Mapping the Catalytic Landscape of a Novel Phytase

Lead Research Organisation: University of East Anglia
Department Name: Biological Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description We have developed engineered enzymes that possess desirable qualities in terms of their degradation of phytate found in animal feeds. In terms of our original objectives:
1. Undertake an exhaustive analysis of the catalytic repertoire of the native enzyme by identifying the major intermediates arising from hydrolysis of phytic acid - By means of HPLC separation of mixtures of hydrolytic intermediates we were able to identify unambiguously the major intermediates accumulating during the early stages of the action of MINPP and of commercial phytases on phytic acid in vitro. Although we did not seek to measure their inhibitory activities, it was possible to identify the major accumulating intermediates by HPLC.
2. Employ mutagenesis strategies to investigate the roles of active site residues on specificity and activity - We have identified active site residues which directly influence the positional specificity of the enzyme. The major inositol polyphosphate intermediates generated by the action of these mutants were identified by HPLC.
3. Seek active site variants of MINPP with altered pH-activity profiles, enhanced thermostability and reduced sensitivity to protease activity - We generated and assayed for enhanced thermal stability a range of mutants of MINPP. Initial screening for melting temperature shift was performed by thermofluor. This was followed by differential scanning calorimetry for mutants showing stability enhancement.
4. Investigate the capacity of exemplar 3- and 6-phytases to accommodate MINPP-like active site structures with the aim of broadening catalytic specificity while minimizing loss of activity - We have carried out exhaustive analysis of the mutability of active site residues of the 3-phytase from Aspergillus niger and (to a lesser extent) the 6-phytase from Escherichia coli. These represent models for 1st and 3rd generation animal feed phytases in use by our industrial partners and others.
5. Investigate the synergistic effects of combination of wild type and engineered MINPP with 3- and 6-phytases - We have performed time-course analysis of the composition of partially-dephosphorylated phytic acid intermediates resulting from the synergistic action of mixtures of MINPPs with commercial 3- and 6-phytases. Unfortunately, we did not observe full dephosphorylation of phytic acid. This work is being taken further through of a DTP iCASE PhD studentship held with the industrial partners and which started in October 2019.
Exploitation Route Our industrial partners will take forward our findings to test them in animal feed trials.
Sectors Agriculture, Food and Drink

 
Description Our findings are available to our industrial partners to assess for ultilization in generation of new animal feed additive enzymes.
First Year Of Impact 2016
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description 17ALERT: Mid-Range Equipment Initiative
Amount £349,442 (GBP)
Funding ID A high sensitivity elemental mass spectrometry facility to support metallo-biology research on the Norwich Research Park 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2018 
 
Description Norwich Research Park iCASE DTP
Amount £150,000 (GBP)
Funding ID A Mechanistic Explanation of the Benefits of Phytase to Sustainable Agriculture 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2018 
End 09/2022
 
Description iCASE DTP
Amount £69,000 (GBP)
Funding ID The biodiversity of organic phosphate (phytate) cycling in soils 
Organisation University of East Anglia 
Department EnvEast NERC Doctoral Training Partnership
Sector Academic/University
Country United Kingdom
Start 10/2017 
End 09/2021
 
Title 6GIT PURPLE ACID PHYTASE FROM WHEAT ISOFORM B2 - PRODUCT COMPLEX 
Description X-RAY CRYSTAL STRUCTURE OF PURPLE ACID PHYTASE FROM WHEAT ISOFORM B2 - PRODUCT COMPLEX 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact First crystal structure of a member of the PAP phytase family 
URL https://www.rcsb.org/structure/6git
 
Title 6GIZ PURPLE ACID PHYTASE FROM WHEAT ISOFORM B2 - SUBSTRATE COMPLEX 
Description X-RAY CRYSTAL STRUCTURE OF THE PURPLE ACID PHYTASE FROM WHEAT ISOFORM B2 - SUBSTRATE COMPLEX 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact First crystal structure of a member of the PAP phytase family 
URL https://www.rcsb.org/structure/6giz
 
Title 6GJ2 PURPLE ACID PHYTASE FROM WHEAT ISOFORM B2 - COMPLEX WITH INOSITOL HEXASULPHATE 
Description X-RAY CRYSTAL STRUCTURE OF THE PURPLE ACID PHYTASE FROM WHEAT ISOFORM B2 - COMPLEX WITH INOSITOL HEXASULPHATE 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact First crystal structure of a member of the PAP phytase family 
URL https://www.rcsb.org/structure/6GJ2
 
Title 6GJ9 PURPLE ACID PHYTASE FROM WHEAT ISOFORM B2 - REGENERATION COMPLEX 
Description X-RAY CRYSTAL STRUCTURE OF THE PURPLE ACID PHYTASE FROM WHEAT ISOFORM B2 - REGENERATION COMPLEX 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact First crystal structure of a member of the PAP phytase family 
URL https://www.rcsb.org/structure/6GJ9
 
Title 6GJA PURPLE ACID PHYTASE FROM WHEAT ISOFORM B2 - H229A MUTANT 
Description X-RAY CRYSTAL STRUCTURE OF THE PURPLE ACID PHYTASE FROM WHEAT ISOFORM B2 - H229A MUTANT 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact First crystal structure of a member of the PAP phytase family 
URL https://www.rcsb.org/structure/6GJA
 
Title PDB 5CU7 Crystal structure of Bacteroides Thetaiotaomicron Multiple Inositol Polyphosphate Phosphatase A324D Mutant 
Description X-ray crystal structure of Bacteroides Thetaiotaomicron Multiple Inositol Polyphosphate Phosphatase A324D Mutant 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Awaiting publication 
URL https://www.rcsb.org/structure/5cu7
 
Title PDB 5CX4 Crystal structure of a Multiple Inositol Polyphosphate Phosphatase 
Description X-ray Crystal Structure of a Multiple Inositol Polyphosphate Phosphatase 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact Awaiting publication. 
URL https://www.rcsb.org/structure/5cx4
 
Description Collaboration with Prof Zhao Yong, Shanghai Ocean University 
Organisation Shanghai Ocean University
Country China 
Sector Academic/University 
PI Contribution A collaboration has begun to engineer phytases suitable for application as fish feed enzymes.
Collaborator Contribution A visit was made to the University of East Anglia in December 2018 by Professor Zhao Yong to discuss collaborative research efforts. This was followed up by visits to Shanghai Ocean University by Andrew Hemmings in May and September, 2019. All expenses were paid by Shanghai Ocean University. PhD student Zhang Zhaohuan, visiting from Prof Zhao Yong's laboratory at Shanghai Ocean University, spent 12 months working at the University of East Anglia as part of his doctoral training. This visit was funded by the China Scholarship Council.
Impact Award of a visiting professorship at Shanghai Ocean University to Andrew Hemmings. This collaboration is multidisciplinary. Hemmings lab (enzyme engineering, structural biology), Zhao lab (Food science and technology).
Start Year 2018
 
Description Display at the Norwich Science Festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The Norwich Science Festival is an annual event held to improve public understanding of local science. Our group presented an interactive display 'Better Enzymes for Animal Nutrition' to inform the general public and school children about our BBSRC-funded research.
Year(s) Of Engagement Activity 2016,2017,2018
URL https://norwichsciencefestival.co.uk/
 
Description Display presented at Royal Norfolk Show 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The Royal Norfolk Show is an annual agricultural show held by the Royal Norfolk Agricultural Association. It is the largest agricultural show in England, and has been held almost every year since 1847. The research group presented an interactive display at the Royal Norfolk Show on the theme 'Better Enzymes for Animal Nutrition'. The purpose of the display was to educate the wider public and farmers around the motivation for our BBSRC-funded research. Over the course of the show we estimate that several hundred individuals visited our stand and engaged with the materials or the demonstrators.
Year(s) Of Engagement Activity 2016,2017
 
Description Explaining enzyme engineering research to year 9 pupils 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact As part of a bigger outreach event and activity, a member of the research group explained enzyme engineering research to two different groups of 20 Year 9 pupils from regional schools that have low university participation.
Year(s) Of Engagement Activity 2020
 
Description Play-testing an educational board game on the topic of enzyme engineering 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact The activity was based on a fun board game designed by a member of the research group that uses toy-bricks to explain how an enzyme recognises a substrate through complementary of shape and through chemical nature. The game makes both players engineer the enzyme to fit a new substrate. This was play-tested with other local postgraduate students from a range of fields. This will then be improved to be run with the general public at science festivals, game festivals and outreach opportunities.
Year(s) Of Engagement Activity 2020
 
Description Speak Up for Food Security qualifiers 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact During a storytelling masterclass organized by Global Food Security, an emotional presentation was delivered explaining food additive enzyme engineering research as well as impacts on GM policies and its aplications. The audience was composed of GFS employees, and postgraduate students and postdocs working in a wide variety of fields in the UK.
Year(s) Of Engagement Activity 2020
URL https://www.foodsecurity.ac.uk/activities/speakup/#post-16143