L/h) (2014)
1.3-fold in ethanol titer*
E. coli GatCS184L X (20) Lactate 18.96* 0.95 7.18-fold in lactate productivity* Utrilla et al.
(0.79 g/L/h) (2012)
2.75-fold in specific xylose
consumption rate (2.70 ± 0.22 g/
g/h)
Manipulating S. cerevisiae RsXIN337C X (50) Ethanol 12.48* 0.39 2.5-fold in xylose consumption Katahira et al.
enzyme activity rate (0.44 g/L/h) (2017)
S. cerevisiae LpXIT63I/V162A G + X (85 + 35) Ethanol 53.3 0.44 1.67-fold in Vmax for xylose* Seike et al.
(0.107 ± 0.002 μmol mg protein− 1 (2019)
min− 1)
1.13-fold in ethanol yield*
Global regulation S. cerevisiae Med2*432Y G (20) Farnesene 0.035** 0.002* 1.47-fold in specific growth rate Dai et al. (2018)
(0.218 ± 0.006 h− 1)
S. cerevisiae ΔGCR2 X (40) Ethanol 9.2* 0.23 2.1-fold in xylose consumption Shin et al.
rate (0.37 g/L/h)** (2021)
3.83-fold in ethanol yield*
S. cerevisiae ΔTHI2 X (20) Ethanol N/A 0.36* 1.27-fold in specific xylose Wei et al.
consumption rate (0.407 ± 0.010 (2018)
g/g DCW/h)
1.32-fold in specific ethanol
production rate (0.147 ± 0.010 g/
g DCW/h)
S. cerevisiae HXK2S14A X (20) Ethanol 6** 0.28 1.24-fold in xylose consumption Zheng et al.
rate (0.341 ± 0.027 g/L/h) (2020)
1.43-fold in ethanol yield
E. coli ΔPGI, CRP G + X (10 + 10) Methyl- 2 0.1* 2.7-fold in xylose utilization rate Wang et al.
downregulation ketone (0.11 g/L/h) ** (2018)
1.33-fold in methyl ketone titer
E. coli XylRR121C/P363S G + X (50 + 50) Lactate 86 0.91 4-fold in xylose utilization rate* Sievert et al.
(0.422 g/L/h) (2017)
1.5-fold in lactate tite

Abbreviations: G, glucose; X, xylose; A, arabinose; Hxt, hexose transporter; Gal2, galactose permease; XI, xylose isomerase; XylE, D-xylose-proton symporter; Glf,
glucose facilitated diffusion protein; GatC, glutamyl-tRNA amidotransferase; Med2, RNA polymerase II mediator complex subunit 2; GCR2, glycolytic genes tran­
scriptional activator; THI2, thiamine biosynthesis regulatory protein; HXK2, hexokinase; CRP, cAMP receptor protein; Pgi, glucose-6-phosphate isomerase; XylR,
xylose transcriptional activator; *Calculation values from the numbers presented in the reference paper; **Estimated values from the graphs presented in the reference
paper.

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J. Kim et al.
3.1. Improving carbon flux inside the cells: transporter engineering
Cells have evolved to effectively take up glucose transported through
numerous sugar transporters embedded in the cell membrane. However,
with little demand for pentose catabolism, the transport of xylose and
arabinose across the cell membrane is often limited by the low affinity of
native sugar transporters and/or regulatory mechanisms limiting the
uptake of these sugars. Therefore, transporter engineering has improved
the carbon flux inside the cells to better support conferred pentose
catabolic pathways without the loss of efficiency in glucose uptake.
In yeast, S. cerevisiae lacking a xylose-specific transporter, low-
affinity hexose transporters have been targeted to allow the trans­
portation of xylose. Modification of the transmembrane region of native
hexose transporters, such as Hxt7, Hxt11, and Gal2, has increased the
affinity for pentose, allowing more efficient delivery of these sugars
inside cells (Farwick et al., 2014; Wang et al., 2017a; Young et al.,
2014). A chimeric hexose transporter of Hxt2 with the N-terminal tail of
Hxt11, Hxt11/2, preventing glucose-induced degradation supported a
58% increase in xylose transport rates with reversed specificity for
xylose over glucose (Shin et al., 2017). In bacteria, the overexpression of
an endogenous and heterologous transporters, and a transporter variant,
such as XylE from Zymomonas mobilis, and GatCS184L, respectively, have
improved the rate of xylose metabolism and the production of
value-added materials (Dunn and Rao, 2014; Utrilla et al., 2012).
In addition, the discovery of a novel xylose transporter, Cs4130, from
Candida sojae has expanded the choice of transporter to convey xylose
inside the cell with 30% faster rate than that with Gxf1 from Candida
intermedia, which is one of the most efficient xylose transporter heter­
ologously expressed in S. cerevisiae (Runquist et al., 2010; Bueno et al.,
2020). Further, an artificial membrane transport metabolon, a complex
of a native sugar transporter of Gal2 and a heterologous xylose isom­
erase (XI), has been adapted to improve xylose catabolism by channeling
xylose across the cell membrane. During high cell density fermentation,
cells expressing a fusion protein of Gal2 and XI as SZ2-H-Gal2 and
WHIL-XI showed about 1.7 and 2.4-fold increased xylose consumption
and ethanol production rates (0.125 and 0.37 g/L/h), respectively
(Thomik et al., 2017). In the thermotolerant methylotrophic yeast
Ogataea polymorpha, introduction of a heterologous sugar transporter,
Hxt1 from S. cerevisiae, conferred improved xylose consumption rate
(0.324 g/L/h) and ethanol yield (0.35 g/g) by 1.73 and 1.3-fold,
respectively (Vasylyshyn et al., 2020) (Table 2).
3.2. Improving carbon flux within a sugar catabolic pathway:
manipulating enzyme activity
Extensive accumulation of enzyme database and computational tools
for comparative genomics has advanced the discovery of numerous
enzymes supporting novel mechanisms for improving the efficiency of a
catabolic pathway for sugar metabolism. The newly discovered enzymes
are often heterologously expressed in microbial hosts for lignocellulosic
biorefineries. With concerns such as codon bias, substrate specificity,
and folding issues, however, heterologous enzymes are often re-
engineered through directed evolution to better support sugar meta­
bolism in microbial hosts.
Heterologous expression of XI has enabled the cofactor neutral
conversion of xylose in S. cerevisiae (Table 1). After the first successful
expression and directed evolution of XI from Piromyces sp., XIs sourced
from various strains have been adapted to confer xylose catabolism in
S. cerevisiae. Recently, RsXI from the hindgut of termite Reticulitermes
speratus has shown to be an appropriate heterologous enzyme for the
efficient xylose catabolism in S. cerevisiae. Through the directed evolu­
tion of RsXI, N337C was confirmed as a core mutation for significantly
improving enzyme activity, which also improved enzyme activities of
previously reported XIs from Piromyces sp. and Clostridium phyto­
fermentans (Katahira et al., 2017). A mutant XI from Lachnoclostridium
phytofermentans ISDg (LpXI), LpXI*T63I/V162A, developed through
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Metabolic Engineering 71 (2022) 2–12
directed evolution, has also been added as an enzyme source for xylose
catabolism in S. cerevisiae (Seike et al., 2019).
3.3. Improving carbon flux in a cellular network: global regulations and
novel targets
The application of high-throughput next-generation sequencing has
enabled the investigation of genome-wide profiling of gene expression
and the discovery of novel genes or unknown regulons of regulatory
proteins, thereby permitting new perspectives on metabolic pathways to
optimize strains for more efficient bioconversion of sugars derived from
lignocellulosic biomass.
Whole genome sequencing revealed novel engineering targets such
as ASC1 for increased xylose conversion (Tran Nguyen Hoang et al.,
2018), and GAL2 for glucose-insensitive L-arabinose uptake (Verhoeven
et al., 2018) in evolved xylose or arabinose-utilizing strains obtained
through adaptive laboratory evolution (ALE). A mutation in MED2 that
regulates the transcription of RNA polymerase II-dependent genes has
been shown to relieve the Crabtree effect in S. cerevisiae, offering an
effective engineering strategy to rewire the central carbon metabolism
(Dai et al., 2018). Transcriptome analysis by RNA-seq could also lead
future research to divert carbon flux toward a biosynthetic pathway by
offering a genome-wide gene expression landscape. In a lignocellulosic
biomass utilizer, anaerobic bacterium, Clostridium acetobutylicum,
RNA-seq was used to understand the hierarchical pentose utilization
between arabinose and xylose with a metabolic shift in pentose catab­
olism from the PP pathway to the PK pathway. In this study, the phos­
phocarrier protein, Crh, has been suggested as a key player in the
pentose hierarchy (Servinsky et al., 2018).
Engineering of transcriptional regulators for efficient carbon utili­
zation has also been applied. Deletion of global transcriptional factors,
such as GCR2, which is involved in glucose metabolism (Shin et al.,
2021), and THI2 (Wei et al., 2019), has improved xylose catabolism in
S. cerevisiae resulting in about 2.1 and 1.27-fold increases in xylose
consumption rates, respectively. In addition, a mutation in the glycolytic
enzyme of hexokinase 2, Hxk2pS14A, which also functions as an intra­
cellular glucose sensor, has been shown to improve xylose catabolic flux
towards ethanol in S. cerevisiae, leading to 1.24 and 1.43-fold increased
xylose consumption rate and ethanol yield, respectively (Zheng et al.,
2020) (Table 2).
In prokaryotes, CCR is a major challenge to be overcome for simul­
taneous use of hexose and pentose sugars. Since microorganisms
consume glucose preferentially over xylose, E. coli strains have been
engineered to constitutively express genes involved in xylose catabo­
lism, such as xylF, a peripheral D-xylose ABC transporter, and xylA, an XI
(Wang et al., 2018). However, this tactic was proven ineffective in
achieving high-yield and productivity, suggesting that sophisticated
metabolic flux and modification are still needed. Instead, the
de-repressed genes in xylose metabolism by the modification of the
ribosome binding site of a key transcriptional regulator for CCR, CRP
(cAMP receptor protein), have been shown to be effective in achieving
higher sugar conversion during simultaneous co-fermentation of glucose
and xylose, during which an increased intracellular NADPH supply was
provided through the deletion of pgi, which encodes
glucose-6-phosphate isomerase (Wang et al., 2018). In addition,
CCR-independent activation of xylose catabolic operons including xylAB
and xylFGH was observed with the variant of transcriptional regulator
XylR (R121C/P363S) and this improved the lactic acid titer by 50%
during glucose/xylose co-fermentation (Sievert et al., 2017).
4. Improving energy balance in sugar catabolism
Cofactors, non-protein compounds of redox carriers, such as NAD+/
NADH and NADP+/NADPH, play important roles in cellular catabolic
and anabolic reactions. A balanced intracellular redox status is essential
for platform microorganisms to control the carbon flux and generate
J. Kim et al. Metabolic Engineering 71 (2022) 2–12

products. Redox balance is often destroyed by the refactoring pathway,
including/excluding enzyme to gain more products or to utilize new
substrates, for example, the cofactor imbalance from an oxidoreductase-
based xylose catabolic pathway heterologously introduced in
S. cerevisiae for lignocellulosic biorefinery.
Many efforts have been made to alleviate and solve the cofactor
imbalance using metabolic engineering approaches. First, the cofactor
balance can be maintained by inactivating competitive genes or path­
ways. NADH generated during the glycolytic pathway is consumed in
several intracellular metabolisms, such as butanol and lactate produc­
tion. Disrupting native NADH-required pathways, such as biosynthetic
pathways for succinate, lactate, ethanol, and acetate, by deleting lactate
dehydrogenase has been shown to be effective in increasing NADH
availability, thus supplying reducing power to the biosynthesis of the
final target product, n-butanol, in E. coli (Lim et al., 2013). Alternatively,
carbon flux can be diverted into an alternative pathway with an
advantage of extra cofactor supply. In E. coli, an engineered heterolo­
gous ED pathway from Zymomonas mobilis was successfully imple­
mented to improve the regeneration of NADPH and thus increase a
terpenoid titer by 97% (Ng et al., 2015). In the cyanobacterium Syn­
echocystis sp., the increased NADPH concentration by overexpression of
endogenous ZWF, encoding glucose-6-phosphate dehydrogenase within
the PP pathway, increased the ethanol titer by 33% (Choi and Park,
2016). Redox-responsive molecules can also be supplied to a biosyn­
thetic pathway for a target product by adopting a cofactor regeneration
system. In E. coli, NADPH-regeneration by overexpressing membrane
bound pyridine nucleotide transhydrogenase, PtnAB, increased xylose
conversion to glycolic acid via the Dahms pathway (Cabulong et al.,
2019). In S. cerevisiae, overexpression of a water-forming NADH oxidase
encoded by nox from Streptococcus pneumoniae restored the deficiency of
cytosolic NADH, leading to efficient glucose fermentation with
repressed respiration (Vemuri et al., 2007). The cofactor imbalance
caused by the introduction of an oxidoreductase-based xylose utilization
pathway has been resolved in S. cerevisiae by expressing the same
water-forming NADH oxidase (noxE), but from a different source, Lac­
tococcus lactis (Zhang et al., 2017). During high cell density fermenta­
tion, which minimized the effect of growth defects by the noxE
overexpression, the ethanol yield from xylose increased from 0.392 to
0.431 g/g. The same approach has been taken to achieve a high titer of
2,3-butanediol (96.8 g/L) during xylose fermentation by S. cerevisiae
expressing an oxidoreductase-based xylose-utilizing pathway with
NADH specific XRm (Kim et al., 2017). Conversion of NADH into
NADPH by NADH kinase, POSΔ517, and glucose supply also improved
the redox balance during the production of 1,2,4-butanetriol in
S. cerevisiae (Yukawa et al., 2021). In Y. lipolytica, NADH preferring XR
(K270R/N272D) supported more balanced cofactor availability during
protopanaxadiol production from xylose (Wu et al., 2019).
5. Directed carbon flux through a biosynthetic pathway
The conversion of all sugar components derived from lignocellulosic
biomass offers economic benefits for lignocellulosic biorefinery by
increasing the overall conversion yield. In addition, expanded substrate
profiles often provide more efficient redirection of carbon flux through a
heterologous biosynthetic pathway in a cell evolved to distribute most
resources for the production of cell biomass instead of target products
(summarized in Table 3). Interestingly, lignocellulosic biorefinery with
engineered strains capable of converting xylose into a non-native/
primary product creates synergistic effects on high-yield production
by having carbon flux from an additional carbon source and more easily
rerouting carbon flux toward a heterologous biosynthetic pathway. In
S. cerevisiae, redirecting carbon flux from ethanol to isobutanol was
more effective during xylose fermentation, resulting in six-fold higher
isobutanol yield than that during glucose fermentation (Lane et al.,
2020), increasing the potential for lignocellulosic biorefinery. Similarly,
coupling the xylose assimilation pathway with a biosynthetic pathway
for a non-native product, protopanaxadiol, yielded a two-fold increased
titer in Y. lipolytica. The higher product yield during xylose fermentation
could be explained by slower carbon metabolism, which is attributed to
a sufficient supply of precursors for a biosynthetic pathway, such as
acetyl-CoA and NADPH, preventing overflow metabolism (Wu et al.,
2019). The 2.6-fold higher carotenoid production in Y. lipolytica during
glucose-xylose co-fermentation compared to glucose fermentation has
also been reported, underlining the efficient divergence of carbon flux

Table 3
Summary of engineering approaches to enhance chemical production from monomeric sugars composing lignocellulosic carbohydrates.
Sugars Main Catabolic Intermediate Strain Product name Engineering Performance Note
pathway
Titer Yields
(g/L) (g/g)

Xylose OR-PP Pyruvate S. cerevisiae Isobutanol XR-XDH, XK, Δpho13, Δald6, mitochondrial- 2.6a 0.036 Lane et al.
localized isobutanol pathway (2020)
Xylose OR-PP Acetyl-CoA Y. lipolytica Protopanaxadiol Adaptation, XRK270R/N272D-XDH, XK, Δku70, 0.300 a
0.002 Wu et al.
HMG, DS, PPDS, ATR1, ERG9, ERG20, TAL1, (2019)
TKL1, XK
Glucose OR-PK Acetyl-CoA S. cerevisiae Carotenoid XRK271N-XDH, xPK, PTA, Δpho13, Δald6, CrtB, 0.903a 0.02 Su et al.
+ CrtI, CrtE, HMG, Gal2N376F (2020)
Xylose
Glucose Iso-PP Acetyl-CoA C. tyrobutyricum Butyrate XI, XK, XylT, adhE2, Δack 46.4b 0.43 Fu et al.
+ (2017)
Xylose
Arabinose Weimberg 2- E. coli 1,4-Butanediol araCDAB, ipdC, yqhD, ΔaraBADAH33, 15.6a 0.22* Tai et al.
Xylose ketoglutarate ΔrhaBADLD78 (2016)
XylBCDX, kivDV461I, yqhD ΔaraBADAH33, 12.0a 0.26*
ΔrhaBADLD78, ΔXI, ΔyjhH, ΔyagE

Abbreviations: XR, xylose reductase; XDH, xylitol dehydrogenase; XK, xylulose kinase; XI, xylose isomerase; Pho13, para-nitrophenyl phosphatase; Ald6, aldehyde
dehydrogenase; KU70, ATP-dependent DNA helicase II subunit ku70; HMG, 3-hydroxy-3-methylglutaryl-CoA reductase; DS, dammarenediol-II synthase; PPDS,
protopanaxadiol synthase; ATR, NADPH-P450 reductase; ERG9, squalene synthase; ERG20, farnesyl pyrophosphate synthase; TAL1, transaldolase; TKL1, trans­
ketolase; XK, xylose transporter; xPK, xylulose-5-phosphate phosphoketolase; PTA, phosphotransacetylase; CrtB, phytoene synthase; CrtI, phytoene desaturase; CrtE,
geranylgeranyl diphosphate synthase; Gal2, galactose permease; GMD, GDP-mannose 4,6-dehydratase; WcaG, GDP-4-keto-6-deoxymannose 3,5-epimerase 4-reduc­
tase; Lac12, lactose permease; WbgL, α-1,2-fucosyltransferase; XylT, D-xylose-proton symporter; AdhE2, aldehyde/alcohol dehydrogenase; Ack, acetate kinase; XylB,
xylose dehydrogenase; XylC, xylonolactonase; XylD, D-xylonate dehydratase; XylX, 2-keto-3-deoxy-D-xylonate dehydratase; AraA, L-arabinose dehydrogenase; AraB,
L-arabinollactonase; AraC, L-arabonate dehydratase; AraD, 2-keto-3-deoxy-L-arabonate dehydratase; RhaB, L-rhamnulokinase; RhaA, L-rhamnose isomerase; RhaD,
rhamnulose-1-phosphate aldolase; IpdC, indolepyruvate decarboxylase; YqhD, alcohol dehydrogenase; YjhH, 2-dehydro-3-deoxy-D-pentonate aldolase; YagE, 2-dehy­
dro-3-deoxy-D-gluconate aldolase; KivD, 2-ketoacid decarboxylase. aFed-batch in bioreactor; bBatch in bioreactor; *Calculation value from the reference papers.

8
J. Kim et al. Metabolic Engineering 71 (2022) 2–12

toward a biosynthetic pathway by incorporating xylose catabolism in a
microbial cell factory (Su et al., 2020).
6. Moving forward to an economically feasible industrial
lignocellulosic biorefinery
Recently, metabolic engineering efforts to efficiently convert the
carbohydrate portion of lignocellulosic biomass have become increas­
ingly validated during fermentation with lignocellulosic hydrolysates
(Ko et al., 2020) (Table 4). The conversion yield of lignocellulosic hy­
drolysates into bioethanol (0.48 g/g) is close to reaching the theoretical
maximum with increasing productivity (0.31 g/g/h) (Hoang Nguyen
Tran et al., 2020). The prospect of lignocellulosic biodiesel production
has also been demonstrated by an engineered glucose/xylose
co-fermenting strain of Y. lipolytica producing 12.01 g/L of lipids from
Miscanthus sacchariflorus (Yook et al., 2020). Though still limited by
product yields, bioconversion of lignocellulosic hydrolysates into
non-natural products, such as muconic acid (53.4 mg/L) and limonene
(0.44 mg/L) by engineered glucose/xylose co-fermenting strains of
S. cerevisiae and Y. lipolytica, respectively, have also been reported (Liu
et al., 2020; Yao et al., 2020). Bioconversion of lignocellulosic hydro­
lysates by bacterial strains with relaxed CCR has also been reported for
ethanol production (Flores et al., 2019; Sun et al., 2018).
The above-mentioned metabolic engineering strategies have also
been applied for developing industrial strains for lignocellulosic bio­
refineries. Xylose catabolism has been shown in industrial strains of
S. cerevisiae, P-2ΔGRE3, and CA11, by expressing both isomerase and
oxidoreductase-based xylose utilization pathways (Cunha et al., 2019).
Interestingly, the co-expression of two different xylose catabolic path­
ways provided high-yield ethanol production supported by cofactor
equilibrium between oxidoreductase and detoxifying enzymes. In
addition, during co-fermentation by a xylose-fermenting diploid strain,
NRRL Y-50362, it was revealed that the high level of XI activity along
with transketolases (TKL1 and TKL2) and transaldolase (TAL1)
contributed to efficient sugar conversion to ethanol through glycolysis in
S. cerevisiae (Feng et al., 2018).
Although these approaches are encouraging, xylose conversion effi­
ciency is still lagging behind glucose conversion. Here, the above-
mentioned metabolic engineering strategies could be collectively
applied to develop the strains utilizing lignocellulosic carbohydrates as a
substrate with maximized carbon conversion rates and yields. The
development of efficient xylose fermenting yeasts, for example, could be
achieved by adapting the following engineering strategies; 1) intro­
ducing an isomerase-based initial xylose catabolic pathway, which is a
cofactor neutral, with increased functionality by expressing a mutant
version of XIN337C, 2) channeling xylose flux across the cell membrane
by implementing an artificial transport metabolon of a Gal2-XI complex,
3) distributing xylose flux through both PP, in which selected genes such
as TKL and TAL are overexpressed, and PK pathways, 4) improving
xylose catabolism even in the presence of glucose by deleting ASC1,
PHO13, and THI2. Based on this configuration, more engineering stra­
tegies could be further applied with the continuous progress in meta­
bolic engineering, and the aid of systems biology and bioinformatics.
7. Future perspectives
We reviewed recent metabolic engineering efforts to improve the
bioconversion of the sugar portion of lignocellulosic biomass, especially
focusing on glucose and xylose metabolism. The natural pathways that
distinctively exist in various microorganisms could be adapted as a
heterologous system with improved efficiency, so that they can serve as
a promising route for producing fuels and chemicals (Fig. 2 and Tables 3
and 4). To improve carbon conversion and energy balance, rewiring
central carbon metabolism appears to be highly necessary, and this can
be achieved by adapting heterologous systems from other bacteria or
eukaryotic strains. With recent developments in sequencing technology
and pipelines for data analysis, metagenomics has been initiated,
allowing for a detailed study of microbial diversity and metabolic po­
tentials without the incubation and isolation of microorganisms. This
provides possibilities for the discovery of new enzymes or novel meta­
bolic pathways that can be applied to metabolic engineering (Ceja-Na­
varro et al., 2019; Li et al., 2019; Yang et al., 2016). In a recent study, a
novel XI was identified by revisiting the previous metagenomics data
set, which better supported faster cell growth of S. cerevisiae when
compared to that from Piromyces sp., one of the best-performing XIs
introduced in S. cerevisiae (Silva et al., 2021). This study suggests that
the exploration of microbial communities using a metagenomics
approach sheds light on new types of enzymes and expands the target
range for metabolic engineering.
With the increasing availability of whole genome sequencing data,
genome-scale models (GEMs) could be more easily applied in 1) guiding
target enzymes for deletion or substitution, 2) redirecting carbon flux

Table 4
Summary of engineering approaches to produce chemicals through a direct utilization of lignocellulosic hydrolysates.
Sugars Main Intermediate Strain Product Engineering Performance Note
Catabolic
Titer Yields
pathway
(g/L) (g/g)

Miscanthus Iso-PP Acetyl-CoA Y. lipolytica Lipids Adaptive laboratory evolution (ALE), XI, 12.01a 0.16 Yook et al.
hydrolysate XK, Δpex10, DGA1 (2020)
Miscanthus Iso-PP Pyruvate S. cerevisiae Ethanol Adaptive laboratory evolution (ALE), XI, 30.1b 0.48 Hoang Nguyen
hydrolysate XK, Δgre3, Δpho13, Δasc1, TAL1, RPE1 Tran et al., 2020
c
Oil palm empty fruit OR-PP PEP, E4P S. cerevisiae Muconic Adaptive laboratory evolution (ALE), AroZ, 0.053 0.001 Liu et al. (2020)
bunch hydrolysate acid CatA, XI, XK, RPE1, RKI1, TAL1, TKL1,
Aro1AroEΔ, Aro4K229L
Corn stover OR-PP Acetyl-CoA Y. lipolytica Limonene XR-XDH, XK, LS, NDPS, HMG, ERG12 0.020c 0.0004 Yao et al. (2020)
hydrolysate
Corncob hydrolysate Iso-PP Pyruvate E. coli Ethanol Δpta-ack, ΔpflB, ΔldhA, Δglk, ΔptsG, ΔmanZ, 39.9*a 0.49* Sun et al. (2018)
XylH, pdc, adhB, pR-pL-ptsG
Corncob hydrolysate OR-PP + Iso- Pyruvate S. cerevisiae Ethanol XDH, XI, XK, Δgre3, XRN272D, TAL1 8.55c
0.384 Cunha et al.
PP (2019)

Abbreviations: PEP, phosphophenolpyruvate; E4P, erythrose-4-phosphate; Gre3, aldose reductase; Asc1, G-protein beta subunit; RPE1, ribulose-phosphate 3-epim­
erase; PEX10, peroxisome biogenesis factor 10; DGA1, diacylglycerol O-acyltransferase; AroZ, 3-dehydroshikimate dehydratase; CatA, catechol 1,2-dioxygenase;
RKI1, ribose-5-phosphate isomerase; Aro1, arom multifunctional enzyme; Aro4, 3-deoxy-D-arabino-heptulosonate-7-phosphate; LS, limonene synthase; NDPS,
neryl diphosphate synthase; ERG12, mevalonate kinase; PflB, formate acetyltransferase; LdhA, lactate dehydrogenase; PtsG, glucose PTS system EIICB component;
ManZ, mannose PTS system EIID component; Glk, glucokinase; XylH, xylose transport system permease protein; Pdc, pyruvate decarboxylase; AdhB, alcohol dehy­
drogenase; aFed-batch-Bioreactor; bBatch-Serum bottle; cBatch-Flask; *Calculation value from the reference papers. Abbreviation of pathways is identical to Fig. 2.
Repeated gene names in Table 3 are omitted in legend.

9
J. Kim et al.
through metabolic pathways, and 3) balancing energy/cofactors for
more efficient bioconversion of lignocellulosic biomass. This approach
promotes engineering non-conventional microorganisms with a poten­
tial for industrial applications but with limited genetic engineering
tools, as shown in the engineering of Y. lipolytica for the production of
dicarboxylic acid and triacylglycerol (Kavscek et al., 2015; Kerkhoven
et al., 2016; Loira et al., 2012; Mishra et al., 2018; Pan and Hua, 2012).
Although this review focused on bioconversion by a single strain,
complete utilization of the carbohydrate portion of lignocellulosic
biomass can also be achieved using a microbial consortium composed of
glucose- and xylose-selective strains, respectively. The consortium of
E. coli strains utilizing only glucose or xylose, by deletion of xylA or glk,
respectively, resulted in 53% higher n-butanol production than that of a
single strain scheme (Saini et al., 2017). A similar approach with a
“Y-shaped” microbial consortium with distinctive sugar catabolic
pathways and shared biosynthetic pathways resulted in efficient butanol
production with high titer (21 g/L) and yield (0.35 g/g) during simul­
taneous co-fermentation of glucose and xylose (Zhao et al., 2019).
8. Conclusion
Lignocellulosic biomass is one of the most promising supplies in
terms of availability and renewability. Through metabolic engineering
efforts, monomeric sugars such as glucose and xylose derived from the
carbohydrate portion of lignocellulosic biomass are more efficiently
converted into fuels and chemicals through carbon catabolic pathways.
Here, we reviewed the challenges and opportunities in several natural
carbon catabolic pathways in bacterial and eukaryotic microorganisms,
which can be a target for metabolic engineering and synthetic biology.
Based on this comprehensive investigation, the following engineering
approaches can be considered to maximize carbon conversion with
balanced energy supply for biosynthesis of target products: 1) manipu­
lation of the sugar transport system for pentose uptake, 2) improvement
of the activities of enzymes directly and indirectly involved in sugar
catabolism; 3) modification of the apparatus for transcription and
translation; 4) use of cofactor recycling system for precise energy bal­
ance; and 5) expansion of the application of these approaches to engi­
neer pathways beyond central carbon metabolism. Progress in these
approaches could be accelerated with the aid of next-generation
sequencing and computational flux modeling through which critical
enzymes and reactions in the pathways can be more sophisticatedly
identified and improved, yielding economically feasible and environ­
mentally sustainable value-added products from lignocellulosic biomass
using microbial cell factories.
CRediT authorship contribution statement
Jiwon Kim: Conceptualization, Visualization, Writing – original
draft. Sungmin Hwang: Conceptualization, Writing – original draft.
Sun-Mi Lee: Conceptualization, Visualization, Supervision, Writing –
original draft, All authors read and approved the final manuscript.
Declaration of competing interest
The authors declare no competing interests.
Acknowledgements
This research was supported by the National Research Foundation of
Korea (NRF) funded by the Ministry of Science and ICT (Information &
Communication Technology) [grant number NRF-
2020M1A2A2080847] and the Korea Institute of Science and Technol­
ogy (KIST) Institutional Program [grant number 2E31272].
10
Metabolic Engineering 71 (2022) 2–12
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