Project Description

Describe how and why you chose your iGEM project.


PROBLEM

Striving towards sustainable agriculture and guaranteed food security is integral to the international efforts described in the Sustainable Development Goals (SDGs)1,2, specifically SDG 2 (Zero Hunger) and SDG 12 (Responsible Consumption and Production). Enhancing the digestibility and nutritional efficiency of animal feed is a critical step in addressing these goals. Through doing so, we can improve the efficiency and uptake of nutrients in livestock, which will have a significant impact on the sustainability and efficiency of food production systems. Our project focuses on using Pichia pastoris GS115 to surface-display xylanase, aiming to provide a sustainable, efficient and economically viable method for direct enzyme production in animal feed.

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Xylanase Benefits on Animal Feed Additives

Xylanases3 are enzymes that break down xylan, a major component of plant cell walls. They help in the breakdown of complex polysaccharides present in animal diets when added to feed, especially for non- ruminants like pigs and poultry. By breaking down the polysaccharides, nutrient absorption is improved, thus leading to better feed efficiency, growth performance, and the animals’ general health. Moreover, because xylanase enhances nutrient digestion and decreases nutrient excretion, it can also lower feed costs and impact on the environment.

Recent innovations in novel xylanase enzymes, such as those detailed in patent filings5,6 WO2020009964A1 and GB2585029A, have compositions with an enhanced stability and performance. It is shown in these documents that the modified xylanases enable further optimization in the nutritional value and digestibility of animal feed by breaking down hemicellulose components. Their efficacy in varied feed formulations are ensured by the improved stability of the enzymes under various conditions, like temperature and pH level. Hence, it’s able to maximize the utilization of nutrients and reduce the impact on the environment typically associated with animal husbandry.

In short, xylanase enzymes demonstrate extreme potential as an additive to animal feed because it allows improved nutrient digestibility and reduced environmental impact. Additionally, advancements in xylanase technology highlights the enzyme’s capability to enhance livestock feed efficiency and sustainability. Using yeast surface display systems with Pichia pastoris to produce recombinant xylanase proteins could further reduce costs and therefore increase its viability. For these reasons, xylanase is an ideal option in animal feed applications.


INSPIRATION

2015 iGEM Team Marburg NUTRInity project

Our team's initiative is inspired by numerous noteworthy projects that preceded us like iGEM Team Marburg 2015's NUTRInity project7 Their project aimed to tackle malnutrition and overconsumption by developing modular tools for the human gut. In order to target and reduce specific nutrient concentrations in the gut, they engineered cell-based particles to produce dietary supplements and designed a cell-free protein matrix. They also created a delivery method that is contact-dependent to alter the gut microbiota. Their project addressed significant nutritional concerns and provided novel approaches to address global health issues using synthetic biology. Our project's objective of improving nutritional and economic value through animal feed additives in animal husbandry aligns with this foundational work on food security.

2022 iGEM Team Shanghai United's Feed Family project

The 2022 iGEM Team Shanghai United’s Feed Family project8 explored the use of engineered enzymes to enhance food digestion. They focused on improving the nutritional absorption of animal feed through multi-enzyme synergistic degradation. They engineered a system to express four enzymes (β-xylosidase, cellulase, xylanase, and acetylxylanesterase) in E. coli to break down complex polysaccharides in silage and cereal feed. They addressed the inefficiency of indigestible components in animal feed by aiming to improve the digestibility and nutrient availability for livestock.

2022 iGEM Team LZU-HS-China-C Whole-cell Biocatalysts project

The 2022 iGEM Team LZU-HS-China-C Whole-cell Biocatalysts project9 attempted to break down sulfadiazine residues in animal excrement in order to combat antibiotic pollution. They inserted the antibiotic-breaking laccase from Pleurotus ostreatus into E. coli. Their approach uses ice nucleoprotein to bind laccase to the cell membrane, effectively breaking down sulfadiazine in the environment. This biological method provides a sustainable remedy to antibiotic contamination from agricultural sources. They created a method that allows enzymes to be visible on the surface of E. coli cells, simplifying the process of harvesting the enzymes without requiring a lot of purification.

We were prompted to look for a safer substitute, however, because of the inherent safety issues with using E. coli for items meant for human or animal consumption. Therefore, our goal is to replace E. coli with the yeast surface display with Pichia pastoris as the vector to carry xylanase. This has several advantages in terms of cost-effectiveness and safety.


SPECIFIC AIMS

Yeast Surface Display System: GCW61 vs. Pir1

Enzymes are anchored to the yeast cell wall by the Yeast Surface Display System10, which streamlines and lowers the cost of production. GCW61, a GPI-modified cell wall protein from Pichia pastoris, and Pir1, a protein from Saccharomyces cerevisiae containing internal repeats, are the two anchor proteins we evaluated11,12. These two anchor proteins are both well-known for their robust attachment and enzyme durability. GCW61 is essential for preserving the normal yeast cell morphology, whereas Pir1 increases the durability of enzymes in varied conditions. We aim to evaluate their performance and viability in our yeast surface display system.

Gene Promoter Selection: GTH1 vs. AOX1

The more commonly used AOX1 promoter13, despite its effectiveness in driving high levels of protein expression, poses safety concerns because it uses methanol as an inducer. On the other hand, the GTH1 promoter14 presents an alternative that is safer. Because it is finely tuned by glucose levels, it allows for high- level expression without the dangers of methanol, which makes it especially suitable for our application.

Evaluation Procedures

We will utilize the GTH1 promoter to drive the expression of xylanase and compare the functionalities of GCW61 and Pir1 anchor proteins. We would use DNS assays15 to measure the amount of reducing glucose produced by xylanase activity and apply 3D modeling techniques16 (I-TASSER, YASARA with FoldX plugin, and PyMOL) for the prediction of the protein structures in order to evaluate the stability and active region of the engineered protein to determine which option is more effective. Following this, we plan to investigate enzyme activity under optimal conditions and assess its resistance to gastric proteases and its stability during feed processing and storage.


ANTICIPATION

In order to express enzymes in yeast, we will create a construct containing the GTH1 promoter, xylanase, and either GCW61 or Pir1 as the anchor protein. We will then use the pZAHR vector, which was developed by Professor Hung-Jen Liu's lab at National Chung Hsing University, to integrate the construct into Pichia pastoris. We will utilize colony PCR and DNA sequencing to confirm the constructions. Significant xylanase activity in glucose-induced P. pastoris expressing the Xylanase-GCW61 and Xylanase-Pir1 constructs should be used to measure the effectiveness of anchor proteins.

Our goal is to create an efficient animal feed additive by examining optimum pH levels and temperature ranges, the capacity of xylanase to withstand stomach proteases, and the stability of its shelf life when freeze- dried. These thorough assessments are intended to provide a strong proof-of-concept for the use of Xylanase- GCW61 in commercial feed additives, with a focus on the enzyme's operational benefits and practical feasibility.

The objective of our project is to increase the nutritional value of animal feed by using synthetic biology to express xylanase in P. pastoric. Reprogramming yeast to produce and express xylanase on its surface will increase digestibility and improve feed's ability to absorb nutrients. This will enable yeast to break down complex polysaccharides in feed more efficiently. Yeast can more efficiently break down complex polysaccharides, improving feed's digestibility and nutrient absorption, by being genetically modified to produce and express xylanase on its surface. With the application of synthetic biology, this method tackles a major agricultural problem and provides the industry with a practical and sustainable answer.

The stability and activity of the xylanase under different conditions, along with its successful integration and expression in yeast, will demonstrate the benefits and use of our established system. We hope to show how this innovative synthetic biology approach can improve animal health, save feed costs, and lessen environmental impact by demonstrating the enzyme's resilience and usefulness.


REFERENCE

  1. Schneider F, Tarawali S. Sustainable Development Goals and livestock systems. Revue Scientifique et Technique (International Office of Epizootics). 2021 Aug 1;40(2):585-95.
  2. Rout PK, Behera BK, Rout PK, Behera BK. Sustainable livestock farming. Sustainability in Ruminant Livestock: Management and Marketing. 2021:137-70.
  3. Chakdar H, Kumar M, Pandiyan K, Singh A, Nanjappan K, Kashyap PL, Srivastava AK. Bacterial xylanases: biology to biotechnology. 3 Biotech. 2016 Dec;6(2):150. doi: 10.1007/s13205-016-0457-z. Epub 2016 Jun 30. PMID: 28330222; PMCID: PMC4929084.
  4. Ramatsui L, Sithole T, Mzimkulu-Ncoyi NH, Malgas S, Pletschke BI. The use of xylanases as additives to feeds: A mini-review of their effect on feed digestion and growth performance of monogastric animals. Microbial Bioprocesses. 2023 Jan 1:83-105
  5. Patent: WO2020009964A1 https://patents.google.com/patent/WO2020009964A1/en
  6. Patent: GB2585029A https://patents.google.com/patent/GB2585029A/en
  7. iGEM Team Marburg 2015 Project: NUTRInity https://2015.igem.org/Team:Marburg
  8. iGEM Team Shanghai United 2022 Project: Feed Family https://2022.igem.wiki/shanghai-united/
  9. iGEM Team Shanghai United 2022 Project: Feed Family https://2022.igem.wiki/lzu-hs-china-c/
  10. Teymennet-Ramírez KV, Martínez-Morales F, Trejo-Hernández MR. Yeast Surface Display System: Strategies for Improvement and Biotechnological Applications. Front Bioeng Biotechnol. 2022 Jan 10;9:794742. doi: 10.3389/fbioe.2021.794742. PMID: 35083204; PMCID: PMC8784408.
  11. Yang J, Huang K, Xu X, Miao Y, Lin Y, Han S. Cell Surface Display of Thermomyces lanuginosus Lipase in Pichia pastoris. Front Bioeng Biotechnol. 2020 Oct 28;8:544058. doi: 10.3389/fbioe.2020.544058. PMID: 33195113; PMCID: PMC7656992.
  12. Martinić Cezar T, Lozančić M, Novačić A, Matičević A, Matijević D, Vallée B, Mrša V, Teparić R, Žunar B. Streamlining N-terminally anchored yeast surface display via structural insights into S. cerevisiae Pir proteins. Microb Cell Fact. 2023 Sep 7;22(1):174. doi: 10.1186/s12934-023-02183-2. PMID: 37679759; PMCID: PMC10483737.
  13. Kielkopf CL, Bauer W, Urbatsch IL. Expression of Cloned Genes in Pichia pastoris Using the Methanol- Inducible Promoter AOX1. Cold Spring Harb Protoc. 2021 Jan 4;2021(1). doi: 10.1101/pdb.prot102160. PMID: 33397779.
  14. Prielhofer R, Reichinger M, Wagner N, Claes K, Kiziak C, Gasser B, Mattanovich D. Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose-regulated GTH1 promoter of Pichia pastoris. Biotechnol Bioeng. 2018 Oct;115(10):2479-2488. doi: 10.1002/bit.26800. Epub 2018 Aug 8. PMID: 30016537; PMCID: PMC6221138.
  15. Sinegani AA, Emtiazi G. The relative effects of some elements on the DNS method in cellulase assay. Journal of Applied Sciences and Environmental Management. 2006 Oct 31;10(3):93-6.
  16. Agnihotry S, Pathak RK, Singh DB, Tiwari A, Hussain I. Protein structure prediction. InBioinformatics 2022 Jan 1 (pp. 177-188). Academic Press.