Genetic engineering ACEI peptides
production
Large-scale production of ACEIp for functional foods and food
supplements is still a challenge. Industrial production of ACEIp has
been mainly based on enzymatic proteolysis of whole food proteins to
release peptides with ACE inhibitory activity (Pihlanto and Mäkinen,
2013). However, such methods generate a complex mixture of compounds and
lead to challenging target peptide isolation processes (Losacco et al.,
2007). The high cost, low recovery and bioavailability of ACEIp produced
by such current methods pushed the need to develop alternative
approaches, as the production of ACEIp via heterologous expression
platforms. Most recombinant ACEIp have been produced inEscherichia coli, the most widely studied heterologous expression
host. Emerging platforms for ACEIp production include lactic acid
bacteria (LAB), plants (predominantly cereals and legumes) and the
microalgae Chlamydomonas reinhardtii .
The main bottleneck in the heterologous expression of short peptide
chains as ACEIp is their susceptibility to proteolytic degradation
inside the host system. Accordingly, direct expression of genetically
engineered ACEIp has been proven troublesome. This section describes
strategies to tackle this problem (design of efficient expression
constructs, e.g. fusion proteins, tandem ACEIp; protein targeting
approaches; and use of protease-deficient hosts such as E. coliBL21 (Rao et al., 2009)) and demonstrates the potential of heterologous
expression systems to produce ACEIp at large-scale, low-cost and in
convenient formulations.
Recombinant ACEI peptide expression in
bacteria
Bacteria are well-established expression systems for high-level
production of recombinant proteins and peptides, and have been the
preferred systems for heterologous ACEIp expression (Losacco et al.,
2007). E. coli is particularly used in new ACEIp production
systems, and the use of LAB is an emerging alternative, given their
Generally Recognized as Safe (GRAS) status.
Four main strategies have been adopted for ACEIp production in bacterial
hosts: 1) generation of multimeric polypeptides containing tandem
repeats of an ACEIp; 2) fusion of ACEIp to proteins; 3) generation of
bioactive polypeptides containing multivariate ACEIp; and 4) mutation of
plant storage proteins to include ACEIp. In all these approaches, the
aim is to protect the ACEIp against degradation, as polypeptides and
fusion proteins are less susceptible to degradation by host peptidases
than single ACEIp. Table 2 presents several recombinant ACEIp produced
in bacterial platforms, their production strategy, yield and
bioactivity.
Tandem repeats of ACEIp
The expression of chimeric configurations containing tandem repeats of a
desired ACEIp, flanked by protease recognition sequences that allow the
peptide release after in vitro protease cleavage or
gastrointestinal digestion showed significant advantages at the
expression level (Kim et al., 2008).
One of the early examples of the use of ACEIp in tandem consisted of ten
amino acids derived from the yeast GAPDH enzyme (YG-1 gene) in 9, 18, or
27 tandem repeats of the gene separated by clostripain cleavage sites
(Park et al., 1998). Contrary to expectations, the highest expression
level of YG-1 was observed for the 9-mer (67% of total proteins), a
fact related to the use of the T7 promoter (Park et al., 1998).
Fida et al. (2009) first reported the direct insertion of a small tandem
peptide multimer ‘gene’ into an expression vector without a fusion
protein tag. In this example, the ACEIp ‘fragmented peptide B’
(PTHIKWGD), retrieved from thermal hydrolysates of tuna meat, was
produced in E. coli as 6-mer. Although bypassing digestion steps
to remove the fusion tag, this approach required a prohibitively
expensive anti-peptide antibody affinity chromatography. Recently, the
expression levels of this peptide B in E. coli have been improved
by multimerization of the tandem sequence. The original 4-mer peptide B
‘genes’ were further assembled as 1, 2, 4 and 8x tandem repeats and
fused to a his-tag. A correlation between the degree of multimerization
and the expression level of these multimer ‘genes’ was observed, with
the 32 multimer (8 x 4mer) presenting the highest expression (45.2% of
total protein). The purified peptide monomers presented antihypertensive
activity in SHR, decreasing systolic BP by 36.5 mm Hg upon 4 h of oral
administration (Li et al., 2015).
Another example is the expression of a sequence encoding IYPR, an ACEIp
isolated from sake and sake lees, as a 7-copy tandem repeat, linked by
trypsin cleavage sites. Following cleavage with trypsin and purification
by affinity chromatography, the resultant ACEIp presented
antihypertensive activity in 10-week old SHR, significantly reducing
systolic BP by 50 mm Hg, upon 4h of a single oral administration (Huang
et al., 2012). Similarly, the DNA-coding sequence for GVYPHK, derived
from a partially purified autolysate of bonito bowels, was linked by a
trypsin cleavage site to form a 10-mer tandem protein (Wang et al.,
2015). A single oral administration to SHR of the ACEIp obtained by
trypsin cleavage, significantly reduced systolic BP already after 2 h of
ingestion.
ACEI peptides fused to
proteins/polypeptides
In a different approach, single or tandem ACEIp can be fused to proteins
and expressed as high molecular weight fusion proteins. The use of
fusion protein partners has several advantages: boosts peptide
expression by increasing mRNA translation; helps short polypeptides
stabilization; and facilitates the purification of the peptides by
affinity chromatography. An additional proteolytic step is usually
required after translation to release the ACEIp from the fusion partner.
The enzyme glutathione S-transferase (GST) has been the most commonly
used fusion partner, but ubiquitin, dihydrofolate reductase (DHFR) and
maltose-binding protein (MBP) have also been used.
The GST fusion protein system has been used both in single and tandem
ACEIp production. A 6-mer tandem of KVLPVP, derived from the milk
β-casein hydrolysate, was linked by clostripain cleavage sites and fused
to GST (Liu et al., 2007). The pure peptide presented an
IC50 of 4.6 μ M and showed antihypertensive
activity in SHR, dramatically decreasing systolic BP in a dose-dependent
manner. Another example comprises recombinant concatemers of multiple
IYVKY copies, fused to GST. Tandems of 2, 4 and 6-mer of the IYVKY
sequence, linked by the chymotrypsin cleavage site and fused
N-terminally to a His-tag and C-terminally to GST, were produced. Their
expression in E. coli BL21 lead to a good yield production of
bioactive single ACEIp IY and VKY (Oh et al., 2002).
The GST expression system was also used to produce single peptides, such
as the ACEI BP1-3, derived from bovine β-casein (Losacco et al., 2007),
and their precursors (Pro-BP1-3) (Losacco et al., 2007). Each Pro-BP or
BP-coding DNA sequences were cloned downstream the GST sequence and
flanked in both sides by the cleavage sites (3-5 aa long) of a membrane
proteinase from Lactobacillus helveticus PR4. The use of this
membrane proteinase was strategic, given the common use of this LAB in
manufacturing dairy products that naturally contain similar ACEIp.
However, the IC50 of BP1-3 was relatively high (Table 2).
Besides GST, other fusion proteins have been used in recombinant ACEIp
production. BP1-3 and ProBP1-3 were also expressed in a probiotic strain
of Bifidobacterium pseudocatenulatum , fused at the 5’ end to a
Shine-Dalgarno ribosome binding site (RBS) consensus sequence (Losurdo
et al., 2013). Although the ACE-inhibitory activity increased in
cell-free extracts of all recombinant hosts, the expression of BP1 and
BP3 and their precursor forms was not detectable by RP-HPLC. This was
attributed to their hydrolysis by intracellular endopeptidases, such as
aminopeptidases and iminopeptidases. Other example is the production of
a single ACEIp from αs1-casein FFVAPFPEVFGK (known as
CEI12), fused to DHFR. Nevertheless, relatively low
yields (0.5 mg.L-1) of the CEI12-DHFR
fusion protein were obtained upon IPTG induction in E. coli (Lv
et al., 2003).
These last reports demonstrate the limitations of single ACEIp
expression approaches, which may be partially overcome by using tandem
ACEIp. The synthetic gene coding for the ACEIp HHL, derived from a
Korean soybean paste, was tandemly multimerized to a 40-mer and ligated
to ubiquitin as a fusion gene (UH40). HHL monomers were recovered at 6.2
mg. L-1 yield (Jeong et al., 2007), showing the
advantages of fusing proteins with ACEI tandem multimers, in comparison
to single peptide approaches. Sixteen tandem repeats of the
α-lactalbumin-derived ACEIp IW were N-terminally fused to MBP, and
expressed in E. coli BL21 (Michelke et al., 2018). This MBP-IW
fusion protein was recovered at low yield (0.52 mg soluble protein/g ofE. coli ) and, after hydrolysis with α-chymotrypsin, only 50.78 µg
of IW monomers were released. Still, the ACEI activity of the
recombinant IW was indistinguishable from that of the chemically
synthesized dipeptides.
Despite the moderate success of fusion protein approaches in E.
coli , most require expensive and time-consuming purification steps to
remove protein tags and the use of non-food-grade inducer molecules,
such as IPTG. A recent study, based on the initial CEI12expression in E. coli (Lv et al., 2003) and Streptococcus
thermophilus (Renye and Somkuti, 2008; Renye and Somkuti, 2015)
attempted to surpass these shortcomings by employing nisin-induced
CEI12 expression in three LAB strains: S.
thermophilus ST128, Lactococcus lactis subsp. lactis ML3,
and L. casei C2. A synthetic CEI12-coding gene
was cloned under the nisA promoter, in-frame with the pediocin leader
peptide to direct the secretion of the resultant fusion peptide by LAB
hosts. Both L. lactis ML3 and L. casei C2 secreted the
recombinant peptide, as confirmed by SDS-PAGE (Renye and Somkuti, 2015).
Although recovered recombinant peptide yields were low, this study is a
landmark, as it first reported the use of GRAS LAB species and of nisin
as “food-grade” inducer, prospecting the use of LAB as ACEIP
production platforms for the functional foods industry.
Multivariate ACEI peptides in bioactive
polypeptides
A third strategy for bacterial expression of bioactive ACEIp relies on
engineering synthetic genes with different ACEIp in tandem. Rao et al.
(2009) described the design and production of a tandem antihypertensive
peptide multimer (AHPM), as a precursor of 11 different ACEIp, joined by
1-3 aa-long linkers corresponding to cleavage sites of gastrointestinal
proteases. The recombinant AHPM polypeptide, fused to a GST tag, was
expressed in E. coli mostly as inclusion bodies, and reached a
maximal production of 35% of total intracellular protein. Although the
yield of multimer peptide recovery was low, simulated gastrointestinal
digestion confirmed the release of highly active fragments from AHPM
(Rao et al., 2009). This research team has also reported the design of a
new polypeptide (BPP-1) composed of several ACEI and antioxidant
peptides, tandemly linked by gastrointestinal proteases cleavage sites.
The BPP-1 precursor consisted of a tandem multimer of the ACEIp: MRW,
WIR, IRA, AMK, MKR, RGY, VAW, DGL, IPP, IKP, IKPFR, IKPVA, AKF, IW, VAF,
VSV, IQY and IVY, and the antioxidant peptides DTHK, YPIL, FLEPDY,
YLEPFR, YLEPDY, YDEPEW, HYRPFW, YEPDY and IWAPFY. To improve the yield
of soluble form, BPP-1 was further fused to the tag ‘cationic
elastin-like polypeptide and SUMO’ (cELP-SUMO). This tag both enhances
the solubility of fusion proteins and allows its cleavage to efficiently
release peptides (Rao et al., 2016). As a result, cELP-SUMO-BPP-1 was
highly expressed in a soluble form in E. coli , consisting of
approximately 52% of the total soluble proteins, with more than 70% of
the fusion protein being expressed in a soluble form. After simulated
gastrointestinal digestion of the purified BPP-1 the resulting
hydrolysates exhibited notable in vitro ACE inhibitory and
antioxidant activities (Rao et al., 2016).
Modified plant storage proteins containing ACEI
peptides
The feasibility of modifying subunits of plant storage proteins to
contain ACEIp has been widely tested in E. coli as primary
evaluation step, envisaging their application in food crops. Two plant
storage proteins have been used for ACEIp production: the soybean
β-conglycinin α’ subunit and the Amaranthus amarantin acidic
subunit.
β-conglycinin α’ subunit (Soybean). Matoba et
al. (2001) introduced Novokinin (the RPLKPW ACEIp, a potent analogue of
ovokinin) into three homologous sites of the soybean β-conglycinin α’
subunit, by site-directed mutagenesis. This modified RPLKPW-containing
α’ subunit was first expressed in E. coli and recovered in a
soluble fraction at yields of 15% of total protein (Matoba et al.,
2001a). When orally administered, the undigested RPLKPW-containing α’
subunit demonstrated a potent antihypertensive effect and a time-course
behavior similar to the one of free RPLKPW peptide, denoting the rapid
release of the peptide from the protein after ingestion. This study
first attested the in vivo functionality of a modified storage
protein containing ACEIp. Nevertheless, the release of the RPLKPW
sequences from the modified subunit by gastrointestinal digestion was
only about 30% in SHR. To overcome this limitation, Onishi and
colleagues (Onishi et al., 2004) optimized the aa residues surrounding
the three RPLKPW peptide units, to facilitate in vivo release.
Furthermore, a fourth RPLKPW sequence was also introduced to improve
RPLKPW peptides yield. This new modified RPLKPW-containing α’ subunit
was efficiently expressed in E. coli as ~25% of
total bacterial proteins (a 10% yield increased relative to previously
reported). Further, it significantly lowered systolic BP in SHR 4h after
oral administration of a 2.5 mg.kg-1 dose, one-fourth
of the previously reported dose, proving the benefits of inserting a
supplementary RPLKPW unit. Finally, a more potent antihypertensive
protein was produced in E. coli , as an extension domain
corresponding to residues 1-143 of the modified α’ subunit, containing
four RPLKPW sequences, with 1.0 mg.kg-1 as the minimum
effective dose.
Acidic subunit of amarantin (Amaranthus).
Another plant storage protein that has been modified to produce active
ACEIp is amarantin, the main seed storage protein of Amaranthus
hypochondriacus . This modified amarantin acidic subunit has been
extensively evaluated in E. coli , using different site directed
mutagenesis strategies. Luna-Suárez and colleagues (Luna-Suárez et al.,
2010) first reported the insertion of 4 tandem repeats of the ACEIp VY
into the third hypervariable region of the amarantin acidic subunit, and
named this chimeric protein ‘bioamarantin’. The same group improved
bioamarantin design by further inserting one copy of RIPP
(Castro-Martínez et al., 2012) or IPP (Medina-Godoy et al., 2013) into
the forth hypervariable region of the amarantin acidic subunit. All
modified subunits showed higher ACEI activities than the native protein.
Furthermore, the enzymatic hydrolysates of AMC3-containing ACEIp
sequences (4xVY and IPP) presented a significant antihypertensive action
at 100 mg.kg-1 dose, 4.5h after oral administration in
SHR (Medina-Godoy et al., 2013). Finally, this team modified the acidic
subunit of amarantin by inserting 4 VY peptides into the fourth
hypervariable region of the acidic subunit, (‘AACM.4’) (Morales-Camacho
et al., 2016). AACM.4 showed highest expression levels, greater in
vivo or kinetic stability and higher percentage of soluble form
(~5% of total protein), compared to the native protein
or previously reported variants (Luna-Suárez et al., 2010;
Castro-Martínez et al., 2012). Moreover, AACM.4 was also the most
thermostable protein, suggesting that the fourth hypervariable region
modification improves the subunit thermal stability. These results
confirmed AACM.4 as a potential target for future plant transformation
and food additive production. Importantly, the improvement of thermal
stability can be a critical factor during functional food processing
procedures.
Recombinant ACEI expression in plants and
microalgae
Plant proteins are precursors of numerous ACEIp that can be released
during gastrointestinal digestion or plant crops processing. However,
given their low content in natural ACEIp, plant/plant-derived food
consumption is usually insufficient to significantly lower BP.
Furthermore, the industrial production of ACEIp, through enzymatic
hydrolysis of plant proteins, can be troublesome and economically
unviable due to high process costs and low ACEIp yield. The use of plant
biotechnology (Figure 2 and Table 3) can thus expand the ACEI properties
of plant crops, envisaging the establishment of novel plant-derived
functional foods and food supplements.
Until now, rice (Oryza sativa ) and soybean were the favored plant
expression hosts for producing ACEIp. Some advantages include the
existence of well-established transformation systems, high level of
recombinant proteins production and high grain yield. Furthermore, the
possibility of ACEIp’ accumulation in rice and soybean seeds is highly
beneficial for peptide stability and storage, providing a direct peptide
delivery route (Twyman et al., 2003). Other reported systems of ACEIp
production include cell suspension cultures and transplastomic platforms
based on C. reinhardtii .
Figure 2 summarizes the main strategies for ACEIp production in plants
and algae, and Table 3 provides detailed information. In Table 3 three
main approaches are presented by chronological order of report: 1) the
modification of storage proteins to carry ACEIp; 2) the generation of
chimeras containing tandem repeats of ACEIp; 3) the generation of
bioactive polypeptides containing multivariate ACEIp (Kim et al., 2008;
Rosales-Mendoza et al., 2013).
Modified plant storage proteins containing ACEI
peptides
Expression of ACEIp in plant storage proteins has the advantage of
ensuring long-term protein stability and storage. Storage proteins are
generally located in specialized compartments, such as protein bodies
and vacuoles, which provide appropriate biochemical environments for
protein/peptide accumulation, protecting those from proteolytic
degradation (Twyman et al., 2003). ACEIp-coding sequences are generally
introduced into homologous sites of the protein’s variable regions to
minimize changes in protein folding, productivity and localization in
plants. Examples are the modified subunits of glutelin, β-conglycinin
and amarantin.
Glutelin. Modified subunits of the rice storage protein
glutelin, containing the potent ACEIp novokinin (RPLKPW), have been
expressed in transgenic rice (Yang et al., 2006). Fusion proteins were
expressed under the control of endosperm-specific glutelin promoters and
specifically accumulated in seeds. Oral administration in SHR of either
the RPLKPW-glutelin fraction or the transgenic rice seeds significantly
reduced systolic BP, confirming the potential as valid nutraceutical
delivery systems for antihypertensive peptides.
β-conglycinin α’ subunit (Soybean). Based on
the findings above described for E. coli (Matoba et al., 2001a;
Onishi et al., 2004), a modified β-conglycinin α’ subunit carrying 4
novokinin peptide units has been expressed in soybean (Nishizawa et al.,
2008). However, the chimeric protein was only 0.2% of the total
extracted protein from the transgenic soybean seeds, a low value to
assess the seeds’ in vivo effects. More recently, novokinin was
expressed in transgenic soybean seeds as a 4 tandem multimer of
novokinin fused to the β-conglycinin α’ subunit. The chimeric protein
produced in transgenic soybean seeds comprised 0.5 % of total soluble
protein and 5 % of total β-conglycinin α’ subunit. This chimeric
protein was shown to possess antihypertensive activity, reducing
systolic BP in SHR after 0.15 g.kg-1 protein extract
oral administration. A similar effect was attained following
administration of 0.25 g.kg-1 dose of defatted flour
(Yamada et al., 2008).
Acidic subunit of amarantin (Amaranthus).
Based on previous studies in E. coli (Luna-Suárez et al., 2010),
bioamarantin was also expressed in cell suspension cultures ofNicotiana tabacum L. NT1. Protein hydrolysates of transgeniccalli showed high ACEI activity, with 3.5
μg.ml-1 IC50 value , 10-fold lower than protein
extracts of wild-type cells (IC50 of 29.0 μg.ml-1)
(Santos-Ballardo et al., 2013). This was the first report of the
production of a chimeric protein comprising ACEIp in plant cell
suspension cultures. More recently, bioamarantin was also expressed in
transgenic tomato fruits, and stably accumulated at levels up to 12.71%
of total protein content. Furthermore, a remarkable increase (5–22 %)
in total protein content was also observed in transgenic tomato fruits,
when compared to non-transformed ones. Protein hydrolysates from
transgenic tomato fruits showed 0.376 to 3.241 μg.ml-1in vitro IC50 values, corresponding to an increase of up to
13-fold in ACEI activity, when compared to the non-transformed fruits
(Germán-Báez et al., 2014). Positive reports of the expression of
modified amarantin variants in E. coli (Luna-Suárez et al., 2010;
Medina-Godoy et al., 2013; Morales-Camacho et al., 2016) along with
their sustained expression in tomato (Germán-Báez et al., 2014) and
tobacco (Santos-Ballardo et al., 2013), prospect the high scale
production of ACEIp-containing modified amarantin in heterologous
hosts.
Tandem repeats of ACEI peptides
Following the first attempts of novokinin production in modified storage
proteins in soybean (Nishizawa et al., 2008; Yamada et al., 2008) and
rice (Yang et al., 2006); Wakasa et al. (2011) aimed to generate
transgenic rice seeds that would accumulate higher amounts of novokinin.
Their strategy comprised the expression of 10 or 18 tandemly repeated
novokinin sequences with a KDEL endoplasmic reticulum-retention signal
at the C-terminus, using the glutelin promoter and its signal peptide.
Although the chimeric protein was unexpectedly accumulating in the
nucleolus and at a low level, significant antihypertensive activity was
detected after a single oral dose in SHR. More importantly, this effect
was observed over 5-wk at doses as low as 0.0625
g.kg-1.
More recently, a synthetic gene containing tandem repeats of the VLPVP
ACEIp has been expressed in a transplastomic C. reinhardtiistrain. VLPVP-coding sequences were linked by cleavage sites of pepsin,
trypsin, and chymotrypsin. Biomass of recombinant C. reinhardtiiwas in vitro digested, and the VLVPV peptide was identified and
quantified by HPLC. The highest expression line produced 0.292 mg
recombinant protein/mg freeze-dried biomass. Intragastric administration
to SHR, at a dose of 30 mg.kg-1, significantly reduced
rats systolic BP (Ochoa-Mendez et al., 2016). This in vivoantihypertensive effect first provided a functional validation of using
ACEIp-producing microalgae as food supplements for hypertension
patients.
Multivariate ACEI peptides in bioactive
polypeptides
Campos-Quevedo et al. (2013) reported the design and production of a
milk-derived chimeric protein containing sequences for multifunctional
bioactive peptides, including peptides with hypocholesterolemic,
antihypertensive, opioid, and antimicrobial activities. The precursor
chimeric protein contained 20 different bioactive peptides linked by
gastrointestinal protease cleavage sites. The synthetic gene coding for
the milk-derived chimeric protein was transferred to C.
reinhardtii using biolistic bombardment. Transplastomic transformants
containing the target synthetic gene were identified, and ELISA
quantification assay revealed that the expressed chimeric protein
accumulated at levels ranging between 0.16 and 2.4% of total soluble
protein. Although no functional studies (in vitro ACEI activity
or in vivo antihypertensive effect) were performed, this study
first showed the potential of C. reinhardtii as expression
platform for the production of ACEIp in multifunctional bioactive
polypeptides.