Soybean Functional Proteins and the Synthetic Biology

Abstract

Recently, soybean consumption has increased, not only because of its potential for industrial and livestock use but also due to its beneficial effects on human health in the treatment and prevention of various diseases because soy can produce a wide number of functional proteins. Despite the soybean-producing high, elevated, nutritive and functional proteins, it also produces allergenic proteins, harmful secondary metabolites, and carcinogenic elements. So, recombinant protein systems that mimic the structures and functions of the natural proteins supply a single tunable and valuable source of advanced materials. But the availability of the technology to produce synthetic functional proteins is still limited. Therefore, Synthetic Biology is a powerful and promising science field for the development of new devices and systems able to tackle the challenges that exist in conventional studies on the development of functional protein systems. Thus, representing a new disruptive frontier that will allow better use of soybean functional proteins, both for animal and human food and for the pharmaceutical and chemistry industry.

1. Introduction

Synthetic Biology allows for more sophisticated and complex engineering than the old genetic modification techniques. Involving scientific subjects and non-biological engineering, including information technology, bioinformatics, and nanotechnology, synthetic biology strives to alter the organism’s genes on a scale much bigger – their genome! – rewriting your genetic code, all chemical instructions needed to project, assemble, and operate a living organism. To invent new ways of life via biochemistry, created in the computer and made from off-the-shelf chemicals, will not just revolutionize biology, but it will also profoundly influence the definition of life, including what it means to be human [1]. The creation of new life forms could be a little bit threatening, although this is just a possible approach of Synthetic Biology that follows all the ethical precepts of modern science and that just has advanced in the field of microbiology, through research involving the development of the minimal genomes of a living organism, like, for example, the minimal genome of the bacteria Mycoplasma mycoides (JCV-Syn3.0) [2] and the project Sc2.0 – Synthetic yeast Genome Project. Another approach less threatening and much more promising is the possibility of modifying existing organisms for “more useful” and economically applicable purposes, such as developing biofabrics to produce medicines or biofuels [3].

One of the oldest leguminous consumed by humankind, Soybean occupies an important place in the world food industry, offering oil and protein source consumption and bran-rich in proteins for animal feed. Generally composed of ~35–40% protein, ~20% lipids, ~9% dietary fibers, and ~ 8.5% moisture based on the dry weight of mature raw seed [4]. Soybean as a protein biofactory/bioreactor for various industrial purposes (cosmetic, pharmaceutical, biofuel, food) is deeply studied due to several aspects inherent to its easy cultivation in a greenhouse and its rich genetic variability [5, 6]. Two possibilities are explored for protein production in soybean, one involves the production of cisgenic proteins and the other consists of transgenic protein production. A typical example of the potential to produce bioactive proteins using soybean is in the therapeutic market. This market moves over 100 billion dollars worldwide and grows 20% annually. However, it is a market where production depends mainly on microorganisms and animals to sustain itself. Obtaining proteins from these organisms presents low productivity and high production cost, in the case of animals which involves debatable ethical issues [7]. However, with the advancement of scientific advances in biotechnology, bioinformatics, and omics technologies, the soybean has been shown a more sustainable, safe, and cheaper alternative to producing bioactive proteins when compared to production by organisms already used [7, 8].

Soybean-based agriculture faces several productivity and global sustainability challenges, including emerging threats from climate change and diseases, forcing the rapid adoption of short-to-long-term genetic innovation methods. Thus, the field of Synthetic Biology (SB or SynBio) is prepared to offer several technological solutions to rapidly improve the development of new soybean cultivars through genetic circuits, biosensors, metabolic engineering, and genome editing techniques. SynBio is a field of scientific research that integrates principles from mathematics, physics, engineering, and chemistry and applies genetic tools to develop bottom-up and top-down strategies to design technological products for industry, medicine, and agriculture (Figure 1). Thus, regarding the sustainable and technical management of soybeans (Glycine max), SynBio can usefully increase biomass production and harvest performance and dramatically transform existing genetic modification techniques. Furthermore, the omics tools have made remarkable progress [9, 10, 11] because there are few technical difficulties in obtaining complete soybean genomic sequences. However, the factor to be overcome is understanding the complex functioning and organization of the genome itself [12, 13, 14, 15, 16, 17, 18]. Thus, the combination of current genomic prediction, design, and synthesis techniques and the recently proposed genome editing methodologies could allow the rapid development of new bioreactor chassis for protein production [19, 20, 21, 22, 23].

Figure 1.

Schematic representation of the concepts and applications of synthetic biology.

Protein is one of the most important components in an essential diet for the survival of organisms; they supply adequate amounts of amino acids to the body. The availability of amino acids from food depends on various factors such as the source of the protein, prior protein processing treatments, interactions with other components of nutrition, their digestibility, absorption, and utilization in the organism [24]. Soybean is the most important source of low-cost proteins, producing more protein and oil per unit than any other leguminous crop, and it’s the most consumed legume worldwide due to its functionality and nutritional value [25]. According to the USDA nutritional database, soybean seeds consist of about 36.5% protein, 19.9% lipids, 30% carbohydrates, and 9.3% dietary fiber. Moreover, soybean is the largest source of protein used in livestock, 98% of soybean meal is used for animal feed (poultry, hogs, and cattle mostly), and only 1% is used to produce food consumed by the human population. Recently, the consumption of soybean proteins has increased due to some beneficial effects of their ingestion for human health in treating and preventing various diseases, like cardiovascular diseases and various forms of cancer. Soybean-based agriculture faces several productivity and global sustainability challenges, forcing the rapid adoption of short-to-long-term genetic innovation methods. Thus, the research field of Synthetic Biology is prepared to offer several technological solutions to rapidly improve the development of new soybean cultivars through genetic circuits, biosensors, metabolic engineering, and genome editing techniques.

2. Synthetic biology applied to soybean crops

Plant Synthetic Biology has a wide range of applications in agriculture and the pharmaceutical and energy industry. In agriculture, genetic engineering can be applied to develop new cultivars that are resistant to herbicides, bugs, illness, and drought and can be used to alter the nutritional profile of a cultivar of interest. In the energy and pharmaceutical industry, SynBio allows the production of plant biofabrics for different compounds like remedies, vaccines, biofuel, etc. The major defiance of the Synthetic Biology implementation in agriculture is the time and the extensive outgoing involved in the propagation, transformation, and screening of the superior plants. Although there is an impulse in the plant biotechnology, following the development of new technologies like genome editing based on CRISPR/Cas9 [26], speed breeding [27], key genomes sequencing [28, 29, 30], and the SynBio growth as a scientific field [31], the challenge goes on. For example, the huge size of the plant genomes and their polyploidy (wheat, for example, has a hexaploidy genome >15Gb [29]) has so far limited the efficiency of the site-specific genetic manipulations. Besides that, plants usually have fewer direct homolog recombination mechanisms (HDR) than the microbial [32]. However, some works have shown the homolog recombination mechanism mediated by the CRISPR/Cas9 for obtaining genetically modified plants [33]. Thus, despite all prominent challenges for SynBio in Agriculture/Plant Biotechnology, new approaches have arisen to overcome the old problems.

Historically, the speed of productivity increase through classical breeding was not enough to meet the world’s demand for food. This lack has required genetic improvement through biotechnology, thus by the 1980s, the development of molecular and plant transformation technologies delivered the first bioengineered genes into plant genomes. Limited yields due to climate stress, changes in pests and pathogens, heat waves, and other weather extremes − the new world reality due to global warming – forces biotechnology and molecular biology to evolve in a new disruptive and fast technology that allows the creation of a new productive and functional crop. Rapid crop improvement must influence naturally grown traits and transformative engineering driven by mechanistic understanding to produce the resilient production systems needed to secure future crops [34]– this will be the new Green Revolution. Currently, the most adopted genetically modified traits are herbicide and insect resistance in crops with large markets, such as soybean [35, 36, 37], canola [38] cotton [39], and corn [40, 41].

The challenges of modern agriculture are not restricted to the increment in production to attend to the huge world population growth. Therefore, actually, agriculture faces industry adaptations to digital and genetic technologies, carbon constraints, environmental and animal welfare legislation, the growing focus on “food as medicine” and their ethical production, risks associated with globalization and climate change, a global shift in diet and more discriminating customers in search of a wealthier world [30]. SynBio can overcome these obstacles, like the industry is a pioneer in the use of technological innovations, it is also the biggest beneficiary of advances in SynBio, but in recent years, primary sectors such as agriculture have benefited from this technological evolution.

According to the United States Department of Agriculture, most commercial releases of bioengineered soybeans aim to provide herbicide tolerance, biotic and abiotic factors, improved oil quality, improved yield, and growth (USDA, 2021; CTNBio, 2021). However, many other traits need to be explored, such as superior nutritional contents and the capacity of cultivars to act as biofabrics of industrial products. Primary industries such as agriculture, fisheries, and forestry have benefited directly from advances in genetic research. It is estimated that about half of the 1−3% annual increase in productivity in crops and livestock to date has been driven by improved genetics, with genetic gain rates predicted to more than double with the implementation of emerging molecular technologies [42].

Soybean [Glycine max (L.) Merr.], the most consumed legume in the world, originated and domesticated in North-Eastern Asian regions, especially China and Korea, its consumption has been disseminated worldwide since it arrived in American colonies in 1765 [31]. Growing demand for a nutritious, quality, low-cost, low-environment impact, source of protein to feed the growing human population turned soybean into one of the most important global agricultural commodities [43]. Because it is one of the major protein sources, such as food, for animal nutrition, including humans, livestock, pets, and fish, thus soybean seeds of commercial crops contain about 40% protein and about 20% oil [44]. In addition to being a source of protein, soy has recently been used to produce biofuels. Currently, the United States, Brazil, and Argentina together produce more than 80% of the world’s soybean crop. On the other hand, China is the largest soybean importer in the world, consuming 30% of the world’s soybean production [45]. Thus, the global soy market is governed by two major producers (the United States and Brazil, respectively), which produce around 68% of the world’s crop, and a major consumer (China) [46].

The agriculture sector has a heavy history of fast improving new transformative techniques innovations, for example, in 2005−2006 the worldwide soybean production was 220.809 million tons, already in 2021−2022 the production reached 385.524 million tons (data from https://www.sopa.org/statistics/world-soybean-production), this increment can be attributed to the development of new disruptive genetic technologies. In the past, mutagenesis is presented as an alternative for classical plant breeding to increase genetic variation in soybean germplasm. Random mutagens techniques were normally used to introduce changes in genes aleatorily, including radiation (such as X-ray), fast neutrons, and gamma rays, chemicals (such as EMS (ethyl methanesulfonate)), and biological mutagenesis (such as T-DNA insertion and transposons) [47]. Although it is hereditary and stable, random mutagenesis demand intensive, specific, time-consuming, and expensive techniques to identify the intended phenotypes in the mutants [48], and in the most cases, it is impossible to locate and obtains the specific allele to determinate function due to the imprecision of the random mutation [43].

In this way, to solve this challenge, in the last 20 years, with the advent of the SynBio, new biotechnologies based on site-directed nucleases (SNDs) or site-specific nucleases (SSNs), such as Zinc Finger Nucleases (ZFNs) [49, 50]. Transcription Activator-Like Effector Nucleases (TALENs) [51, 52] or the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) [53, 54] has been developed for generation of site-specific mutagenesis and, as the multiple SDN platforms are a very useful tool, they have been integrated into the new and actual plant breeding programs[55, 56]. Thus, with the rise of the SynBio tools and techniques, there was an exponential acceleration in the speed, quality, and several launches of new commercially interesting crops and, on the other hand, a great reduction in costs and time spent. These new approaches also increased the scope and size of genetic variability available for crop improvement, allowing the creation of diverse new engineered soybean crops for a wider range of traits (Table 1).

	Target trait	Function target gene and technology	Ref.
Yield
Plant Architecture	Alters plant height and internode length	CRISPR/Cas9 multiplex knockout of GmLHY / (GmLHY1a, GmLHY1b, GmLHY2a, GmLHY2b)	[58]
	Shorter plastochron length	CRISPR/Cas9 multiplex knockout of GmSPL9/ (GmSPL9a, GmSPL9b, GmSPL9c, GmSPL9d)	[59]
	Control flowering time and plant height	CRISPR/Cas9 multiplex knockout of GmAP1/ (GmAP1a, GmAP1b, GmAP1c, GmAP1d)	[60]
Photoperiod	Expand the regional adaptability	CRISPR/Cas9 multiplex knockout of GmFT2a e GmFT5a	[61]
	phenotypic diversity associated with important agronomic traits	BE base editor of GMFT2a and GmFT4	[62]
	Affect photoperiodic flowering	CRISPR/Cas9 knockout of GmPRR37	[63]
Nutrition and Quality
Storage Protein		CRISPR/Cas9 knockout:
	Altered expression pattern of three storage proteins	Glyma.20 g148400	[64]
		Glyma.03 g163500
		Glyma.19 g164900
Seed Oil	Increase of oleic acid rate and decrease linoleic acid content	TALENs targeted mutagenesis of GmFAD2- 1A and GmFAD2-1B	[60]
	High oleic, low linoleic and α-linolenic acid	CRISPR/Cas9 multiplex knockout of GmFAD2- 1A and GmFAD2-1B	[65]
	Increase in oleic acid, a decrease of linoleic acid, and a higher protein rate	CRISPR/Cas9 multiplex knockout of GmFAD2-1A and GmFAD2-2A	[66]
	Increase of oleic acid rate and decrease linoleic acid content	TALENs directly delivery of mutation in GmFAD3A in fad2-1a fad2-1b soybean plants.	[67]
	High oleic and low linoleic acid rate	CRISPR/Cas9 mediated targeted disruption GmFAD2-2	[68]
Bean Flavor-free	Reduction of beany flavor	CRISPR/Cas9 knockout GmLox1, GmLox2, and GmLox3	[69]
Abiotic Stress Tolerance
Herbicide Resistance	Chlorsulfuron-resistant	CRISPR/Cas9 knock-in (HDR) of GmAls	[70]
	Fertile transgenic soybean with herbicide tolerance	ZFNs knock-in (NHEJ): aad-1 (2,4D tolerance maker)	[71]
		dgt-28 (glyphosate tolerance maker) and dsm-2 (glufosinate tolerance maker) at GmFAD2-1a locus
Nitrogen Fixation
Root Nodulation	gmric1/gmric2 double mutants with increased nodule numbers and gmrdn1-1/1-2/1-3 triple mutant lines with decreased nodulation	CRISPR/Cas9 multiplex knockout of GmRIC1 (Glyma.13 g292300) and GmRIC2 (Glyma.06 g284100), GmRDN1-1 (Glyma.02g279600), GmRDN1-2 (Glyma.14 g035100) and GmRDN1-3 (Glyma.20 g040500)	[72]

Table 1.

Roll some examples of engineered soybean crops with improved traits by synthetic biology. Based on [57].

3. Soybean as a protein source

According to the Poverty and Shared Prosperity Report, after the pandemic, the estimative world percentage of people in extreme poverty (characterized by a daily income up to U$1,90) will reach about 9.1% to 9.4% of the world population. This estimate is alarming because, before the pandemic, it was estimated that poverty would fall to 7.9% in 2020. Unfortunately, people in poverty have a low caloric intake and nutritional deficiencies, especially regarding access to micronutrients and essential amino acids [73]. Food proteins, from animal or vegetable sources, supply the essential amino acids important for the construction and maintenance of the basic body structures therefore, they are fundamental for the right physical and mental development of children. Vegetable proteins represent a low-cost source of nutrients and energy, but, in many cases, they are poor in highly digestible essential amino acids. Experts predict a large increase in the world population. Consequently, the demand for plant proteins will increase in the same proportion due to the low cost and lower environmental impact of their production [74].

For centuries, humankind has cultivated seed crops as a protein source, legumes and cereals, as the principal cultivated crops. These cultures currently provide more than 70% of the protein for human consumption. Nonetheless, legumes accumulate higher quantities of protein in contrast to cereal, and among the legume crops, soybean is the one with the highest percentage of proteins [75]. Soybean seeds are a rich source of high-quality digestible proteins and contain all the essential amino acids found in animal proteins, without cholesterol and with a low level of saturated fatty acids [76]. Most soybean seed components, including proteins and peptides, isoflavones, saponins, and protease inhibitors, have been shown to have biological activity [77, 78].

The principal advantages of soybean as a protein source are (1) good balance in amino acids composition, containing all the essential amino acids; (2) presence of components physiologically beneficial to human health, which are shown to lower the cholesterol and reduce the risk of hyperlipidemia and cardiovascular diseases; (3) excellent processing ability, as emulsification, gelling, water- and oil- holding capacity; and (4) excellent nutritional and functional properties of their proteins, for example, solubility, emulsifying, film-forming and foaming properties[79, 80]. Their composition varies according to the variety, location, and conditions of its planting, such as climate and farming practices [76] and besides that, stimuli, like genetic modification, can also modify protein profile, expression, and accumulation rates [81].

Soybean proteins can be classified into four main groups: albumins, globulins, prolamins, and glutelins [82]. Based on solubility patterns, soybean proteins can be classified into two categories, albumins (water-soluble) and globulins (salt solution-soluble), which represent the primary protein type [83]. When separated with ultracentrifugation, two major storage proteins can be identified, glycinin (11S) and β-conglycinin (βCG, 7S), corresponding, respectively, to ~40% and ~ 50% of soy proteins amount [81] corresponding to the largest mass of the soy seed. The sedimentation coefficient values are a more precise pattern for identifying soybean proteins, where larger S (Svedberg units) numbers correspond to a larger protein. By ultracentrifugation under appropriate buffer conditions (0.5 ionic strength and pH 7.6), soybean proteins can be separated into four main groups, 2S, 7S, 11S, and 15S fractions [84]. While the 2S fraction (20% of total proteins) contains the most albumins, such as 2S globulin, cytochrome C, Kunitz trypsin inhibitor, and Bowman-Birk trypsin inhibitor, both the inhibitors are associated with delayed growth in children [84, 85]. The globulins are mainly present in 7S, 11S, and 15S fractions of soybean proteins. Like the 2S fraction, the 7S fraction (40% of total protein) is also highly heterogeneous, containing β-conglycinin, α-amylase, lipoxygenase, and hemagglutinin [86], and the 11S fraction (30% of total protein) consists of only the glycinin, which is the major protein in soybean seeds. The β-conglycinin and the glycinin correspond to the major component of storage proteins. The minor component, typically about 10% of the total proteins, the 15S fraction, maybe be a polymer (possibly a dimer of glycinin) [84].

3.1 Soybean-based meat

The plant-based meat industry is focused on developing burgers, patties, mince, and sausages. Besides that, at the moment, the production of the primary meat cuts, such as steak, is not the primary search worry, due to their structure composition complexity, many groups have progressed in the research of synthetic beef. Between the commercially available plant-based meat, the Beyond Burger (BB) and the Impossible Burger (IB) can be highlighted. The main ingredients of this burger can vary, but usually, it contains soy protein, wheat gluten, egg protein, or milk proteins. In the specific case of BB, non-genetically engineered ingredients such as beetroot are incorporated to give red color to meat analogs and promote the feeling of a “bleeding” meat when cooked [87, 88].

At the beginning of the 21st century, meat analogs entered the mainstream due to the demand for healthy foods, and the worry about the sustainability implications of the consumer’s diet continued to increase. Also called meat substitute, meat alternatives, fake or mock meat, and imitation meat, the meat analog is, for definition, the product of replacing the main ingredient with meat [89]. The search for sustainable, healthy, and tasty meat analogs started in the early 1960s [90].

Usually, the meat analogs found in the markets are advanced plant-based meat. The texture and taste are like the conventional meat, which uses plant-derived ingredients that have attributed almost exactly to animal-derived meat and can be indistinguishable from their animal-based equivalents. The biggest challenge for food producers is developing acceptable quality meat analogs because their characteristics depend on the ingredients used. Plant-based meat needs to be unfolded, cross-linked, and realigned to form microscopic and macroscopic fibers [89]. Different techniques are applied to plant-based meat proteins to improve their “meat qualities” to texture, processes such as extrusion, spinning, and simple shear flow have been used [91]. To solidify the structure, following the previous treatment, heating, cooling, drying, or coagulation can be applied [92].

Among the vegetables used to produce plant-based meat, the soybean stands out due to the presence of the leghemoglobin protein that mimics animal myoglobin. The soybean-based meat was the first kind of plant-based meat; in the early 1960s, traditionally, soybean proteins were used as ingredients for food analogs such as tofu and tempeh (fermented soybean cake). These products were made basically by simple processing/fermentation techniques and have been highly consumed in southeast Asia countries for centuries since 965 BCE [93]. In addition to these traditional Asian products, in the mid to late 20th century, the Texturized Vegetable Protein (TVP) was introduced as meat alternative, obtained from the extruded defatted soybean meal soybean proteins concentrates or wheat gluten, made most from soybeans [94, 95].

Meanwhile, Soybean Leghemoglobin (SLH) is used in IB, whose function is not only to provide red-colored liquid mimicking the ‘bleeding’ of minced meat but can also impart a meat flavor profile in plant-based products of meat [96]. SLH is a close structural ortholog of animal myoglobin that plays a crucial role in the consumption of animal-based meat because, during the cooking, this especially abundant heme protein unfolds and exposes the heme cofactor, responsible for the catalyzes the transformation of the amino acids, nucleotides, vitamins, and sugars naturally present in animal muscle tissue, into a mainly specific and diverse set of flavor and aroma compounds, which combination creates the distinctive and unmistakable meat flavor [96]. SLH, in your turn, acts the parallel role of unfolding under cooking, releasing their heme cofactor to catalyze the transformation of the same ubiquitous biomolecules, isolated from plant sources, into a wide range of compounds that mimic the unique meat flavor and aroma [97].

Besides all these advantages, in many cases, the plant-based meat can present insufficient rates of essential amino acids and trace elements, which can become more challenging to produce plant-based products that perfectly mimic the meat’s nutritional values, such as the meat flavor and aroma [89]. The SynBio rises like an efficient approach that will allow the perfect plant-based meat production. By engineering the plant’s existing compounds, like SLH, into more “animal-like” compounds and by creating and introducing artificial and synthetic compounds that can improve the meat quality. This development can be achieved by protein and metabolic engineering with the aim to produce the needed ingredients to create a synthetic mimic of plant-based meat.

3.2 Proteomics studies in soybean

Proteomics is a useful tool for examining changes in protein profile generated by the response to various external or internal stimuli such as salt concentration, drought, desiccation, cold, heat, mineral toxicity, mineral deficiency, mutations, and gene introduction or silencing. In addition, proteomics can analyze differences in nutrition-relevant food proteomes, such as identifying marks for the quality of processed foods [98].

It is safe to work with soybean because several materials about their genetic information are available in the literature. A high-quality soybean genome (Wm82) is currently available in databases and is used as a reference for several studies involving omics [12]. Genomes of wild and cultivated soybeans are also available. Recently, the genome and proteome of a highly productive tropical Brazilian species (BRS 537) (EMBRAPA, 2021) are deposited in NCBI in accession GCA_012273815.2. In addition, data from several soybean proteomes under several conditions are also available for prospecting proteins. All these data provide solid information about the genetics and behavior of the crop, facilitating the identification of targets of interest with precision. Species proteomic sets are one of the richest materials for finding these targets, as they indicate that genes present in the genome are being translated into functional proteins.

The fact that soybean is naturally rich in protein content (40%) demonstrates a wide range of protein material to be explored, which is why soybean proteomes are widely studied worldwide. The search for new targets and potential uses is getting bigger every day. Currently, with large-scale proteomics, the isolation of a greater number of proteins is made possible, and much remains to be explored in this crop [99]. In addition to the genetic variability and availability of data on the culture, its importance in producing recombinant proteins for industrial purposes is also due to several other factors. How reducing production costs, easy cultivation in the greenhouse, high protein/biomass ratio, production safety, dosage accuracy allow generating of marketable formulations that may not require purification, low technology sustainability for the production line, reducing the risk of contamination, scalability, and minimal waste production. No other protein expression technology is as efficient as the soybean system [7].

The state-of-the-art studies involving proteomic analysis of soybean seeds in the last 16 years are described in Table 2. Since 2005, 50 articles that evaluated the set of proteins in soybean seeds were computed, these studies involve a wide range of proteomic scenarios that come helping in the screening for proteins that can play roles in metabolic pathways for the synthesis of essential amino acids, bioactive proteins, tolerance to various environmental factors, production of sustainable fuels, and others [139], besides providing increasingly solid knowledge about the behavior of the crop under different conditions and stages of development.

Objective	Tissue	Methodology	Ref.
Protein profile in conventional soybean	Mature Seed	2-DE; MALDI-TOF-MS; LC/MS-MS	[100, 101, 102, 103, 104]
	All seed stages	2-DE; MALDI-TOF-MS	[105]
	Mature Seed	2-DE; MALDI-TOF-MS	[106, 107, 108]
		NanoUPLC-MS	[109]
		SDS-PAGE; MALDI-TOF-MS; ESI-Q-TOF MS	[110]
		NanoHPLC-MS/MS	[111]
		SDS-PAGE; LC-MS/MS	[112]
	Seed in development	2-DE; LC-ESI-MS/MS	[113]
		2-DE; nESI-LC-MS/MS; Sec-MudPIT	[114]
		2-DE; LC-MS-MS	[115]
		MS/MS	[116]
	Seed cotyledons	2-DE; MALDI-TOF-MS	[117]
	Germinating Seeds	2-DE; MALDI-TOF/MS	[118]
		SDS-PAGE; LC-MS/MS	[119]
Protein profile in soybean with high protein content	Mature Seed	2-DE; MALDI-TOF-MS	[120, 121, 122]
		2-DE; SDS-PAGE; LC/MS-MS	[123]
		Tandem Mass Tag -TMT; LC-MS/MS	[124]
Protein profile in transgenic soybean	Mature Seed	2-DE; MALDI-TOF-MS	[125]
		SDS-PAGE; 2-D PAGE; MALDI-QTOF MS/MS	[126]
		SDS-PAGE	[127]
	Seed Cotyledons	ITRAQ; LC-MS/MS	[128]
	Germinating Seeds	LC-MS-MS	[129]
Protein profile of mutated soybean	Mature Seed	SDS-PAGE	[130]
		2-DE; MS/MS	[131]
		Tandem Mass Tag -TMT	[132]
Protein profile of Chernobyl area soybean	Mature Seed	2-DE; MS/MS	[133]
Protein profile under High temperature	Mature Seed	DIGE; MALDI-TOF MS; MS/MS	[134]
Protein profile under salt stress	Germinating Seeds	2-DE PAGE; MALDI-TOF-MS	[135]
Protein profile under high temperature and humidity stress	Seed in development	2-DE; MALDI-TOF-MS	[136]
Protein profile under biological fermentation	Meal – SBM	2-DE; LC-MS-MS	[137]
Protein profile under chilling temperature	Germinating Seeds	2-DE; MALDI-TOF/MS	[138]

Table 2.

Studies involving proteomics analysis in soybean seeds.

Proteomes for studies of nutritional factors are also widely observed in the literature, as they directly influence protein digestibility. Natarajan and team [107]investigated protein and genetic profiles of Kunitz trypsin inhibitors (KTIs) in seeds of 16 different soybean genotypes that included four groups consisting of wild soybean (Glycine soja), ancestor of cultivated soybean. They identified that KTI exists as multiple isoforms in soybean. The authors noted that the number and intensity of proteins between wild and cultivated genotypes varied. These data suggest that the greatest variation in protein profiles occurred between wild and cultivated soybean genotypes rather than between genotypes in the same group. However, genetic variation of genes related to KTI1, KTI2, and KTI3 was detected within and between groups (Figure 2).

Figure 2.

Examples of conventional soybean seeds are characterized by functional annotation. Label: BR16; BRS 257, BRS 258, Embrapa 48, BRS 267; Mindou 6; Maverick. Numbers represent the percentage of protein in each category.

A proteomic study on developing soybean seeds (G. max var. Mindou 6) showed 48 differentially expressed proteins [109]. Among these proteins, 25% were related to protein destination and storage, 42% to energy and metabolism, 15% to disease/defense, 6% to transporters, 4% to secondary metabolism, 4% to transcription, 2% to the synthesis of proteins and 2% for cell growth/division. It was observed that with the maturity of the seeds, the number of proteins varied, some decreased, and others increased their concentrations. The sucrose-binding protein (SBP) 2 precursors, which can contribute to improving the digestibility, nutritional value, and food quality of seeds, were increased with maturity (Figure 2).

4. Soybean functional proteins

The function proprieties of proteins are physicochemical aspects that influence the behavior of proteins in food preparations, for example. Based on epidemiological studies, soybean consumption has been associated, for many years ago, with several potential health benefits in reducing chronic diseases such as insulin resistance/type II diabetes, cardiovascular disease, obesity, certain types of cancer, and immune disorders [76]. Nowadays, it is already proven that soybeans are a rich source of phytochemicals, and many of these compounds have important benefits to human and animal health. Among these phytochemicals, phytoestrogens, mainly isoflavones (genistein and daidzein) and lignans, usually get more attention [140]. Nonetheless, in recent years, those physiological functions have been attributed to soybean proteins intact or more commonly bioactive and functional peptides derivate from soybean processing. These bioactive peptides are small protein fragments produced by enzymatic hydrolysis, fermentation, food processing, and gastrointestinal digestion of larger soybean proteins [141], showing multiple beneficial metabolic effects [78, 142].

The soybean seeds contain ~38% protein, ~18% oil, ~30% carbohydrates, ~14% moisture, ash, and secondary metabolites, are a considerable source of vitamins (A, thiamin, riboflavin, pyridoxine, and folic acid) and minerals (Fe, Zn, Mg, K, Ca, Mn, and Se), phytoestrogens and fibers, as well as a widely important source of protein [143]. More important than quantity is the quality of the proteins found in soybean, all eight essential amino acids, which are necessary to human nutrition, but are not produced by the human body, are found in soybean. While the sulfur-containing amino acids (methionine and cysteine) are a limiting factor with a chemical score of 47, compared to 100, as ideal protein for human nutrition [144], soybean proteins are an extraordinary source of lysine.

The two major storage proteins, glycinin (11S) and β-conglycinin (βCG, 7S), are considered naturally bio-inactive, but different ratios of βCG and glycinin may have other nutritional and physiological effects. However, many bioactive peptides are inert while still constituting a larger protein but become activated when released from the original structure by gastrointestinal digestion, enzyme and food processing, or fermentation. These peptides common are 2 to 20 amino acids in length and can be absorbed by the human intestine, falling into the bloodstream, where they can exercise systemic or local physiological effects in target tissues [76]. It has shown a difference in the human intestinal absorption of 11S peptides compared to 11S globulin or amino acids mixture, being that the 11S peptides take to a significantly greater increase in venous blood amino acids concentration. This difference is more notable for aromatic and branched-chain amino acids, which could indicate that hydrolyzed soybean proteins are faster and more efficiently absorbed in the human intestine [145].

Thus, in the last decade, the focus of research on the functionality of soy-based foods has shifted from proteins to bioactive peptides. Moreover, numerous bioactive soybean peptides have been identified with widespread beneficial physiological effects, such as anti-diabetic, anti-cancer, hypotensive, anti-inflammatory, antioxidant, and lipid-lowering (hypocholesterolemic, hypotriglyceridemic, anti-obesity) (Table 3) [76]. Among these is the lunasin, one chemopreventive peptide that consists of 43 amino acids residues with a C-terminal of nine aspartic acid and cell adhesion motif, enabling the binding to non-acetylated H3 and H4 histones, preventing their acetylation, which gives they the anti-carcinogenic activity [160, 169].

Soybean Protein Source	Bioactive Peptide	Properties	Ref.
βCG	YVVNPDNDEN	Hypocholesterolemic	[146, 147]
	YVVNPDNNEN
	LAIPVNKP	ACE inhibition	[77, 148]
	LPHF
Glycinin	IAVPGEVA	Hypocholesterolemic Anti-diabetic	[147, 149, 150, 151]
	IAVPTGVA		[147, 149, 152, 153]
	LPYP		[146, 147, 150, 154, 155]
	VLIVP	ACE inhibition	[77]
	SPYP
	WL
	SFGVAE	Hypocholesterolemic	[150]
	HCQRPR	Phagocytosis stimulatory peptide	[155, 156, 157]
	QRPR
Lunasin	SKWQHQQDSCRKQKQ GVNLTPCEKHIMEKIQ GRGDDDDDDDDD	Antioxidative Anti-inflammatory Anti-cancer Hypocholesterolemic	[77, 141, 157, 158, 159]
Bowman-Birk Inhibitor		Anti-cancer Proteinase inhibition Chemoprevention	[160, 161, 162, 163, 164, 165, 166, 167, 168]
Soybean Protein	YVVFK; IPPGVPYWT; PNNKFPQ; NWGLPV; TPRVF	Hypotensive	[157, 169, 170]
	WGAPSL; VAWWMY; FVVNATSN	Hypocholesterolemic	[77, 155, 157]

Table 3.

Some examples of the principal soybean bioactive peptides and their properties. Adapted from [76].

Lectin (hemagglutinin or agglutinin), a highly specific carbohydrate-binding protein with an important role in biological recognition, can be founded in soybean seed, ~0.2–1% of total protein [171]. Trypsin and protease inhibitors encompass several proteins and peptides, such as the Bowman-Birk protease inhibitor (BBI), the Kunitz trypsin inhibitor (KTI), and lunasin. BBI is a small protein of ~10 kDa, belonging to the serine protease inhibitor family, that mightily interacts with trypsin and/or chymotrypsin and strongly inhibits their enzymatic function [172]. The soybean KTI consists of a protein of ~20 kDa, with a single polypeptide chain cross-linked by two disulfide bridges, which inhibits trypsin and, at a lesser rate, chymotrypsin [171]. Both soybean lectins and protease inhibitors usually have been classified as antinutrients because they may lower the nutritional value of soybean. Therefore, their consumption has shown preventing effects against many diseases, such as cancer. The BBI already demonstrated an anti-inflammatory function that prevents the development of cancer and coronary diseases [171, 173].

In addition to these proteins with known functions and characteristics, several aspects of human health are attributed to soybean proteins. The proteins of many animal species show a high-fat content that can be implicated in increasing blood cholesterol, triglycerides, and Low-Density Lipoprotein (LDL-c), which stimulated the search for other protein sources [143]. Studies about the soybean protein consumption effect on subjects with hypercholesterolemia concluded that it could reduce total blood cholesterol, triglycerides, and LDL-c levels [174]. In 1999, the US Food and Drug Administration (FDA) approved the label for foods containing soybean proteins as protection against coronary heart disease. Its potential role in reducing risk factors for cardiovascular disease is one of the highest causes of death worldwide [143]. Soybean proteins can positively impact the angiotensin-converting enzyme (ACE) activity, acting as ACE inhibitor peptides that can be released enzymatically from a larger protein precursor in vivo during gastrointestinal digestion and in vitro by food processing. These peptides can reduce blood pressure by limiting the effects of ACE II in vasoconstriction and improving the vasodilatory effects of bradykinin, a potent endothelium-dependent vasodilator and mild diuretic [175, 176].

4.1 Synthetic biology applied to functional proteins

Being a wide broad domain with many new and emerging fields, SynBio can give the necessary tools to face many of the challenges of the modern world. The inherent complexity and redundancy of the plant genome represent a problem to be solved by SynBio, too, for this, it applies the most important engineering principles: decoupling, abstraction, and standardization [177]. Decoupling is the simplification of complex problems into smaller ones that can be solved individually. Abstraction divides the topology of information into hierarchical levels, allowing limited and selected data to be exchanged between levels. Standardization is used to determine and characterize orthogonal parts and standardized conditions for testing. Engineering a biological system is one method to manipulate information, process chemicals, provide food, constructing materials, and help to maintain or enhance human health and our environment [178].

Plants are the great chemist of nature, being a perfect platform for SynBio approaches. The rise of SynBio broadened the horizons of plant engineering. However, as SynBio is still dependent on existing transformation techniques, the major challenge to implementing SynBio in the production of modified interest plants is the time and expense involved in the propagation, transformation, and screening of higher plants [39]. But with, the application of the SynBio approach in other fields of functional, protein production, like food production systems, will save water resources, improve land-use efficiency, and avoid the use of pesticides and fertilizers [179]. In addition, the SynBio based functional proteins and food manufacturing systems are less affected by uncontrollable environmental factors and are easier to carry out according to high-quality standards and scale at an industrial level. By constructing cell-based food factories, foods such as plant-based meat analogs, animal-free bioengineered milk, and sugar substitutes can be created from completely renewable resources [179].

Soybean proteins have been widely used to produce many protein-based food formulations due to their excellent nutritional and functional properties. Physical modification, chemical modification, and enzymatic modification have been applied to improve the functional aspects of soybean proteins [80]. But to overcome the future food and global climate challenges, just improvement in processing techniques is not enough, it is a critical step in the development of new soybean varieties that can be able to meet both the demands of the consumer market and the producers. In many cases, the traditional plant breeding cannot attend to this demand, proving to be necessary classical and, mainly, new genetic engineering techniques to add value to the soybean crop, such as reduction of allergens and antinutrients factors along with the increase of quantity and quality proteins, oil, and carbohydrates [98].

The first option for the improved soybean crops in a “functional way” is the generation of genetically modified varieties. For example, the genetic engineering techniques, such as CRISPR-Cas9, represents a great opportunity to improve the nutritional value of soybean-based foods, for instance, by developing carotenoid-enriched functional crops and oilseeds crops with elevated levels of omega 3 fatty acid [39]. Besides the increment/silencing of the expression of target genes, soybean can also represent an important vehicle for the creation of bio fabric, to produce a wide range of bioactive compounds by the heterology expression of desirable genes or by the metabolic engineering of the plant. For example, based on classical genetic transformation techniques, soybean has already been used to produce functional human growth hormone and coagulation factor IX [180]; and anti-HIV Cyanovirin-N [181].

5. Conclusion

Soybean seeds are an excellent source of proteins, as they provide all essential amino acids and a promising source of biologically active proteins/peptides with a wide range of effects such as anti-diabetic, anti-hypertensive, anti-cancer, antioxidant, anti-inflammatory, hypolipidemic, immunostimulatory, and neuromodulatory properties. However, soybean has a low content of sulfur amino acids, and many consumers may exhibit allergenic and antinutritional reactions due to the presence of certain proteins and peptides, such as protease inhibitors. But the same inhibitors, like KTI and BBI, show anti-cancer and anti-inflammatory activity, respectively. Thus, the future of soybean-based foods is not just about the classic plant breeding and/or new processing techniques to remove undesirable characters because they may be interesting for other applications. SynBio rises as a modern solution to create a more “consumable” soybean, through protein and metabolic engineering, to remove just the exact allergenic and antinutritional factors. In addition, soybean can be a great platform to create biofabrics combined with SynBio techniques.