Microbial Phytases and Phytate: Exploring Opportunities for Sustainable Phosphorus Management in Agriculture

Myo-inositol phosphates (phytates) are important biological molecules produced largely by plants to store phosphorus. Phytate is very abundant in many different soils making up a large portion of all soil phosphorus. This review assesses current phytase science from the perspective of its substrate, phytate, by examining the intricate relationship between the phytate-hydrolyzing enzymes and phytate as their substrate. Specifically, we examine available data on phytate’s structural features, distribution in nature and functional roles. The role of phytases and their localization in soil and plant tissues are evaluated. We provide a summary of the current biotechnological advances in using industrial or recombinant phytases to improve plant growth and animal nutrition. The prospects of future discovery of novel phytases with improved biochemical properties and bioengineering of existing enzymes are also discussed. Two alternative but complementary directions to increase phosphorus bioavailability through the more efficient utilization of soil phytate are currently being developed. These approaches take advantage of microbial phytases secreted into rhizosphere either by phytase-producing microbes (biofertilizers) or by genetically engineered plants. More research on phytate metabolism in soils and plants is needed to promote environmentally friendly, more productive and sustainable agriculture.


Introduction
Phosphorus is one of the key elements necessary for growth and development of all liv-ing organisms. It is essential for biogenesis of phospholipids in cell membranes, nucleic acids, ATP and plays an important role in many regulatory and metabolic processes [1] [2]. Phosphorus is also an important component of many soils, where it is often found in both organic and inorganic forms. Plants utilize mostly inorganic soil phosphates for growth and development, but phosphate concentration in many agricultural soils rapidly declines as the demand for more agricultural products intensifies. Insufficient amounts of easily extractable inorganic phosphorus is one of the most critical factors limiting agricultural yields, a problem that is typically solved by widespread application of rock phosphate fertilizer. However, such approach is not sustainable long-term and will eventually lead to the depletion of world phosphorus reserves [3]. Indeed, some reports predict that rock phosphate deposits will be exhausted by the end of this century [4]. In addition, application of rock phosphate is not very efficient, as up to 80% of all fertilizer is quickly modified, immobilized or transformed into insoluble organic phosphorus derivatives and thus, becomes unavailable to plants. Furthermore, the massive use of phosphate fertilizers has a substantial negative impact on the environment, as runoff from the fields pollutes natural water reservoirs, where it can destabilize ecosystems through eutrophication and waterlogging.
In addition to inorganic phosphates, a substantial fraction of total soil phosphorus is present in organic form [5] [6]. While the exact numbers may vary from one soil type to another, many authors estimate that various organic forms of phosphorus may constitute 30% -80% of the total soil P [7] [8]. In certain soil types, myo-inositol phosphate (phytate) is one of the major forms of organic soil phosphorus making up to 50% of all organic P in soil [8] [9] [10] [11]. For example, phytate concentration was shown to vary from 3.9% to 25.3% of total extractable P in carbonate-free Cambisol soils and calcareous czernosems, respectively [12]. Phytate is a relatively stable compound and is often found in precipitated forms and in immobilized aggregates. Such precipitates are difficult to solubilize due to phytate's chelating activity and the formation of inaccessible complexes with metal cations, amino acids, peptides and various mineral soil components [13]. The phytate-peptide complexes are at least partially resistant to proteolytic degradation in gastrointestinal tract of non-ruminant animals [14], which prevents extraction of these valuable nutritional factors from plant seeds. Hence, phytate is often considered an anti-nutritional factor for animals. Undigested by animals, insoluble complexes formed by myo-inositol phosphate and other compounds are excreted and accumulate in soil and water, shifting their ecological balance.
Although inositol phosphate can be viewed as a rich source of phosphorus in soil, plants are largely unable to utilize it from the rhizosphere due to the low phytatehydrolyzing (phytase) activity in plant roots [15]. Phytases release inorganic phosphate from phytate to generate low-phosphorylated myo-inositols. Phytases are synthesized by many microorganisms, including various bacteria, fungi, micromycetes and other microbes often collectively called biofertilizers due to their ability to promote plant growth. Secreted microbial phytases hold a high potential for biotechnology due to their often higher specific activity towards phytate [16] [17] and can potentially be used to increase soil phosphorus availability for plant nutrition. In light of this, a promising approach in plant biotechnology is to generate transgenic plants engineered to secrete microbial phytases into rhizosphere. In theory, this approach can provide substantial amounts of phosphorus for plant nutrition, which would in turn increase plant productivity and their nutritional value for animal consumption [18]. In addition, such genetically modified plants could potentially help solve ecological problems by reducing phytate accumulation in soil and water [19] [20].
Several excellent reviews on microbial phytases have recently been published and are highly recommended [5] [6] [19]- [25]. This review focuses on phytase science from the perspective of its substrate, phytate. Specifically, we evaluate the most recent data on phytate's structure, distribution in nature and function, its role in plant nutrition. We further discuss promising environmentally friendly and cost effective strategies to increase soil phosphorus bioavailability through the use of biofertilizers or generation of transgenic plants capable of secreting microbial phytases into rhizosphere. The advantages and drawbacks of each approach, as well as the likely direction of future research, are also discussed.

Structural Features of Inositol Phosphates
Inositol phosphates were first identified in biological systems over 100 years ago [26] [27]. For a long time they remained the subject of intense scientific debates largely because of lack of clear understanding of their dimensional structures. Specifically, the exact three-dimensional model of inositol was unclear until studies using nuclear magnetic resonance and X-ray diffraction analysis demonstrated the vast structural diversity of different isoforms [28]. We now know that the exact conformation of inositol (cyclohexane-1,2,3,4,5,6-hexol) varies depending on bond location, leading to the formation of multiple stereoisomers [1] [29]. Hydroxyl groups of stereoisomers are oriented either axially or equatorially, thus resulting in nine possible inositol conformations [29]. The names of all nine inositol stereoisomers are typically highlighted in italics. Myo-inositol with one axial and five equatorial hydroxyl groups has the most stable conformation (the "chair") ( When hydroxyl groups in inositol ring are replaced by phosphate residues the molecule becomes a phosphorylated alcohol-inositol phosphate. Depending on the number of phosphates, several different compounds can be formed-from inositol-monophosphate to inositol-hexakisphosphates. For example, myo-inositol hexakisphosphate is the phosphate salt of myo-inositol, in which all six hydroxyl groups are substituted by phosphate residues (Figure 2). According to the official nomenclature, this compound is myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen) phosphate, but often it is also called myo-inositol hexakisphosphate or simply phytate. The official abbreviation for this Figure 1. Myo-inositol "chair" conformation. compound is InsP 6 or IP 6 . While sometimes phytate is also called phytic acid, this terminology is not generally applicable to other stereoisomers of phosphorylated inositol. with Са 2+ -phytate complex being the most common. Interestingly, phytate complexes with bivalent cations are formed in both acid and alkaline conditions and are found in either dissolved or precipitated states [23]. Phytate may also form complexes with trivalent iron and aluminum cations in vivo [36] [37].
Negatively charged phosphate residues in phytate can also bind positively charged  amino acid residues in peptides and native proteins leading to the formation of stable protein-phytate complexes. A trimeric protein-metal cation-phytate complex can also be formed both in acid and alkaline pH conditions [23] [38]. Overall stability of such complexes largely depends on pH, concentration and cation type.

Phytate Distribution in Soil
The most wide-spread phosphorylated inositols in soil are myo-, scyllo-, chiroand neo-inositol phosphates, of which myo-inositol hexakisphosphate is the most common.
The other three phosphorylated stereoisomers can be ranked from more to less abundant in the order of scyllo > chiro > neo, but these are generally very rare in biological systems [39] [40]. Inositol phosphates with low phosphate group content (from monophosphate to tetrakisphosphate) are also uncommon in soil.
Inositol phosphates can undergo epimerization, when two stereoisomers (for example, myo-inositol and scyllo-inositol) that differ by spatial location of only one chemical bond can turn into each other. Experiments with labeled carbon isotopes established that myo-inositol in certain soil conditions can be phosphorylated by soil microorganisms to yield myo-inositol hexakisphosphate [39]. However, natural chemical synthesis of phosphorylated inositol stereoisomers in soil is thought to be rare, mainly because such chemical reactions require prolonged heating in strongly acid or alkaline conditions. Instead, most soil inositol phosphates are assumed to be synthesized by living organisms, including plants and soil microbes.
Indeed, myo-inositol hexakisphosphates are widely distributed in various plant tissues (especially seeds), as well as in other eukaryotes. Scyllo-inositols mostly in monophosphate form are found in aleurone layer of barley seeds. D-chiro-inositol hexakisphosphates are present in small quantities in pine needles and in leaves of flowering trees [39]. Other inositol phosphate stereoisomers have not yet been found in plants.
Phytate appears to be the main storage form of phosphorus and inositol in plants. Phytate makes up to 30% of all phosphorus fractions in roots, while its fraction increases up to 80% in seeds and cereal grains [21] [41] [42]. Phytate mostly accumulates at the last stages of plant life cycle and is returned to soil with seeds, where it is again made available to plants during germination via intrinsic phytases [43] [44]. Seed phytate can often be found in association with insoluble salts of potassium, magnesium and other metals and is stored in globular inclusions (globoids) of aleurone layer and in embryo vacuoles. For example, more than 80% of phytate in maize is present in embryo [45].
Typically, seed ripening and germination are accompanied by changes in pH, temperature, metal cation concentration that promote conversion of phytate complexes to a more soluble form [46].

Phytases as Biological Tools to Harvest Inorganic Phosphorus from Phytate
The enzymes phytases belong to the general class of phosphatases (EC 3.1.3) and hydrolyze phytate to release inorganic phosphorus [47]. Phytases are classified into several families with important differences in structure, substrate specificity, pH-optimum and mechanism of hydrolysis. Some of the histidine acid phosphatases (HAPs) have low pH-optimum and a broad substrate specificity, while most known alkaline β-propeller phytases (BPPs, mostly of bacillar origin) are specific only to phytate molecule and its complexes. Phytate-degrading enzymes are commonly found in oilseeds and nuts, legumes and in cereal pollen grains. Phytase activity is abundant in cereal seeds (rye, triticale, barley, wheat) and in pseudo-cereal fruits (amaranth, buckwheat). Legumes and oilseeds have about 10 times lower phytase activity than grain seeds [48].
Phytases from plant seeds are often associated with membrane structures and are present in aleurone layer in cereals and in cotyledons in legumes, where large quantities of phytate are also found [48] [49]. Some stages of plant life cycle, such as seed germination, are characterized by increased level of phytase activity which is necessary to promote fast growth of seedlings [47]. Most purified and characterized plant phytases have acidic pH-optimum averaging around pH 5.0 and are stable up to 55˚C -60˚C.
For example, a maize phytase has maximum activity at 55˚C and pH 4.5, while a soybean phytase GmPhy has pH-optimum at pH 4.5 -5.0 and is stable up to 60˚C [49].
The few plant phytases that can be found in roots are characterized by low hydrolytic activity and are not secreted into rhizosphere. Phytase activity of Arabidopsis thaliana roots represents less than 0.8% of total root acid phosphomonoesterase (phosphatase) activity [15]. Furthermore, this phytase does not appear to be an extracellular enzyme.
Therefore, A. thaliana and other plants are mostly unable to grow on phytate as the only source of phosphorus in the agar medium [15]. Similarly, experiments with wheat in laboratory conditions established that low phytase activity in plant roots is the main factor limiting wheat ability to obtain phosphorus from phytate [50].
While many soil bacteria and fungi are known to produce extracellular phytate-hy-  [55]. All these features make bacterial phytases a very attractive option for animal feed additives.

Microbial Phytases as Molecular Biofertilizers
High agricultural productivity in the future will largely depend on the continued technological advances to reduce fertilizer application rates and the cost of food production.  [50]. Other known bacterial biofertilizers include species of Azotobacter, Rhizobium, Pseudomonas, Azospirillum and Burkholderia [56].
Indeed, soil microorganisms are often viewed as an abundant source of biofertilizers [57]. Enterobacter strains selected from the rhizosphere of legumes have a positive effect on plant growth and phosphorus nutrition and are known to produce phytases [58]. Due to the presence of high phytase activity a strain of Pseudomonas sp. from Australian agricultural soils was able to release up to 80% of all phosphate from phytate, thus positively affecting plant development [59]. A number of bacteria with phytate-hydrolyzing activity that are able to improve plant phosphorus nutrition was isolated from white lupine (Lupinus albus) rhizosphere in Japan [60]. Almost all of these strains were classified as representatives of Burkholderia family. In addition, some Pseudomonas, Enterobacter, and Pantoea isolates are also able to release inorganic phosphate from phytate [61]. Overall, it is now becoming increasingly clear that phytase-producing soil bacteria (mostly belonging to gamma-proteobacteria) are widespread in the rhizosphere of different plant species.
In general, bacterial biofertilizers containing live microorganisms are widely used to improve crop yield in India, China, Iran and other countries. Specifically, the use of nodule bacteria, Azotobactor/Azosporillum and Phosphobacteria-based biofertilizers is relatively common [62]. Microbial biofertilizers are typically produced in liquid, powder and granular formats [63]. Biofertilizers as a highly efficient alternative to chemical fertilizers are praised for their relative ease of application, non-toxic and eco-friendly nature, and cost effectiveness [64]. regulators, and reduction of ethylene levels in root cells [65]. However, the more widespread commercial use of bacterial biofertilizers is to some degree limited by our insufficient knowledge of the ecological, molecular and physiological impact of microbial communities on plant growth [66]. Nevertheless, the use of natural soil bioresources, including soil microorganisms, can serve as a promising alternative to the currently standard application of inorganic fertilizers.
The latest scientific findings are consistent with the notion that microbial phytases play a fundamental role in soil phosphorus life cycle. Indeed, due to their potentially substantial agronomic and ecological value for plant growth during periods of longterm phosphorus deprivation, microbial phytases become an appealing target for industrial use [61]. For example, treatment of seeds with a fungal extracellular phytase promoted plant phosphorus nutrition in soils with high phytate content [67]. Similarly, enrichment of phosphate-limited soils with phytase-producing bacteria, such as Bacillus mucilaginosus and B. amyloliquefaciens, was shown to improve growth of tobacco and corn, respectively [65] [68]. Finally, bacterial phytases also positively impact plant nutrition by freeing up important soil microelements typically chelated by phytate.
Thus, the use of biofertilizers in the form of either bacterial culture liquid purified microbial phytases or live phytase-producing bacterial strains can be viewed as an efficient and environmentally friendly approach to increase bioavailability of soil phosphorus and reduce the currently widespread use of inorganic phosphate fertilizers.

Transgenic Plants as a Promising Alternative to Phosphate Fertilizers
Several new biotechnological advances now make it possible to utilize phytate as an abundant source of phosphorus, especially for farm animals. Many phytases of bacterial or fungal origin are traditionally used as animal feed supplements to improve phosphorus balance in monogastric farm animals, such as pigs, chicken and fish. These include phytases from Aspergillus ficuum (niger) (sold as Natuphos), Aspergillus niger (All-zyme), Aspergillus awamori (Finase and Avizyme), Aspergillus oryzae (SP, TP, SF, AMA-FERM, and Phyzyme), and Peniophora lycii (Ronozyme, Roxazyme, and Bio-Feed phytase) [69]. Additional technologies include: pre-processing grains to activate endogenous phytases; mutations in phytate metabolism genes that decrease phytate synthesis rate in plant seeds; the use of genetically modified farm animals that produce phytases in saliva or genetic modification of plants to express microbial phytases [19].  [72]. Similarly, expression of B. subtilis 168PhyA phytase in Arabidopsis thaliana led to a higher shoot dry weight and an increase in phosphorus content by 100% compared to the wild type [75].
Despite these encouraging results in laboratory conditions, the situation in actual soils may nevertheless turn out to be less promising. In fact, to date there is limited evidence that such transgenic plants are indeed characterized by improved P acquisition and plant growth in natural soils. One clear example of improved P acquisition from natural agricultural soils was the transgenic expression of phytase and APase genes in alfalfa using a root-specific promoter, though the effect appeared to vary with the type of soil tested [85]. On the other hand, most published reports up to date support the notion that the situation in real soils may be very different from exciting results obtained in laboratory media. For example, experiments with transgenic Trifolium subterraneum expressing phytase phyA established that improved P uptake and increased plant growth previously observed in agar was compromised when the same plants were grown in real soil [86]. Furthermore, transgenic tobacco plants expressing a fungal phytase gene did not show improved P acquisition when grown in P-deficient soils [87]. Similarly, expression of B. subtilis phytase transgene in tobacco resulted in improved phosphorus uptake from phytate only in sterile laboratory conditions but not in real soil [88]. In another report, while constitutive overexpression of AtPAP15 gene in soybeans did result in higher yield, this phenotype was mainly achieved by increased internal P use efficiency rather than by enhanced soil P acquisition [89]. Taken together, these data clearly suggest that effectiveness of plants expressing transgenic phytases might be limited. One potential explanation is that biochemical properties of recombinant phytases, such as stability or optimum pH, could be substantially modified because of local soil conditions. Indeed, microbial community in the rhizosphere may not support physiological and nutrient changes introduced by plants with transgenic phytases [90]. This apparent failure of genetically engineered plants secreting bacterial phytases to demonstrate improved growth in real soil conditions may also stem from the fact that secreted phytases quickly lose activity in soil possibly due to adsorption of the enzymes (though this process appears slower in the rhizosphere) [87]. This rapid enzyme immobilization may thus quickly limit phytase capacity to interact with phytate in soil and undermine all previously envisioned advantages of such transgenic approach to improving plant phosphorus metabolism.

Towards Future Strategies to Improve Plant Phosphorus Metabolism
To feed the ever-growing world population, modern agriculture will continue to rely on improvements in biotechnology. Two alternative but often overlapping strategies that rely on the use of bacterial phytases should be considered as potentially viable options.
Bacterial phytases can either be genetically introduced into crops or supplied to soil as purified enzymes or through application of microbial biofertilizers. In comparison to their eukaryotic counterparts, bacterial phytases are often cheaper to manufacture and easier to express in plants using modern molecular and genetic tools. In addition, bacterial biofertilizers are often easy to cultivate in large volumes and subsequently use to treat plant roots or seeds. When searching for the best way to take advantage of microbial phytases, several factors should first be carefully considered. The first aspect is the need to identify, either bioinformatically or through careful microbiological and biochemical screening of soil microorganisms, producers of highly active and thermo-sta-  [102]. In addition to organic acids, bacteria can produce indole acetic acid (IAA), siderophores, vitamins, amino acids, ammonia and cyanide. Furthermore, microorganisms provided in the context of biofertilizers can compete with other microbes for colonization of plant roots, reduce ethylene production and suppress diseases caused by pathogenic bacteria and fungi [56].
While biofertilizers clearly represent a promising route for modern agriculture, many obstacles still exist to their successful application in the field. Despite the well-documented positive effects of bacterial biofertilizers on plant growth in some settings, a giant gap needs to be bridged between these successful greenhouse experiments and field studies, where many biofertilizers often fail to substantially improve plant growth [103] [104]. This discrepancy may be due to several factors, such as unfavorable interaction with other rhizosphere organisms, adverse physical and chemical soil properties (e.g., low pH), poor ability of strains to colonize plant roots and other environmental factors, such as high ambient temperature and low rainfall during the growing season.
Many of these factors can negatively affect the outcome of biofertilizer application. One possible strategy to overcome these limitations is the use of microorganisms adapted to the particular climatic conditions of agricultural region [105].

Conclusion
In the last few decades our reliance on non-renewable rock phosphate fertilizers has become a major limiting factor affecting environmental, political, and economic aspects of modern agriculture. To sustain current and future agricultural needs, several novel approaches to phosphorus management in the field have been proposed, including the use of biofertilizers and genetically engineered plants. A particularly useful synergistic effect could potentially be achieved by the combined use of genetically-modified plants secreting efficient bacterial phytases into rhizosphere and simultaneous application of biofertilizers that contain microorganisms adapted for local environmental conditions.
While the ultimate goal of many researchers is to create the best conditions for efficient phosphorus nutrition, increased biomass and yield, more studies are clearly necessary to chart the best strategies and to develop advanced biotechnologies that rely on microbial or plant-produced phytases. Overall, better mechanistic understanding of the relationship between phytase properties, phytate availability and roles in plant physiology will be required to improve plant nutrition.