Download Constitutive expression of RyhB regulates the heme biosynthesis

RESEARCH LETTER
Constitutive expression of RyhB regulates the heme
biosynthesis pathway and increases the 5-aminolevulinic acid
accumulation in Escherichia coli
Fangfang Li1, Yang Wang1, Kai Gong1, Qian Wang2, Quanfeng Liang1 & Qingsheng Qi1
1
State Key Laboratory of Microbial Technology, Shandong University, Jinan, China; and 2National Glycoengineering Research Center, Shandong
University, Jinan, China
Correspondence: Qingsheng Qi, State Key
Laboratory of Microbial Technology,
Shandong University, Jinan 250100, China.
Tel./fax: +86 531 88365628;
e-mail: [email protected]
Received 30 July 2013; revised 24 October
2013; accepted 25 October 2013. Final
version published online 9 November 2013.
DOI: 10.1111/1574-6968.12322
Editor: Olga Ozoline
MICROBIOLOGY LETTERS
Keywords
iron-containing protein; metabolic
engineering; regulating sRNA.
Abstract
In the current study, the small RNA ryhB, which regulates the metabolism of
iron in Escherichia coli, was constitutively expressed in engineered E. coli
DALA. The resulting strain E. coli DALRA produced 16% more 5-aminolevulinic acid (ALA) than the parent strain E. coli DALA in batch fermentation.
Meanwhile, we found that addition of iron in the medium increased heme formation and reduced ALA yield, whereas the presence of iron chelator in the
medium decreased heme concentration and increased the ALA production efficiency (ALA yield per OD600). The qRT-PCR analysis showed that the mRNA
levels of hemB and hemH were also decreased as well as the known RyhB target
genes of acnAB, sdhAB, fumA, and cydAB in E. coli DALRA. These results indicated that small RNA can be used as a tool for regulating ALA accumulation
in E. coli.
Introduction
Heme is an essential iron-containing component of proteins in the electron transport chain that drives aerobic and
anaerobic respiration, for example CydAB (Pomka, 1999),
and is an important prosthetic group in many sensory
regulatory proteins and enzymes. In heme biosynthesis
pathway, 5-aminolevulinic acid (ALA) is a key intermediate that determines the accumulation of heme (Fig. 1).
Recently, ALA received much attention as a pharmaceutical
for cancer therapy and tumour diagnosis (Bhowmick &
Girotti, 2010; Mikolajewska et al., 2010; Sakamoto et al.,
2010). It can also be used as a kind of selective and biodegradable herbicide, insecticide and growth-promoting
factor (Rebeiz et al., 1988; Sasaki et al., 2002).
Two metabolic pathways have been described for ALA
biosynthesis (Sasaki et al., 2002). One is the C4 pathway,
which exists in mammals, birds, yeasts, some protozoa
and purple non-sulphur photosynthetic bacteria such as
Rhodobacter sphaeroides. In this pathway, ALA is formed
through the condensation of glycine and succinyl-CoA, a
C4 intermediate of the tricarboxylic acid (TCA) cycle.
The other pathway is the C5 pathway, which utilizes
FEMS Microbiol Lett 350 (2014) 209–215
a-ketoglutarate, a C5 intermediate of the TCA cycle, as
the carbon skeleton. This pathway is mainly present in
higher plants, algae, and many bacteria. In our previous
study, we engineered an Escherichia coli strain DALA that
can accumulate ALA using glucose as sole carbon source
through the C5 pathway (Kang et al., 2011). However,
further regulation of this pathway for high ALA production met with many problems because C5 pathway is
already highly regulated. For example, HemA and HemL
work synergistically, whereas HemA is not stable when
heme is present in excess (Wang et al., 1999). Our initial
study also indicated that down-regulation of porphobilinogen synthase (HemB), an enzyme that catabolizes ALA
in the heme biosynthesis pathway(Jaffe et al., 1995), did
not lead to high ALA production as expected, but instead
resulted in a decreased accumulation of ALA. Therefore,
other strategies will have to be employed to improve the
production of ALA.
Recently, a large amount of small non-coding RNA
(sRNA) has been identified in bacteria, especially in
E. coli, by comparative genomics, bioinformatics and
other screening strategies (Wassarman et al., 2001; Vogel
et al., 2003; Kawano et al., 2005). The majority of sRNAs
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210
F. Li et al.
Fig. 1. Schematic presentation of TCA cycle and heme (including ALA) biosynthesis pathway in Escherichia coli. The genes noted were
investigated by qRT-PCR. The dotted arrows present feedback inhibition of heme. The regulation sites (acnAB, sdhAB, fumA and cydAB) and
possible regulation sites (hemB, hemH) of RyhB are indicated. Genes: acnA, aconitate hydratase 1; acnB, bifunctional aconitate hydratase 2;
sdhAB, subunits of succinate dehydrogenase; gltX, glutamyl-tRNA synthetase; hemA, glutamyl-tRNA reductase; hemL, glutamate-1-semialdehyde
aminotransferase; hemB, porphobilinogen synthase; hemH, ferrochelatase. GSA, glutamate-1-semialdehyde; PBG, porphobilinogen.
play important roles in a variety of essential physiological
processes in vivo. Among these, RyhB, a small RNA of
90 nt in length, is involved in iron consumption under
iron-limiting conditions by down-regulating the expression of iron-containing proteins, including the enzymes
of the TCA cycle and the aerobic respiratory chain (Masse
& Gottesman, 2002; Masse et al., 2005; Semsey et al.,
2006). The last step of the heme biosynthesis pathway in
E. coli is the insertion of ferrous iron into the protoporphyrin IX, forming heme. As RyhB plays an important
role in iron homeostasis and can reduce iron-binding
proteins expression under low-iron conditions, it is very
likely to down-regulate the heme synthesis pathway if it is
overexpressed. Based on this supposition, we overexpressed ryhB in E. coli DALA and investigated its role in ALA
biosynthesis.
Material and methods
Bacterial strains and plasmids
The strains, plasmids and primers used in this research are
summarized in Supporting Information, Tables S1 and S2.
Molecular cloning was done using E. coli DH5a according
to the standard protocol. The ryhB gene was cloned from
the plasmid pKK102-ryhB (Kang et al., 2012). Only the
RNA polymerase binding region of PBAD and the ryhB
gene region were amplified to allow constitutive expression of ryhB. The amplified ryhB gene fragment was
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Published by John Wiley & Sons Ltd. All rights reserved
digested with KpnI and BamHI, and ligated into pCLRA
(Kang et al., 2011), which was digested by the same
restriction enzymes. The resulting plasmid was named
pCLRA-ryhB. The cloning scheme of ryhB and the construction procedure of E. coli DALRA are shown in Fig. 2.
Medium and culture conditions
Luria–Bertani medium (10 g L 1 tryptone, 5 g L 1 yeast
extract and 10 g L 1 NaCl, pH 7.2) was used for bacterial
cultivation during genetic operation. The modified minimal medium (16 g L 1 (NH4)2SO4, 3 g L 1 KH2PO4,
16 g L 1 Na2HPO412H2O, 1 g L 1 MgSO47H2O, 0.01 g
L 1 MnSO47H2O and 2 g L 1 yeast extract, pH 7.0;
Kang et al., 2011) was used for cultivation and fermentation. Glucose (20 g L 1) was added initially as carbon
source. Ampicillin (100 lg mL 1) and spectinomycin
(25 lg mL 1) were added to provide selective pressure
for plasmid stability during cultivation. Isopropyl-b-D-thiogalactopyranoside was used at a final concentration of
0.1 mM to induce the expression of plasmid-borne
genes whose expression was under the control of the lac
promoter.
Bacterial pre-culture was done in modified minimal
medium using 2 g L 1 glucose as carbon source for 12 h.
The pre-culture was then inoculated at 1% (v/v) volume
into 50 mL fresh medium containing 20 g L 1 glucose
and cultivated at 37 °C, 250 r.p.m. for 32 h. When necessary, 100 lM FeSO4 or 250 lM 2, 2′-dipyridyl was added
FEMS Microbiol Lett 350 (2014) 209–215
211
RyhB expression in E. coli
Fig. 2. Cloning scheme of ryhB and construction procedure of Escherichia coli DALRA.
after 4 h cultivation. The pH was monitored every 4 h
and 5 M NaOH was added to maintain the pH at 7.0.
Fermentation samples were taken at 4-h intervals to measure OD600, ALA and heme concentration.
Quantitative real-time PCR
The primers used in quantitative real-time PCR (qRTPCR) are listed in Table S2. Total cellular mRNA was
FEMS Microbiol Lett 350 (2014) 209–215
extracted with RNAeasy Mini Kit (Tiangen) after strains
were cultivated in a shake flask for 4 h. The cDNA was
obtained by reverse transcription using PrimeScript RT
reagent Kit (TaKaRa) and qRT-PCR was carried out
using SYBR Premix Ex TaqTM II (TaKaRa) with the
LightCycler 480 Real-Time PCR system (Roche). The
mRNA level of each genes was measured using three biological repeats and three technical repeats. Gene gapA was
used as the control because it was stable and was
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212
F. Li et al.
expressed constantly. The qRT-PCR data were analyzed
using the 2 DDCt protocol reported previously (Livak &
Schmittgen, 2001).
Analytical methods
Optical density was monitored at a wavelength of
600 nm with a spectrophotometer (Shimadzu). The concentration of organic acids and glucose was analyzed by
high-performance liquid chromatography (HPLC). Samples were prepared by centrifugation (12 000 g for 5 min
at 4 °C) and filtration (0.22 lm syringe filter). The
HPLC system (Shimadzu) was equipped with a cation
exchange column (HPX-87 H; BioRad Labs) and a
differential refractive index detector (Shimadzu RID-10
A). H2SO4 (0.5 mM) was used as the mobile phase at
the rate of 0.6 mL min 1. The exchange column was
operated at 65 °C. To analyze the concentration of extracellular ALA and intracellular heme, 1 mL of each culture sample was centrifuged (12 000 g for 5 min at
4 °C). The supernatant was used to measure ALA concentration using modified Ehrlich’s reagent (Mauzerall &
Granick, 1956). The cell pellet was used to analyze heme
according to the method described by Sassa (1976).
Glutamate was analyzed by a SBA-40C biosensor (developed by Biology Institute of Shandong Academy of Sciences) equipped with an immobilized glutamate oxidase
membrane.
Results
Constitutive overexpression of ryhB increased
ALA production and decreased heme
accumulation
To investigate the effect of sRNA RyhB on the heme
biosynthesis pathway, we subcloned ryhB into the plasmid pCLRA downstream of a constitutive promoter,
generating plasmid pCLRA-ryhB (Fig. 2). The recombinant E. coli strain harbouring pCLRA-ryhB, named
DALRA, was then evaluated with respect to its growth
and ALA accumulation by cultivation in modified minimum medium supplemented with 20 g L 1 glucose. As
expected, we found that overexpression of ryhB led to
increased ALA production from 1.54 to 1.78 g L 1 in
batch fermentation, 16% more than that of the control
(Fig. 3). However, the accumulation of heme, which is
located downstream of ALA in the heme biosynthesis
pathway, decreased significantly in E. coli DALRA. The
heme accumulation in strain DALRA was only
0.232 nmol OD 1, 56.6% less than that of the control.
In addition, the cell growth was also slightly affected. At
32 h, the OD600 of E. coli DALA and DALRA was 13.13
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Fig. 3. Effect of ryhB overexpression on cell growth, ALA production
and heme accumulation. The pre-culture was inoculated at 1% (v/v)
into 50 mL medium with 20 g L 1 glucose and cultivated at 37 °C,
250 r.p.m. for 32 h. Fermentation samples were taken at an interval
of 4 h for measuring OD600, ALA and heme concentration. Results
are the average of three individual experiments.
and 13.95, respectively. As several metabolic pathways
are affected by RyhB, overexpression of this sRNA shall
have overall influence on the host, especially the heme
biosynthesis pathway.
Overexpression of RyhB changed metabolic
flux and down-regulated iron-containing
proteins
As previous studies suggested that the key genes in the
TCA cycle (acnAB, sdhAB, fumA), all of which encoded
iron-containing proteins, were targets of RyhB (Masse &
Gottesman, 2002; Masse et al., 2005), we measured the
concentration of the metabolic intermediates of E. coli
DALA and DALRA (Table 1) and analyzed the transcription of these target genes by qRT-PCR (Fig. 4). When glucose was depleted, E. coli DALRA accumulated 0.237 g
L 1 succinate and 0.325 g L 1 glutamate, respectively,
whereas DALA accumulated 0.170 g L 1 succinate and
0.148 g L 1 glutamate. There was no significant difference
in lactate and acetate accumulation between E. coli DALA
Table 1. Analysis of metabolic intermediates of Escherichia coli DALA
and E. coli DALRA
Metabolic
intermediates
Concentration (g L 1)
DALA
Glutamate
Succinate
Lactate
Acetate
0.148
0.170
0.070
4.968
0.002
0.006
0.003
0.233
Concentration (g L 1)
DALRA
0.325
0.237
0.071
4.776
0.025
0.001
0.021
0.117
Data shown were measured from the samples taken at the time glucose was depleted. Results are the average and standard deviation of
three duplicates.
FEMS Microbiol Lett 350 (2014) 209–215
213
RyhB expression in E. coli
Influence of iron and 2, 2′-dipyridyl on the
production of ALA and heme accumulation
Fig. 4. Effect of ryhB overexpression on relative gene transcription in
Escherichia coli DALRA compared with the control E. coli DALA. Gene
gapA was selected as standard and the error bars indicate the
standard deviation of the mean of three replicates.
and DALRA and no citrate was detected in the fermentation broth of either strain. In E. coli, the transcription of
ryhB is activated under iron-limitation conditions, which
makes the mRNA of sdhCDAB unstable (Masse & Gottesman, 2002). Artificial overexpression of ryhB probably
destabilized the mRNA of sdhCDAB and blocked the
sdhCDAB site of the TCA cycle, resulting in the accumulation of succinate and glutamate in E. coli DALRA. To
confirm it, we performed a qRT-PCR analysis. Among the
genes analyzed in the TCA cycle, sdhB was the most
affected (Fig. 4). The relative transcription level of sdhB
was down-regulated to only 0.3-fold of the control, which
explained why the succinate was accumulated. The
overexpression of ryhB also down-regulated the transcription of hemB and hemH genes in the heme biosynthesis
pathway, but the key gene gltX, which is involved in the
ALA biosynthesis, was up-regulated, which partly explains
the improvement in ALA production. The proteins in the
aerobic respiration chain which contain heme as essential
prosthetic group, such as CydAB, were also down-regulated in E. coli DALRA (Fig. 4). We did a bioinformatics
analysis of RyhB with hemB and hemH to determine
possible modes of RyhB interaction with these two genes
using CLUSTALW2. Sequence alignment showed that part of
RyhB could pair with the sequence within hemB and
hemH mRNA. This part of the RyhB sequence is very similar to the sequence that participates in the interaction
with sdhC (Masse & Gottesman, 2002) (see Fig. S1). Thus,
RyhB may interact with hemB and hemH by binding to
the specific site of DNA, inhibiting the transcription of
these genes.
FEMS Microbiol Lett 350 (2014) 209–215
As reported previously, the transcription of ryhB was
repressed when iron was present and induced when iron
chelator was present (Masse & Gottesman, 2002). Thus, the
addition of iron chelator appears to facilitate the ALA production, whereas addition of extra iron represses ryhB
more. To measure the effect of iron and iron chelator on
ALA production and heme accumulation, we cultured
E. coli DALA and DALRA in the presence of 100 lM
FeSO4 or 250 lM 2, 2′-dipyridyl. When FeSO4 was added,
only about 0.9 g L 1 ALA was produced, but the accumulation of heme increased to about 0.55 nmol OD 1 in both
E. coli DALA and E. coli DALRA (Fig. 5a). The addition of
FeSO4 in the culture may switch the in vivo metabolic flux
from ALA to heme formation by chelating ferrous ion with
protoporphyrin IX, resulting in decreased ALA accumulation and increased heme accumulation. This implies that
the effect of ryhB overexpression on heme biosynthesis and
ALA accumulation is blocked by extra iron. Treatment of
E. coli DALRA culture with 2, 2′-dipyridyl only led to
1.5 g L 1 ALA production (Fig. 5a). However, we found
that the cell growth was obviously affected in the 2, 2′-dipyridyl treatment group due to the harmfulness of 2, 2′-dipyridyl (Fig. 5b). The OD value of E. coli DALA and
DALRA of this group was 10.35 and 9.68, respectively. Calculation of the ALA production per cell density in E. coli
DALRA was 0.172 g L 1 OD 1, 25% percent higher than
in the control (Fig. 5c). In the presence of 2, 2′-dipyridyl,
protoporphyrin IX may compete with the iron, reducing
the actual concentration of iron for heme formation.
Discussion
The application of microRNA (micRNA) and short interfering RNA (siRNA) has increased greatly in eukaryotic
cells in the last two decades due to the development of
gene silence technology (Ashrafi et al., 2003; Lum et al.,
2003). Nonetheless, the technology remains to be developed/optimized for bacteria in which gene knockout technology is already well established. Gene silence technology
does have a potential application in necessary genes that
cannot be knocked out, and the technology should be
fine-tuned. Recently, inspired by the modular architecture
observation of the natural sRNA genes in bacteria (Bouvier et al., 2008; Papenfort et al., 2010), researchers
designed artificial synthetic sRNA genes to regulate bacteria gene expression (Man et al., 2011; Sharma et al., 2011;
Na et al., 2013). Together with natural sRNA gene, artificial synthetic sRNA gene has become an effective and convenient tool for fine-tuning the expression of specific
genes.
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Published by John Wiley & Sons Ltd. All rights reserved
214
F. Li et al.
(a)
In this study, we found that the constitutive overexpression of natural small RNA RyhB influenced the transcription of iron-containing enzymes and drove the metabolic
flux to ALA production. Interestingly, overexpression of
ryhB down-regulated the transcription of hemB and hemH.
The presence of possible targeting sites in the mRNA of
hemB and hemH indicated that they could be targets of
RyhB. The down-regulation of hemB and hemH by ryhB
reduced the metabolic flux from ALA to heme, resulting in
increased ALA production and decreased heme accumulation. Although the mechanism of the regulation of hemB
and hemH by RyhB was not confirmed, there are at least
three reasons for the increased ALA accumulation in E. coli
DALRA: (1) RyhB up-regulated gltX expression involved in
ALA biosynthesis; (2) RyhB down-regulated the gene transcription involved in ALA metabolism; (3) the smaller
amount of heme accumulation caused de-repression of the
feedback inhibition of ALA synthesis genes (Levican et al.,
2007; Jones & Elliott, 2010). The down-regulation of the
heme-containing protein CydAB in E. coli DALRA was
probably due to less heme provision in vivo. A previous
study showed that cydAB was directly regulated by RyhB
(Masse et al., 2005). It is possible that RyhB repressed the
heme biosynthesis pathway during iron-limiting conditions
and released free iron for other essential biological processes. Nevertheless, the heme accumulation in both E. coli
DALA and DALRA was increased compared with the wildtype E. coli strain. As the anabolic heme biosynthesis is
coupled to catabolic electron chain-driven ATP synthesis,
the regulation of the heme biosynthesis pathway shall facilitate
the overall metabolism of the host (M€
obius et al., 2010).
In E. coli, the biosynthesis of ALA and heme is highly
regulated. To promote ALA production, a strategy that
can fine-tune the gene expression of this pathway is necessary. Application of native sRNA gene in this pathway
is an attractive strategy and can be further exploited.
(b)
(c)
Acknowledgements
Fig. 5. Effect of iron and chelator on heme accumulation, ALA
production and cell growth with ryhB overexpression. (a) ALA
production and heme accumulation in FeSO4 or 2, 2′-dipyridyl-treated
Escherichia coli DALA and DALRA. (b) OD value of FeSO4 or 2, 2′dipyridyl-treated E. coli DALA and DALRA. (c) ALA concentration per
OD in 2, 2′-dipyridyl-treated E. coli DALA and DALRA and control.
Cultivation was carried out in a 300-mL Erlenmeyer flask, containing
50 mL modified minimal medium, at 37 °C, 250 r.p.m. for 32 h.
Fermentation samples were taken at 4-h intervals to measure OD600,
ALA and heme concentration. Results are the average of three
individual experiments.
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
This work was financially supported by a research grant
from National High-Tech Research and Development Plan
of China (2012AA022104) and a grant from the National
Natural Science Foundation of China (31370085) and
Independent Innovation Foundation of Shandong University (IIFSDU) (2012ZD029). The authors do not have any
conflicts of interest.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. Putative interaction of RyhB with its targets, hemB,
hemH and sdhC (Masse & Gottesman, 2002). Nucleotides
participating in the interaction are shown in bold.
Table S1. Strains and plasmids used in this study.
Table S2. Primers used in this study.
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