Bioresource Technology 384 (2023) 129280

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Microbial itaconic acid bioproduction towards sustainable development:

Insights, challenges, and prospects
Priskila Adjani Diankristanti 1, I-Son Ng 2, *
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Biocatalyst improvement is essential for

industrial production of itaconic acid.
• Titers, productivities, and yields vary
widely but improved in the last 10
years.
• Ustilago maydis, aside from A. terreus,
shows potentials as native IA producer.
• In vitro IA production obtained remark­
able yield in shorter reaction time.
• Biomass waste and one-carbon com­
pounds can be an economical produc­
tion pathway.

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

Keywords: Microbial biomanufacturing is a promising approach to produce high-value compounds with low-carbon foot­
Itaconic acid print and significant economic benefits. Among twelve “Top Value-Added Chemicals from Biomass”, itaconic
Aspergillus terreus acid (IA) stands out as a versatile platform chemical with numerous applications. IA is naturally produced by
Cis-aconitic acid decarboxylase
Aspergillus and Ustilago species through a cascade enzymatic reaction between aconitase (EC 4.2.1.3) and cis-
Bioproduction
aconitic acid decarboxylase (EC 4.1.1.6). Recently, non-native hosts such as Escherichia coli, Corynebacterium
Sustainable development goals
glutamicum, Saccharomyces cerevisiae, and Yarrowia lipolytica have been genetically engineered to produce IA
through the introduction of key enzymes. This review provides an up-to-date summary of the progress made in IA
bioproduction, from native to engineered hosts, covers in vivo and in vitro approaches, and highlights the
prospects of combination tactics. Current challenges and recent endeavors are also addressed to envision
comprehensive strategies for renewable IA production in the future towards sustainable development goals
(SDGs).

* Corresponding author.
E-mail address: yswu@mail.ncku.edu.tw (I.-S. Ng).
1
ORCID: 0000-0002-2006-5747.
2
ORCID: 0000-0003-1659-5814.

https://doi.org/10.1016/j.biortech.2023.129280
Received 3 May 2023; Received in revised form 30 May 2023; Accepted 1 June 2023
Available online 7 June 2023
0960-8524/© 2023 Elsevier Ltd. All rights reserved.
P.A. Diankristanti and I.-S. Ng Bioresource Technology 384 (2023) 129280

Fig. 1. Metabolic pathway of IA production. Blue color illustrates the pathway existing only in A. terreus, pink color illustrates the pathway existing only in U. maydis,
orange color illustrates the pathway existing only in E. coli, and black color illustrates the pathway existing in all hosts. Glucose as substrate undergoes glycolysis
pathway in cytosol to produce pyruvate and begin the TCA cycle in mitochondria. Other possible substrates, xylose and arabinose, undergo the pentose phosphate
pathway to produce pyruvate and begin the TCA cycle. CAA will be transported back to the cytosol and initiate IA synthesis.

1. Introduction
Overdependency on fossil fuels accounts for 85% of global primary
energy consumption (Kabeyi and Olanrewaju, 2022), given that they
currently are the major aspects to produce chemicals, electricity, and
transportation fuels. Shifting to renewable sources is certainly a way to
combat climate change, leading to the awakening of renewable chemical
production. In 2015, the United Nation set up the 17 Sustainable
Development Goals (SDGs) as a perspective of protecting the planet by
interconnecting environment protection, economic growth, and social
integration (Olabi et al., 2023). Nevertheless, to completely substitute
fossil-based resources and achieve the goals, it is mandatory to provide
an economically viable way to convert renewable resources into high-
titer and high-value chemicals.
In 2004, the United States Department of Energy introduced twelve
promising bio-based building blocks as “Top Value-Added Chemicals
from Biomass” (Werpy and Petersen, 2004), in which itaconic acid (IA)
is listed. Among all, IA is the only one that provides possibilities to
substitute acrylic acid and methacrylic acid. Furthermore, it can be used
as a building block in producing essential polymers, such as resins, fi­
bers, and rubber (Okabe et al., 2009), and even applied for medical
industry and agriculture sector. Widespread application of IA is ranging
from dietary supplement in animal feed (Zhu et al., 2022), drug-delivery
system (Veerabagu et al., 2019), intelligent food packaging (Cottet et al.,
2021), dye removal agent (Gnanasekaran et al., 2019), and slow-release
fertilizer system (Bora and Karak, 2022). One of the most valuable de­
rivatives of IA is methyltetrahydrofuran (MTHF), an emerging compo­
nent in biofuel synthesis and an eco-friendly solvent for chemical
reactions (Ashokkumar et al., 2022). The market size of IA in 2021 is
80,000 tons with an estimated price up to $2 per kilogram. Global
market value is expected to rise from $96 million to $116.6 million in
2026 (Narisetty et al., 2021), indicating the importance of IA for in­
dustrial usage.
IA-producing pathway relies on the conversion of citric acid into the
intermediate cis-aconitic acid (CAA) by aconitase (acn, EC 4.2.1.3) and
finally into IA by cis-aconitic acid decarboxylase (cadA, EC 4.1.1.6)

2
P.A. Diankristanti and I.-S. Ng Bioresource Technology 384 (2023) 129280

(Klement and Büchs, 2013), as pointed by bold lines in Fig. 1. Two-step
enzymatic conversion requires right compartmentalization of both en­
zymes to obtain high itaconic acid accumulation. Although Aspergillus is
known as the most prevalent microbial cell factory for IA production,
further optimization of production process remains critical, as it has
been reported that fermentation using filamentous fungi undergoes a
few drawbacks. Being highly aerobic, Aspergillus demands sufficient
oxygen supply for itaconic acid production, which is considered as cost
ineffective (Gopaliya et al., 2021; Narisetty et al., 2023). Therefore,
engineered heterologous hosts, such as Escherichia coli (Harder et al.,
2018; Harder et al., 2016), Saccharomyces cerevisiae (Blazeck et al.,
2014), Corynebacterium glutamicum (Merkel et al., 2022), and Yarrowia
lipolytica (Blazeck et al., 2015), are used to solve deficiencies of native
hosts in the view of tight oxygen supply. Overexpression of cadA from
A. terreus has started to take place ever since its first comprehensive
identification and characterization (Li et al., 2022; Kanamasa et al.,
2008), allowing non-native hosts to obtain high level IA through
metabolic engineering and synthetic biology.
This review provides a summary of the recent advancements in IA
bioconversion, including the utilization of native and engineered hosts.
It also covers the benefits of employing in vivo processes using commonly
available substrates (such as glucose or glycerol) and waste materials, as
well as the application of whole-cell bioconversion of citrate to IA.
Additionally, the review discusses the current challenges and limitations
associated with IA bioconversion, providing clear insight of future
expansion in a comprehensive direction in achieving the renewable
material toward low carbon emission.
2. In vivo IA production from native hosts: Aspergillus or
Ustilago?
Microbial fermentation using native hosts is the most predominant
way of IA production, initiated by substrate uptake and conversion into
two pyruvate molecules through glycolysis or pentose phosphate
pathway. One pyruvate molecule undergoes an anaplerotic reaction in
the cytosol to replenish the TCA intermediates (oxaloacetate and ma­
late), while the other one is further decarboxylated into acetyl-CoA and
directly enters TCA cycle to produce citrate, the precursor of IA
(Klement and Büchs, 2013; Zhu et al., 2021). IA production pathway
involves two key enzymes, aconitase (acn, EC 4.2.1.3) which converts
citrate into CAA, and cis-aconitic acid decarboxylase (cadA, EC 4.1.16)
which transforms CAA into IA. CAA is synthesized in the mitochondria
and transported into the cytosol by mitochondrial tricarboxylate trans­
porter (mttA), where the following decarboxylation step and IA forma­
tion take place.
Complete gene cluster of IA bioconversion in A. terreus has been
encoded through transcriptome analysis (Li et al., 2011; Deng et al.,
2020), where cadA is proven to be an essential enzyme in IA biocon­
version. Compartmentalization of the key enzymes and intermediates
plays a vital role as IA bioproduction requires the shuttling of CAA from
mitochondria to cytosol in the presence of mttA, a native mitochondrial
transporter in A. terreus. Attempts to maximize the power of mttA have
been reported, which the high copy number of mttA in A. terreus showed
no improvement (Wierckx et al., 2020), while introduction of mttA in
A. niger boosted IA yield significantly (Wang et al., 2022). To elevate the
quality of downstream processing and diminish product inhibition ef­
fect, Kreyenschulte et al. (2018) carried out an in situ reactive extrac­
tion, where IA is extracted using trioctylamine as solvent, and glucose is
supplemented to maintain substrate availability. It showed a way to
integrate production and recovery steps of IA, yielding up to 105 g/L IA.
Concerning further development of IA production in A. terreus as well
as cost reduction, Huang et al. (2014) overexpressed glucoamylase gene
from A. niger to allow direct conversion from liquefied starch into IA,
preventing gelatinization on high-temperature sterilization and omit­
ting the saccharification step. Xylose, another alternative sugar, has
been reported to give comparable result to glucose-grown microbial cell
factories. Magalhães Jr et al. (2020) assessed the use of xylose in
A. terreus NRRL 1960 as an alternative substrate, although the final IA
titer was lower compared to that used by glucose. Aside from sugar
compounds, other possibilities of substrate such as biomass hydrolysate
(Krull et al., 2017; Gnanasekaran et al., 2019; Narisetty et al., 2021) and
agricultural waste (Huang et al., 2014; Bafana and Pandey, 2019; Yang
et al., 2020) are reported.
In contrast to IA production pathway in A. terreus, IA production in
U. maydis involves the decarboxylation of trans-aconitic acid, the ther­
modynamic isomer of CAA (Kuenz and Krull, 2018; Hosseinpour Tehrani

Table 1
Summary of in vivo IA production in Aspergillus terreus and Ustilago maydis.
Strains Carbon source IA titer (g/ Time Yield (g/ Strategy References
L) (h) g)

A. terreus XH86-8 Liquefied corn 77.6 72 0.55 - Expression of glucoamylase gene under citA promoter Huang et al., 2014
starch - Expression cassette using strong signal peptide
A. terreus DSM WCHa 27.7 146 0.41 - Utilization of WCH Krull et al., 2017
23081 - Detoxification via ion exchanger
A. terreus DSM Glucose 105 115 0.58 - In situ product recovery using trioctylamine as substrate Kreyenschulte et al., 2018
23801 - Glucose supplementation
A. terreus C1 Potato starch 29.7 168 0.2 - Utilization of potato starch waste under optimized physical Bafana and Pandey, 2019
waste properties
A. niveus Glycerol, ABH b 31.55 168 0.45 - Utilization of ABHb and purified glycerol Gnanasekaran et al., 2019
A. niger AB1.13 Glucose 42.7 240 0.26 - Overexpression of acl12 to improve glycolytic flux Hossain et al., 2019
- Nitrogen source feeding
A. terreus NRRL Xylose, glucose 25.96 240 0.35 - Utilization of xylose for glucose-grown biocatalyst Magalhães Jr et al., 2020
1960
A. terreus 2433 Bamboo residue 41.54 240 N/D - Utilization of bamboo residue in fed-batch fermentation Yang et al., 2020
A. terreus BD Glucose, FWH c 41.1 216 0.27 - Utilization of FWHc for itaconic acid production in a Narisetty et al., 2021
thermal-tolerant strain
U. maydis MB215 Glycerol, glucose 220 480 0.33 - Oversxpression of ria1 and AtMttA Hosseinpour Tehrani et al.,
- fuz7 and cyp3 deletion 2019a
- Optimized fermentation and in situ product crystallization
U. maydis MB215 Cellulose, glucose 33.8 480 0.16 - Co-culture with T. reesei RUT C-30, a cellulose producer Schlembach et al., 2020
- By-product reduction through cyp3, MEL, and UA deletion
U. rabenhorstiana Glucose 50.3 360 0.4 - Optimization of physical properties Krull et al., 2020
U. maydis MB215 Glucose 75.7 140 0.66 - By-product reduction through cyp3, MEL, and UA deletion Becker et al., 2021
- fuz7 deletion
- Overexpression of ria1 & AtMttA
a
wheat chaff hydrolysate; b algal biomass hydrolysate; c food waste hydrolysate.

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P.A. Diankristanti and I.-S. Ng
et al., 2019b). As shown in Fig. 1, CAA shuttling from mitochondria to
cytosol is existed in U. maydis, in the presence of mitochondrial trans­
porter mtt1. This step is essential for IA bioproduction, proven by a
strong increase of IA titer with the overexpression of mtt1. Mitochondrial
transporter mtt1 also facilitates the shuttling of cytosolic oxaloacetate
into mitochondria, which is closely related to intracellular anaplerotic
reaction to replenish TCA intermediates (Wierckx et al., 2020).
Furthermore, heterologous expression of mitochondrial transporter mttA
from A. terreus in U. maydis showed increased IA production, with the
overexpression of the transcriptional regulator gene ria1 and deletion of
fuz7 and cyp3 (Geiser et al., 2016b). Hosseinpour Tehrani et al. (2019)
integrated optimized fermentation using engineered strain and in situ
product crystallization to reduce product inhibition. This approach
successfully increased the productivity up to three folds, leading to 220
g/L IA, the current highest titer achieved through fermentation in native
host. Another strategy worth mentioning to boost IA bioproduction is
blocking the formation of downstream product. In U. maydis, IA is
further being oxidized into (S)-2-hydroxyparaconate by cyp3, a cyto­
chrome P450 monooxygenase (Geiser et al., 2016a). Deletion of cyp3 is
reported to be beneficial for IA bioproduction, while the overexpression
of it resulted in drastic decrease of IA (Schlembach et al., 2020). Aside
from cyp3 deletion, Becker et al. (2021) reported that deletion of the
whole gene cluster for glycolipid biosynthesis, mannosylerythritol lipids
(MEL) and ustilagic acid (UA), is beneficial for IA production, as it re­
duces the amount of side reactions and byproducts accumulation. Under
this condition, U. maydis produced IA by 1.3-fold higher and increased
the production rate by 12%.
Commercial production of IA nowadays is relying on A. terreus as the
most impressive native producer with high yield of end-product up to
146 g/L (Hevekerl et al., 2014). With A. terreus being the sole host for
commercial production of IA, industries are undoubtedly facing several
limitations: high initial sugar concentration and oxygen supply, low pH
condition, and strictly controlled manganese content (Wu et al., 2017).
Although it was reported that phosphate limitation attenuates the
inhibitory effect of manganese (Saha and Kennedy, 2019), a manganese-
tolerant host seems more beneficial. Being capable of producing IA at a
more neutral pH range (4.5 to 6.5) and tolerant to manganese, U. maydis
offers a better opportunity for IA production, especially in a non-purified
lignocellulose-based fermentation. Low pH might inhibit growth and
reduce carbon intake, resulting in the drop of biomass accumulation and
final IA titer. Schlembach et al. (2020) proposed a consolidated bio­
process through the co-culture fermentation of U. maydis as the host for
IA bioproduction and T. reesei RUT C-30 which converts cellulose
feedstock into glucose, to be consumed by U. maydis for IA production.
This study has shown a breakthrough by proving the compatibility of
U. maydis and T. reesei to undergo an integrated saccharification-
fermentation process, yielding up to 33.8 g/L IA in 480 h fed-batch
cultivation. Other Ustilago species, such as U. rabenhorstiana (Krull
et al., 2020), U. vetiveriae (Zambanini et al., 2017), U. cynodontis (Hos­
seinpour Tehrani et al., 2019b) have also been explored as alternatives,
reported to obtain more than 50 g/L IA. Taken together, these findings
present solution to overcome drawbacks from A. terreus and expand the
prospects of utilizing Ustilago species in large-scale IA production. All
the results have been summarized in Table 1. Moreover, compared to the
filamentous Aspergillus, the yeast-like morphology of Ustilago is showing
advantages for large-scale production, not to mention the insensitivity to
impurities. Comprehensive understanding on fungal morphology is
essential to fully exploit promising potentials, as fungi undergoes sig­
nificant change under fluctuation in oxygen content and shearing force
(Nemestóthy et al., 2019; Cairns et al., 2019).
3. Engineered E. coli for in vivo IA production
Known as a superior host for recombinant protein expression, E. coli
stands out as a promising non-native host for IA production, owing to the
rapid growth, well-characterized genome, and accessible genetic
4
Bioresource Technology 384 (2023) 129280
engineering tools (Wang et al., 2021; Feng et al., 2022). Since cadA is not
naturally present in E. coli, overexpression of cadA from A. terreus is an
essential step in utilizing E. coli to synthesize IA (Kim et al., 2017; Xu and
Li, 2023). In addition to acn and cadA, IA production also relies on the
activity of gltA which converts oxaloacetate into citric acid. Vuoristo
et al. (2015) obtained 690 mg/L IA through the overexpression of gltA
and acnA from C. glutamicum along with cadA from A. terreus, and
deletion of pta and ldhA to accumulate pyruvate, which is the precursor
for IA bioproduction. Utilization of synonymous codon variants (scv) is a
method to upgrade functional expression level of cadA in E. coli. Jeon
et al. (2016) screened scv of cadA in the first 10 codons to engineer
E. coli. Among all positive clones, one expressed more than 95% of cadA
in soluble form, leading to threefold improvement of IA production
under the condition of nitrogen limitation and glycerol feeding. Ye et al.
(2022) introduced an unnatural chassis to mimic spatial compartmen­
talization in E. coli, where instead of acn, prpD is employed to convert
citrate to cis-aconitate. This strategy allowed intermediates release
without physical barrier, resulting in 5.06 g/L IA from acetate and a
yield of 0.33 g/g.
Beside existing as the intermediate product from the conversion of
citric acid into IA, CAA is also the intermediate between the reversible
conversion of citrate and isocitrate catalyzed by aconitase (acnB).
However, the presence of isocitrate dehydrogenase (icd) prevents aco­
nitase from converting back into CAA and drags it away from the IA
production pathway (Chang et al., 2017; Harder et al., 2018). As a result,
the irreversible conversion of isocitrate decarboxylates into α-ketoglu­
tarate must be inhibited to conserve CAA. Nevertheless, high IA pro­
ductivity might be obtained through icd inhibition, while glutamate
auxotrophy is inevitable.
To minimize trade-off effect between cell growth and IA production,
dynamic control of icd is examined, as it allows the cell to grow and
accumulate sufficient biomass for the production phase. Harder et al.
(2018) applied cI857, a heat-inducible repressor used in the production
of recombinant protein in E. coli, to construct a temperature-sensitive
two-stage process. Enabling fast formation of biomass and augmented
productivity, they obtained 47 g/L IA. However, precise temperature
control in a large-scale bioreactor is undoubtedly a hassle, thus it is
necessary to dynamically regulate metabolic flux. YpItcR/Pccl, an IA-
responsive promoters from from Yersinia pseudotuberculosis, has been
reported to effectively regulate cellular activity between biomass phase
and production phase without deleting any vital gene for growth (Hanko
et al., 2018). The presence of itaconic acid activated YpItcR/Pccl, thus
repressed the growth phase and further directed the metabolic flux into
the IA synthesis pathway. Zhao et al. (2022) designed a CRISPRi-
mediated self-inducible system (CiMS) using YpItcR/Pccl, which then
was used to regulate the expression of icd, pykA, and sucCD. By utilizing
CiMS, increase in itaconic acid titer by 23% was observed. A
fluorescence-based biosensor has also been developed using YpItcR/Pccl
and is effective to identify the optimum expression level of cadA. This
system can also detect the presence of mesaconate, cis-, and trans-aco­
nitate (Hanko et al., 2018).
Another photocontrol strategy to regulate icd expression level was
reported, where AsLOV2, a light-sensing photoreceptor of Avena sativa is
inserted into different sites of icd, allowing precise control of TCA cycle
flux and redirection into IA-producing pathway (Li et al., 2022b). Out of
other strategies of knocking down or inhibiting icd, the recombinant
Weimberg pathway from Bacillus xenovorans was introduced to over­
come glutamate auxotrophy by producing ketoglutarate needed in TCA
cycle. Due to the independency of existing native pathways in E. coli,
Weimberg pathway is able supply ketoglutarate using xylose as sub­
strate without competing against IA production pool (Lu et al., 2021).
Two major long-overdue problems in engineered hosts with heter­
ologous protein expression are inclusion body formation due to the low
protein solubility (Vuoristo et al., 2015), and metabolic flux imbalance
which leads to low product titer (Cha et al., 2020). Several attempts to
balance cellular pathway flux have been reported, namely modulation of
P.A. Diankristanti and I.-S. Ng Bioresource Technology 384 (2023) 129280

Table 2
Summary of in vivo IA production in non-native hosts.
Strains Carbon source IA titer Time Yield (g/ Strategy References
(g/L) (h) g)

In vivo
E. coli BW25113(DE3) Glucose 0.69 72 0.06 - Overexpression of AtCadA and CgGltA Vuoristo et al.,
- Deletion of pta and ldhA 2015
E. coli XL1-Blue Glycerol 7.2 90 0.1 - Expression of codon variants (scv) of AtCadA Jeon et al., 2016
- Feed glycerol and citric acid
E. coli Rosetta(DE3) N/D 0.22 24 N/D - Self-assembled reaction of Acn and AtCadA via protein-peptide Yang et al., 2017
interaction
E. coli BW25113 Glycerol 43.0 32 0.6 - Overexpression of gltA, acnB, CgPyc, AtCadA Chang et al.,
- icd deletion 2017
E. coli W3110 Acetate 3.6 88 0.09 - Overexpression of aceA, gltA (ACS pathway) Noh et al., 2018
- iclR deletion (glyoxylate shunt pathway)
E. coli MG1655 Glucose 46.9 120 0.45 - Dynamic regulation of icd expression through heat-inducible Harder et al.,
repressor (cI857) 2018
E. coli XL1-Blue Glucose 6.6 48 0.66 - Construction of synthetic protein scaffold between gltA, acnA, Tran et al., 2019
and AtCadA
E. coli BW25113 Glycerol, xylose 20 100 0.32 - Expression of Weimberg pathway from B. xenovorans Lu et al., 2021
- High aeration and fine-tuning of xylose supply
E. coli BL21(DE3) Glucose 3.3 24 0.33 - Dynamic regulation of icd using AsLOV2, a light-sensitive Li et al., 2022b
biosensor
E. coli MG1655 Glucose 3.9 N/D N/D - Dynamic regulation of icd expression using itaconic acid- Zhao et al., 2022
responding biosensor (YpItcR)
E. coli W3110 Acetate 5.06 80 0.33 - Utilization of prpD for kinetic compartmentalization under Ye et al., 2022
strong promoter
Corynebacterium glutamicum Acetate 29.2 46 0.16 - Overexpression of codon optimized AtCadA Merkel et al.,
ATCC 13032 - Coupled pH and DO control 2022
Halomonas bluephagenesis TD01 Citrate 63.6 36 0.63 - Expression of CadA and Can Zhang et al.,
- Assistance of GroESL 2021
- Downregulation of icd
Methylorubrum extorquens AM1 Methanol 0.03 192 N/D - Overexpression of codon optimized AtCadA Lim et al., 2019
- Impediment of PHB through mutation of phaR
Pichia kudriavzevii Glucose 1.2 24 0.03 - Overexpression of PkMttA and AtCadA Sun et al., 2020
- Downregulation of icd
Pseudomonas putida JE3719 Alkali-treated 1.4 48 0.79 - Dynamic two-stage bioconversion using nitrogen-limiting Elmore et al.,
lignin biosensor cassette 2021
Yarrowia lipolytica Glucose 22.0 460 0.22 - Expression of mitochondrial transporter AtMttA Zhao et al., 2019
Yarrowia lipolytica WCO a 54.6 96 0.3 - Overexpression of AtCadA and POT1 Rong et al., 2022
- icl deletion
Saccharomyces cerevisiae Ethanol 0.14 72 0.01 - Overexpression of AAC2 Xu et al., 2021
- Utilization of ethanol
Saccharomyces cerevisiae Glucose 0.17 72 0.01 - Construction of synthetic hybrid promoter PGPD Blazeck et al.,
2014
Schizosaccharomyces pombe Glucose 1.55 72 0.03 - Overexpression of AtCadA Fujie et al., 2022
- Deletion of idh2, mae1, acl2, mce1
- High cell density culture

enzyme expression level (Noh et al., 2017), directed evolution to in­
crease enzymatic expression level or turnover activities (Lv et al., 2016),
cell surface display to enhance stability (Guo et al., 2018), fusion tag
protein (Lin et al., 2022), and protein scaffold mechanism to colocalize
multiple enzymes (Xu and Li, 2021). Tran et al. (2019) developed a
synthetic protein scaffold between gltA, acnA, and cadA by channeling
three protein–protein ligands to the C-terminus of each enzyme, which
are GBD-gltA, SH3-acnA, and PDZ-cadA. Another innovative strategy
was proposed by Yang et al. (2017), where acnA and cadA are fused
through the protein-peptide interactional force of PDZ domain and PDZ
ligand, allowing a self-assembly interaction to take place. Chang et al.
(2017) supplemented citrate upon induction to increase precursor sup­
ply for IA pool and minimize CAA loss from side reactions. Combined
with high aeration and pH maintenance in fed-batch reactor, 43 g/L IA
was obtained in 32 h of fermentation using glycerol, reaching a final
yield of 0.6 g IA/g glycerol.
Among all other carbon sources, glucose serves as the most beneficial
carbon source in fermentation, which is then dissimilated into pyruvate.
When a substrate dissimilation involves PEP-dependent phosphoryla­
tion, such as glucose and glycerol, overexpression of pyruvate carbox­
ylase from C. glutamicum (CgPyc) is more beneficial than the native PEP-
carboxylase from E. coli (EcPpc) (Chang et al., 2017).Acetate is the
predominant product of overflow metabolism in E. coli, generated
through the utilization of acetyl-CoA (Szenk et al., 2017). E. coli owns
the capacity to ingest acetate when there is no other carbon source in the
culture medium (Sakuntala and Kim, 2022). The uptake and conversion
of acetate from acetyl-CoA involves either ACK-PTA (acetate kinase-
phosphotransacetylase), or ACS (acetyl-CoA synthase) pathway which
is driven by ATP (Li et al., 2016). Nevertheless, acetate significantly
inhibits cell growth despite the potential it holds to achieve economi­
cally viable microbial process (Pinhal et al., 2019; Kiefer et al., 2021). To
overcome this limitation, E. coli W, an acetate-tolerant strain, is pro­
posed as a host through the activation of ACS pathway along with
glyoxylate shunt pathway (Noh et al., 2018). ACS pathway allows cell to
grow on acetate as the sole carbon source via anaplerosis reaction, while
glyoxylate shunt pathway enables the conversion of acetyl-coA into
malate and succinate (C4 compounds), which further yield oxaloacetate,
the precursor of IA pool (Dolan and Welch, 2018). By utilizing this
strategy, Noh et al. (2018) obtained 3.57 g/L IA in 88 h fermentation.
The aforementioned study also demonstrated excellent acetate tolerance
and assimilation as shown by 38.7 g/L acetate consumed during culti­
vation. Although the final titer is considered low (5.31 mol IA/mol ac­
etate, 0.09 g IA/g acetate), this finding is a proof of acetate feasibility for
low-cost IA production.
Employing E. coli as a non-native host is a propitious strategy, owing
to the comprehensive genetic background, rapid growth rate, and the

5
P.A. Diankristanti and I.-S. Ng
Fig. 2. Scheme of in vitro whole-cell IA bioconversion. Four critical concerns are (1) genetic design, (2) biocatalyst cultivation, (3) bioconversion optimization, and
(4) biocatalyst recycle.
feasibility to overexpress heterologous protein, albeit still limited by the
solubility issue. Reflecting from all the aforementioned potentials, in­
vestigations are going further to explore other prospects from a wide
range of non-native hosts. Other prospects, such as new biosynthesis
route, unconventional substrate, or novel hosts, are worth exploring
(Table 2).
4. Exploration of diverse non-native hosts and various carbon
sources
To investigate the prospect of using methanol as carbon source, en­
gineering methylotrophic hosts is considerably favorable. Methyloru­
brum sp, which is capable of converting C-1 molecules such as methanol
and formate, was reported as a heterologous host for IA production. Lim
et al. (2019) utilized methanol to produce IA in M. extorquens and
attempted to regulate carbon flux through phaR mutation, resulting in
0.03 g/L IA. Although further study to characterize related gene and
enhance final titer is still required, this report shows a comprehensive
possibility of both host and substrate. Alkali-treated lignin was reported
by Elmore et al. (2021) as carbon source, along a nitrogen-limiting
biosensor to control nitrogen starvation phase. This strategy was re­
ported to obtain 1.4 g/L IA in 48 h. Acetate, which severely inhibits cell
growth in E. coli, was used as the sole carbon sole in IA production using
C. glutamicum ATCC 13032, where 29.2 g/L IA was achieve in 46 h
(Merkel et al., 2022). Compared to acetate-based fermentation using
E. coli which was reported to reach 3.57 g/L IA in 88 h (Noh et al., 2018),
C. glutamicum holds a higher potential as a model organism in chemical
production from acetate, as shown by higher IA titer and acetate
consumption.
Yeast is another alternative for cell factory aside from bacteria,
which owns more robust growth in extreme environment, such as low
pH, low temperature, high oxygen concentration, and high shear stress
(Cheng et al., 2017). Yeast is capable of utilizing a wide range of sub­
strates, varying from sugar such as glucose and glycerol (Zeng et al.,
2020; Bansal et al., 2020), up to other nonfermentable substrates such as
acetate, lactate, and ethanol (Xu and Li, 2021). Among other yeast,
6
Bioresource Technology 384 (2023) 129280
S. cerevisiae has gained increasing attention due to the “fully sequenced
genome” as a potential host for IA production, either using glucose or
ethanol as carbon source. Blazeck et al. (2014) constructed a synthetic
hybrid promoter PGPD, which is the modification of the fastest consti­
tutive promoter in yeast. Under the control of galactose that enabled
fine-tuning, they obtained 0.17 g/L IA. However, yeast respiration is
substantially inhibited by the presence of glucose in high concentration,
resulting in insufficient ATP for cellular metabolism, a phenomenon
known as the Crabtree effect (Malina et al., 2021; Rothman et al., 2021).
Intending to suppress the Crabtree effect, Xu and Li (2021) attempted to
use ethanol which then is converted into acetyl-CoA, the precursor of
citric acid synthesis. To increase ethanol uptake rate and accelerate
acetyl-CoA synthesis, AAC2, the major mitochondrial ADP/ATP trans­
porter, was overexpressed. Upregulation of AAC2 reduced acetate
accumulation by enhanced final IA titer from 77 mg/L to 142 mg/L.
Yarrowia lipolytica, an oleaginous yeast, has been explored as a great
industrial platform for its potential to produce lipid (Wang et al., 2020),
terpenoids (Ma et al., 2019), and IA (Rong et al., 2022). By over­
expressing cadA from A. terreus and blocking downstream pathway,
Rong et al. (2022) reported the production of 54.55 g/L IA in stirred-
tank reactor. As the compartmentalization of CAA between mitochon­
dria and cytosol plays a crucial role, a mitochondrial transporter mttA
from A. terreus was introduced into Y. lipolytica along with cadA.
Augmentation of CAA synthesis greatly affected IA production by
providing overflowing supply of precursor, leading to about 60-fold
increase in final IA titer. Schizosaccharomyces pombe, as known as
fission yeast, was reported to be able in producing up to 1.55 g/L IA from
glucose (Fujie et al., 2022). Moreover, P. kudriavzevii, as known as
I. orientalis, was engineered as a host owing to better ability to effectively
maintain its membrane integrity and higher tolerance to acid. Sun et al.
(2020) overexpressed cadA and mttA from A. terreus in P. kudriavzevii for
efficient transport of CAA to cytosol, along with icd deletion, and ob­
tained 1.2 g/L IA from glucose in 24 h with a yield of 29 mg IA/g
glucose.
Another prospective host comes from alkaliphiles, H. bluephagenesis,
which has shown rapid growth in alkaline environment (pH > 8.5) as
P.A. Diankristanti and I.-S. Ng
Table 3
Summary of in vitro IA production using citrate as substrate.
Strains IA titer (g/L) Time (h) Yield (g/
g)
E. coli Rosetta(DE3) 8.7 30 0.1
E. coli BL21(DE3) 41.6 24 0.48
E. coli BL21(DE3) 47.5 24 0.55
E. coli BL21(DE3) 67.0 8 0.35
E. coli Lemo21(DE3) 98.7 10 0.51
Shewanella livingstonensis 1.41 1 0.16
Ac10
well as in open nonsterile condition. Its high tolerance to organic salt
including IA is advantageous to diminish negative effect of product in­
hibition. With the overexpression of cadA from A. terreus and acnA from
C. glutamicum, icd deletion, and co-expression of GroESL, Zhang et al.
(2021) attained 63.6 g/L IA from 500 mM citrate in 36 h, which is the
highest IA titer obtained from a heterologous host (Table 2). Nonethe­
less, in vivo IA production is still holding a few drawbacks, long culti­
vation time to reach favorable titer remains a challenge. On the lookout
to shorten fermentation period, in vitro strategy is gaining attraction, by
utilizing whole-cell biocatalysts where the bioproduction pathway is
engineered.
5. Whole-cell bioconversion for high-level IA production
In vitro approach, in contrast to in vivo, employs cell post-cultivation
as biocatalyst carrying related genes to undergo the reaction outside the
cell itself (Honda et al., 2017). Cells are cultivated and maintained in
suitable medium with sufficient nutrient to ensure growth and metabolic
activity. After cultivation stage, cells are harvested and exposed to
desired substrate (in this case, citric acid) to initiate the bioconversion of
IA. Utilization of whole-cell biocatalysts in in vitro production offers
trouble-free control over physical parameters (i.e., enzyme content, pH,
temperature), faster reaction rate, simpler product isolation, and
excellent reusability. Being highly specific by eliminating competing
pathway, whole-cell biocatalysts possess the capability of sidestepping
off-target reactions, thus allowing the theoretical yield to approach
100%. On top of that, through metabolic engineering, synthetic pathway
and heterologous enzymes can be introduced to hosts, enabling high-end
chemical production from low-cost feedstock (Lin et al., 2017).
Numerous value-added chemicals have been produced by using whole-
cell biocatalysts, namely cadaverine (Ting et al., 2021), gamma-
aminobutyric acid (GABA) (Xue et al., 2021), putrescine (Yang et al.,
2022), p-coumaric acid (Effendi et al., 2021), and IA (Diankristanti
et al., 2023). The scheme of in vitro IA production is illustrated in Fig. 2,
from the design step, biocatalysts production and optimization, and the
recycling attempts to reach an economically viable system.
As IA bioconversion is involving two enzymes, enzymatic balance,
efficacy, and effectivity of the cascade reaction must be taken into
consideration when designing the biocatalysts. In the aspect of enzyme
kinetics, deciphering kinetic constants of enzyme is essential to under­
stand how quickly the enzyme can convert the substrate and how easily
the enzyme can get saturated. Hsiang et al. (2022) reported that cadA,
compared to acnA, has a much higher substrate affinity and lower re­
action rate, implying the conversion of CAA to IA is the rate-limiting
step. In view of recombinant protein production, accessing data from
enzyme database such as BRENDA (BRaunschweig ENzyme DAtabase)
and employing computational codon optimization strategies are essen­
tial to accentuate the structural and functional design, since they enable
deeper understanding of cellular mechanism and phenotype-to-
genotype correlations.
Possessing the combination of rapid growth rate, well-studied
Bioresource Technology 384 (2023) 129280
Strategy References
- Self-assembled reaction of Acn and AtCadA via protein-peptide Yang et al., 2017
interaction
- Increasing AtCadA copy number Kim et al., 2017
- Increase AtCadA copy number Moon et al., 2018
- Immobilization using Na-alginate and BaCl2
- Overexpression of AtCadA and CgAcnA under dual T7 promoters Hsiang et al., 2022
- Cold treatment for 24 h Diankristanti et al., 2023
- Integration of GroELS
- Heat at 45 ◦ C to increase membrane permeability Luo et al., 2020
genetic background, and inexpensive culture medium, E. coli is a
preferred host for recombinant protein production, specifically as
whole-cell biocatalysts in IA production. Expression of acnA from
C. glutamicum ATCC 13032 and codon-optimized cadA from A. terreus
into E. coli BL21(DE3) under dual T7 promoter is reported for further
investigations (Kim et al., 2017; Hsiang et al., 2022). The effect of gene
copy number was found out to increasing cadA copy number up to three
times, IA production was enhanced by 25.7% (Kim et al. 2017). A self-
assembly enzymatic cascade was designed through the fusion of cadA
with PDZ ligand and acnA with PDZ domain Yang et al. (2017), there­
fore, the protein-peptide interactional force enables these two enzymes
to co-localize and connect, leading to lower steric hindrance and higher
IA titer. Aside from E. coli as whole-cell biocatalysts, Luo et al. (2020)
investigated the potential of Shewanella livingstonensis Ac10 by
expressing acnB from E. coli and cadA from A. terreus under the tac
promoter. This study generates a psychrophile-based simple biocatalyst
(PSCats), in which desired pathways from mesophilic or thermophilic
bacteria are overexpressed. By increasing the temperature, intrinsic
metabolic pathways are inactivated, while the targeted recombinant
genes remain (Mojarrad et al., 2021).
There are two major stages of in vitro IA production: the growth when
the metabolic flux is used for replication, and the production when
cellular energy is driven into IA-producing pathway. Rich medium such
as Terrific Broth (TB) medium (Kim et al., 2017) and Triptic Soy Broth
(Luo et al., 2020) are used to elevate biomass. However, it is quite
inapplicable commercially, thus it is necessary to formulate an economic
culture media without compromising on cell, such as by employing
lignocellulosic feedstock (Nieder-Heitmann et al., 2018). Utilization of
glycerol instead of glucose is also an attempt to deal with acetate
overflow which usually occurs when glucose is being consumed.
At the end of cultivation, biocatalysts are washed using deionized
water or phosphate-buffered saline (PBS) to remove remaining compo­
nents of culture medium. Citric acid, the sole substrate, is added into the
cell pellet to let the reaction begin at defined incubation temperature. To
attain an industrially favored and economically feasible process, opti­
mizing titer, productivity, and yield of the desired product is indis­
pensable. Hence, it is favorable to optimize related parameters, namely
pH, temperature, reaction time, cell density, and substrate concentra­
tion. Kim et al. (2017) found the optimum pH and temperature for in
vitro IA production are 5.5 and 35 ◦ C, respectively. Hsiang et al. (2022)
carried out a Taguchi L9 array to further optimize biocatalyst density in
term of OD600 and found that biocatalysts activity increased along with
the increasing cell density from 40 to 90, but it started to diminish when
the value of OD600 elevated from 90 to 130. This phenomenon is related
to the availability of enzyme-substrate active binding site which allow
IA bioconversion to take place.
Cell permeability is a factor which influences the rate of substrate
uptake and product release through the cell membrane (Osire et al.,
2021), thus closely affects IA conversion rate and final titer. Surfactant,
such as hexadecyltrimethylammonium bromide, polyvinyl alcohol, and
Tween 80, has been reported to enhance membrane permeability of
7
P.A. Diankristanti and I.-S. Ng
Fig. 3. Comparison of IA production in vivo and in vitro, considering three critical factors which are feedstock cost, reaction time, and purification cost.
E. coli thus allow rapid mass transfer. On the other hand, Luo et al.
(2020) applied heat treatment at 45 ◦ C for 15 min in prior to the reac­
tion, boosting IA productivity by 6 folds (Table 3).
In addition to high level of IA, commercial production also calls for
highly stable and reusable whole-cell biocatalysts. A certain level of
whole-cell stability and reusability can be approached through enzyme
immobilization, which also mitigates product separation process for
continuous production. Moon et al. (2018) immobilized engineered
E. coli JY001 with barium-alginate beads and optimized various related
conditions, such as components proportion in the beads, permeabiliza­
tion agent (i.e., CTAB), temperature, and pH. Immobilized biocatalysts
could maintain initial activity even after four times of repeated used,
while the activity of free cells dramatically decreased after 2 cycles.
Heat-treated S. livingstonensis could also remained stable in four cycles
(Luo et al., 2020), confirming an excellent cell membrane integrity
which is a critical parameter in whole-cell bioconversion. It is clear that
in vitro bioconversion is an advantage with maximum productivity of
9.8 g/L/h (Diankristanti et al., 2023), while in vivo approach using
A. terreus could only reach around 1 g/L/h (Huang et al., 2014).
6. Opportunities, challenges, and perspectives
Microbial cell factories sometimes suffer from low productivity,
limited native pathways, and denaturation as a result of withstanding
environmental tensions. Oxygen and pH controls are essential to main­
tain growth and ensure stable productivity, as cellular metabolism often
leads to acid generation and oxygen depletion. When chemical is pro­
duced inside the cell, the production pool competes with growth-related
pathway, leading to a trade-off between growth and yield, even to severe
cellular imbalance and crashes.
Heterologous expression of recombinant genes faces the difficulties
of protein misfolding and inclusion body formation, that can be over­
come by utilizing the proper promoter of vector, optimizing codon usage
of DNA sequence, and controlling the cultural temperature. To upgrade
enzymatic properties, protein engineering serves as an approach
through the modification of amino acid sequences. Kim et al. (2022)
performed an activity-enhancing mutation using SCANEER, a web-based
tool which identifies the evolution of amino acid sequence, to amplify
the activity of cadA. Based on the evolutionary information, three
8
Bioresource Technology 384 (2023) 129280
mutants of cadA successfully enhanced IA production by 1.86 folds.
Owing to the capacity to expand the number of engineered enzyme
properties, machine learning might be an invaluable strategy to control
heterologous protein expression and beat the roadblocks. To further
enhance cellular robustness, modification of global metabolic regulatory
in fungi is worth exploring. Microbes often alter its gene expression and
carbon flux as a respond to non-ideal bioconversion process, leading to a
failure in IA production pathway. Pomraning et al. (2022) examined
laeA gene, putative methyltransferase gene which exists in secondary
metabolite clusters, and found out that it could enhance IA production.
Higher yield was observed as a result of increased enzymatic activity in
IA gene cluster, but phosphate limitation led to decreased growth.
However, higher activity of secondary metabolites might also lead to
troublesome purification process, even contamination.
In vitro whole-cell bioconversion strives to mitigate the drawbacks
present in in vivo process, such as competing side reaction, by-product
formation, complicated product extraction, and intractable down­
stream process. Nonetheless, in vitro IA production is facing the problem
of restricted mass transport due to the impediment from cell membrane.
Xue et al. (2021) demonstrated the effect of cold treatment in acceler­
ating substrate uptake rate and productivity through the migration of
glutamate decarboxylase (GadB) in whole-cell E. coli, and further
improving cell reusability up to 10 times of repeated use. Diankristanti
et al. (2023) exploited the aforementioned cold treatment to develop a
seeding strategy, where biocatalyst is kept in rich medium for quality
maintenance and stored in low temperature for long-term usage, and
finally obtained 98.7 g/L IA, which is the highest record for IA pro­
duction when using in vitro method. To maximize the performance of
whole-cell biocatalysts, Sakkos et al. (2019) constructed a microbial
exoskeleton consisting of silica and poly-diallyldimethylammonium
chloride. Layer-by-layer nanocoating on cell surface was found to
elevate catalysis rate up to 5 folds. This finding offers an insight on how
a microbial exoskeleton could protect cell from environmental stresses
such as extreme heat and cold, osmotic shock, and desiccation, while at
the same time enhance reactivity. From industrial point of view, in vitro
IA production is restricted by the relatively high cost of citric acid, which
is only half of IA market price. Revealing alternative cascade pathways
to utilize cheaper substrate in the whole-cell bioconversion is a major
focus of future research in this field.
P.A. Diankristanti and I.-S. Ng
Holding a great lot of potentials for high-end application, commer­
cial production of IA is still gaining attention. However, the barricade of
purification process is a challenge to conquer. Although in vivo strategy
requires lower price of feedstock since it allows the utilization of various
carbon source, the purification process will definitely cost a pretty
penny, given that multiple side reactions exist alongside the desired IA
pathway. While in vitro is highly dependent on citric acid whose price is
relatively high, it offers little to no purification process as the conversion
from citric acid to IA is the only reaction taking place. The trade-off of IA
production through in vivo and in vitro approaches is shown in Fig. 3. All
untangled considerations provide blunt perspectives when it comes to
choosing the direction of IA bioproduction from total consideration of
feedstock cost, reaction time, and purification cost.
7. Conclusion
Producing IA in vivo or using whole-cell biocatalyst (in vitro) has its
own pain and gain, either dodging competing side reactions and long
cultivation time, or sidestepping barricade of cell wall and mass trans­
port limitation. Stability and reactivity are vital to meet industrial needs,
where microbes are expected to work under dynamic conditions, steady
supply chain, and well-organized recycle set-up. Engineering de novo
strains through computational modeling and machine learning paves a
way to maximize performance of microorganisms. By tailoring good
genetic design and reaction chassis, microbial and low-carbon IA bio­
production is strikingly moving forward to circular economy and
sustainability.
CRediT authorship contribution statement
Priskila Adjani Diankristanti: Writing – original draft, Conceptu­
alization. I-Son Ng: Conceptualization, Resources, Supervision, Writing
– original draft, Writing – review & editing.
Declaration of Competing Interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
The authors are grateful for the financial support received from the
Ministry of Science and Technology (MOST 111-2221-E-006-012-MY3
and MOST 110-2221-E-006-030-MY3) in Taiwan.
Data availability
Data will be made available on request.
Acknowledgments
The authors are grateful for the financial support received from the
Ministry of Science and Technology (MOST 111-2221-E-006-012-MY3
and MOST 110-2221-E-006-030-MY3) in Taiwan.
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