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.