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Metabolic engineering for 4-aminophenylalanine
production from lignocellulosic biomass byrecombinant Escherichia coli†
Hideo Kawaguchi, ab Shunsuke Masuo,
Tomohisa Hasunma, ab Tatsuo Kaneko,
Chiaki Ogino *f and Akihiko KondoafgcdKeiko Wakai,a Naoki Takaya, cSatoshi Okada,e Takashi Sazuka,e

To synthesize a high-performance biopolyimide bioplastic from lignocellulosic feedstock, Escherichia coli wasmetabolically engineered to produce 4-amino-L-phenylalanine (4APhe) as a diamine monomer. A highbiomass sorghum cultivar was used as the model lignocellulosic feedstock, and the enzymatic hydrolysate
was used as a substrate for 4APhe production in fed-batch culture. When using the ldh mutant strain,
HKE6027, 4APhe production from glucose was increased by over three-fold compared to the parent strain.
This increase was due to the disruption of biosynthetic pathways that produce either acetate or lactate as
by-products. Comparative metabolomic analysis revealed increased ﬂux in both the pentose phosphate andshikimate pathways in HKE6027, resulting in the highest yields. However, 5.7 g L−1 of 4APhe was produced
from enzymatic hydrolysis by HKE6027, which was 24% lower than that obtained from glucose. The
concentrations of 14 potential fermentation inhibitors present in the enzymatic hydrolysate were
determined, and their inhibitory eﬀects on both cell growth and 4APhe production were examined. The
addition of groups of potential inhibitors present in the enzymatic hydrolysate of sorghum bagasse showedthat benzaldehyde- and cinnamic acid derivatives inhibited 4APhe fermentation at low concentrations (halfReceived 13th February 2023
Accepted 23rd May 2023

maximum inhibitor concentration IC50 = 16 mg L−1), whereas furfural and 5-hydroxymethylfurfural, which
are well-known fermentation inhibitors, inhibited 4APhe fermentation at relatively high concentrations (IC50

DOI: 10.1039/d3su00053b= 640 mg L−1). These results provide insight into the design of metabolic pathways tailored for the

rsc.li/rscsus

utilization of lignocellulosic biomass to produce aromatic compounds through the shikimate pathway.

Sustainability spotlight
This study produced a starting material for the synthesis of polyimide from an inedible and renewable feedstock of lignocellulosic biomass using microbial
fermentation. This process can produce an organic molecule from plant biomass for CO2 xation and convert it into a high-performance bioplastic, resulting in
the long-term mitigation of greenhouse gas emissions in the industrial sector. Therefore, this study contributes to the circular economy and alternative plastic
raw material production for a sustainable society. Furthermore, this work aligns with goals 12 of “Responsible Consumption and Production” and 13 of “Climate
Action” of the United Nation's Sustainable Development Goals.

Introduction
Bioplastics are becoming increasingly popular as alternatives topetroleum-derived plastics due to their bio-based or

biodegradable nature.1,2 Bioplastics made from renewablebiomasses, such as lignocellulosic feedstocks, have the potential to mitigate carbon dioxide emissions. This is especially
important as global plastic production has risen from 1.5

aGraduate School of Science, Technology and Innovation, Kobe University, 1-1
Rokkodai, Nada, Kobe 657-8501, Japan

e

b

f

c
Faculty of Life and Environmental Sciences, Microbiology Research Center for
Sustainability, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 3058572, JapangEngineering Biology Research Center, Kobe University, 1-1 Rokkodai, Nada, Kobe
657-8501, Japand

Energy and Environmental Area, Graduate School of Advanced Science andTechnology, Japan Advanced Institute of Science and Technology, 1-1 Asahidai,
Nomi, Ishikawa 923-1292, Japan© 2023 The Author(s). Published by the Royal Society of Chemistry

Bioscience and Biotechnology Center, Nagoya University, Furo, Chikusa, Nagoya
464-8601, Japan

Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 6578501, Japan. E-mail: ochiaki@port.kobe-u.ac.jp; Fax: +81-78-803-6192; Tel: +8178-803-6193

Biomass Engineering Research Division, RIKEN, 1-7-22 Suehiro, Turumi,
Yokohama, Kanagawa 230-0045, Japan† Electronic supplementary information
https://doi.org/10.1039/d3su00053b(ESI)

available.

See

DOI:

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million tons in the 1950s to 335 million tons in 2016.3 Currently,
some bioplastics, such as poly(lactic acid) (PLA) and poly(butylene succinate) (PBS) are commercially available; however,the lower limits of thermostability (glass transition temperature, Tg # 70 °C)4 and mechanical strength (tensile strength, s# 50 MPa)5 of these bio-based aliphatic polyesters compared tothose of petroleum-based plastics hinder their extensive utilization. To replace petroleum-based plastics with bioplastics,
new molecular structures must be designed to improve thephysicochemical limitations of bioplastics.
4-Amino-L-phenylalanine (4APhe) is a metabolic intermediate in the biosynthesis of chloramphenicol and pristinamycin
I in Streptomyces venezuelae and S. pristinaespiralis, respectively.6,7 In the biosynthesis process involving S. pristinaespiralis, the chorismate metabolic intermediate in the shikimate
pathway is converted into 4-aminophenylpyruvate (4APP) ina three-step enzymatic reaction encoded by the papABC genes.
The resulting 4APP is then transformed into 4APhe by transaminase7 (Fig. 1). Recently, 4APhe has been used as a diamine
monomer to synthesize unique high-performance bio-based
polymers, such as gelable polypeptide8 and particulate

Paperpolyimide.9 These biopolymers exhibit signicantly higher
thermostability (Tg > 300 °C) compared to PLA and PBS and arecomparable to commercially available petroleum-based
plastics.
Lignocellulose is mainly composed of cellulose, hemicellulose, and lignin,10 which are renewable and non-edible
bioresources that can serve as alternative feedstocks for thesynthesis of sustainable plastics in society.1 The hydrolysate of
lignocellulosic biomass, which contains glucose released from
cellulose, is a preferred carbon source for fermentation.
However, this hydrolysate also contains organic contaminants
derived from the decomposition of cellulose, hemicellulose,
and lignin, which can act as fermentation inhibitors that hindercell growth and metabolism during fermentation.1,11,12 Althougha metabolically engineered Escherichia coli strain expressing
papABC derived from Pseudomonas uorescens was developed
and found to successfully produce 4APhe from glucose,13–16 it
was not able to produce 4APhe from lignocellulosic biomass(Table 1).
In this study, we aimed to produce 4APhe from lignocellulosic biomass. To achieve this, we developed metabolicallyFig. 1 Engineered metabolic pathway in a recombinant Escherichia coli strain capable of producing 4-amino-phenylalanine (4APhe) fromglucose through the shikimate pathway. In the host strain E. coli NST37DpheLA, which lacks both chorismate mutase/prephenate dehydratase(tyrA) and a pheLA locus [in the leader peptide for pheA expression (pheL) and chorismate mutase/prephenate dehydratase (pheA)] and possesses
two genes of code for feedback-inhibition-resistant (fbr) 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHP) synthases (aroFfbr
and aroGfbr) and fbr shikimate kinase (aroKfbr) and three genes, namely papA, papB, and papC encoding 4-amino-4-deoxychorismate synthase,
4-amino-4-deoxychorismate mutase, and 4-amino-4-deoxyprephenate dehydrogenase, respectively, derived from P. ﬂuorescens SBW25,
were overexpressed. To eliminate synthetic pathways for the formation of lactate and acetate as by-products, lactate dehydrogenase (ldh) and/or ackA_pta locus [acetate kinase (ackA) and phosphate acetyltransferase (pta)] were deleted from the chromosome of the host strain E. coli
NST37DpheLA in this study.

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Table 1RSC Sustainability
4-Amino-L-phenylalanine produced from various carbon sources by microbial fermentation

StrainSubstrate

Process

Titer (g L−1)

Reference

Recombinant Escherichia coli
Recombinant E. coli
Recombinant E. coli
Recombinant E. coli
Recombinant E. coli

GlucoseGlucoseGlucoseGlucoseEnzymatic hydrolysate of sorghum
bagasseFed-batchFed-batchFed-batchFed-batchFed-batch

2.2
4.4
2.9
7.4
5.7

Tateyama et al., (2019)14
Masuo et al., (2019)16
Minakawa et al., (2019)15
This study
This study

engineered E. coli strains that can eliminate by-productformation pathways. We compared their metabolic prolesduring 4APhe production from glucose to determine thelimiting step for 4APhe production (Fig. 1). Using therecombinant strain with the highest 4APhe yield, we performed
fed-batch culture for 4APhe production from the enzymatichydrolysate of pretreated sorghum bagasse as a model lignocellulosic biomass. Additionally, we identied the types ofmolecules present in the hydrolysate of pretreated sorghum
bagasse that inhibit 4APhe fermentation while not aﬀecting cell
growth.

Experimental
Microorganisms and media
The bacterial strains and plasmids used in this study are listed
in Table 2. For genetic manipulation, E. coli strains were grown
at 37 °C in Luria–Bertani (LB) medium.17 The E. coli strain
NST37DpheLA (DE3), which has feedback-resistant alleles ofaroGr encoding 3-deoxy-D-arabino-heptulosonate 7-phosphatesynthetase and lacks a pheLA locus both in the leader peptide for

Table 2

Preparation of the enzymatic hydrolysate of sorghum bagasse
The hybrid sorghum cultivar Tentaka18 was grown in 2020 in anexperimental eld at Nagoya University, Japan. Whole plants
were harvested at the heading stage and fully dried in a greenhouse. Aer removal of the panicles, the culms were ground

Strains and plasmids used in this studya

Name
Strains
Escherichia coli
NST37 (ATCC 31882)
E. coliNST(DE3)DpheLA
HKE1002
HKE6042
HKE6021
HKE6054
HKE6027
HKE6046
HKE6057
Plasmids
pET-pfpapA
pCDF-pfpapBC
pACYC-aroG4apheA expression (pheL) and chorismate mutase (pheA),13 wasused as a host strain to develop a metabolically engineered
strain for 4APhe production. For 4APhe production, modied
M9 medium containing the following was used: 6 g Na2HPO4,
3 g KH2PO4, 0.5 g NaCl, 10 g (NH4)2SO4, 0.5 g MgSO4$7H2O,
15 mg CaCl2, 50 mg thiamine-HCl, 5.0 g peptone, 2.5 g yeast
extract, 20 mg phenylalanine (Phe), 20 mg tyrosine (Tyr), 20 mg
tryptophan (Trp), 4 mg ZnSO4$7H2O, 2.2 mg H3BO3, 1 mg
MnCl2$4H2O, 1 mg FeSO4$7H2O, 0.32 mg CoCl2$6H2O, 0.32 mg
CuSO4$5H2O, 0.22 mg (NH4)6Mo7O24$4H2O, and 10 mg EDTA
per liter, supplemented with 40 g L−1 of either glucose or
enzymatic hydrolysate of sorghum bagasse as the carbon
source, unless otherwise described. When appropriate, media
were supplemented with 50 mg mL−1 ampicillin, 50 mg mL−1
streptomycin, and 34 mg mL−1 chloramphenicol for E. coli.

Relevant characteristics

Reference or source

aroFr, aroGr, tyrA, tyrR, pheAr, pheAo, trpE

ATCC

aroFr, aroGr, tyrA, tyrR, pheAr, pheAo, trpE, dcm(DE3)DpheLA

Tateyama, et al. (2016)14

E. coli NST(DE3)DpheLA bearing pACYC-aroG4, pET-pfpapA and pCDF-pfpapBC
Mutant of Dldh of strain E. coli NST(DE3)DpheLA
Mutant of DackA_ptaDpoxB of strain E. coli NST(DE3)DpheLA
Mutant of DldhDackA_ptaDpoxB of strain E. coli NST(DE3)DpheLA
HKE6042 bearing pACYC-aroG4, pET-pfpapA and pCDF-pfpapBC
HKE6021 bearing pACYC-aroG4, pET-pfpapA and pCDF-pfpapBC
HKE6054 bearing pACYC-aroG4, pET-pfpapA and pCDF-pfpapBC

Masuo, et al. (2016)13
This work
This work
This work
This work
This work
This work

Ampr; E. coli vector harboring papA gene derived from Pseudomonas
uorescens for 4APhe biosynthesis
Smr, E. coli vector harboring papBC genes derived fromP. uorescens for 4APhe biosynthesis
Cmr; E. coli vector harboring the aroG4 gene encoding
the feedback-resistant isozyme DAHP synthase

Masuo, et al. (2016)13,14
Masuo, et al. (2016)13,14
Masuo, et al. (2016)13,14

4APhe, 4-amino-L-phenylalanine; DAHP, 3-deoxy-D-arabino-heptulosonate 7-phosphate.

© 2023 The Author(s). Published by the Royal Society of Chemistry

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into a ne powder using a blender (WB-1; TGK, Hachioji, Japan)
tted with a 2 mm screen. A solid extract of sorghum bagasse
was prepared by treating it with dilute acid as described previously.12 For preparing the enzymatic hydrolysate of the pretreated sorghum bagasse, the commercially available cellulase,
enzyme blend (Sigma-Aldrich, Burlington, MA, USA) was usedfollowing a previous method.12 Briey, sorghum bagasse was
enzymatically hydrolyzed using the cellulase enzyme blend at
a load of 50 lter paper units per gram of dry biomass. Aer 72 hof incubation at 50 °C, the hydrolysate was collected and subjected to centrifugation (10 000g, 4 °C, 15 min); the resulting
supernatant was aseptically ltered for compositional analysis
with 4APhe fermentation as the carbon source.
DNA manipulation
PrimeSTAR Max DNA Polymerase (Takara Bio, Kusatsu, Japan)
was used for polymerase chain reaction (PCR) to amplify DNA
fragments, according to the manufacturer's instructions. The
PCR fragments were puried using a QIAquick PCR Purication
Kit (Qiagen, Hilden, Germany). Electroporation was used totransform E. coli, as previously described.19 Plasmid DNA was
isolated from E. coli, as per a previously described procedure.20
Construction of metabolically engineered E. coli strains for
4APhe production
E. coli strain NST37DpheLA(DE)13 (Table 2) was metabolicallyengineered via homologous recombination to suppress byproduct formation by lambda Red recombination21 usinga Quick & Easy E. coli Gene Deletion Kit (Gene Bridges GmbH,
Heidelberg, Germany) according to the manufacturer's
instructions. To inactivate the ldhA and poxB genes and ackA-pta
locus, pairs of primers 1 and 2, 5 and 6, and 3 and 4, respectively, were used to generate linear DNA fragments for homologous recombination (Table 3). The resulting deletion mutantsof HKE6042, HKE6021, and HKE6054 (Table 2) were transformed by electroporation with the plasmids pACYC-aroG4,
pCDF-pfpapBC, and pET-pfpapA for 4APhe biosynthesis (Fig. 1).
Transformants were selected based on their resistance toampicillin, chloramphenicol, and streptomycin and subsequently screened for their ability to produce 4APhe from glucose
as the sole carbon source, thereby yielding recombinant strainsof HKE6027, HKE6046, and HKE6057 and eliminating thebiosynthetic pathway of lactate, acetate, or both of them
(Table 2).

Table 34APhe fermentation by recombinant E. coli strains
Comparative 4APhe production using four recombinant E. coli
strains was conducted in batch culture with glucose (40 g L−1)
as the sole carbon source. Recombinant E. coli strains weregrown aerobically until the late log phase in a 10 mL test tube
containing LB broth with glucose (10 g L−1) under constantagitation (180 rpm) for 18 h at 30 °C. The pre-culture was
inoculated into 90 mL of modied M9 medium containing
glucose (40 g L−1) in a 200 mL jar fermentor Bio Jr.8 BJR25NA1S-8M (ABLE Co. & Biott Co., Tokyo, Japan) to obtaina cell concentration corresponding to an optical density at
600 nm (OD600) of 0.2. The cultivation conditions were as
follows: temperature, 30 °C; pH, 7.2, maintained by the additionof ammonia; stirrer speed, 600 rpm; and aeration withcompressed air, 1.0 vvm (all unless indicated otherwise). Aer
5 h of cultivation, 80 mM isopropyl-b-D-1-thiogalactopyranoside(0.8 mM, nal concentration) was added to each of the four
culture vessels. For the fed-batch culture, either glucose or
enzymatic hydrolysate of sorghum bagasse was used as the solecarbon source and added to each culture batch to obtain an
initial glucose concentration of 30 g L−1. These carbon sourceswere added to the culture batches four times in increments of
20 g L−1 glucose during the cultivation period. To identify
fermentation inhibitors for 4APhe production, potential
fermentation inhibitors detected in the enzymatic hydrolysateof sorghum bagasse were added to the fermentation medium atvarious concentrations. Modied M9 medium (2 mL) containing glucose as the sole carbon source was added to a Microplate
24 Deep-well (Porvair Science, Norfolk, UK), and the potential
inhibitors were added at various concentrations (0 to1000 mg L−1) before inoculation. Pre-culture (20 mL) wastransferred to the medium and incubated under constantagitation (180 rpm) for 28 h at 30 °C in a BioShaker M BR-022UP(TAITEC, Saitama, Japan) aer being sealed with the gaspermeable lm Breathe-EASIER (Diversie Biotech, Dedham,
MA, USA).
Metabolomic analysis
Metabolomic analysis of E. coli cells was conducted as previously described.12 Major metabolites of the central metabolic
pathways (e.g., glycolysis, pentose phosphate pathway [PPP],
tricarboxylic acid [TCA] cycle, and shikimate pathway) wereanalyzed using an ion-pair liquid chromatography-tandem
mass spectrometry (LC-MS/MS) method.22 Dried cell extracts

Oligonucleotides used in this study

Name

Target gene

Sequence (5′–3′)a

Primer 1
Primer 2
Primer 3
Primer 4
Primer 5
Primer 6

ldh
ldh
ackA_pta
ackA_pta
poxB
poxB

AACGTCGACCTTGACGCGGCAAAAGAACTGGGGCTGAAAGTAGTCCGTGTAATTAACCCTCACTAAAGGGCGGGCAGACAGGCGACGGAATACGTCATCCTGGATCACGTCGTTGGATTTATCTAATACGACTCACTATAGGGCTCGTGGCTCCCTGACGTTTTTTTAGCCACGTATCAATTATAGGTACTTCCATGAATTAACCCTCACTAAAGGGCGG
ACGCTCTTTCAGCTGTTCCGGGGTGTCAGTGCCCTGAGACATAACGAAGATAATACGACTCACTATAGGGCTCG
CTCTCTGAACGGTCTTAGTGACAGTCTTAATCGCATGGGCACCATCGAGTAATTAACCCTCACTAAAGGGCGGTATACAGGCTGAAACCTTTGGCCTGTTCGAGTTTGATCTGCGGTGGAATGTAATACGACTCACTATAGGGCTCGa

The homology arm for homologous recombination with the red/ET system is indicated with underlines.

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were dissolved in 50 mL MilliQ water for LC-MS/MS proling andquantication of 30 intracellular E. coli metabolites. The
following metabolites were analyzed: sugar phosphates(glucose-6-phosphate [G6P], fructose-6-phosphate [F6P],
fructose-1,6-bisphosphate [FBP], dihydroxyacetone phosphate[DHAP], glyceraldehyde-3-phosphate [GAP], 2- and 3-phosphoglycerate [2PG + 3PG], phosphoenolpyruvate [PEP], 6-phosphoD-gluconate [6PG], ribulose-5-phosphate [Rib5P], ribose-5phosphate [R5P], xylose-5-phosphate [X5P], erythrose-4phosphate [E4P], and sedoheptulose-7-phosphate [S7P]);
organic acids (aconitate [Aco], citrate [Cit], fumarate [Fum],isocitrate [IsoCit], lactate [LA], malate [Mal], oxaloacetate [OXA],
2-oxoglutarate [AKG], pyruvate, and succinate [Suc]); shikimate
derivatives (4-aminobenzoic acid [PABA], 4APhe, 4APP, andshikimate-3-phosphate [Sh3P]); nucleotides (adenosine
diphosphate [ADP] and adenosine triphosphate [ATP]); coenzymes (acetyl-CoA [AcCoA], oxidized and reduced nicotinamide
adenine dinucleotide [NAD+ and NADH, respectively], oxidized
and reduced NAD-phosphate [NADP+ and NADPH, respectively]); and amino acids (glutamate [Glu], glutamine [Gln], Phe,
Trp, and Tyr). Metabolites were quantied as previously
described23 using an Agilent 1200 series MS and Agilent 6460
with a Jet Stream Technology liquid chromatography withtandem mass spectrometry (LC-MS/MS) system (Agilent Technologies, Waldbronn, Germany) equipped with a Maestro C18
column (2.1 × 150 mm, 3 mm particle size; Shimadzu, Kyoto,
Japan). For quantitative metabolomics, E. coli cells were subjected to cold methanol quenching followed by metabolite
extraction from the cells, as previously described.12

Analytical procedures
Culture samples were centrifuged (15 000g, 4 °C, 10 min), andthe concentrations of glucose, 4APhe and its derivative PABA,
and organic acids (acetate, lactate, and Suc) in the resulting
supernatants were measured. Glucose concentration was
assayed enzymatically using the Glucose CII test kit (Wako,
Osaka, Japan). The concentrations of 4APhe and PABA were
determined via high-performance liquid chromatography(HPLC) using an LC-20A apparatus (Shimadzu, Kyoto, Japan)
equipped with a COSMOSIL(R) HILIC Packed Column (4.6 mm
I.D. × 250 mm; Nacalai Tesque, Tokyo, Japan) and an SPDM20A diode array detector (Shimadzu). The column oventemperature was set at 30 °C, and the mobile phase wasacetonitrile/10 mM ammonium acetate = 70 : 30 (v/v) at a ow
rate of 1.25 mL min−1. 4APhe and PABA were detected atwavelengths of 241 and 270 nm, respectively, and the concentrations of 4APhe and PABA were calculated based on standard
curves of standard substances. Cell mass was estimated bymeasuring optical density at 600 nm (OD600) using a spectrophotometer (U-3010; Hitachi, Tokyo, Japan). Suc concentration
was determined via HPLC, as described previously.24 To
improve the selectivity and accuracy of analysis for trace organic
acids in contaminants derived from lignocellulosic biomass,
the concentrations of acetate and lactate were enzymaticallydetermined using F-kit acetic acid and D-lactic acid/L-lactic acid(Roche, Penzberg, Bavaria, Germany), respectively, according to© 2023 The Author(s). Published by the Royal Society of Chemistry

RSC Sustainability
the manufacturer's instructions. The concentrations of potential fermentation inhibitors dissolved in the enzymatic hydrolysate of sorghum bagasse were determined via gas
chromatography/mass spectrometry, as described previously.24
Based on GC/MS analysis, 18 potential inhibitors were selectedbased on their abundance, inhibition strength, and/or structurefrom previous reports,10–12,25,26 and the concentration of thesecompounds in the enzymatic hydrolysate of sorghum bagasse
was determined.
Statistical analysis
Diﬀerences in metabolite concentrations and cell densities
between fermentation media were compared using paired
Student's t-tests. Results with p values <0.05 were considered
statistically signicant.

Results and discussion
Metabolic engineering for improved 4APhe production byrecombinant E. coli
A recombinant E. coli strain HKE1002, expressing papABC
derived from P. uorescens SBW25 in E. coli NST37(DE3)DpheLA,13 was used as the parent strain for 4APhe production
from glucose (Table 2). Using this strain, three metabolicallyengineered strains were developed to improve 4APhe production by eliminating the formation of acetate and lactate byproducts (Table 2). Lactate formation was completely lost
because of the elimination of the ldh gene encoding lactate
dehydrogenase from the chromosome in the recombinantstrains (HKE6027 and HKE6057); transient lactate accumulation was signicantly increased by the disruption of DackA_ptaDpoxB (HKE6046), encoding acetate kinase, phosphate
acetyltransferase, and pyruvate oxidase,27 compared to that seen
with the parent strain (HKE1002) (Fig. 2). Acetate accumulationaer 96 h of cultivation was signicantly lower in a culture witheither of the three recombinant strains (<1.5 g L−1) than ina culture with the parent strain (HKE1002) (3.6 g L−1). All threerecombinant strains consumed the entire available glucose in
the culture media aer 78 h of cultivation, whereas the parent
strain consumed the entire available glucose aer 96 h ofcultivation. Furthermore, 4APhe concentration aer 78 h ofcultivation was more than three-fold higher in the culture witha recombinant strain than in that with the parent strain(Fig. 2B).
Of the recombinant strains, the Dldh strain (HKE6027)
showed the highest 4APhe yield (4.1 g L−1), followed by thatseen with the DldhDackA_ptaDpoxB (HKE6057) and DackA_ptaDpoxB (HKE6046) strains. In addition to 4APhe, anotherchorismate derivative of PABA28 also accumulated in the culture
media as a by-product (Fig. 1). PABA accumulation aer 78 h ofcultivation was two-fold higher in the culture with the Dldhstrain (HKE6027) (620 mg L−1) than in that with the otherstrains, including the parent strain (<300 mg L−1) (Fig. 2D).
Consequently, the total amounts of 4APhe and PABA aer 78 hof cultivation were 0.6 (with HKE1002), 4.7 (with HKE6027), 3.4(with HKE6046), and 3.9 g L−1 (with HKE6057), respectively. In

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4-Amino-phenylalanine (4APhe) production from glucose by recombinant Escherichia coli strains HKE1002 (parent; open squares),
HKE6027 (Dldh; closed circles), HKE6046 (DackA_ptaDpoxB; closed triangles), HKE6057 (DldhDackA_ptaDpoxB; closed diamonds). The
concentrations of glucose (A), 4APhe (B), p-aminobenzoic acid (PABA) (D), acetate (E), and D-lactate (F) in culture broth are presented. Cell
growth is presented in panel (C). Each E. coli strain was grown under aerobic conditions until the late log phase in Luria–Bertani medium;
thereafter, the culture was inoculated to obtain an initial OD600 of 0.2 in modiﬁed M9 medium containing D-glucose as the sole carbon source(40 g L−1). Data are plotted as the average ± standard deviation calculated from the results of triplicate individual experiments.
Fig. 2a previous study, 4.4 g L−1 of 4APhe was produced in glucose-fed
batch culture using a recombinant E. coli strain without eliminating the acetate and lactate biosynthesis pathway.13 In thepresent study, a comparable concentration of 4APhe (4.1 g L−1)
was produced from glucose in the bath culture using HKE6027(Dldh) upon eliminating the lactate biosynthesis pathway(Fig. 2B). These results suggest that carbon ux via chorismite
in the shikimate pathway was increased by the disruption of the
Dldh or DackA_ptaDpoxB strains and that further enhanced ux
was not observed with the combined strain DldhDackA_ptaDpoxB. A small amount of D-lactate (0.15 g L−1) was
produced by the parent strain (HKE1002) whereas its accumulation reduced signicantly in the culture of the Dldh strain(HKE6027) (Fig. 2F). The concentration of L-lactate was below
the detection limit under all cultivation conditions (data notshown).

1048 | RSC Sustainability, 2023, 1, 1043–1054

Comparative metabolome analysis using three 4APheproducing strains
The metabolic proles of the three metabolically engineered
strains during 4APhe production were compared, thereby
revealing metabolic shis associated with enhanced 4APheproduction (parent, Dldh, and DackA_ptaDpoxB strains). Timecourse metabolomics of the central metabolic pathway clearly
showed strain-specic proles associated with improved 4APheproductivity (Fig. 3). In the PPP, which plays a major role in
NADPH regeneration in E. coli cells, intracellular levels of 6PG,
Rib5P, R5P, and NADPH were relatively higher in both HKE6027(Dldh) and HKE6046 (DackA_ptaDpoxB) strains with higher
4APhe productivity than in the parent strain (HKE1002). In theglycolytic pathway, the levels of F6P and 2PG + 3PG aer 29 and48 h of cultivation were signicantly higher in HKE6046(DackA_ptaDpoxB) than those in the other two strains.

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Fig. 3 Time-course metabolomics of the central metabolic pathway. Relative abundances of metabolic intermediates in three recombinant
Escherichia coli strains HKE1002 (parent), HKE6027 (Dldh), and HKE6046 (DackA_ptaDpoxB) during 4-amino-L-phenylalanine (4APhe)production in modiﬁed M9 medium containing D-glucose as the sole carbon source is shown. The X-axis indicates cultivation time (h). For the
proﬁling experiment, each strain was cultured under aerobic conditions until the late log phase in Luria–Bertani medium, and the culture wasthen inoculated to an initial OD600 of 0.2 into modiﬁed M9 medium containing D-glucose as the sole carbon source (40 g L−1). The cells were
harvested at 29, 48, and 54 h of cultivation and subsequently subjected to metabolome analysis. Data are presented as the average ± standard
deviation calculated from the results of triplicate individual experiments.

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Compared to the parent strain with low 4APhe productivity, in
addition, PEP levels were signicantly higher in HKE6046(DackA_ptaDpoxB) and signicantly lower in HKE6027 (Dldh).
Knockout of the acetate synthesis pathway exerted a signicant
impact on pyruvate metabolism. Lactate levels aer 48 h ofcultivation and AcCoA levels during 29 to 59 h of cultivation
were signicantly higher in HKE6046 (DackA_ptaDpoxB) than in
the other two strains. In addition, compared to the other two
strains, HKE6046 (DackA_ptaDpoxB) showed signicantly
higher ADP levels and comparable ATP levels. Consequently,
the ATP/ADP ratio was signicantly lower in HKE6046 than in
the other two strains during 29 to 59 h of cultivation.
For 4APhe production from glucose, HKE6027 (Dldh) showed
the highest productivity, followed by HKE6057 (DldhDackA_ptaDpoxB) and HKE6046 (DackA_ptaDpoxB) (Fig. 2). HKE6027(Dldh) exhibited distinct metabolic proles. Aer 48 h of cultivation, the intracellular levels of Sh3P and 4APhe were signicantly higher and PABA and 4APP levels were markedly higher
than those of other strains, whereas the PEP level was signicantly lower, suggesting an increased ux toward 4APhebiosynthesis via the shikimate pathway. In contrast, HKE6046(DackA_ptaDpoxB), which eliminates the acetate biosynthesis
pathway, accumulated glycolytic intermediates including F6P,
FBP, 2PG or 3PG, PEP, pyruvate, and AcCoA, in addition tohaving a markedly low ATP/ADP ratio (Fig. 3). Although theintracellular levels of PEP and the NADPH/NAPD+ ratio, which
serve as precursors and cofactors to synthesize shikimate for
4APhe production, respectively,29 were the highest of all intracellular metabolite levels in all three strains, 4APhe production
by HKE6046 (DackA_ptaDpoxB) was lower than that by HKE6027(Dldh) (Fig. 2). Unlike HKE6027 (Dldh), HKE6046 showedcomparable levels of intracellular metabolites, including Sh3P,
PABA, 4APP, and 4APhe, in the shikimate pathway and subsequent 4APhe biosynthesis with the parent strain. In E. coli,acetate kinase encoded by the ackA gene is responsible for ATP
generation,30 and ATP is a cofactor for Sh3P synthesis by shikimate kinase in the shikimate pathway.31 These results suggestthat the knockout of ackA_pta genes results in the overow of
AcCoA and its upstream metabolites and reduces ATP generation required for the shikimate pathway, leading to attenuated4APhe production by HKE6046. Compared to the parent strain,
the ldh mutant showed four-hold higher 4APhe production(Fig. 2B) and signicantly lower intracellular PEP levels withcomparable pyruvate levels (Fig. 3). These results suggest that
more PEP was utilized for 4APhe production rather than acetateproduction aer the deletion of ldh.

Fed-batch culture for 4APhe production from the enzymatichydrolysate of lignocellulosic biomass
The concentrations of glucose and potential fermentation
inhibitors in the enzymatic hydrolysate of sorghum bagasse
were determined to utilize the enzymatic hydrolysate as the solecarbon source for 4APhe production. The enzymatic hydrolysate
contained glucose and xylose at concentrations of 160 and 0.7 g
L−1, respectively (Table 4). Of the potential fermentation
inhibitors, sinapinic acid was the most abundant1050 | RSC Sustainability, 2023, 1, 1043–1054

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Table 4 Concentrations of glucose and potential fermentation
inhibitors present in the enzymatic hydrolysate of sorghum bagasse

Component

Unit

Concentrationa

Monosaccharides
GlucoseXylose

g L−1
g L−1

160.1  13.7
0.7  0.1

Potential inhibitors
Furfural
5-Hydroxymethylfurfural
5-Methyl-2-furaldehyde
4-Hydroxybenzaldehyde
Vanillin
Syringaldehyde
Phenylacetaldehyde
Benzoic acid
p-Hydroxybenzoic acid
Syringic acid
Sinapinic acidp-Coumaric acidtrans-Ferulic acid
Levulinic acidmg L−1
mg L−1
mg L−1
mg L−1
mg L−1
mg L−1
mg L−1
mg L−1
mg L−1
mg L−1
mg L−1
mg L−1
mg L−1
mg L−1227.3  73.8
2728.3  339.9
66.6  37.8
30.9  14.3
21.1  2.7
82.8  1.3
6.3  2.7
337.1  101.7
363.2  106.6
1224.0  313.8
3135.5  553.5
525.4  65.0
1370.1  106.9
311.5  177.8

aConcentrations of monosaccharides and potential inhibitors arepresented as the average ± standard deviation calculated from theresults of triplicate individual experiments.

(3136 mg L−1), followed by 5-hydroxymethylfurfural (5HMF),
trans-ferulic acid, and syringic acid at concentrations>1000 mg L−1 (Table 4). In addition, major fermentation
inhibitors of furfural and p-coumaric acid were also present atconcentrations of 227 and 525 mg L−1, respectively, whereas theconcentrations of other potential inhibitors, such as benzaldehydes of 4-hydroxybenzaldehyde, vanillin, and syringaldehyde,
were relatively low, ranging from 21 to 83 mg L−1. The enzymatic hydrolysate of sorghum bagasse was used as the solecarbon source for subsequent 4APhe fermentation.
In the fed-batch culture for 4APhe production, either glucoseor the enzymatic hydrolysate of sorghum bagasse was fed as thesole carbon source to increase the glucose concentration from10 to 15 g L−1 in the culture at each feeding point. Within 48 hof cultivation, both glucose consumption and cell growth werehigher in cultures with the enzymatic hydrolysate than in thosewith pure glucose (Fig. 4). Within 45 h of cultivation before thesecond feeding, volumetric productivity of 4APhe was 0.09 and0.08 g h−1 L−1 from glucose and enzymatic hydrolysate ofsorghum bagasse, respectively, while its yields based on theamount of glucose consumed were 0.098 and 0.103 g 4APhe/g
glucose from glucose and the enzymatic hydrolysate, respectively. Aer 142 h of cultivation, the glucose-fed culture yielded
7.4 g L−1 of 4APhe and accumulated 2.3 and 7.1 g L−1 of Suc andacetate, respectively, as by-products (Fig. 4A). During thisperiod, the cell concentration in each culture continuously
increased and reached an OD600 of 21.3. However, in the culture
with sorghum hydrolysate, cell growth and 4APhe production
almost ceased aer 48 h of cultivation, although the sorghum
hydrolysate was fed even aer that (Fig. 4B). Consequently, the4APhe concentration transiently reached 5.7 g L−1 aer 91.5 h ofcultivation and then gradually decreased. In the culture with© 2023 The Author(s). Published by the Royal Society of ChemistryView Article Online

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Fig. 4 Fed-batch culture for 4-aminophenyalanine (4APhe) production from glucose (A) and enzymatic hydrolysate of sorghum bagasse (B) by
the recombinant Escherichia coli strain HKE6027 (Dldh). The concentrations of glucose (open squares), 4APhe (closed circles), cells (closed
triangles), acetate (AA; crosses), lactate (LA; open triangles), and succinate (Suc; open diamonds) are presented. HKE6027 was grown underaerobic conditions until the late log phase in Luria–Bertani medium, and the culture was then inoculated to an initial OD600 of 0.2 into modiﬁed
M9 medium containing either glucose or sorghum bagasse hydrolysate as the sole carbon source to yield an initial glucose concentration of 20 g
L−1. Arrows indicate the points at which the carbon sources should be fed. Data are plotted as the average ± standard deviation, calculated fromthe results of triplicate individual experiments.

sorghum hydrolysate, Suc production was signicantly
increased compared with that seen in the culture with glucose,and the concentrations of Suc and acetate were 18.2 and 1.0 g
L−1, respectively, aer 116.5 h of cultivation. Global eﬀorts are
increasingly being made to use second-generation feedstock.22
Second-generation feedstocks of lignocellulosic biomass
predominantly contain glucose followed by xylose with trace
amounts of other monosaccharides such as arabinose andmannose;10 diluted acid-pretreated sorghum bagasse contains
60% and 7% glucose and xylose as dry biomass, respectively,12
and adaptive mutant of E. coli can simultaneously utilize bothxylose and glucose along with arabinose.32 These results suggestthat the metabolic engineering of E. coli for the co-utilization ofhexose and pentose could improve 4APhe in further studies.
In previous studies, 4.4 g L−1 of 4APhe was produced fromglucose using recombinant E. coli (Table 1).13,14 In the present
study, a high concentration (5.7 g L−1) of 4APhe was obtained
from lignocellulosic hydrolysate by a metabolically engineered
E. coli strain that eliminated by-product synthesis. However,
4APhe yield from the lignocellulosic hydrolysate was 24% lessthan that from pure glucose. The enzymatic hydrolysate ofsorghum bagasse contained various contaminants, such as
furfural, 5HMF, p-coumaric acid, and trans-ferulic acid, which
served as fermentation inhibitors for E. coli in previous
studies33–35 (Table 4). These results suggest that 4APhe production from the enzymatic hydrolysate of sorghum bagasse bymetabolically engineered E. coli was subjected to fermentation
inhibition.11,36
In previous studies, methods for the purication of 4APhe14
and the direct use for the synthesis of high-performance polymer37 have been developed. For the downstream process,a higher concentration of 4APhe aer fermentation can achieve
higher recovery eﬃciency for the following polymer synthesis.
In the present study, over 2.2 g L−1 of 4APhe that was requiredfor purication14 was produced from lignocellulosic biomass(Table 1). Although annual production of plastics globally is© 2023 The Author(s). Published by the Royal Society of Chemistryover millions of tonnes,2 most of them are commodity plastics,
and the share of engineering and high-performance plastics
that are synthesized from aromatic monomers, e.g. 4APhe, isonly 7%.38 Based on the share of the engineering plastics, theirproduction capacity is estimated at 15 kg per h per plant.39 Dueto the low demand and high price of the engineering and highperformance plastics which require 4APhe as an aromatic
monomer, the product concentration of 4APhe demonstrated in
the present study is expected to be economically viable.

Identication of fermentation inhibitors for 4APheproduction
To identify the fermentation inhibitor(s) present in the enzymatic hydrolysate of sorghum bagasse, groups of potential
inhibitors were individually or collectively added to the medium
in a dose-dependent manner before starting 4APhe production
from glucose. The potential fermentation inhibitors wereselected from the results shown in Table 4 and classied into
ve groups based on their structures: group A, furan aldehydes(furfural, 5HMF, and 5-methyl-2-furaldehyde); group B,
hydroxybenzaldehydes (4-hydroxybenzaldehyde, vanillin, andsyringaldehyde); group C, (hydroxy)benzoic acids (benzoic acid,syringic acid, and p-hydroxybenzoic acid); group D, cinnamic
acid derivatives (trans-ferulic acid and p-coumaric acid); andgroup E, aliphatic acid (levulinic acid). Group E consisted ofonly levulinic acid, and potential inhibitors of acetate andlactate25 were not detected in the enzymatic hydrolysate ofsorghum bagasse. Dose–response curves were measured for thealdehydes and acids or their mixture, and half and fullmaximum inhibitory concentrations (IC50 and IC100, respectively) were determined for cell growth and 4APhe production(Table 5 and Fig. S1†).
Both hydroxybenzaldehydes and cinnamic acid derivatives
exerted the highest inhibitory eﬀects on 4APhe production (IC50= 20 mg L−1), whereas the IC50 of these inhibitors regarding cell
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Table 5 Concentrations of potential fermentation inhibitors required to inhibit 4-amino-L-phenylalanine (4APhe) production by recombinant
Escherichia coli strain HKE6027

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Cell growthb

4APhe productionb

Specicproductivity of
4APhebGroup of inhibitorsa (mg L−1)

IC50

IC100

IC50

IC100

IC50

IC100

Concentrationsof inhibitorc

A
B
C
D
E
Mixture

>1000
240
240
240
600
90

>1000
1000
320
1000
>1000
320

640
20
80
20
170
20

>1000
320
320
640
1000
160

840
20
160
20
240
45

>1000
>1000
>1000
640
>1000
160

377.5
16.8
240.4
236.8
38.9
910.4

a

Group: A, furan aldehydes (furfural, 5HMF, and 5-methyl-2-furaldehyde); B, benzaldehydes (4-hydroxybenzaldehyde, vanillin, andsyringaldehyde); C, benzoic acids (benzoic acid, syringic acid, and p-hydroxybenzoic acid); D, cinnamic acid derivatives (trans-ferulic acid and pcoumaric acid); E, levulinic acid; all, a mixture of all of the aforementioned compounds. Each group of inhibitors was individually added to theculture medium before inoculation in a dose-dependent manner, and cell growth and 4APhe production were measured aer 72 h ofcultivation. b IC100 is the concentration of inhibitors where cell growth or 4APhe production is completely suppressed. IC50 is the concentrationof inhibitors where cell growth or 4APhe production is reduced by half. These values were determined from the results of dose–response curvesof cell density and 4APhe concentration aer 72 h of cultivation (shown in Fig. S1, ESI). c Initial concentration of inhibitors present in fed-batch
culture with the enzymatic hydrolysate of sorghum bagasse for 4APhe production, as shown in Fig. 4B.

growth was relatively high (240 mg L−1). In 4APhe production,
benzoic acids also demonstrated a notable inhibitory eﬀect(IC50 = 80 mg L−1), whereas furan aldehydes, which are some of
the major fermentation inhibitors, exhibited a relatively low
inhibitory eﬀect (IC50 = 640 mg L−1). A synergetic eﬀect of thesefermentation inhibitors was observed for cell growth only but
not for 4APhe production. The concentrations of benzoic acids
and cinnamic acid derivatives present in sorghum bagassehydrolysate were more than three-fold higher than the IC50
values for 4APhe production, whereas the concentration of
benzaldehydes in the hydrolysate was comparable to the IC50
values for 4APhe production. Diﬀerent levels of inhibitory
eﬀects of potential fermentation inhibitors derived fromlignocellulosic biomass on microbial production by E. coli cells
have been reported previously.12,24,26 These results suggest that
benzoic acid and cinnamic acid derivatives present in sorghum
bagasse hydrolysate play a major role in the inhibition of 4APheproduction from the hydrolysate.
Compared to pure glucose as the sole carbon source, theenzymatic hydrolysate of sorghum bagasse reduced 4APheproduction by 23% and increased Suc formation (Fig. 4).
Increased Suc production by an engineered E. coli strain was
also observed in the presence of furfural present in lignocellulosic hydrolysate.40 Fermentation inhibition assay revealed thathydroxybenzaldehydes, cinnamic acid derivatives, and(hydroxy)benzoic acids served as strong inhibitors for 4APheproduction, whereas the inhibitory eﬀect of well-known
fermentation inhibitors furan aldehydes, such as furfural and
5HMF,33 on 4APhe production was limited (Table 5). In
a previous study, the cinnamic acid derivatives p-coumaric acidand trans-ferulic acid inhibited fermentation by E. coli.34 In the
enzymatic hydrolysate of sorghum bagasse, the total concentrations of cinnamic acid derivatives and (hydroxy)benzoic acids
were markedly higher than the IC50 values for 4APhe production, whereas the total concentrations of furan aldehydes andbenzaldehydes were lower and comparable to the IC50 values

1052 | RSC Sustainability, 2023, 1, 1043–1054(Tables 4 and 5). In addition, p-coumaric acid present inlignocellulosic hydrolysate inhibits the enzymatic activities of
shikimate kinase from Sorghum bicolor,41 and chorismate
mutase or prephenate dehydrogenase from Alcaligenes eutrophus42 is responsible for 4APhe production in the shikimate
pathway, although the inhibitory eﬀects of these enzymes havenot been reported in E. coli. In previous studies, the eﬀects ofcomponents of lignocellulosic hydrolysate on microbial activity
were evaluated based on the levels of metabolic intermediates
and gene expression as well as cell growth.11,12 These results
suggest that benzoic acids and cinnamic acid derivatives are
critical fermentation inhibitors for 4APhe production from the
enzymatic hydrolysate of sorghum bagasse and that the inhibitory eﬀect of furfural and 5HMF on 4APhe production by E. coli
is limited, even aer repeated feeding of the hydrolysate.

Conclusions
Microbial production of starting materials for the synthesis of
bio-based polymers exclusively depends on the use of edible
glucose but not non-edible lignocellulosic feedstocks. In thisstudy, 4APhe, which serves as a diamine monomer to synthesize
unique biopolyimides,9,43 was produced from the inedible
feedstock of sorghum bagasse instead of glucose, as reported
previously. Comparable metabolome analysis revealed specic
metabolic proles in E. coli cells with high 4APhe productivity,
which was improved by metabolic engineering. Although 5.7 g
L−1 of 4APhe produced from lignocellulosic biomass was thehighest concentration recorded, it was 24% lower than that
produced from glucose. Among the potential inhibitors present
in the enzymatic hydrolysate of sorghum bagasse, benzaldehydes, and cinnamic acid derivatives inhibited 4APhe fermentation at low concentrations (IC50 = 16 mg L−1), whereas the
predominant and well-known fermentation inhibitors furfuraland 5HMF inhibited 4APhe fermentation at relatively highconcentrations (IC50 = 640 mg L−1). These results provide© 2023 The Author(s). Published by the Royal Society of ChemistryView Article Online

Paper
insight into the design of metabolic pathways tailored for theutilization of lignocellulosic biomass to produce aromaticcompounds via the shikimate pathway by attenuating fermentation inhibition.

Conﬂicts of interest
Open Access Article. Published on 27 May 2023. Downloaded on 10/14/2024 11:30:28 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

There are no conicts of interest to declare.

Acknowledgements
This work was supported by the JST ALCA Program, Japan(Grant Number JPMJAL1010), the JST Mirai Program (Grant
Number PMJMI17EG), and the New Energy and Industrial
Technology Development Organization (NEDO), Japan (Grant
Number JPNP18016). We gratefully acknowledge the contribution of Junko Imada, Kumiko Yoshihara, Sharif Moinul Hasan,
Taeko Fukumoto, Takanobu Yoshida, and Yoshimi Hori toward
technical support.

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