Efficient Biotransformation of Luteolin to Isoorientin through Adjusting Induction Strategy, Controlling Acetic Acid, and Increasing UDP-Glucose Supply in Escherichia coli

Article abstract of DOI:10.1021/acs.jafc.8b05958

Isoorientin is a C-glycosylated derivative of luteolin and exhibits a number of biological properties. In this study, multiple strategies were adopted to improve isoorientin production from luteolin in Escherichia coli. Isoorientin production was improved substantially by adjusting induction strategies and controlling acetic acid accumulation, with maximum isoorientin production reaching 826 mg/L. Additionally, a novel UDP-glucose synthesis pathway was reconstructed in E. coli through cellobiose phosphorylase-catalyzed phosphorolysis of cellobiose for the production of glucose 1-phosphate, which serves as a precursor in UDP-glucose formation. The results from two mechanisms of UDP-glucose formation in E. coli, cellobiose phosphorolysis and sucrose phosphorolysis, were compared. Increasing the UDP-glucose supply resulted in maximal isoorientin production reaching 1371 mg/L. Finally, isoorientin (1059 mg) was obtained from 1 L of fermentation broth by simple purification steps with a yield of 81.5%. Therefore, this study provides an efficient method for isoorientin production and a novel UDP-glucose synthesis pathway.

Full text of DOI:10.1021/acs.jafc.8b05958

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Biotechnology and Biological Transformations
Efficient biotransformation of luteolin to isoorientin
through adjusting induction strategy, controlling acetic
acid and increasing UDP-glucose supply in Escherichia coli
Jianjun Pei, Qing Sun, Linguo Zhao, Hao Shi, Feng Tang, and Fuliang Cao
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05958 • Publication Date (Web): 10 Dec 2018
Downloaded from http://pubs.acs.org on December 18, 2018
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Efficient biotransformation of luteolin to isoorientin through adjusting induction
strategy, controlling acetic acid and increasing UDP-glucose supply in
Escherichia coli
Jianjun Pei1, 3#, Qing Sun1,3#, Linguo Zhao1, 3*, Hao Shi , Feng Tang , Fuliang Cao
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1, 3
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College of Chemical Engineering, Nanjing Forestry University, Nanjing, China;
2Huanyin Institute of Technology, Huanyin, China
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Jiangsu Key Lab of Biomass-Based Green Fuels and Chemicals, Nanjing, China
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International Centre for Bamboo and Rattan, Beijing, China
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Corresponding author, Linguo Zhao. College of Chemical Engineering, Nanjing
Forestry University, Nanjing 210037, China. Phone: +86-025-85427962. E-mail:
njfu2302@163.com, lgzhao@njfu.edu.cn.
#
These authors contributed equally to this work
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Abstract
Isoorientin is a C-glycosylated derivative of luteolin and exhibits a number of
biological properties. In this study, multiple strategies were adopted to improve
isoorientin production from luteolin in Escherichia coli. Isoorientin production was
improved substantially by adjusting induction strategies and controlling acetic acid
accumulation, with maximum isoorientin production reaching 826 mg/L. Additionally,
a novel UDP-glucose synthesis pathway was reconstructed in E. coli through
cellobiose phosphorylase-catalyzed phosphorolysis of cellobiose for the production of
glucose 1-phosphate, which serves as a precursor in UDP-glucose formation. The
results from two mechanisms of UDP-glucose formation in E. coli, cellobiose
phosphorolysis and sucrose phosphorolysis, were compared. Increasing the
UDP-glucose supply resulted in maximal isoorientin production reaching 1371 mg/L.
Finally, isoorientin (1059 mg) was obtained from 1 L of fermentation broth by simple
purification steps with a yield of 81.5%. Therefore, this study provides an efficient
method for isoorientin production and a novel UDP-glucose synthesis pathway.
Keywords: Isoorientin; C-Glucosyltransferase; Cellobiose phosphorylase; Acetic acid;
UDP-glucose
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Introduction
C-glycosylflavones, which are conjugated with sugar residues through C-glycosidic
bonds, are present in food and health-care products such as wheat, maize, rice, mung
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bean, blueberries, lemon juice, acai pulp, rooibos tea and bamboo leave extracts .
Compared with O-glycosylation, C-glycosylation is highly stable to acid and resistant
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to glycosidase hydrolysis . Isoorientin (3’,4’,5,7-tetrahydroxy-6-C-glucopyranosyl
flavone), a naturally occurring C-glycosylflavone, is found in many plants7, 9-11
.
Isoorientin has been reported to have many pharmacological activities, such as
anticancer, antioxidant, hypotensive, anti-inflammatory, antispasmodic, antimicrobial,
hepatoprotection, and antiglycation activities12-19. Although some C-glycosylflavones,
including isoorientin, have been extracted from bamboo leaves and Atractylodes
japonica7, 20, it is difficult to prepare them because of their low concentration and
complex composition in plants. Therefore, large-scale and low-cost C-glycosylflavone
production has become important.
In plants, C-glycosylflavones are synthesized by C-glucosyltransferases.
C-glycosylflavone-related genes from different plants have been reported21-26
.
C-glucosyltransferases can be divided into two groups on the basis of their substrate
specificity. The C-glucosyltransferases in the first group, which catalyze the
conversion of only the open-chain form of 2-hydroxyflavanone to C-glycosylflavone,
were identified in rice, maize, and buckwheat21-24. 2-Hydroxyflavanone is generated
from flavanone by the cytochrome P450 flavanone 2-hydroxylase in plants27-28
.
Subsequent to this discovery, Brazier-Hicks reconstructed a biosynthetic pathway of
C-glycosylflavone in yeast by introducing a cytochrome P450 flavanone
2-hydroxylase gene and a C-glucosyltransferase gene from Oryza sativa. The
recombinant yeast biosynthesized 2-hydroxynaringenin-C-glucoside from flavanone,
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with a maximum titer of 7.2 mg/L . Because it is difficult to express cytochrome
P450 flavanone 2-hydroxylase genes in Escherichia coli and to prepare the
open-chain form of 2-hydroxyflavanone, recombinant E. coli was not engineered to
produce orientin, vitexin, isoorientin, or isovitexin using the first group of
C-glucosyltransferases.
The second group of C-glucosyltransferases directly C-glucosylates the C-6
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position of flavones, which has been identified only in Gentiana triflora . A
C-glucosyltransferase (Gt6CGT) gene from G. triflora was introduced to first
construct a recombinant E. coli strain to optimize the UDP-glucose synthetic pathway,
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and the maximum titer of isoorientin in this strain reached 34 mg/L . Thus, it is
necessary to improve C-glycosylflavone production towards low-cost production.
The soluble expression of key enzymes, enzymatic activity, supply of UDP-glucose,
regulation of metabolic processes, and fermentation conditions all affect
C-glycosylflavone production in E. coli. Many recombinant proteins from plants often
fail to fold into their native state and accumulate as inclusion bodies when the
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respective genes expressed in E. coli . Thus, expression of genes encoding
C-glucosyltransferases with high specific activity is essential to improve the
production of C-glycosylflavone in E. coli. On the other hand, the most common
byproduct, acetic acid, which is synthesized by phosphotransacetylase/acetate kinase
and pyruvate oxidase in E. coli, can reduce the level of recombinant enzymes and
inhibit the growth of E. coli31-32. Therefore, avoiding the accumulation of acetic acid
in the fermentation process is crucial to the expression of Gt6CGT and
C-glycosylflavone production.
In addition, a reasonable supply of UDP-glucose in vivo is also important to
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produce C-glycosylflavone . Supply of UDP-glucose must meet the catalytic
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efficiency of C-glucosyltransferase. Two methods, strengthening the endogenous
UDP-glucose synthesis pathway and introducing the exogenous UDP-glucose
synthesis pathway, have been used to improve the UDP-glucose supply in E. coli30,
34-35. In this study, a novel UDP-glucose synthetic pathway was reconstructed in E.
coli by introducing the cellobiose phosphorylase gene (cep) from Saccharophagus
degradans and the UTP-glucose-1-phosphate uridylyltransferase gene (ugpA) from
Bifidobacterium bifidum33, 36. Cellobiose phosphorylase can split cellobiose into
glucose and glucose 1-phosphate. Subsequently, glucose 1-phosphate will be
converted into UDP-glucose by UgpA. Cellobiose phosphorylase-catalyzed
phosphorolysis of cellobiose has many advantages such as its circumvention of
catabolite repression, better tolerance of common inhibitors under both anaerobic and
aerobic conditions, control of favorable energy metabolism and increases in the
expression of heterologous genes36-37. Thus, introducing cep and ugpA genes into E.
coli might improve isoorientin production.
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In this paper, the aim was to increase isoorientin biosynthesis in recombinant E.
coli by optimizing the soluble expression of C-glucosyltransferase (Gt6CGT),
adjusting induction strategies, controlling acetic acid accumulation and increasing the
supply of UDP-glucose, which resulted in successful improvement in isoorientin
production on the fermentation level (Fig. 1).
Materials and methods
Strains, plasmids, media, and chemicals
All plasmids and strains used in this research are listed in Table 1. The strains were
grown at 37°C in Luria-Bertani (LB) medium that was supplemented with antibiotics
when required. Luteolin was purchased from Shaanxi Huike Botanical Development
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Co., Ltd (Xian, China). Isoorientin was purchased from MUST Bio-Technology
(Chengdu, China). LB medium contained 10 g/L tryptone, 5 g/L yeast extract, and 10
g/L NaCl; TB-Gly medium contained 12 g/L tryptone, 24 g/L yeast extract, 2.32 g/L
KH PO , 12.54 g/L K HPO , and 10 g/L glycerol; TB-Glc contained 12 g/L tryptone,
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24 g/L yeast extract, 2.32 g/L KH PO , 12.54 g/L K HPO , and 10 g/L glucose;
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TB-Dex contained 12 g/L tryptone, 24 g/L yeast extract, 2.32 g/L KH PO , 12.54 g/L
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K HPO , and 10 g/L dextrin; and TB-Mal contained 12 g/L tryptone, 24 g/L yeast
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extract, 2.32 g/L KH PO , 12.54 g/L K HPO , and 10 g/L maltodextrin.
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Plasmid construction
Gt6CGT (AB985754.1) was synthesized to incorporate E. coli codons. The NcoI site
was added to the 5ʹ ends of the gene, the BamHI site was added to the 3ʹ ends of the
gene, and six histidine residues were fused to the C-termini of recombinant enzyme.
Gt6CGT was digested with NcoI and BamHI and subcloned into the expression vector
pCDFDuet-1 at the NcoI and BamHI sites to create pCDFDuet-Gt6CGT.
A cellobiose phosphorylase gene (cep, GenBank no. ABD80580.1) was synthesized
to incorporate E. coli codons. An NcoI site was added to the 5’ end of the gene, and an
EcoRI site was added to the 3’ end of the gene. The synthesized gene (cep) was
subcloned into pACYCDuet-1 at the NcoI and EcoRI sites to create
pACYCDuet-Cep.
A UTP-glucose-1-phosphate uridylyltransferase gene (ugpA, GenBank no.
YP_003971086.1) was obtained from pACYCDuet-cscB-Basp-UgpA and digested
with NdeI and KpnI. Then, ugpA was subcloned into pACYCDuet-Cep at the NdeI
and KpnI sites to produce pACYCDuet-Cep-UgpA.
Expression of Gt6CGT
The plasmid pCDFDuet-Gt6CGT was use for the transformation of E. coli BL21(DE3)
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to obtain the recombinant strain BL21-CGT-I. The plasmids pCDFDuet-Gt6CGT and
pG-KJE8 were used for the co-transformation of E. coli BL21 (DE3) cells to obtain
the recombinant strain BL21-CGT-KJE8. The plasmids pCDFDuet-Gt6CGT and
pGro7 were used for the co-transformation of E. coli BL21 (DE3) cells to obtain the
recombinant strain BL21-CGT-pGro7. The plasmids pCDFDuet-Gt6CGT and pKJE7
were used for the co-transformation of E. coli BL21 (DE3) cells to obtain the
recombinant strain BL21-CGT-pKJE7. The plasmids pCDFDuet-Gt6CGT and
pG-Tf2 were used for the co-transformation of E. coli BL21 (DE3) cells to obtain the
recombinant strain BL21-CGT-Tf2. The plasmids pCDFDuet-Gt6CGT and pTf16
were used for the co-transformation of E. coli BL21 (DE3) cells to obtain the
recombinant strain BL21-CGT-pTf16. Recombinant strains were cultured in 5 mL of
LB with appropriate antibiotics overnight. Seed culture (1 mL) was added to 50 mL of
fresh LB medium with appropriate antibiotics. Cultures were incubated at 37°C until
the OD600 reached 0.5-0.6, then induced to express recombinant Gt6CGT by adding
isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration between 0.1 and
0.2 mM, and incubated further at 20°C for 24 h, 30°C for 12 h or 37°C for 8 h.
Expression of Gt6CGT was examined by SDS-PAGE and the detection of
C-glucosyltransferase activity.
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C-glucosyltransferase activity
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C-glucosyltransferase activity was measured as described previously . The reaction
mixture, which contained 0.5 mM luteolin as a substrate, 1 mM UDP-glucose, 50 mM
phosphate buffer (pH 7.5), and various amounts of Gt6CGT in 100 μL, was incubated
for 30 min at 50°C. The reaction was stopped by adding 400 μL of methanol, and its
products were assayed via high-performance liquid chromatography (HPLC). One
unit of enzyme activity was defined as the amount of enzyme necessary to synthesize
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1 μmol of isoorientin per min under the assay conditions.
Isoorientin production by recombinant strains
The recombinant strains (BL21-CGT-I) were added to 50 mL of fresh medium in
250-mL shake flasks containing appropriate antibiotics and grown at 37°C until the
OD600 reached 0.6. A total of 900 mg/L luteolin and 0.1 mM IPTG were added to
BL21-CGT-I. Fermentation broths were incubated at 20°C and 180 rpm for 24 h or 48
h. Methanol (five volumes) was added directly to the fermentation broths. The
supernatant was harvested by centrifugation at 12,000 x g for 5 min and analyzed
using HPLC.
The effects of the induction temperature on isoorientin production were determined
by induction at different temperatures (20°C, 30°C, and 37°C) with 0.1 mM or 0.2
mM IPTG. To determine the effects of induction strategies on isoorientin production,
BL21-CGT-I was first induced at 20°C with 0.1 mM IPTG for 3 h, 6 h, or 9 h, and
then 900 mg/L luteolin was added to produce isoorientin for 48 h. The effects of
different carbon sources on isoorientin production were determined by growth in
different media (LB, TB-Gly, TB-Glc, TB-Dex, TB-Mal) in 250-mL shake flasks.
The samples were measured using a method similar to that described above.
Effects of UDP-glucose supply on isoorientin production
To determine the effects of UDP-glucose supply on isoorientin production in E. coli,
the plasmids pCDFDuet-Gt6CGT and pACYCDuet-cscB-Basp-UgpA were used for
the co-transformation of E. coli BL21 (DE3) cells to obtain the recombinant strain
BL21-CGT-II. The plasmids pCDFDuet-Gt6CGT and pACYCDuet-Cep-UgpA were
used for the co-transformation of E. coli BL21(DE3) cells to obtain the recombinant
strain BL21-CGT-III. The recombinant strains were cultured in 5 mL of LB with
streptomycin (50 μg/mL) and chloramphenicol (40 μg/mL) overnight. Seed culture (1
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mL) was added to 50 mL of fresh TB-Gly medium containing streptomycin and
chloramphenicol and was grown at 37°C until the OD600 reached 0.6. The broth was
incubated at 20°C for 6 h after the addition of 0.1 mM IPTG. Then, 900 mg/L luteolin
was added to the broth, which was incubated at 20°C for 116 h, and different
concentrations of cellobiose were added to the broth at different times after the
addition of luteolin.
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Preparation and structural identification of isoorientin
BL21-CGT-III was cultured in 50 mL of LB with streptomycin and chloramphenicol
overnight. Seed culture (50 mL) was added to 1000 mL of fresh TB-Gly medium in
3000-mL shake flasks containing 50 μg/mL streptomycin and 40 μg/mL
chloramphenicol and was grown at 37°C until the OD600 reached 0.6. The broth was
incubated at 20°C for 6 h after the addition of 0.1 mM IPTG. Then, 900 mg/L luteolin
was added to the broth, which was incubated at 20°C for 116 h, and 2% cellobiose
was added to the broth at 12 h after adding luteolin. The broth was harvested by
centrifugation (20,000 g, 4°C, 10 min) to remove the cells and then applied to an
AB-8 column macroporous resin (2.5 x 30 cm, Jianghua, China) equilibrated with
distilled water (10 volumes) and eluted with 100 mL of different concentrations of
ethanol (5%, 30%, 50% and 80%). The sample was evaporated to dryness and
analyzed by LC/MS and HPLC.
Acetic acid quantitation
The acetic acid concentration of the culture was determined by Agilent 7890A gas
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chromatography, and the sample was prepared as described previously . The column
temperature began at an initial temperature of 70°C for 3 min; then, it was raised to
230°C at an incremental rate of 8°C per minute, and it was held for 3 min (30 m x
0.25 mm x 0.25 μm, HP-INNOWAX, Agilent) with N (2 mL/min) as the carrier gas.
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The detector temperature was 300°C. The flow rate of the tail gas (N ) was 25
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mL/min, the H flow rate was 30 mL/min, and the air flow rate was 400 mL/min.
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Sugar analysis
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Maltodextrin and dextrin were determined by the phenol-sulfuric acid method .
Glucose and sucrose were determined with an HPLC 1200 system (Agilent, USA) and
a Prevail Carbohydrate ES 5 μm column (250 mm×4.6 mm; Grace, USA) according
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to a previous method . Glycerol was determined using the Glycerol Assay Kit
(Jiancheng Bioengineering Institute, Nanjing, China).
HPLC and LC/MS analysis
HPLC analysis of luteolin and isoorientin was performed using an HPLC 1200 system
(Agilent, USA) and a C18 (250 × 4.6 mm; i.d., 5 m) column with methanol (A) and
distilled water (B) at an A/B ratio of 55:45 for 15 min. The flow rate was 0.8 mL/min,
and detection was performed by monitoring the absorbance at 368 nm. LC/MS
analysis for luteolin and isoorientin was performed with an LTQ Orbitrap XL
(ThermoFisher Scienticfic, USA) in negative mode with an ion trap analyzer. The ion
spray was operated at 25 arbitrary units N /min, 3.5 kV, and 300°C.
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Results and discussion
Effects of soluble expression of Gt6CGT on isoorientin production
Recently, several C-glucosyltransferase genes have been cloned and characterized
from plants21-26. C-glucosyltransferase from Oryza sativa (rice) has been used for
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synthesizing natural and novel C-glycosylflavones from UDP-glucose in vitro .
Brazier-Hicks reconstructed the recombinant yeast to produce C-glycosylflavones by
co-expressing the rice C-glucosyltransferase gene with the flavanone 2-hydroxylase
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gene derived from either licorice, or rice . But most of C-glucosyltransferases
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catalyze the conversion of only the open-chain form of 2-hydroxyflavanone to
C-glycosylflavone. C-glucosyltransferase (Gt6CGT, AB985754.1) catalyzes the
generation of C-glycosylflavones (isoorientin and isovitexin) using flavones (luteolin,
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apigenin) as substrates and has been reported only in G. triflora . Luteolin is
inexpensive and comparatively easy to prepare. Thus, it is feasible to reconstruct a
recombinant strain for producing C-glycosylflavone from luteolin. A recombinant E.
coli strain was successfully constructed by introducing the Gt6CGT gene, and the
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maximum titer of isoorientin was 34 mg/L . Because the expression and activities of
key enzymes were closely related to metabolite production in E. coli, to increase the
expression of Gt6CGT, Gt6CGT was optimized by utilizing the codons preferred by E.
coli.
The recombinant Gt6CGT protein was expressed by adding 0.1 mM IPTG and
incubating the culture at 37°C for approximately 6 h. However, the overexpression of
Gt6CGT resulted in the production of large amounts of inclusion bodies. The activity
of Gt6CGT was only 0.1 nmol/min/mg in the recombinant strain BL21-CGT-I (Fig.
2A, lane 3-4). Molecular chaperones were introduced by plasmids pG-KJE8, pGro7,
pKJE7, pG-Tf2, and pTf16 to reduce inclusion body formation by Gt6CGT in
BL21-CGT40-41. However, compared to those in BL21-CGT, the activity of Gt6CGT
and isoorientin production in these recombinant strains did not improve (data not
shown). To reduce inclusion body formation by Gt6CGT, we expressed the gene
using different strategies such as the use of low-temperature induction and low
inducing agent concentrations30, 42. Only a small proportion of Gt6CGT was soluble in
the cell-free extracts, and most of the enzyme was in inclusion bodies under induction
at 30°C with 0.1 mM IPTG (Fig. 2A). Expression at 20°C with 0.1 mM IPTG
decreased the inclusion body formation, and the activity of Gt6CGT reached 3.2
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nmol/min/mg, which was approximately 32 times higher than that at 37°C with 0.1
mM IPTG (Fig. 2A). But expression at 16°C with 0.05 or 0.1 mM IPTG did not
improve the activity of Gt6CGT. These results suggested that it was difficult to
further improve the activity of Gt6CGT by reducing the expression of Gt6CGT in E.
coli. Isoorientin production under different induction conditions indicated the same
behavior in BL21-CGT. The highest isoorientin production was 47 mg/L, which
occurred when the recombinant strain was induced at 20°C with 0.1 mM IPTG; this
production was higher than that at 30°C or 37°C with 0.1 mM IPTG (Fig. 2B). These
results indicated that the activity of Gt6CGT was closely related to isoorientin
production, and it was difficult to further improve isoorientin production by reducing
the amount of Gt6CGT inclusion bodies in E. coli. Some fusion tags such as
maltose-binding protein (MBP), thioredoxin (Trx), N-utilization substance (NusA)
and glutathione-S-transferase (GST) have been used to enhance the solubility of
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expressed proteins in E. coli . Want et al. developed an activity-independent form of
SE-PACE (soluble expression phage-assisted continuous evolution) to correct
folding-defective variants of maltose-binding protein (MBP) and to improve the
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solubility of proteins . These methods provide opportunities for further improving the
soluble expression of Gt6CGT in E. coli.
Effects of induction strategies on isoorientin production
Flavonoids always have antibacterial activity and inhibit the growth of E. coli45-46, so
adding luteolin might affect the expression of Gt6CGT in the recombinant strain. The
expression of Gt6CGT would be inhibited if luteolin is added too early, and
recombinant strains would enter the death phase if luteolin is added too late. Thus, to
improve isoorientin production, a reasonable induction and conversion strategy was
adopted. The recombinant strain BL21-CGT-I was first induced for a period of time to
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express Gt6CGT, after which luteolin was added for the production of isoorientin. As
shown in Fig. 3, isoorientin production significantly increased when the recombinant
strain was induced according to this strategy. The best strategy was performing the
first induction for 6 h, and then adding luteolin. The maximum isoorientin production
was 168 mg/L in LB medium for 48 h of bioconversion; this amount was
approximately 2 times higher than that of the control (Fig. 3A). The same results were
found in TB-Gly medium, and the maximum isoorientin production according to this
strategy was 545 mg/L, which was approximately 4.3 times higher than that of the
control (Fig. 3B). These results indicated that adjusting the timing of luteolin addition
to improve isoorientin production is an effective method despite being very simple.
Effects of carbon sources on the accumulation of acetic acid and isoorientin
production
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Acetic acid inhibits the expression of enzymes and the growth of E. coli . Because
the activity of Gt6CGT was closely related to isoorientin production, acetic acid could
decrease isoorientin production through inhibiting the expression of Gt6CGT. The
main cause of acetic acid production was that the capacity of the tricarboxylic acid
cycle or cellular respiration could not keep up with the carbon flooding into the
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central metabolic system .
Different carbon sources could affect acetic acid production through adjusting the
carbon metabolism rate in recombinant strains. Thus, the effects of different carbon
sources (those in TB-Gly, TB-Glc, TB-Dex, and TB-Mal) on isoorientin production
were determined. As shown in Fig. 4, the highest isoorientin production was 826
mg/L, which was found at 80 h in TB-Gly and was approximately 730% of that (113
mg/L) in TB-Glc (Fig. 4C,D). Isoorientin production reached 376 and 358 mg/L at 80
h in TB-Mal and TB-Dex, respectively, which were also higher than that in TB-Glc
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(Fig. 4A,B,D). In contrast, the consumption rate of glucose was far higher than those
of dextrin and maltodextrin in E. coli (Fig. 4A,B), and the concentration of acetic acid
in TB-Glc was 1655 mg/L at 32 h, which was higher than those in TB-Dex (371 mg/L)
and TB-Mal (317 mg/L) (Fig. 4A,B,D). Glycerol as the carbon source could improve
the expression of recombinant enzymes by decreasing specific growth rate and acetic
acid accumulation.
The concentration of glycerol could also affect acetic acid accumulation in E. coli.
As shown in Fig. 5, acetic acid accumulation increased significantly with increasing
concentrations of glycerol, which resulted in a reduction in isoorientin production.
Isoorientin production was only 265 mg/L in TB-Gly medium containing 4% glycerol,
which was 33% of that in TB-Gly medium containing 1% glycerol. However, the
concentration of acetic acid in TB-Gly medium containing 4% glycerol was
approximately 2.4 times higher than that in medium containing 1% glycerol (Fig. 5).
The flux of additional carbon into the central metabolic system could cause
metabolic imbalance, which results in excessive acetic acid production by E. coli32
and acetic acid can inhibit the expression of recombinant enzymes. These results
indicated that decreasing the rate of carbon source consumption to inhibit acetic acid
accumulation is critical to improving isoorientin production in E. coli. However,
consuming carbon sources too slowly could also not be conducive to improving
isoorientin production in E. coli. The consumption rates of dextrin and maltodextrin
were slower than those of glycerol, and isoorientin production in TB-Dex and TB-Mal
was only 50% of that in TB-Gly, although the concentration of acetic acid in TB-Dex
and TB-Mal was lower than that in TB-Gly. Thus, controlling the concentration of
acetic acid by adding appropriate carbon sources was crucial to improving isoorientin
production in E. coli.
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Effects of UDP-glucose supply on isoorientin production
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The UDP-glucose supply in E. coli is another important factor in isoorientin
production because Gt6CGT catalyzes the transfer of a glucose residue from its
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activated form (UDP-glucose) to flavone to form isoorientin . The intracellular
UDP-glucose pool can be efficiently increased by introducing sucrose permease
(cscB), sucrose phosphorylase (Basp) and uridylyltransferase (UgpA) genes30, 33-34
.
These enzymes use sucrose to synthesize UDP-glucose. The plasmids
pCDFDuet-Gt6CGT and pACYCDuet-cscB-Basp-UgpA were used for the
co-transformation of E. coli to obtain the recombinant strain BL21-CGT-II. However,
isoorientin production in BL21-CGT-II was only 445 mg/L in TB-Gly medium
without the addition of sucrose, which was far lower than that in BL21-CGT-I (Fig.
6A, 4C). These results indicated that the expression of cscB, BasP and ugpA could
affect the expression of Gt6CGT or the physiological state of E. coli. With increasing
addition of sucrose, isoorientin production decreased continuously, and the
concentration of acetic acid increased continuously (Fig. 6A). The recombinant strains
harboring the cscB, Basp and ugpA genes have proven that sucrose could be rapidly
transferred and metabolized, resulting in an accumulation of acetic acid and a
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reduction in isoorientin production . Thus, manipulating the UDP-glucose synthesis
pathway in this manner was not suitable for isoorientin production in this study.
Cellobiose phosphorylase-catalyzed cellobiose assimilation has many advantages
such as its circumvention of catabolite repression, tolerance of common inhibitors,
and ability to increase the expression of recombinant protein36-37. Thus, a novel
UDP-glucose synthesis pathway was reconstructed in the recombinant strain by
introducing the cellobiose phosphorylase gene (cep) from S. degradans and the
uridylyltransferase gene (ugpA) from B. bifidum33, 36-37. As shown in Fig. 6B,
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isoorientin production decreased when the UDP-glucose synthesis pathway was
introduced into E. coli, resulting in 610 mg/L titer in BL21-CGT-III without the
addition of cellobiose. However, as cellobiose was added into fermentation broth,
isoorientin production increased significantly. Isoorientin production reached 1176
mg/L with the addition of 2% cellobiose, which was 193% of that in samples without
added cellobiose (Fig. 6B). Furthermore, the concentration of acetic acid was low,
even when the cellobiose concentration reached 40 g/L. These results suggested that
the reconstruction of the UDP-glucose synthesis pathway by introducing cep and
ugpA genes could enhance the supply of UDP-glucose and was conducive to
increasing isoorientin production in BL21-CGT-III. On the other hand, the merits of
cellobiose phosphorylase-catalyzed cellobiose assimilation could also be helpful for
the metabolism and growth of bacteria36-37, which is crucial for metabolite production
in E. coli. Therefore, the method described herein for the generation of UDP-glucose
from cellobiose could be widely used for the glycosylation of flavonoids and other
bioactive substances.
Subsequently, the optimal time to add cellobiose was determined in BL21-CGT-III.
As shown in Table 2, isoorientin production changed according to the time at which
cellobiose was added. The maximum isoorientin production reached 1206 mg/L in
BL21-CGT-III when cellobiose was added 12 h after the luteolin had been added.
Isoorientin production increased slightly with the addition of cellobiose at the end of
fermentation, reaching only 766 mg/L when cellobiose was added 60 h after luteolin
had been added.
Time courses for isoorientin production in BL21-CGT-III under the optimal
conditions are shown in Fig. 7. The concentration of acetic acid remained moderately
low for the whole period of growth in BL21-CGT-III with the addition of cellobiose,
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and the consumption rate of cellobiose in BL21-CGT-III was 0.13 g/L/h. These
results indicated that the recombinant strain harboring the novel UDP-glucose
synthesis pathway could slowly phosphorylate cellobiose to produce UDP-glucose
and avoid acetic acid accumulation. At the beginning of bioconversion, the specific
productivity was 3.6 mg/L/h during hours 8-32 of bioconversion time. With the
addition of cellobiose, the specific productivity gradually increased. The specific
productivity was 15.3 mg/L/h during hours 32-116 of bioconversion, which was
approximately 4 times higher than that at the beginning of bioconversion. Finally,
maximal isoorientin production reached 1371 mg/L at 116 h with a corresponding
molar conversion of 97.4%, which was the highest titer of isoorientin reported to date
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and the first to reach the gram scale in E. coli . Thus, this study presented a novel
UDP-glucose synthesis pathway with two distinct characteristics: it avoided acetic
acid accumulation and slowly and continuously generated UDP-glucose.
In the previous decade, several flavonoid glycosides have already been produced in
recombinant strains, which were constructed by introducing UDP-dependent
glycosyltransferase genes and the UDP-glucose synthesis pathway30, 33-34. De Bruyn et
al. developed in vivo glycosylation platform in E. coli W, resulting in production of
hyperoside and quercitrin reaching 0.94 g/L and 1.12 g/L, respectively33-34. But most
research has focused on O-glycosylflavones and the production of C-glycosylflavones
was also low in recombinant strains. In this study, isoorientin production was the first
to reach the gram scale by adjusting induction strategies, controlling acetic acid
accumulation and increasing UDP-glucose supply in E. coli. The general
phenylpropanoid pathway has been clarified in plants and some intermediate
metabolites such as pinocembrin (429 mg/L) and naringenin (329 mg/L) have been
produced in E. coli by constructing the phenylpropanoid biosynthesis pathway47-49
.
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C-glycosylflavones are synthesized from flavones by flavone synthases and
C-glucosyltransferases. Thus, based on this result, an efficient isoorientin synthesis
pathway from glucose could be reconstructed in E. coli.
Preparation of isoorientin from fermentation broth
The broth was harvested by centrifugation to remove the cells and residual luteolin.
Most of the residual luteolin was removed by centrifugation, and the yield of
isoorientin reached 90.1% (Table 3, Fig. 8C) because luteolin had poor water
solubility compared with isoorientin. Then, the sample was applied to an AB-8
macroporous resin column equilibrated with distilled water, and isoorientin was eluted
with 30% ethanol and evaporated to dryness (Fig. 8D). Isoorientin (1059 mg) was
obtained by these simple purification steps with a yield of 81.5% (Table 3, Fig. 8).
The purity of the product was more than 95% as determined by LC/MS and HPLC
-
(Fig. 8). An analysis of the m/z value of the molecular ion [M-H] of the
bioconversion product (447.0930) showed that its value corresponded to a D-glucose
adduct of luteolin (Fig. 8E), and the product had a retention time similar to that of
authentic isoorientin (Fig. 8). These results confirmed that the bioconversion product
was isoorientin.
Acknowledgements
This work was supported by the National Key R&D Program of China
(2017YFD0600805), the National Natural Science Foundation of China (31570565),
the Open Foundation of Jiangsu Provincial Engineering Laboratory for Biomass
Conversion and Process Integration (JPELBCPI2017002), the Qing Lan Project and
the Priority Academic Program Development of Jiangsu Higher Education Institutions
(PAPD).
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Figure legends
Fig. 1 The schematic diagram for improvement of isoorientin production from
luteolin in E. coli. Abbreviations for enzymes: LacY, lactose permease; Cep,
cellobiose phosphorylase; UgpA, uridylyltransferase.
Fig. 2 Effects of soluble expression of Gt6CGT on isoorientin production. (A)
SDS-PAGE analysis of recombinant Gt6CGT in BL21-CGT-I. Lane M: protein
marker, lane 1, 3, 5, 7, 9: total protein in BL21-CGT-I; lane 2, 4, 6, 8, 10: soluble
protein in BL21-CGT-I. (B) Isoorientin production in BL21-CGT-I. Arrow
represented recombinant Gt6CGT.
Fig. 3 Effects of induction strategies on isoorientin production in LB medium (A) and
TB-Gly medium (B).
Fig. 4 Effects of carbon sources on accumulation of acetic acid and isoorientin
production. (A) Accumulation of acetic acid and isoorientin production in TB-Mal. (B)
Accumulation of acetic acid and isoorientin production in TB-Dex. (C) Accumulation
of acetic acid and isoorientin production in TB-Gly. (D) Accumulation of acetic acid
and isoorientin production in TB-Glc.
Fig. 5 Effects of different concentrations of glycerol on accumulation of acetic acid
and isoorientin production.
Fig. 6 Effects of different concentrations of sucrose and cellobiose on accumulation of
acetic acid and isoorientin production. (A) Effects of sucrose on accumulation of
acetic acid and isoorientin production in BL21-CGT-II at 104 h. (B) Effects of
cellobiose on accumulation of acetic acid and isoorientin production in BL21-CGT-III
at 104 h.
Fig. 7 Time course for isoorientin production under optimal conditions.
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Fig. 8 HPLC and LC/MS analysis of isoorientin production. (A) Authentic isoorientin
and luteolin. (B) HPLC analysis of fermentation broth. (C) HPLC analysis of the
sample by centrifugation. (D) HPLC analysis of the sample by AB-8 column. (E)
LC/MS analysis of isoorientin production.
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Table 1 Plasmids and strains used in this study
Descriptions
Plasmids/strains
Plasmids
References
pCDFDuet-1
pACYCDuet-1
CDF ori; Strepr
P15A ori; Cmr
Novagen
Novagen
pCDFDuet-Gt6CGT pCDFDuet-1 carrying Gt6CGT from Gentiana This study
triflora, T7 promoter.
pACYCDuet-Cep
pACYCDuet-1
carrying
cellobiose This study
phosphorylase gene (cep) from Saccharophagus
degradans, T7 promoter.
pACYCDuet-Cep-U pACYCDuet-1
gpA Saccharophagus
carrying
cep
from This study
and
degradans
UTP-glucose-1-phosphate uridylyltransferase
gene (ugpA) from Bifidobacterium bifidum, T7
promoter.
3
0
pACYCDuet-cscB-B pACYCDuet carrying sucrose permease gene
asp-UgpA
(cscB) from E. coli W, sucrose phosphorylase
gene (Basp) from B. adolescentis, and ugpA
from B. bifidum, T7 promoter.
pG-KJE8
Carrying chaperone protein DnaK gene (dnaK), TaKaRa
chaperone protein DnaJ gene (dnaJ), chaperone
protein GrpE gene (grpE), chaperone protein
GroES gene (groES) and chaperone protein
GroEL gene (groEL); dnaK, dnaJ and grpE
were promoted by araB promoter. grpES and
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groEL were promoted by tetR promoter.
Carrying groES and groEL genes; groES and TaKaRa
groEL were promoted by araB promoter.
Carrying dnaK, dnaJ and grpE genes; dnaK, TaKaRa
dnaJ and grpE were promoted by araB
promoter.
pGro7
pKJE7
pG-Tf2
pTf16
Carrying groES and groEL genes, and trigger TaKaRa
factor gene (tig); groES, groEL and tig were
promoted by Pzt-1 promoter.
Carrying tig; tig was promoted by araB TaKaRa
promoter.
Strains
BL21 (DE3)
BL21-CGT-KJE8
Escherichia coli BL21 (DE3)
Novagen
BL21(DE3) harboring pCDFDuet-Gt6CGT and This study
pG-KJE8
BL21-CGT-pGro7
BL21-CGT-pKJE7
BL21-CGT-Tf2
BL21(DE3) harboring pCDFDuet-Gt6CGT and This study
pGro7
BL21(DE3) harboring pCDFDuet-Gt6CGT and This study
pKJE7
BL21(DE3) harboring pCDFDuet-Gt6CGT and This study
pG-Tf2
BL21-CGT-pTf16
BL21(DE3) harboring pCDFDuet-Gt6CGT and This study
pTf16
BL21-CGT-I
BL21-CGT-II
BL21(DE3) harboring pCDFDuet-Gt6CGT
This study
BL21(DE3) harboring pCDFDuet-Gt6CGT and This study
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pACYCDuet-cscB-Basp-UgpA
BL21-CGT-III
BL21(DE3) harboring pCDFDuet-Gt6CGT and This study
pACYCDuet-Cep-UgpA
Table 2 Effects of cellobiose addition on isoorientin production in BL21-CGT-III
Strains
Cellobiose
g/L)
BL21-CGT-III 10
Time of cellobiose addition
Isoorientin (mg/L) a
(
0 h after adding luteolin
12 h after adding luteolin
24 h after adding luteolin
36 h after adding luteolin
48 h after adding luteolin
60 h after adding luteolin
1003 ± 19
1206 ± 21
998 ± 20
934 ± 17
831 ± 16
766 ± 18
1
1
1
1
1
0
0
0
0
0
a
The concentration of isoorientin was determined at 104 h.
Table 3 Purification of isoorientin from fermentation broth
Step
Isoorientin (mg)
Yield (%)
100%
Purity
ND
Fermentation broth 1300 ± 28
Centrifugation
1171 ± 16
1059 ± 15
90.1 ± 1.2%
81.5 ± 1.2%
ND
AB-8 column
95 ± 0.8%
ND: not determined.
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Figure 1
Figure 2
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