Mapping the Catalytic Landscape of a Novel Phytase

Phytic acid is the major source of inorganic phosphate needed for animal growth and is found in the grains, oil seeds and beans of common animal feeds. However, animals do not produce the enzymes necessary to digest phytic acid, relying instead on enzymes (generically termed phytases) produced by the trillions of bacteria that reside in the animal's gut. In non-ruminants such as pigs and chickens, the capacity of these enzymes to digest the phytic acid of their diet is limited. As a result their feed is supplemented with inorganic phosphorous, most commonly applied in the form of a non-renewable resource, dicalcium phosphate (DCP), which is mined at great cost and environmental expense. To increase the extent of conversion of phytic acid into available dietary phosphate, phytases are routinely added to animal feeds. Efficient breakdown of phytic acid by added phytase is cost effective and enables a reduction in the supplementation of feed with DCP. It is estimated that addition of phytase saves 4-5 £/tonne of feed in the cost of DCP supplement. As a result, approximately 90% of the poultry and 85% of the pig feeds manufactured world-wide contain an added phytases. All phytases currently in use as animal feed supplements are modified derivatives of enzymes from either bacteria or fungi belonging to a class called histidine acid phosphatases (HAP). These HAP enzymes are specific in the way they attack phytic acid: they are either 3-phytases which remove the phosphate from position 3 first, or are 6-phytases which first remove that in the 6-position. The former are found in fungi while the latter are found in bacteria. While both classes of enzyme are efficient, they are susceptible to the build up of concentrations of partially-digested phytic acid and therefore other less susceptible types of enzymes are sought by the many industrial producers of animal feed enzymes.
We have recently discovered a new HAP phytase, MINPP, which is secreted by a major human gut bacterium, Bacteroides thetaiotaomicron. This enzyme is the first representative of a new class of HAPs which are characterized by a subtle change in the arrangement of the amino acid residues which endow it with its catalytic activity. Accordingly, the change in position and identity of a catalytic residue combined with an unusual active site character has marked consequences for the properties of the enzyme. The enzyme active site is more open leading to greater flexibility in how it digests phytic acid, i.e. in the position from which it removes the first phosphate. Building on this discovery and in collaboration with a major producer of animal feed enzymes, we propose a programme of research which seeks to develop MINPP as the first of a new generation of phytases with enhanced catalytic flexibility for use in the animal feed industry. To achieve this objective we will firstly map out a complete picture of the biochemical and enzymological properties of the enzyme including a detailed analysis of the role(s) of active site residues on the digestion of phytic acid. We will then go on to engineer the amino acid sequence of the enzyme to imbue it with enhanced thermal stability and other commercially attractive properties. In the second part of the project, we will perform an evaluation of the effects on their catalytic properties of substituting MINPP-like active site residues into the active sites of fungal 3- and bacterial 6-phytases already employed as industrial enzymes. This results of this analysis may prove attractive to feed enzyme manufacturers (who have already invested substantial funds in optimizing the enzymes they have in the market) as a cost-effective means of extending the catalytic flexibility, and therefore utility, of their respective enzymes.

A primary objective of this proposal is a complete description of the catalytic repertoire of a novel phytase from a major human commensal bacterium with unusual hydrolytic flexibility. In the process we will also seek to develop a set of generic tools which may be employed to guide the engineering of phytases to introduce hitherto unrealized catalytic flexibility suitable for animal feed applications.
Our available high resolution crystal structures for MINPP will be used to identify residues for site directed and saturation mutagenesis. We will screen mutants and assay their activity against (1) phytic acid, (2) a common accumulating intermediate of 6-phytase activity, D-Ins(2,3,4,5)P4 and (3) the typical end product of 6-phytase activity, Ins(2)P1. Mutagenesis strategies will also be employed in attempts to enhance the thermostability of the enzyme. These experiments will be guided by bioinformatics analysis and large-scale molecular dynamics simulations. Thermal stability of positives from our screens will be fully characterized by differential scanning calorimetry. We will also use these approaches in experiments aimed at reducing sensitivity to protease activity. Mutants will be routinely characterized by high resolution crystal structure analysis making use of MX beamlines at the Diamond Synchrotron.
Exemplar phytases (1) 3-phytase (PhyA from A.niger) and (2) 6-phytase (AppA2 from an E.coli strain isolated from pig colon) will be engineered with the objective of endowing these with elements of the catalytic flexibility displayed by MINPP. This will be performed using a semi-rational approach based on crystal structure data and site directed or site saturation mutagenesis. To accomplish these objectives we will need a rapid and sensitive assay of phytase activity and so we propose will employ a novel method developed in the laboratory of one of the applicants. This method is currently awaiting patent protection.

The feed enzyme market was valued at $781.7m in 2012 and is expected to increase to around $1.2bn by 2018. Phytases account for approximately 50% of all sales of feed enzymes as approximately 90% of the poultry and 80% of the pig feeds manufactured world-wide contain an exogenous phytase. Large producers of commercial phytases include AB Agri (Quantum Blue, 6-phytase), DSM/Novozymes (Ronozyme HiPhos, 6-phytase), BASF/Verenium (Natuphos, 3-phytase), DuPont/Danisco (Phyzyme, 6-phytase; Axtra, 6-phytase), Huvepharma (Optiphos, 6-phytase) and many other smaller manufacturers exist. The development of a new generation phytase with enhanced catalytic flexibility as proposed, suitable for application as zootechnical agent in animal feeds promises to have a significant direct impact on UK producers of phytases and an indirect impact on producers of pork, poultry and farmed fish through enhanced feed conversion ratios. The research therefore promises to contribute towards economic prosperity by enhancements in business revenue. The development costs for new industrial enzymes are significant. Much of this cost rests with the research needed to identify the modifications necessary to endow the enzyme with the appropriate physicochemical properties needed to survive storage and the pelleting process experienced during feed production. The possibility to transfer MINPP-like catalytic flexibility to a pre-engineered but specific phytases will impact though a shortening of the discovery chain and a reduction in development costs. The research promises to enhance the research capacity, knowledge and skills of businesses developing products in this area.
This proposal involves fundamental and applied research in the area of industrial biotechnology. BBSRC has identified industrial biotechnology as a high-level priority area in its Strategic Plan 2010-2015. It will also directly involve UK industry as the work will be performed in collaboration with AB Vista UK Ltd and AB Enzymes, both part of the Agri group of Associated British Foods, a major UK-based producer of animal feed enzymes. The potential impacts of this work on UK industry and the environment in areas of intensive pig and poultry production are significant. The use of phytases in animal feeds has been shown to significantly improve the mobilization of phosphorous from phytic acid and thus helps ameliorate the effects of excreted phytate on the environment. An engineered enzyme with the ability to more efficiently mobilize phosphorous from feed phytate will have a direct impact on the levels of phytate in excreta and thus reduce the likelihood of negative environmental effects. The research will therefore contribute to environmental sustainability, protection and impact reduction.
This project will provide excellent training opportunities for the postdoctoral scientist in modern technologies in analytical biochemistry, molecular biology and structural and computational biology. Regular opportunities are available to participate in seminars within the University of East Anglia or elsewhere within the Norwich Research Park. As such they will have ample opportunities to extend their presentational skills and network within the local scientific community. The collaboration between the UEA groups and the industrial partners will extend these opportunities, with the PDRA spending an extended period (expected to be 3 months) at the enzyme production facilities of AB Enzymes (Darmstadt, Germany) and/or its partner Roal Oy (Finland) in a knowledge-exchange activities. The research will therefore contribute towards the pool of highly skilled researchers able to contribute to the UK economy. The PDRA will also be expected to take part in Science Open Days at UEA, in science outreach activities at local schools and in public events, therefore contributing to increased public awareness and understanding of science.