**5.3 Heterologous production of ACEI peptides in plants**

In recent years, the application of recombinant DNA technologies for the production of ACEI peptides at a large scale and low cost has gathered attention in the biotechnology community. Investigation has been focused on the development of expression methods for antihypertensive peptide production in different plant crops [128]; and here, we tried to provide some promising examples.

Thus far, the main strategies that have been adopted are as follows: the overexpression of ACEI peptide precursor proteins and the production of particular peptides as heterologous components [101], the modification of some storage proteins to produce chimeric proteins carrying ACEI peptides [101], and also the generation of multimer proteins containing tandem repeats of ACEI peptides, flanked by protease recognition sequences that allow the peptide release during gastrointestinal digestion.

#### *5.3.1 Rice*

Transgenic rice plants that accumulate novokinin (RPLKPW), a potent antihypertensive peptide designed according to the structure of ovokinin (2–7) (RADHPF), as a fusion with the rice storage protein glutelin, have been generated. The engineered peptide is expressed under the control of endosperm-specific glutelin promoters and specifically accumulates in seeds. Oral administration of either the RPLKPW-glutelin fraction or transgenic rice seeds to spontaneously hypertensive rats (SHRs)—the main model for assessing the in vivo activity of ACEI peptides (e.g., [108, 111, 122])—significantly reduced systolic blood pressures, suggesting

**63**

*5.3.3 Tomato and tobacco*

IC50 value of 3.5 μg ml<sup>−</sup><sup>1</sup>

wild-type cells (IC50 of 29.0 μg ml<sup>−</sup><sup>1</sup>

*Prospects for the Production of Recombinant Therapeutic Proteins and Peptides in Plants…*

and particularly for administration of antihypertensive peptides [129].

the possible application of transgenic rice seed as a nutraceutical delivery system

Wakasa et al. [130] attempted the generation of transgenic rice seeds that would accumulate higher amounts of novokinin peptide by expressing 10 or 18 tandemly repeated novokinin sequences, with the KDEL endoplasmic reticulum retention signal at the C-terminus, and using the glutelin promoter along with its signal peptide. Although the chimeric protein was unexpectedly located in the nucleolus and the accumulation was low, a significant antihypertensive activity was detected after a single oral dose to SHRs. More importantly, this effect was observed over a relatively longer duration time, with intervals of 5 weeks between doses as low as

Soybean [*Glycine max* (L.) Merr.] is an attractive option for the production of ACEI peptides given that soybean seeds contain a large amount of total protein. Therefore, there has been an effort to generate soybean lines with improved ACEI

Matoba et al. [128], introduced novokinin (RPLKPW) into homologous sequences of a soybean β-conglycinin α' subunit by site-directed mutagenesis. Founded on first achievements from an *E. coli* expressed protein, the muted β-conglycinin α' subunit carrying novokinin repeats were also expressed in soybean. This chimeric protein accumulated at levels of up to 0.2% of extracted protein from transgenic soybean seeds [131]. Still, the levels of expression were too low, and

Novokinin has also been expressed in transgenic soybean seeds in a fusion form along with a β-conglycinin α' subunit. Interestingly, a reduced systolic blood

extracts. A similar effect was attained following administration of a 0.25 g kg<sup>−</sup><sup>1</sup>

of defatted flour. Thus, it was concluded that this chimeric protein produced in

Additionally, a synthetic gene of His-His-Leu (HHL), an ACEI peptide derived from a Korean soybean paste, was tandemly multimerized to a 40-mer, ligated with ubiquitin as a fusion gene (UH40), and subsequently expressed in *E. coli*. Following digestion with leucine aminopeptidase, the 405-Da HHL monomer was recovered by reversephase high-performance liquid chromatography (HPLC). MALDITOF mass spectrometry, glutamine-TOF mass spectrometry, N-terminal sequencing, and measurement of ACE-inhibiting activity confirmed that the resulting peptide was the HHL [133]. The potential use of this antihypertensive chimeric protein in soybean has yet to be assessed.

A modified version of amarantin, the main seed storage protein of *Amaranthus* 

protein comprising an ACEI peptide was produced in plant cell suspension cultures. This modified version of amarantin was also expressed in the fruit of transgenic tomato plants. Protein hydrolysates from transgenic tomato fruits showed in vitro ACE inhibition, with IC50 values ranging from 0.376 to 3.241 μg ml<sup>−</sup><sup>1</sup>

, and 10-fold lower levels than that of protein extracts of

) [134]. This was the first time that a chimeric

*hypochondriacus*, carrying four tandem repeats of the ACEI dipeptide Val-Tyr into the acidic subunit of amarantin, was expressed in cell suspension cultures of *Nicotiana tabacum* L. NT1. Protein hydrolysates obtained from transgenic *calli* showed high levels of inhibition of the angiotensin-converting enzyme, with an

of protein

dose

;

it was not possible to assess the in vivo effects of these soybean seeds.

pressure was observed in SHRs after administering a dose of 0.15 g kg<sup>−</sup><sup>1</sup>

properties foreseeing the creation of novel functional foods.

soybean possessed an antihypertensive activity [132].

*DOI: http://dx.doi.org/10.5772/intechopen.84419*

0.0625 g transgenic seeds per kg.

*5.3.2 Soybean*

#### *Prospects for the Production of Recombinant Therapeutic Proteins and Peptides in Plants… DOI: http://dx.doi.org/10.5772/intechopen.84419*

the possible application of transgenic rice seed as a nutraceutical delivery system and particularly for administration of antihypertensive peptides [129].

Wakasa et al. [130] attempted the generation of transgenic rice seeds that would accumulate higher amounts of novokinin peptide by expressing 10 or 18 tandemly repeated novokinin sequences, with the KDEL endoplasmic reticulum retention signal at the C-terminus, and using the glutelin promoter along with its signal peptide. Although the chimeric protein was unexpectedly located in the nucleolus and the accumulation was low, a significant antihypertensive activity was detected after a single oral dose to SHRs. More importantly, this effect was observed over a relatively longer duration time, with intervals of 5 weeks between doses as low as 0.0625 g transgenic seeds per kg.

### *5.3.2 Soybean*

*Genetic Engineering - A Glimpse of Techniques and Applications*

**Source ACEI activity (IC50; μM) Antihypertensive activity** 

**(mmHg)**

*Chlorella vulgaris* 29.6 Not determined — [114] Chebulic myrobalan 100 Not determined — [115] Bitter melon 8.64 −31.5 to −36.3 2–10 [116] Mung bean 13.4 Not determined — [117] Pea 64 Not determined — [118] Peanut 72 Not determined — [100] Potato 18–86\* Not determined — [119] Rapeseed 28 −11.3 7.5 [120] Rice 18.2 −40 30 [111] Soybean 14–39\* −17.5 2 [121] Soybean 21 Not determined — [122] Soybean 1.69 Not determined — [123] Soybean 17.2 Not determined — [124] Spinach 0.6–4.2\* −13.5 to −20\* 20–100 [112] Wakame 21–213\* −50 50 [113] Walnut 25.7 Not determined — [125] Wheat 20 Not determined — [126]

**Dose (mg/kg) Reference**

**5.3 Heterologous production of ACEI peptides in plants**

*Examples of ACEI peptide activity from different plant origin.*

*Different values for the same plant product related to the ACEI peptide sequence.*

In recent years, the application of recombinant DNA technologies for the production of ACEI peptides at a large scale and low cost has gathered attention in the biotechnology community. Investigation has been focused on the development of expression methods for antihypertensive peptide production in different plant

Thus far, the main strategies that have been adopted are as follows: the overexpression of ACEI peptide precursor proteins and the production of particular peptides as heterologous components [101], the modification of some storage proteins to produce chimeric proteins carrying ACEI peptides [101], and also the generation of multimer proteins containing tandem repeats of ACEI peptides, flanked by protease recognition sequences that allow the peptide release during

Transgenic rice plants that accumulate novokinin (RPLKPW), a potent antihypertensive peptide designed according to the structure of ovokinin (2–7) (RADHPF), as a fusion with the rice storage protein glutelin, have been generated. The engineered peptide is expressed under the control of endosperm-specific glutelin promoters and specifically accumulates in seeds. Oral administration of either the RPLKPW-glutelin fraction or transgenic rice seeds to spontaneously hypertensive rats (SHRs)—the main model for assessing the in vivo activity of ACEI peptides (e.g., [108, 111, 122])—significantly reduced systolic blood pressures, suggesting

crops [128]; and here, we tried to provide some promising examples.

**62**

*5.3.1 Rice*

*\**

**Table 1.**

gastrointestinal digestion.

Soybean [*Glycine max* (L.) Merr.] is an attractive option for the production of ACEI peptides given that soybean seeds contain a large amount of total protein. Therefore, there has been an effort to generate soybean lines with improved ACEI properties foreseeing the creation of novel functional foods.

Matoba et al. [128], introduced novokinin (RPLKPW) into homologous sequences of a soybean β-conglycinin α' subunit by site-directed mutagenesis. Founded on first achievements from an *E. coli* expressed protein, the muted β-conglycinin α' subunit carrying novokinin repeats were also expressed in soybean. This chimeric protein accumulated at levels of up to 0.2% of extracted protein from transgenic soybean seeds [131]. Still, the levels of expression were too low, and it was not possible to assess the in vivo effects of these soybean seeds.

Novokinin has also been expressed in transgenic soybean seeds in a fusion form along with a β-conglycinin α' subunit. Interestingly, a reduced systolic blood pressure was observed in SHRs after administering a dose of 0.15 g kg<sup>−</sup><sup>1</sup> of protein extracts. A similar effect was attained following administration of a 0.25 g kg<sup>−</sup><sup>1</sup> dose of defatted flour. Thus, it was concluded that this chimeric protein produced in soybean possessed an antihypertensive activity [132].

Additionally, a synthetic gene of His-His-Leu (HHL), an ACEI peptide derived from a Korean soybean paste, was tandemly multimerized to a 40-mer, ligated with ubiquitin as a fusion gene (UH40), and subsequently expressed in *E. coli*. Following digestion with leucine aminopeptidase, the 405-Da HHL monomer was recovered by reversephase high-performance liquid chromatography (HPLC). MALDITOF mass spectrometry, glutamine-TOF mass spectrometry, N-terminal sequencing, and measurement of ACE-inhibiting activity confirmed that the resulting peptide was the HHL [133]. The potential use of this antihypertensive chimeric protein in soybean has yet to be assessed.

#### *5.3.3 Tomato and tobacco*

A modified version of amarantin, the main seed storage protein of *Amaranthus hypochondriacus*, carrying four tandem repeats of the ACEI dipeptide Val-Tyr into the acidic subunit of amarantin, was expressed in cell suspension cultures of *Nicotiana tabacum* L. NT1. Protein hydrolysates obtained from transgenic *calli* showed high levels of inhibition of the angiotensin-converting enzyme, with an IC50 value of 3.5 μg ml<sup>−</sup><sup>1</sup> , and 10-fold lower levels than that of protein extracts of wild-type cells (IC50 of 29.0 μg ml<sup>−</sup><sup>1</sup> ) [134]. This was the first time that a chimeric protein comprising an ACEI peptide was produced in plant cell suspension cultures.

This modified version of amarantin was also expressed in the fruit of transgenic tomato plants. Protein hydrolysates from transgenic tomato fruits showed in vitro ACE inhibition, with IC50 values ranging from 0.376 to 3.241 μg ml<sup>−</sup><sup>1</sup> ;

this represented an increase of up to 13-fold in the inhibitory activity when compared with the protein hydrolysates of non-transformed fruits [135]. These two results suggest the possible application of tobacco plant cell suspension cultures and transgenic tomato fruits for massive production of this engineered version of amarantin, which could be especially used as an alternative hypertension therapy [134, 135].
