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\begin{document}

\title{Strain engineering for high-level 5-aminolevulinic acid production in
Escherichia coli}



\author[1]{Dragan Miscevic}%
\author[2]{Ju-Yi Mao}%
\author[1]{Teshager Bitew Kefale}%
\author[1]{Daryoush Abedi}%
\author[1]{Murray Moo young}%
\author[1]{C. Perry Chou}%
\affil[1]{University of Waterloo}%
\affil[2]{National Taiwan Ocean University}%


\vspace{-1em}



  
  \date{June 18, 2024}


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\selectlanguage{english}
\begin{abstract}
As issues surrounding depleting fossil fuels, climate change, and
various other environmental impacts are becoming more prevalent, there
is a growing interest in technological shifts toward a bio-based
economy. Various advanced biotechnological tools have been developed to
customize cell factories for the production of a wide range of complex
fine chemicals from renewable feedstock. Herein, we report development
of a microbial bioprocess for high-level and potentially economical
production of 5-aminolevulinic acid (5-ALA), a valuable
non-proteinogenic amino acid with multiple applications in medical,
agricultural, and food industries, using Escherichia coli as a cell
factory. We first implemented the Shemin (i.e., C4) pathway for
heterologous 5-ALA biosynthesis in E. coli. To reduce, but not to
abolish, the carbon flux toward essential tetrapyrrole/porphyrin
biosynthesis, we applied Clustered Regularly Interspersed Short
Palindromic Repeats interference (CRISPRi) to repress hemB expression,
leading to extracellular 5-ALA accumulation. We then applied metabolic
engineering strategies to direct more dissimilated carbon flux toward
the key precursor of succinyl-CoA for enhanced 5-ALA biosynthesis. Using
these engineered E. coli strains for bioreactor cultivation, we
successfully demonstrated high-level 5-ALA biosynthesis solely from
glycerol (\textasciitilde{}30 g l-1) under both microaerobic and aerobic
conditions, achieving up to 5.95 g l-1 (36.9\% yield) and 6.93 g l-1
(50.9\% yield) 5-ALA, respectively. This study represents one of the
most effective bio-based production of 5-ALA from a structurally
unrelated carbon to date, highlighting the importance of integrated
strain engineering and bioprocessing strategies to enhance bio-based
production.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Increasing concerns over climate change and fossil fuel depletion have
provoked a need for sustainable production of chemicals and fuels. One
approach of particular attractiveness is the use of biological cell
factories, such as microorganisms, as biocatalysts to drive chemical
conversions from renewable and clean carbons, so called ``bio-based
production''. The emergence of novel biotechnological tools for strain
engineering, in particular systems biology, synthetic biology, metabolic
engineering, and genetic/genomic engineering, has promoted the
development of hyper strains for bio-based production (Choi et al.
2019). In addition to the sustainability, bio-based production can have
economical potentials over synthetic organic chemistry particularly for
the production of complex chemicals in the pharmaceutical and fine
chemical industries, such as amino acids, organic acids, and vitamins
(Keasling 2010).

5-Aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid existing
in most living organisms as a metabolic intermediate toward biosynthesis
of essential tetrapyrrole/porphyrin pigment compounds, such as heme
(Schlicke et al. 2015) (Fig. 1). In nature, there are two major
metabolic routes for 5-ALA biosynthesis, i.e., (1) C4 (also known as
Shemin) pathway (mainly existing in mammals, fungi, and purple sulphur
bacteria), in which succinyl-CoA and glycine are structurally fused by
5-aminolevulinate synthase (ALAS or HemA) to form 5-ALA (Kang et al.
2004), and (2) C5 pathway (existing in most bacteria, all archaea and
plants), in which glutamate is converted to 5-ALA via three enzymatic
reactions of ligation, reduction, and transamination catalyzed by
glutamyl-tRNA synthase (GluTS), glutamyl-tRNA reductase (GluTR), and
glutamate-1-semialdehyde-2,1-aminomutase (GSAM), respectively (Woodard
and Dailey 1995).

Practically, 5-ALA has broad applications in many fields, such as
medicine (Inoue 2017; Juzeniene et al. 2002), agriculture (Hotta et al.
1997), and food preservation (Li et al. 2016). Hence, technologies for
5-ALA production have been developed. While 5-ALA can be chemically
derived from various precursors, such as levulinic acid (MacDonald
1974), tetrahydrofurfurylamine (Kawakami et al. 1991), 5-bromo esters
(Ha et al. 1994), and\emph{N} -furfurylphthalimide (Takeya et al. 1996),
these synthetic approaches are deemed uneconomical and the production
processes are often complicated for implementation with low yields (Kang
et al. 2017). As a result, bio-based production of 5-ALA using either
multi-enzyme systems (Meng et al. 2016) or various cell factories has
been explored (Sasaki et al. 2002), in particular photosynthetic
microorganisms, such as\emph{Rhodobacter sphaeroides} ,
\emph{Rhodopseudomonas palustris} , and\emph{Chlorella} sp. (Sasaki et
al. 1995), \emph{Streptomyces coelicolor}(Tran et al. 2019),
\emph{Corynebacterium glutamicum} (Zhang and Ye 2018), as well as
genetically tractable \emph{Escherichia coli}(Ding et al. 2017; Zhang et
al. 2015; Zhang et al. 2019).

In this study, we explored strain engineering strategies for high-level
5-ALA production in \emph{E. coli} . Native biosynthesis of 5-ALA
in\emph{E. coli} is achieved via the C5 pathway, which is adopted in
most previous studies for 5-ALA biosynthesis using \emph{E. coli} as a
cell factory (Kang et al. 2011; Li et al. 2014; Zhang et al. 2015).
However, this metabolic route is considered mechanistically complex and
energetically ineffective, particularly upon high-level 5-ALA
biosynthesis, as it requires multiple tightly regulated enzymes (Wang et
al. 1999) and utilization of ATP/NADPH as limiting cofactors (Li et al.
1989). On the other hand, for high-level biosynthesis of a target
metabolite, it is critical to ensure intracellular abundance of the
corresponding precursors, i.e., succinyl-CoA/glycine for the Shemin
pathway or glutamate for the C5 pathway in the 5-ALA case. This is
normally achieved by proper metabolic direction of the dissimilated
carbon flux in key pathways, otherwise artificial supplementation of
structurally related carbons, which are often expensive, becomes
necessary. Considering the above technical aspects/limitations, we chose
to implement the Shemin pathway into \emph{E. coli} for heterologous
5-ALA biosynthesis with metabolic direction of the dissimilated carbon
flux toward succinyl-CoA in the tricarboxylic acid (TCA) cycle.
Moreover, without supplementation of structurally related carbons,
glycerol was used as the sole carbon source for cultivation of
engineered \emph{E. coli} strains due to its low cost (Ciriminna et al.
2014) and highly reduced nature, generating approximately twice the
number of reducing equivalents upon its degradation compared to
traditional fermentable sugars (Murarka et al. 2008; Yazdani and
Gonzalez 2007).

By initializing the formation of essential porphyrin compounds, 5-ALA is
among the most conserved metabolites across all biological kingdoms
(Pet\selectlanguage{polish}ř\selectlanguage{ngerman}í\selectlanguage{polish}č\selectlanguage{english}kov\selectlanguage{ngerman}á et al. 2015; Yu et al. 2015). However, 5-ALA normally acts
as a metabolic intermediate toward porphyrin biosynthesis with minimal
accumulation, limiting its overproduction. Given the imperative
physiological role of porphyrin compounds, attempting to accumulate
5-ALA by inactivation of the immediate post-5-ALA conversion catalyzed
by 5-aminolevulinate dehydratase (HemB, encoded by \emph{hemB} ) would
abolish porphyrin biosynthesis (Fig. 1) and, therefore, be detrimental
to the cells. Hence, instead of gene inactivation, we applied Clustered
Regularly Interspersed Short Palindromic Repeats interference (CRISPRi)
to repress \emph{hemB} expression and, therefore, increase 5-ALA
accumulation without imposing physiological impacts to the
5-ALA-producing cells. Using various engineered \emph{E. coli} strains
for bioreactor cultivation, we demonstrated high-level 5-ALA
biosynthesis under both microaerobic and aerobic conditions with
glycerol as a sole carbon source.

\textbf{Materials and Methods}

Bacterial strains and plasmids

Bacterial strains and deoxynucleic acids (DNAs) used in this study are
listed Table 1. Genomic DNA from bacterial cells was isolated using the
Blood \& Tissue DNA Isolation Kit (Qiagen, Hilden, Germany). Standard
recombinant DNA technologies were applied for molecular cloning (Miller
1992). \emph{Taq} DNA polymerase was obtained from New England Biolabs
(Ipswich, MA, USA). All synthesized oligonucleotides were obtained from
Integrated DNA Technologies (Coralville, IA, USA). DNA sequencing was
conducted by the Centre for Applied Genomics at the Hospital for Sick
Children (Toronto, Canada). \emph{E. coli} BW25113 was the parental
strain for derivation of all engineered strains in this study and
\emph{E. coli} DH5\selectlanguage{greek}α \selectlanguage{english}was used as a host for molecular cloning. Note that
the \emph{ldhA} gene (encoding lactate dehydrogenase) was previously
inactivated in BW25113, generating BW[?]\emph{ldhA} (Srirangan et al.
2014).

For genetic implementation of the Shemin pathway in \emph{E. coli} ,
the\emph{hemA} gene was first amplified by polymerase chain reaction
(PCR) using the primer set g-hemA and the genomic DNA of
wild-type\emph{Rhodobacter sphaeroides} DSM 158 as the template. The
amplified\emph{hemA} gene was Gibson--assembled with the PCR-linearized
pK184 using the primer set g-pK-hemA to generate pK-hemA. The expression
of the cloned \emph{hemA} gene in the pK184 vector was under the control
of the P\emph{\textsubscript{lac}} promoter.

Knockouts of the genes, including \emph{sdhA} (encoding succinate
dehydrogenase complex flavoprotein subunit A, SdhA) and
\emph{iclR}(encoding transcriptional AceBAK operon repressor, IclR),
were introduced into BW[?]\emph{ldhA} by P1 phage transduction (Miller
1992) using the appropriate Keio Collection strains (The Coli Genetic
Stock Center, Yale University, New Haven, CT, USA) as donors (Baba et
al. 2006). To eliminate the co-transduced FRT-Kn\textsuperscript{R}-FRT
cassette, the transductants were transformed with pCP20 (Cherepanov and
Wackernagel 1995), a temperature sensitive plasmid expressing a flippase
(Flp) recombinase. Upon Flp-mediated excision of the
Kn\textsuperscript{R} cassette, a single Flp recognition site (FRT
``scar site'') was generated. Plasmid pCP20 was then cured by growing
cells at 42\selectlanguage{ngerman}°C. The genotypes of derived knockout strains were confirmed
by colony PCR using the appropriate verification primer sets listed in
Table 1.

The expression of \emph{hemB} was repressed by CRISPRi using various
derived plasmids from pdcas9-bacteria (Addgene plasmid \# 44249) and
pgRNA-bacteria (Addgene plasmid \# 44251). All synthesized
oligonucleotide pairs have 60 nucleotides (nt), which includes 20
nt\emph{hemB} -targeting sequence, 20 nt upstream and 20 nt downstream
sequences of pgRNA-bacteria vector (Fig. 2). They were ordered from
Integrated DNA Technologies (Coralville, IA, USA) and annealed as
described previously (Pengpumkiat et al. 2016), generating four
double-stranded DNA fragments of\emph{hemB} -gRNA-L1, \emph{hemB}
-gRNA-L2, \emph{hemB} -gRNA-L3, and\emph{hemB} -gRNA-L4 (Table 1). All
these four DNA fragments were individually Gibson-assembled with the
PCR-linearized pgRNA-bacteria using the primer set g-pgRNA to generate
pgRNA-L1, pgRNA-L2, pgRNA-L3, and pgRNA-L4, respectively (Table 1). The
four \emph{hemB} -repressed strains, i.e., DMH-L1, DMH-L2, DMH-L3, and
DMH-L4, were developed by creating a triple-plasmid system (Fig. 2)
containing pK-hemA, pdcas9-bacteria, and the gRNA-containing plasmid
(i.e., pgRNA-L1, pgRNA-L2, pgRNA-L3, or pgRNA-L4). For the control
strain DMH-CT, the original pgRNA-bacteria plasmid without any
\emph{hemB} -targeting sequence was used as the third plasmid.

Media and bacterial cell cultivation

All medium components were obtained from Sigma-Aldrich Co. (St Louis,
MO, USA) except yeast extract and tryptone which were obtained from BD
Diagnostic Systems (Franklin Lakes, NJ, USA). \emph{E. coli} strains,
stored as glycerol stocks at -80°C, were streaked on lysogeny broth (LB;
10 g l\textsuperscript{-1} tryptone, 5 g l\textsuperscript{-1} yeast
extract, and 5 g l\textsuperscript{-1} NaCl) agar plates and incubated
at 37°C for 14-16 hours.

For shake-flask cultivations, single colonies were picked from LB plates
to inoculate 30 ml LB medium in 125 ml conical flasks. The cultures were
shaken at 37°C and 280 rpm in a rotary shaker (New Brunswick Scientific,
NJ, USA) and used as seed cultures to inoculate 220 ml LB media at 1\%
(v/v) in 1 L conical flasks. This second seed culture was shaken at 37°C
and 280 rpm until a cell density of 0.80 OD\textsubscript{600} was
reached. Cells were then harvested by centrifugation at 9,000 ×g and
20°C for 10 minutes and resuspended in 30 ml modified M9 production
media. The suspended culture was transferred into a 125 ml screwed cap
plastic production flasks and incubated at 37°C at 280 rpm in a rotary
shaker. Unless otherwise specified, the modified M9 production medium
contained 20 g l\textsuperscript{-1} glycerol, 5 g
l\textsuperscript{-1}yeast extract, 10 mM NaHCO\textsubscript{3}, 1 mM
MgCl\textsubscript{2,}5\textsuperscript{th} dilution of M9 salts mix
(33.9 g l\textsuperscript{-1} Na\textsubscript{2}HPO\textsubscript{4},
15 g l\textsuperscript{-1} KH\textsubscript{2}PO\textsubscript{4}, 5 g
l\textsuperscript{-1} NH\textsubscript{4}Cl, 2.5 g
l\textsuperscript{-1}NaCl ), 1,000\textsuperscript{th} dilution of Trace
Metal Mix A5 (2.86 g l\textsuperscript{-1}
H\textsubscript{3}BO\textsubscript{3}, 1.81 g l\textsuperscript{-1}
MnCl\textsubscript{2}\selectlanguage{english}*4H\textsubscript{2}O, 0.222 g
l\textsuperscript{-1} ZnSO\textsubscript{4}\selectlanguage{english}*7H\textsubscript{2}O, 0.39 g
l\textsuperscript{-1}Na\textsubscript{2}MoO\textsubscript{4}\selectlanguage{english}*2H\textsubscript{2}O,
79 \selectlanguage{greek}µ\selectlanguage{english}g l\textsuperscript{-1} CuSO\textsubscript{4}*5H\textsubscript{2}O,
49.4 \selectlanguage{greek}µ\selectlanguage{english}g
l\textsuperscript{-1}Co(NO\textsubscript{3})\textsubscript{2}*6H\textsubscript{2}O),
and was supplemented with 0.1 mM isopropyl \selectlanguage{greek}β-\selectlanguage{english}D-1-thiogalactopyranoside
(IPTG). All shake-flask cultivation experiments were performed in
triplicate.

For bioreactor cultivations, single colonies were picked from LB plates
to inoculate 30 mL super broth (SB) medium (32 g
l\textsuperscript{-1}tryptone, 20 g l\textsuperscript{-1} yeast extract,
and 5 g l\textsuperscript{-1} NaCl) in 125 mL conical flasks. The
overnight cultures were shaken at 37\selectlanguage{ngerman}°C and 280 rpm in a rotary shaker
(New Brunswick Scientific, NJ, USA) and used as seed cultures to
inoculate 220 mL SB media at 1\% (v/v) in 1 L conical flasks. This
second seed culture was shaken at 37°C and 280 rpm for 14-16 hours.
Cells were then harvested by centrifugation at 9,000×g and 20°C for 10
minutes and resuspended in 50 mL fresh LB media. The suspended culture
was used to inoculate a 1 L stirred tank bioreactor (containing two
Rushton radial flow disks as impellers) (CelliGen 115, Eppendorf AG,
Hamburg, Germany) at 37°C and 430 rpm. The semi-defined production
medium in the batch bioreactor contained 30 g l\selectlanguage{english}\textsuperscript{-1}
glycerol, 0.23 g l\selectlanguage{english}\textsuperscript{-1}
K\textsubscript{2}HPO\textsubscript{4}, 0.51 g l\selectlanguage{english}\textsuperscript{-1}
NH\textsubscript{4}Cl, 49.8 mg l\selectlanguage{english}\textsuperscript{-1}
MgCl\textsubscript{2}, 48.1 mg l\selectlanguage{english}\textsuperscript{-1}
K\textsubscript{2}SO\textsubscript{4}, 1.52 mg l\selectlanguage{english}\textsuperscript{-1}
FeSO\textsubscript{4}, 0.055 mg l\selectlanguage{english}\textsuperscript{-1}
CaCl\textsubscript{2}, 2.93 g l\selectlanguage{english}\textsuperscript{-1} NaCl, 0.72 g
l\selectlanguage{english}\textsuperscript{-1} tricine, 10 g l\selectlanguage{english}\textsuperscript{-1} yeast extract,
10 mM NaHCO\textsubscript{3}, and 1,000th dilution (i.e., 1 ml
l\selectlanguage{english}\textsuperscript{-1}) trace elements (2.86 g l\selectlanguage{english}\textsuperscript{-1}
H\textsubscript{3}BO\textsubscript{3}, 1.81 g l\selectlanguage{english}\textsuperscript{-1}
MnCl\textsubscript{2}\selectlanguage{english}* 4H\textsubscript{2}O, 0.222 g
l\selectlanguage{english}\textsuperscript{-1} ZnSO\textsubscript{4}\selectlanguage{english}* 7H\textsubscript{2}O, 0.39
g l\selectlanguage{english}\textsuperscript{-1} Na\textsubscript{2}MoO\textsubscript{4}\selectlanguage{english}*
2H\textsubscript{2}O, 79 \selectlanguage{greek}μ\selectlanguage{english}g l\textsuperscript{-1} CuSO\textsubscript{4}*
5H\textsubscript{2}O, 49.4 \selectlanguage{greek}μ\selectlanguage{english}g
l\textsuperscript{-1}Co(NO\textsubscript{3})\textsubscript{2}*
6H\textsubscript{2}O) (Neidhardt et al. 1974), and was supplemented with
0.1 mM isopropyl \selectlanguage{greek}β-\selectlanguage{english}D-1-thiogalactopyranoside (IPTG). Microaerobic and
semiaerobic conditions were maintained by purging air into the headspace
and bulk culture, respectively, at 0.1 vvm, designated as aeration level
I (AL-I) and II (AL-II). Aerobic conditions were maintained by purging
air into the bulk culture at 1 vvm (AL-III). The pH of the production
culture was maintained at 7.0 \selectlanguage{ngerman}± 0.1 with 30\% (v/v)
NH\textsubscript{4}OH and 15\% (v/v)
H\textsubscript{3}PO\textsubscript{4}.

Analysis

Culture samples were appropriately diluted with 0.15 M saline solution
for measuring cell density in OD\textsubscript{600} using a
spectrophotometer (DU520, Beckman Coulter, Fullerton, CA). Cell-free
medium was prepared by centrifugation of the culture sample at 9,000×g
for 5 minutes, followed by filter sterilization using a 0.2 \selectlanguage{greek}µ\selectlanguage{english}M syringe
filter. Extracellular metabolites and glycerol were quantified using
high-performance liquid chromatography (HPLC) (LC-10AT, Shimadzu, Kyoto,
Japan) with a refractive index detector (RID; RID-10A, Shimadzu, Kyoto,
Japan) and a chromatographic column (Aminex HPX-87H, Bio-Rad
Laboratories, CA, USA). The HPLC column temperature was maintained at
35\selectlanguage{ngerman}°C and the mobile phase was 5 mM
H\textsubscript{2}SO\textsubscript{4}(pH 2) running at 0.6 mL
min\textsuperscript{-1}. The RID signal was acquired and processed by a
data processing unit (Clarity Lite, DataApex, Prague, Czech Republic).

The 5-ALA titer in the cell-free medium was measured using a modified
Ehrlich's reagent (Mauzerall and Granick 1956). The percentage yield of
5-ALA was defined as the mole (or mass) ratio of the produced 5-ALA to
the theoretically maximal 5-ALA produced based on the consumed glycerol
with a molar ratio of one-to-three. Note that one-mole succinyl-CoA
(derived from two-mole glycerol) and one-mole glycine (derived from
one-mole glycerol) are required to generate one-mole 5-ALA. The bulk
level of secreted porphyrin compounds in the cell-free medium was
estimated using a spectrophotometer at two specific wavelengths, i.e.,
OD\textsubscript{405} (measuring Soret band) and OD\textsubscript{495}
(measuring Q-band).

Real-time quantitative reverse transcription PCR (qRT-PCR)

Cells used for RNA extraction were cultivated in 30 mL liquid LB medium
at 37°C and harvested in the exponential growth phase. Total RNA
of\emph{E. coli} was extracted using the High Pure RNA Isolation Kit
(Roche Diagnostics, Basel, Switzerland) according to manufacturer's
instructions. Complementary DNAs (cDNAs) were synthesized using the
High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific,
MA). Sequence specific primers were used for reverse transcription
of\emph{hemB} mRNA (i.e., q-hemB) and internal control
\emph{rrsA}(encoding ribosomal RNA 16S) mRNA (i.e., q-rrsA) (Table 1),
at a final concentration of 1 \selectlanguage{greek}µ\selectlanguage{english}M. 100 ng of the total RNA was used in a
20 \selectlanguage{greek}µ\selectlanguage{english}L reaction mixture. Real-time qRT-PCR was carried out using the
Power SYBR\selectlanguage{ngerman}® Green PCR Master Mix (ThermoFisher Scienti\selectlanguage{english}fic; MA) in an
Applied Biosystems StepOnePlus\selectlanguage{english} System as per the manufacturer's
instructions. All experiments were performed in duplicate.

\textbf{Results}

\emph{Repression of} \textbf{hemB \emph{expression for extracellular
5-ALA accumulation}}

We first implemented the Shemin pathway by episomal expression
of\emph{hemA} from \emph{R. sphaeroides} in BW\selectlanguage{english}[?]\emph{ldhA} , deriving
DMH. The effects of the implemented Shemin pathway were observed by
comparing the two cultures of BW\selectlanguage{english}[?]\emph{ldhA} and DMH in screw-cap
shake-flasks containing 20 g l\textsuperscript{-1} glycerol as the sole
carbon source. While BW\selectlanguage{english}[?]\emph{ldhA} generated no detectable levels of
5-ALA or porphyrin, DMH produced 0.44 g l\textsuperscript{-1} 5-ALA
(5.22\% yield) with significant porphyrin biosynthesis (Fig. 3),
suggesting that the Shemin pathway was active in DMH. In particular, the
dark red color of the DMH culture (Fig. 3) suggests that most glycerol
was converted to porphyrin pigments with limited 5-ALA accumulation.

We then aimed to reduce the carbon flux toward porphyrin biosynthesis in
DMH by repressing the expression of \emph{hemB} (Fig. 1), leading to a
limited conversion of two 5-ALA molecules into porphobiliongen (PBG) and
extracellular 5-ALA accumulation. We applied CRISPRi by designing
four\emph{hemB} -targeting gRNAs with different relative expression
efficiencies, generating four corresponding strains of DMH-L1, DMH-L2,
DMH-L3, and DMH-L4 (Fig. 3). Note that DMH-CT is the control strain
without a \emph{hemB} -specific gRNA for CRISPRi. While cell growth and
glycerol consumption were somewhat affected by the presence of the
triple-plasmid system, all \emph{hemB} -repressed strains had
significant extracellular 5-ALA accumulation compared to the control
strain DMH-CT upon shake-flask cultivation. The minimal physiological
impacts suggest that the \emph{hemB} -repressed strains still had
sufficient biosynthesis of essential porphyrins. In particular, DMH-L1
and DMH-L4 had both titers and yields up to 4-5 fold higher than the
control strain DMH-CT (Fig. 3). This significant increase in 5-ALA
accumulation in DMH-L1 (1.26 g l\textsuperscript{-1}) and DMH-L4 (1.61 g
l\textsuperscript{-1}) occurred simultaneous with considerably reduced
levels of the relative\emph{hemB} expression at 67\% and 40\%,
respectively (Fig. 2). Additional supporting evidence for the repressed
\emph{hemB} expression was reflected by the decrease in porphyrin
biosynthesis in all four\emph{hemB} -repressed strains as the degrees of
pigmentation and the corresponding absorbances (in 405 and 495 nm) of
the cell-free media were significantly lower than that of the control
strain DMH-CT (Fig. 3). Given the lowest relative \emph{hemB} expression
and the most superior extracellular 5-ALA accumulation in DMH-L4 among
all\emph{hemB} -repressed strains, we used this strain for all
subsequent experiments.

Effects of oxygenic condition on 5-ALA biosynthesis

5-ALA biosynthesis via the Shemin pathway requires succinyl-CoA as one
of the two key precursors. Several metabolic pathways are involved in
succinyl-CoA formation in \emph{E. coli} , i.e., reductive TCA branch,
oxidative TCA cycle, and glyoxylate shunt (Fig. 1). These metabolic
pathways, along with cell growth and acetogenesis, can be sensitive to
the oxygenic condition, which critically directs the dissimilated carbon
flux toward succinyl-CoA for 5-ALA biosyntheses. To investigate such
oxygenic effects, batch cultivation of the control strain DMH in a
bioreactor was subject to three different levels of aeration, i.e., AL-I
(microaerobic), AL-II (semiaerobic), and AL-III (aerobic) (Fig. 4).
While cell growth and glycerol consumption were favored by oxygen
exposure, 5-ALA biosynthesis was more effective under lower aeration
levels, i.e., 0.53 g l\textsuperscript{-1} (3.40\% yield) under AL-I,
0.39 g l\textsuperscript{-1} (2.44\% yield) under AL-II, and 0.31 g
l\textsuperscript{-1} (2.08\% yield) under AL-III, presumably because
that more carbon was directed toward the succinyl-CoA node under
microaerobic conditions. In addition, the reduced 5-ALA biosynthesis
under aerobic conditions occurred with less porphyrin formation,
reflected by a significantly less pigmentation of the culture medium.
Note that, in addition to low levels of 5-ALA biosynthesis in DMH under
all investigated culture conditions, acetate was the major side
metabolite with high yields up to 75.9\%, implying a significant carbon
spill at the acetyl-CoA node. The results suggest that 5-ALA
biosynthesis in DMH was favored by lower oxygenic levels and potentially
limited by acetogenesis.

Metabolic engineering to enhance 5-ALA biosynthesis under microaerobic
conditions

To alleviate growth limitation and acetogenesis in DMH under AL-I, we
derived a single-knockout mutant DMH\selectlanguage{english}[?]\emph{sdhA} , in which the
oxidative TCA cycle was inactivated (Fig. 5-I). In addition to reduced
acetogenesis, DMH\selectlanguage{english}[?]\emph{sdhA} produced 0.94 g l\textsuperscript{-1}5-ALA
with 6.61\% yield. With an increased carbon flux toward porphyrin
biosynthesis, pigmentation of the DMH\selectlanguage{english}[?]\emph{sdhA} culture medium was
significantly enhanced. On the other hand, while glycerol consumption
and cell growth were significantly retarded upon cultivation of
the\emph{hemB} -repressed strain DMH-L4 under AL-I, 5-ALA biosynthesis
was drastically improved with much reduced acetogenesis, achieving 4.73
g l\textsuperscript{-1} 5-ALA with 32.0\% yield (Fig. 5-II). The
improved 5-ALA biosynthesis was also evidenced by significant reduction
in pigmentation of the culture medium compared to DMH. Notably, compared
to DMH\selectlanguage{english}[?]\emph{sdhA} or DMH-L4, 5-ALA biosynthesis was further improved
upon cultivation of DMH-L4\selectlanguage{english}[?]\emph{sdhA} , in which the \emph{sdhA}
mutation and\emph{hemB} -repression was simultaneously introduced, under
AL-I, achieving 5.95 g l\textsuperscript{-1} 5-ALA with 36.9\% yield
(Fig. 5-III). These results suggest that the dissimilated carbon flux
was directed toward the succinyl-CoA node for 5-ALA biosynthesis
primarily via the reductive TCA branch under microaerobic conditions,
and such carbon flux direction was rather effective upon simultaneous
disruption of the oxidative TCA cycle and \emph{hemB} repression.
Finally, compared to DMH, higher levels of succinate, formate, and
ethanol were observed upon cultivation of all engineered strains, i.e.
DMH\selectlanguage{english}[?]\emph{sdhA} , DMH-L4, and DMH-L4\selectlanguage{english}[?]\emph{sdhA} , under microaerobic
conditions for enhanced 5-ALA biosynthesis.

Metabolic engineering to enhance 5-ALA biosynthesis under aerobic
conditions

We also explore 5-ALA biosynthesis under aerobic conditions, which often
facilitate carbon utilization and cell growth. While the DMH-L4 culture
under AL-III showed effective glycerol dissimilation and cell growth,
biosynthesis of 5-ALA and porphyrins was much lower than that of the
DMH-L4 culture under AL-I (Fig. 6-I vs. Fig. 5-II), suggesting a
potential limitation in succinyl-CoA precursor under aerobic conditions.
Nevertheless, the significant enhancing effects of\emph{hemB}
-repression on 5-ALA biosynthesis were still observable under aerobic
conditions by comparing the two cultures of DMH-L4 and DMH under AL-III
(Fig. 6-I vs. Fig. 4-III). To overcome the limitation in succinyl-CoA
under AL-III, we derived another mutant of DMH-L4\selectlanguage{english}[?]\emph{iclR} with a
deregulated glyoxylate shunt. Compared to the parental strain DMH-L4,
DMH-L4\selectlanguage{english}[?]\emph{iclR} had a much higher 5-ALA biosynthesis with effective
glycerol dissimilation and cell growth under AL-III (Fig. 6-III vs. Fig
6-I), suggesting successful direction of the dissimilated carbon flux
toward succinyl-CoA for 5-ALA biosynthesis via the glyoxylate shunt
under aerobic conditions. For more effective carbon flux direction, we
derived another double mutant of DMH-L4\selectlanguage{english}[?]\emph{iclR} \selectlanguage{english}[?]\emph{sdhA} with a
disruptive oxidative TCA cycle such that the directed carbon flux at the
succinate node via the glyoxylate shunt could be further directed toward
succinyl-CoA via the reductive TCA branch. Compared to the parental
strain DMH-L4\selectlanguage{english}[?]\emph{iclR} , DMH-L4\selectlanguage{english}[?]\emph{iclR} \selectlanguage{english}[?]\emph{sdhA} had even
higher 5-ALA biosynthesis under AL-III (Fig. 6-V vs. Fig. 6-III),
achieving 6.93 g l\textsuperscript{-1} 5-ALA with 50.9\% yield, though
the single mutation of \selectlanguage{english}[?]\emph{sdhA} appeared to be rather harmful to
cell physiology and, therefore, culture performance (Fig. 6-II vs. Fig.
6-I). Note that the 5-ALA yield for DMH-L4\selectlanguage{english}[?]\emph{sdhA} \selectlanguage{english}[?]\emph{iclR} was
3.4-fold that for DMH-L4 and 1.3-fold that for DMH-L4\selectlanguage{english}[?]\emph{iclR} .
Also, note that the significant enhancing effects of \emph{hemB}
-repression on 5-ALA biosynthesis were further confirmed by comparing
the two cultures of DMH-L4\selectlanguage{english}[?]\emph{iclR} \selectlanguage{english}[?]\emph{sdhA} and DMH\selectlanguage{english}[?]\emph{iclR}
\selectlanguage{english}[?]\emph{sdhA} under AL-III (Fig. 6-V vs. Fig. 6-IV). On the other hand,
the successful carbon flux direction toward the Shemin pathway via the
glyoxylate shunt and reductive TCA branch for biosynthesis of both 5-ALA
and porphyrin pigments can be also observed by comparing the two
cultures of DMH\selectlanguage{english}[?]\emph{iclR} \selectlanguage{english}[?]\emph{sdhA} and DMH under AL-III (Fig. 6-IV
vs. Fig. 4-III). These results successfully demonstrated the
consolidated strategy based on carbon flux redirection in the TCA cycle
toward the Shemin pathway with repressed \emph{hemB} expression to
enhance 5-ALA biosynthesis.

\textbf{Discussion}

While \emph{E. coli} has the native C5 pathway for 5-ALA biosynthesis,
we herein adopted the exogenous Shemin pathway since the intracellular
level of the key precursor succinyl-CoA can be metabolically
manipulated. Being a metabolic intermediate in the pathway for
biosynthesis of essential porphyrins, 5-ALA hardly accumulates
in\emph{E. coli} . To prevent intracellular drainage of 5-ALA, we
explored gene knockout of \emph{hemB} , but failed to derive the
corresponding mutant strain (data now shown), confirming that
\emph{hemB} is an essential gene. Hence, CRISPRi was applied to repress
\emph{hemB}expression, particularly in DMH-L1 and DMH-L4, such that
5-ALA could accumulate as a result of its reduced conversion. Our
results further suggest that 5-ALA acts as a committed precursor for
porphyrin biosynthesis. Despite the increased extracellular 5-ALA
accumulation associated with the repressed \emph{hemB} expression,
suppression of carbon flux toward essential porphyrin biosynthesis
somewhat impacted cell growth upon cultivation.

\begin{quote}
While the effects of oxygen supply on 5-ALA biosynthesis were
investigated in several organisms (Nishikawa et al. 1999; Yu et al.
2015), little effort on this front was made for \emph{E. coli}. With the
implemented the Shemin pathway, 5-ALA biosynthesis in \emph{E. coli} can
critically depend on the availability of the key precursor of
succinyl-CoA, whose formation is rather oxygen-sensitive. In \emph{E.
coli}, succinate (and, therefore, succinyl-CoA) can be derived via three
oxygen-dependent pathways in the TCA cycle, i.e., (i) reductive TCA
branch, (ii) oxidative TCA cycle, and (iii) glyoxylate shunt (Fig. 1)
(Cheng et al. 2013). Under anaerobic conditions, succinate accumulates
as an end-product of mixed acid fermentation via the reductive TCA
branch (Thakker et al. 2012). Although the reductive TCA branch can
potentially yield high-level succinate, this pathway is generally
unfavorable due to the limited availability of reducing equivalents
(Skorokhodova et al. 2015). Under aerobic conditions, succinate is
normally used up as a metabolic intermediate of the oxidative TCA cycle
without accumulation, except for the conditions of oxidative stress
and/or acetate/fatty-acid consumption under which succinate can be
aerobically derived via the operational glyoxylate shunt (Thakker et al.
2012). Using the control strain DMH, the effects of oxygenic conditions
on 5-ALA biosynthesis were systematically investigated for cell
cultivation under microaerobic (AL-I), semiaerobic (AL-II), and aerobic
(AL-III) conditions. Note that glycerol utilization and cell growth were
severely retarded when DMH was cultivated under a strict anaerobic
condition (data not shown). Our results show that biosynthesis of 5-ALA
and porphyrins was favored by microaerobic conditions, though the low
oxygenic level could trigger high-level acetogenesis and the associated
physiological impacts. The results suggest that, in DMH, most of the
dissimilated carbon flux was channeled into the Shemin pathway for
biosynthesis of 5-ALA and porphyrins via the reductive TCA branch; and
the oxidative TCA cycle and glyoxylate shunt contributed minimally
toward such carbon flux channeling. Compared to DMH, blocking the
oxidative TCA cycle in DMH\selectlanguage{english}[?]\emph{sdhA}could potentially channel more
dissimilated carbon flux toward succinyl-CoA via the reductive TCA
branch under AL-I and, therefore, improve biosynthesis of 5-ALA and
porphyrins with much reduced acetogenesis. With more dissimilated carbon
flux channeling into the Shemin pathway, porphyrin biosynthesis was
further reduced by repressing\emph{hemB} expression to enhance 5-ALA
accumulation in DMH-L4\selectlanguage{english}[?]\emph{sdhA} under AL-I, achieving 5.95 g
l\textsuperscript{-1}5-ALA and with 36.9\% yield while minimizing
porphyrin biosynthesis. Note that inactivating the oxidative TCA cycle
and/or repressing\emph{hemB} expression resulted in uncommon
accumulation of formate with reduced acetogenesis, compared to the
control strain DMH. Such observation suggests that, under these genetic
backgrounds and microaerobic conditions, pyruvate formate lyase (PFL;
via which formate is coproduced) could be more active than pyruvate
dehydrogenase (PDH) for the conversion of pyruvate to acetyl-CoA. In
\emph{E. coli}, PDH and PFL are responsible for decarboxylation of
pyruvate to form acetyl-CoA under aerobic and anaerobic conditions,
respectively (Wang et al. 2010). It was also reported that acetate and
formate could induce opposite proteome responses in\emph{E. coli} as
most proteins induced by one of these two acids are repressed by the
other (Kirkpatrick et al. 2001). While the control strain DMH had a
low-level biosynthesis of 5-ALA and porphyrins under aerobic conditions
such as AL-III, implying a limited carbon flux contribution from the
oxidative TCA cycle (and, therefore, limited succinyl-CoA precursor)
into the Shemin pathway, repressing\emph{hemB} expression could also
increase 5-ALA accumulation significantly in DMH-L4. Inactivating the
TCA oxidative cycle in DMH-L4\selectlanguage{english}[?]\emph{sdhA} significantly retarded cell
growth with limited glycerol dissimilation, cell growth, and metabolite
production under AL-III, suggesting the critical metabolic roles of the
TCA oxidative cycle for biomass formation and biosynthesis under aerobic
conditions as per previous observations (Guest 1981; Steinsiek et al.
2011). To resolve the apparent succinyl-CoA limitation under AL-III, we
explored channeling of the dissimilated carbon flux via a deregulated
glyoxylate shunt by mutating \emph{iclR} in DMH-L4\selectlanguage{english}[?]\emph{iclR} and
observed a significantly enhanced 5-ALA biosynthesis. Nevertheless, with
an active TCA oxidative cycle in DMH-L4\selectlanguage{english}[?]\emph{iclR}, the carbon flux
arising from the deregulated glyoxylate shunt could divert at the
succinate node to either the oxidative or reductive TCA direction.
Importantly, the flux diversion could be prevented with the dissimilated
carbon being effectively directed into the Shemin pathway by further
mutating\emph{sdhA} for inactivation of the oxidative TCA cycle in
DMH-L4\selectlanguage{english}[?]\emph{sdhA}\selectlanguage{english}[?]\emph{iclR}, achieving 6.93 g
l\textsuperscript{-1}5-ALA with 50.9\% yield upon its cultivation under
AL-III while minimizing porphyrin biosynthesis. Such carbon flux
rerouting effects under AL-III could also be observed by the enhanced
biosynthesis of 5-ALA and porphyrins (reflected by significant
pigmentation of the culture medium in Fig. 6-IV) in
DMH\selectlanguage{english}[?]\emph{sdhA}\selectlanguage{english}[?]\emph{iclR} compared to DMH. Additionally, note that the
retarded glycerol utilization and cell growth for DMH-L4\selectlanguage{greek}Δ\selectlanguage{english}\emph{sdhA}
could be complemented by the \emph{iclR}mutation, suggesting that both
the oxidative TCA cycle and glyoxylate shunt contribute to active TCA
operation for sustained cell growth and metabolic biosynthesis under
aerobic conditions. While this study successfully demonstrated
high-level biosynthesis of 5-ALA from structurally unrelated carbons
under both microaerobic and aerobic conditions, the overall culture
performance was limited by significant acetogenesis, particularly during
extended fedbatch cultivation (data not shown).
\end{quote}

\textbf{Funding information}

This work was supported by the following Government of Canada grants:
(\textbf{1} ) Natural Sciences and Engineering Research Council (NSERC)
Strategic Partnership grant 430106-12; and (\textbf{2} ) Canada Research
Chair (CRC) grant 950-211471

\textbf{Competing financial interests}

The authors declare no competing financial interests.

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\textbf{Table captions}

\emph{E. coli} strains, plasmids, and oligonucleotides used in this
study

\textbf{Figure captions}

\begin{enumerate}
\tightlist
\item
  \textbf{Schematic representation of the natural metabolism and the
  implemented Shemin pathway for 5-ALA and porphyrin biosynthesis
  in\emph{E. coli} from glycerol.} Metabolic pathways outlined:
  glycolysis, glycine biosynthesis, pyruvate carboxylation, and
  oxidative TCA cycle (in black); glyoxylate shunt in the TCA cycle (in
  light brown); reductive branch of TCA cycle (in blue); Shemin pathway
  (in green); porphyrin biosynthesis (in red). Colored proteins:
  mutations (in red); overexpression (in purple). Metabolite
  abbreviations: 5,10-MTH, 5,10-methenyltetrahydrofolic acid; 5-ALA,
  5-aminolevulinic acid; 3-PG, 3-phosphoglycerate; 3-PP,
  3-phosphooxypyruvate; O-P-Serine, O-phospho-L-serine; PBG,
  porphobilinogen; PEP, phosphoenolpyruvate. The number of carbon atoms
  for each metabolite is specified in orange. Protein abbreviations:
  AceA, isocitrate lyase; AceB, malate synthase A; AceK, isocitrate
  dehydrogenase kinase/phosphatase; AckA, acetate kinase; AdhE,
  aldehyde-alcohol dehydrogenase; FHL, formate hydrogenlyase; HemA,
  5-aminolevulinate synthase; HemB, 5-aminolevulinate dehydratase; IclR,
  AceBAK operon repressor; IDH, isocitrate dehydrogenase; IDH-P,
  isocitrate dehydrogenase-phosphate; LdhA, lactate dehydrogenase A; PC,
  pyruvate carboxylase; PckA, phosphoenolpyruvate carboxykinase; PDH,
  pyruvate dehydrogenase; PFL, pyruvate formate-lyase; PK, pyruvate
  kinase; PPC, phosphoenolpyruvate carboxylase; Pta,
  phosphotransacetylase; SdhA, succinate dehydrogenase complex (subunit
  A).
\item
  \textbf{Design strategy for CRISPRi-mediated \emph{hemB} repression
  .}The three plasmids with their major genetic features, such as
  promoters, selection markers, key genes, are shown. The design
  of\emph{hemB} -targeting sequences and their associated interacting
  spots in the \emph{hemB} gene (i.e., L1, L2, L3, and L4) and the
  predicted repression efficiencies (numbers in parenthesis) are shown.
  The resulting \emph{hemB} -repressed strains, i.e., DMH-CT (control),
  DMH-L1, DMH-L2, DMH-L3, and DMH-L4, were characterized for
  quantification of the relative \emph{hemB} expression using qRT-PCR.
  All qRT-PCR values are reported as means \selectlanguage{ngerman}± SD (n = 2).
\item
  \textbf{Shake-flask cultivation of \emph{hemB}-repressed strains for
  5-ALA accumulation.} Strains compared include BW\selectlanguage{english}[?]\emph{ldhA} , DMH,
  DMH-CT, DMH-L1, DMH-L2, DMH-L3, and DMH-L4. Results of the 48h
  shake-flask cultivation in (\textbf{a} ) cell density
  (OD\textsubscript{600}), (\textbf{b} ) glycerol consumption,
  (\textbf{c} ) 5-ALA titer and percentage yield, and (\textbf{d} )
  porphyrin biosynthesis (represented by the absorbance readings of the
  Soret peak (OD\textsubscript{405}) and Q-band (OD\textsubscript{495})
  and the images of cell-free media) are shown. All values are reported
  as means +- SD (n = 3).
\item
  \textbf{Bioreactor cultivation of DMH for 5-ALA biosynthesis under
  different oxygenic conditions.} Time profiles of cell density
  (OD\textsubscript{600}), glycerol consumption and metabolite
  production profiles, acetate and 5-APA percentage yields, and
  porphyrin biosynthesis (represented by the absorbance readings of the
  Soret peak in OD\textsubscript{405} and Q-band in
  OD\textsubscript{495} and the images of cell-free media) are shown.
  The percentage yields of acetate and 5-ALA are calculated based on the
  consumed glycerol at the end of cultivation. (\textbf{I} ) AL-I:
  microaerobic, (\textbf{II} ) AL-II: semi-aerobic, and (\textbf{III} )
  AL-III: aerobic. All values are reported as means +- SD (n = 2).
\item
  \textbf{Bioreactor cultivation of engineered \emph{E. coli} for 5-ALA
  biosynthesis under microaerobic (AL-I) conditions.} Strains compared
  include DMH\selectlanguage{english}[?]\emph{sdhA} , DMH-L4, and DMH-L4\selectlanguage{english}[?]\emph{sdhA} . Time
  profiles of cell density (OD\textsubscript{600}), glycerol consumption
  and metabolite production profiles, acetate and 5-APA percentage
  yields, and porphyrin biosynthesis (represented by the absorbance
  readings of the Soret peak in OD\textsubscript{405} and Q-band in
  OD\textsubscript{495} and the images of cell-free media) are shown.
  The percentage yields of acetate and 5-ALA are calculated based on the
  consumed glycerol at the end of cultivation. (\textbf{I} )
  DMH\selectlanguage{english}[?]\emph{sdhA} , (\textbf{II} ) DMH-L4, and (\textbf{III} )
  DMH-L4\selectlanguage{english}[?]\emph{sdhA} . All values are reported as means +- SD (n = 2).
\item
  \textbf{Bioreactor cultivation of engineered \emph{E. coli} for 5-ALA
  biosynthesis under aerobic (AL-III) conditions.} Strains compared
  include DMH-L4, DMH-L4\selectlanguage{english}[?]\emph{sdhA} , DMH-L4\selectlanguage{english}[?]\emph{iclR} ,
  DMH\selectlanguage{english}[?]\emph{sdhA} \selectlanguage{english}[?]\emph{iclR} , and DMH-L4\selectlanguage{english}[?]\emph{sdhA} \selectlanguage{english}[?]\emph{iclR} .
  Time profiles of cell density (OD\textsubscript{600}), glycerol
  consumption and metabolite production profiles, acetate and 5-APA
  percentage yields, and porphyrin biosynthesis (represented by the
  absorbance readings of the Soret peak in OD\textsubscript{405} and
  Q-band in OD\textsubscript{495} and the images of cell-free media) are
  shown. The percentage yields of acetate and 5-ALA are calculated based
  on the consumed glycerol at the end of cultivation. (\textbf{I} )
  DMH-L4 (\textbf{II} ) DMH-L4\selectlanguage{english}[?]\emph{sdhA} , (\textbf{III} )
  DMH-L4\selectlanguage{english}[?]\emph{iclR} , (\textbf{IV} ) DMH\selectlanguage{english}[?]\emph{sdhA} \selectlanguage{english}[?]\emph{iclR} , and
  (\textbf{V} ) DMH-L4\selectlanguage{english}[?]\emph{sdhA} \selectlanguage{english}[?]\emph{iclR} . All values are
  reported as means +- SD (n = 2).
\end{enumerate}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
\textbf{Name}\strut
\end{minipage} & \begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
\textbf{Description, relevant genotype or primer/oligo sequence (5'
3')}\strut
\end{minipage} & \begin{minipage}[b]{0.30\columnwidth}\raggedright\strut
\textbf{Source}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{E. coli} \textbf{host strains}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DH5\selectlanguage{greek}α\selectlanguage{english}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
F-, \selectlanguage{greek}\emph{endA1, glnV44, thi-1, recA1, relA1, gyrA96, deoR, nupG φ80d
lacZ[?]acZd ladlacZYA -- argF) U169, hsdR17(rK-mK +), λ-}\selectlanguage{english}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Lab stock\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
BW25113\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
F-, [?]\emph{(araD-araB)567, [?]lacZ4787(::rrnB-3), λ-, rph-1,
[?](rhaD-rhaB)568, hsdR514}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
(Datsenko and Wanner 2000)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
BW[?]\emph{ldhA}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
BW25113 \emph{ldhA} null mutant\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
(Srirangan et al. 2014)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
BW[?]\emph{ldhA/}pK-hemA\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH[?]\emph{sdhA} DMH[?]\emph{sdhA}[?]\emph{iclR}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{sdhA} null mutant of DMH \emph{sdhA} and \emph{iclR} mutants of
DMH\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH-CT\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH/pK-hemA/pgRNA-bacteria/pdcas9-bacteria\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH-L1\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH/pK-hemA/pgRNA-L1/pdcas9-bacteria\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH-L2\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH/pK-hemA/pgRNA-L2/pdcas9-bacteria\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH-L3\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH/pK-hemA/pgRNA-L3/pdcas9-bacteria\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH-L4\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH/pK-hemA/pgRNA-L4/pdcas9-bacteria\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH-L4[?]\emph{sdhA}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{sdhA} null mutant of DMH-L4\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH-L4[?]\emph{iclR}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{iclR} null mutant of DMH-L4\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
DMH-L4[?]\emph{sdhA}[?]\emph{iclR}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\emph{sdhA} and \emph{iclR} null mutants of DMH-L4\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Plasmids}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
pCP20 pK184\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Flp+, \selectlanguage{greek}λ \selectlanguage{english}cI857+, \selectlanguage{greek}λ \selectlanguage{english}pR Rep(pSC101 ori)ts, ApR, CmR p15A ori, KmR,
P\emph{lac::lacZ'}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
(Cherepanov and Wackernagel 1995) (Jobling and Holmes 1990)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
pdcas9-bacteria\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
p15A ori, P\textsubscript{Tet}-dCas9\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
(Qi et al. 2013)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
pgRNA-bacteria\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
ColE1 origin, P\textsubscript{J23119}-gRNA\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
(Qi et al. 2013)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
pgRNA-L1\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Derived from pgRNA-bacteria,
P\textsubscript{speI}::\emph{hemB-}gRNA-L1\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
pgRNA-L2\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Derived from pgRNA-bacteria,
P\textsubscript{speI}::\emph{hemB-}gRNA-L2\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
pgRNA-L3\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Derived from pgRNA-bacteria,
P\textsubscript{speI}::\emph{hemB-}gRNA-L3\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
pgRNA-L4\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Derived from pgRNA-bacteria,
P\textsubscript{speI}::\emph{hemB-}gRNA-L4\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
pK-hemA\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
Derived from pTrc99a, P\emph{\textsubscript{trc}}::\emph{phaAB}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\textbf{Primers/oligo sequences}\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
v-ldhA\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
GATAACGGAGATCGGGAATGATTAA; GGTTTAAAAGCGTCGATGTCCAGTA\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
(Akawi et al. 2015)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
v-sdhA\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
CTCTGCGTTCACCAAAGTGT; ACACACCTTCACGGCAGGAG\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
v-iclR\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
GGTGGAATGAGATCTTGCGA; CCGACACGCTCAACCCAGAT\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
c-frt\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
AGATTGCAGCATTACACGTCTTGAG;
CCAGCTGCATTAATGAATCGGGCCATGGTCCATATGAATATCCTCC\strut
\end{minipage} & \begin{minipage}[t]{0.32\columnwidth}\raggedright\strut
(Srirangan et al. 2014)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
c-ptrc cf-gRNA q-hemB q-rrsA g-hemA g-pK-hemA g-pgRNA hemA-gRNA-L1
hemA-gRNA-L2 hemA-gRNA-L3 hemA-gRNA-L4\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
CCGATTCATTAATGCAGCTGG; GGTCTGTTTCCTGTGTGAAATTGTTA
ATCTTTGACAGCTAGCTCAGTCC; CAAGCTTCAAAAAAAGCACCGA GCGCGCTATGTTTGAAGAGA;
TCGGTATGGTGAGAGATGCC TCCAGGTGTAGCGGTGAAAT; TGAGTTTTAACCTTGCGGCC
CACAGGAAACAGCTATGACCATGGACTACAATCTGGCACTCGA;
GAGCTCGAATTCGTAATCATTCAGGCAACGACCTCGGC
GCGCCGAGGTCGTTGCCTGAATGATTACGAATTCGAGCTCGGTAC;
AGTGCCAGATTGTAGTCCATGGTCATAGCTGTTTCCTGTGTG GTTTTAGAGCTAGAAATAGCAAGTT;
ACTAGTATTATACCTAGGACTGAGC
GTCCTAGGTATAATACTAGT\emph{CCCTCGATTCCACAAACATC}GTTTTAGAGCTAGAAATAGC;
GCTATTTCTAGCTCTAAAAC\emph{GATGTTTGTGGAATCGAGGG}ACTAGTATTATACCTAGGAC
GTCCTAGGTATAATACTAGT\emph{AAAGCCGTTGAAGCCATGCC}GTTTTAGAGCTAGAAATAGC;
GCTATTTCTAGCTCTAAAAC\emph{GGCATGGCTTCAACGGCTTT}ACTAGTATTATACCTAGGAC
GTCCTAGGTATAATACTAGT\emph{GCCTGATGTTTGTGGAATCG}GTTTTAGAGCTAGAAATAGC;
GCTATTTCTAGCTCTAAAAC\emph{CGATTCCACAAACATCAGGC}ACTAGTATTATACCTAGGAC
GTCCTAGGTATAATACTAGT\emph{ATGCGCTTCCGGCAGCTTCA}GTTTTAGAGCTAGAAATAGC
GCTATTTCTAGCTCTAAAAC\emph{TGAAGCTGCCGGAAGCGCAT}ACTAGTATTATACCTAGGAC\strut
\end{minipage} & \begin{minipage}[t]{0.30\columnwidth}\raggedright\strut
(Srirangan et al. 2014) This study This study This study This study This
study This study This study This study This study This study\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{gRNA} & \textbf{20 nt targeting-sequence}\tabularnewline
\midrule
\endhead
\emph{hemB-}gRNA-L1 & CCCTCGATTCCACAAACATC\tabularnewline
\emph{hemB-}gRNA-L2 & AAAGCCGTTGAAGCCATGCC\tabularnewline
\emph{hemB-}gRNA-L3 & GCCTGATGTTTGTGGAATCG\tabularnewline
\emph{hemB-}gRNA-L4 & ATGCGCTTCCGGCAGCTTCA\tabularnewline
\bottomrule
\end{longtable}

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/302765/articles/432841-strain-engineering-for-high-level-5-aminolevulinic-acid-production-in-escherichia-coli}

\textbf{Hosted file}

\verb`image11.emf`  available at \url{https://authorea.com/users/302765/articles/432841-strain-engineering-for-high-level-5-aminolevulinic-acid-production-in-escherichia-coli}

\textbf{Hosted file}

\verb`image14.emf`  available at \url{https://authorea.com/users/302765/articles/432841-strain-engineering-for-high-level-5-aminolevulinic-acid-production-in-escherichia-coli}

\selectlanguage{english}
\FloatBarrier
\end{document}

