Team:NIT Warangal/Engineering

iGEM NIT_Warangal

At the beginning of the project, we were deeply intrigued by the engineering cycle and were keen on doing multiple iterations in order to get the best result possible. But due to the ongoing pandemic, we were restricted to doing just one cycle. The lack of computational resources and restrained access to the laboratories hampered our project, but our motivation led us to complete the first iteration successfully, and enabled us to learn more about the challenges we might face, and prompted us to devise suitable solutions to overcome them. On this page, we will explain about how we proceeded with accomplishing the first iteration of the engineering cycle, and the inspiration behind it.

INSPIRATION

As the team members were exploring the iGEM website and wikis of other teams, the team was inspired by iGEM's motto of "Local People solving Local Problems". Motivated by this goal, the team started finding out problems that are faced by the local population and potential solutions to these problems. One problem that marred the local community was that of harm to animals that were consuming cottonseed meal. After diving a bit more into the problem we realized that the presence of gossypol was an inhibitor in widespread use of cotton seed meal or cake for usage in the local community. We also realized the potential of using cottonseed as a protein source, which can be proposed as a solution to solve widespread malnutrition problem across India and other developing nations.

RESEARCH

After an exhaustive study of existing methods for degossypolization of cotton seeds through various research articles, we found four prominent methods which had succeeded in doing so. The methods we came across are as follows:

Two stage solvent extraction process: Utilizing aqueous and anhydrous acetone, a two stage solvent extraction has been developed which decreases free gossypol level in cottonseed and increases protein concentration to about 72.2% ^[1].
Treatment with ferrous sulphate: This study showed that fish food containing 40% cottonseed meal when supplemented with 580 mg/kg Fe, considerably increased growth rate of fish. This was due to inactivation of free gossypol by iron^[2].
Acidic solvent extraction: Ethanol and acetone based solutions were used with phosphoric acid and water to hydrolyse the protein bound gossypol. Total gossypol level in cottonseed meal was reduced by 5-10%^[3].
Solid state fermentation: A mixed fungal culture was prepared by mixing Candida tropicalis with Saccharomyces cerevisiae. After the solid state fermentation process, the cottonseed meal showed 79.5% detoxification rate for free gossypol and 59.5% for bound gossypol. 13.4% increase in crude protein was recorded in the fermented cottonseed cake^[4].

However, we were not fully satisfied with this, and our zeal for innovation led us to think of a novel enzymatic approach to degrade gossypol in a more efficient manner.

DESIGN :

While designing the model of our project, we kept the expected final outcome in mind. The proposed functionality of our model, which was to degrade gossypol, particularly influenced our thinking and aided us in designing the intricacies of the enzymatic prototype.

Once our preliminary research regarding other approaches was completed in the 'Research Phase' of our project, we set out to design our method of enzymatic degradation. For this, we had to decide 2 major components of our project :

Expression organism that should be utilized to produce the enzyme
Media in which the enzyme will be produced

After a round of research and consulting with our PIs, it was determined that Pichia pastoris will be an ideal candidate for expressing our enzyme.

Pichia pastoris is well known as a recombinant expression host system and is largely used for the commercial production of various enzymes and therapeutic proteins. As a yeast, it shares the advantages of molecular and genetic manipulations with Saccharomyces, and it has the added advantage of much higher heterologous protein expression levels. Along with this, Pichia pastoris has the added advantage of showing several post-translational modifications making it suitable for transformation. Cost effective production as well as easy scalability make it suitable for large scale production^[5].

Crude glycerol was identified as a prospective solution as it provides a reliable carbon source to the organism to synthesize the enzyme. Crude glycerol is a low-value byproduct which is primarily obtained from the biodiesel production process and since biodiesel production is predominant all across the world, it would be easily available for a majority of the global stakeholders. Crude glycerol doesn't have any significant usage (until it's purified) in other industries and thus is considered as waste, and so by utilizing crude glycerol we will be recycling a waste product reducing environmental impact.

The enzyme selection process has been extensively discussed in the lab section.

pPIC9K is an expression vector for Pichia strains, which has a strong Alcohol oxidase promoter that helps in higher protein expression and is identical to pPIC9 except for the presence of the kanamycin resistance gene for in vivo screening of multiple copy inserts.

We used EcoRI and NotI restriction sites for double digestion and insertion of the Melanocarpus albomyces laccase gene. The 2 sites are present in frame with the alpha-factor secretion signal.

Addition of the alpha-factor secretion signal to heterologous proteins in Pichia pastoris usually results in the secretion of the intact heterologous protein to the extracellular medium ^[6].

Fig 1: pPIC9k-Laccase plasmid BBa_K4077003

BUILD

Not having access to labs for the majority of the iGEM cycle made it difficult for the team to validate and analyze the design of our project. A robust design which ensures a sustainable and feasible result for our project was designed, along with incorporating aspects that will help us tweak certain components of the design once we identify nuances that need to be addressed during the 'Learn' phase of the Engineering Cycle. A Brief overview of this phase is as follows :

Fig 2: Build phase flow chart

Once we identified our protein of interest,i.e., laccase we carried out propagation of the pPIC9k vector into E.Coli DH5α which is a widely used and popular strain used for recombination.

The entire transformation process has been detailed in the Proof of Concept section.

Once transformation is completed we screen for positive clones and further the phenotype of the organism will be determined.

The two genes, AOX1 and AOX2 in Pichia pastoris code for alcohol oxidase. Majority of the alcohol oxidase activity in the cell is accounted for by the AOX1 gene product. Expression of the AOX1 gene is induced to very high levels by methanol and is tightly regulated. Typically more than 30% of the total soluble protein in cells grown on methanol is accounted for by the AOX1 protein. While AOX2 is around 97% homologous to AOX1, the growth on methanol is much slower than with AOX1.

Loss of the AOX1 gene, and for that reason a lack of maximum of the cell's alcohol oxidase activity, results in a strain with a MutS (methanol utilization slow) phenotype. A MutS strain has a mutant aox1 locus, however is wild-type for AOX2. It has decreased capacity to metabolize methanol and for that reason poor growth on methanol medium^[7].

Since we plan to produce laccase in a medium containing methanol it is important to select only Mut+(methanol utilization plus) plasmid.

TESTING

Fig 3: Flow for testing phase

The recombinant plasmids would be used to produce laccase in crude glycerol as well as pure glycerol to compare performance.

Laccase activity is determined by oxidation of the ABTS method^[8]. ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) can be used to detect the binding of molecules to each other. The absorbance of the ABTS⁺ cation can be used to evaluate the antioxidant capacity of drug solutions. It is commonly used with blue multicopper oxidase enzymes, like laccases^[9]. The non-phenolic dye ABTS is oxidized by the laccase to form a more stable form of the cation radical. The concentration of the cation radicals can be correlated to enzyme activity, and plays a major role in the intense blue-green color. It is observed to be at 420 nm.

After recording laccase activity, we proceeded to determine the protein concentration by the dye-binding method of Bradford using BSA as standard.

Determination of the laccase activity will help us gain an idea of whether the strain is producing sufficient amounts of laccase to degrade the gossypol.

LEARN

The Learn Phase of the engineering cycle takes the outputs from the Test Phase and looks at how certain parameters and factors could be changed in either the Design Phase or Build Phase or both that could lead to a more efficient solution.

Information gained in this phase can be used to predict future scale-up procedures as we seek to make our solution accessible and affordable to all stakeholders involved in the project. Here are some of the possible challenges that we might encounter during the project:

Possible Challenges^[10]:

Is the expression sufficient to produce enough laccase in both pure glycerol as well as crude glycerol used in the "Test Phase"?
Is the final concentration of laccase after purification enough to carry out CSM treatment?
Does the enzyme(laccase) function in the conditions predicted (temperature, pH, agitation, etc)?
Is our laccase capable of removing enough gossypol from CSM for safe consumption ?
When used by stakeholders in a practical scenario, does the laccase interact with other ligands and get used up before binding to gossypol?

Answering these questions will help us understand areas where our design perhaps needs to improve and can also provide insight into aspects of our build which could be optimized.

References :

[1] Gerasimidis K, Fillou DT, Babatzimcpoulou M, Tassou K, Katsikas H. Preparation of an edible cottonseed protein concentrate and evaluation of its functional properties. Int J Food Sci Nutr. 2007 Sep;58(6):486-90. DOI: 10.1080/09637480701288488. PMID: 17710592.
[2] Gaber, M.M. & El-Halfawy, Magdy & Ramadan, Amal. (2012). Utilization of cottonseed meal supplemented with iron for detoxification of gossypol in Nile tilapia, broodstock and their impact on the hatchability of their progenies. Journal of Aquaculture Research and Development. 3.
DOI: 10.4172/2155-9546.1000151.
[3] Pelitire, Scott & Dowd, Michael & Cheng, Huai. (2014). Acidic solvent extraction of gossypol from cottonseed meal. Animal Feed Science and Technology. 195.
DOI: 10.1016/j.anifeedsci.2014.06.005.
[4] Vellaichamy, Mageshwaran & shaikh, A & Kathe, A. (2017). Optimization of process parameters for gossypol detoxification in chemical disinfected cottonseed cake by mixed fungal culture during solid state fermentation. Journal of scientific and industrial research. 76. 714-719.
[5] Karbalaei, M., Rezaee, S. A., & Farsiani, H. (2020). Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins. Journal of cellular physiology, 235(9), 5867-5881. DOI: https://doi.org/10.1002/jcp.29583
[6] Lin-Cereghino, G.P., Stark, C.M., Kim, D., Chang, J., Shaheen, N., Poerwanto, H., Agari, K., Moua, P., Low, L.K., Tran, N., Huang, A.D., Nattestad, M., Oshiro, K.T., Chang, J.W., Chavan, A., Tsai, J.W. and Lin-Cereghino, J. (2013). The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris. Gene, [online] 519(2), pp.311-317.
Available at: http://www.sciencedirect.com/science/article/pii/S0378111913001765
[7] Yeast Expression Support from Thermofisher
[8] Bourbonnais R, Leech D, Paice MG. Electrochemical analysis of the interactions of laccase mediators with lignin model compounds. Biochimica et Biophysica Acta. 1998;1379(3):381-390.
[PubMed] [Google Scholar]
[9] Milena Morandi Vuodslo, ... Mário Roberto Maróstica Junior, in Bioactive Compounds, 2019
[10] Inspired by https://2020.igem.org/Team:ULaval/Engineering