Team:Worldshaper-Nanjing/Contribution
Preface
Library construction is a very important task in synthetic biology. This year we discovered a new method in literature, namely non-homologous end joining (NHEJ), which is expected to be a rapid method for constructing libraries. Here we briefly introduce this method, and attach the references, hoping to provide references for the iGEM teams who will to the corresponding work in the future. In addition, enzyme-constrained model of Y. lipolytica and summary of amylase and biodiesel related iGEM projects are also introduced.
Non-homologous End Joining (NHEJ) Enables Rapid library Construction
Y. lipolytica is an important oleaginous industrial microorganism, which is used to produce biofuels and other high value-added compounds[1]. The integration of homology-independent foreign DNA into the genome is a common event, and its frequency is several orders of magnitude higher than homologous recombination (HR) mediated integration in most eukaryotes[2]. Non-homologous end joining (NHEJ) is also a common repair mechanism. Compared with the HR of DNA requiring a homologous repair template, the NHEJ of the genome does not need a homologous DNA repair template or DNA replication. Instead, NHEJ binds to the broken end of double-stranded DNA through protein complex and recruits corresponding ATP-dependent DNA ligase to complete the connection repair of double-stranded DNA.
It has been reported that a complete LEU2 expression cassette without upstream and downstream homologous arm was introduced into Y. lipolytica and the number of transformants was calculated in the corresponding selected medium. The results showed that exogenous DNA was efficiently and randomly inserted into the genome of Y. lipolytica in a non-homologous dependent manner. Then, the reporter gene hrGFP was fused with LEU2 gene expression cassette and transformed into Y. lipolytica to obtain a series of transformants and detect the fluorescence intensity. The results showed that the expression level of hrGFP was 0.24-3.02 times higher than control. Finally, they used Genome Walking method to obtain the genomic site information of six hrGFP-integrated strains.
The insertion of exogenous DNA fragments into functional genes or regulatory elements in Y. lipolytica genome may result in random deletion of genes. Thereby a large-scale deletion-library was formed, in which protein expression levels were affected according to gene-deleted sites. But in other words, combined with high-throughput screening technologies, such as Nile Red lipid staining, flow cytometry selection, etc., it can screen out better-expressed strains faster, thus greatly reducing the labor and time required for pathway optimization and overexpression of industrial enzymes.
Enzyme-constrained Model of Y. lipolytica
The enzyme-constrained model of Y. lipolytica is primarily used to accurately predict the cellular phenotype of Y. lipolytica, as well as the rate for it to utilize certain carbon sources according to their enzymatic properties.
The enzyme-constrained model of Y. lipolytica is constructed on the basis of the latest metabolic network model, iYLI647. Unlike the others, this model embraces the most comprehensive data. When constructing the enzyme-constrained model, we also modified certain parameters and biomass equations especially to our demands.
After constructing and visualizing the model, we can clearly observe the enzymatic properties of Y. lipolytica. Compared to other traditional models, our constructed model can orient certain key targets within reactions more accurately. What’s more, according to the results of the rate of Y. lipolytica utilizing carbon sources predicted by the model, 29 kinds of carbon sources could be used by Y. lipolytica, among which starch possesses the most efficiency. This result also provided us with solid theoretical evidence for our further experiments that modify Y. lipolytica to utilize starch with higher efficiency.
Summary of Amylase and Biodiesel Related iGEM Projects
Biodiesel can improve global warming caused by carbon dioxide emissions, which is a major environmental problem harmful to human beings. The main component of substandard crops is starch. Amylase plays an important role in the transformation from starch to biodiesel. We collected the relevant research of iGEM, and summarized the related work of amylase and biodiesel, as shown in the Table 1. We hope that iGEMers, which is interested in synthesis of biodiesel and modification of amylase, can provide more effective methods. We also hope that biodiesel, as a clean energy, can be well utilized to benefit the earth.
- Table 1: Previous Projects of iGEM Team on Amylase and Biodiesel
1) Previous Projects on Amylase
Amylase can react with a variety of substrates, making it very suitable for changing conditions in large-scale industrial environment[8]. Amylases are still very active at lower temperatures and higher pH values, which makes them very useful in the detergent industry[9]. Utah State (2014) did the following work: Produce stain-fighting enzymes in E. coli; Immobilize enzymes to bioplastic; Manufacture a reusable bioplastic laundry treatment (cellulase, amylase, and chlorophyllase). The results showed an increase of 3.527 μM in concentration of chlorophyllide over the control. BroadRun-Baltimore (2016) designed six constructs; each one a different combination of 2 different strong constitutive promoters and 3 amylase enzymes. In the end, they use engineered yeast to produce α-amylase and degrade starch, thus controlling anaerobic butyric acid production. TU Dresden (2017) demonstrate the secretion of α-amylase of B. subtilis, sfGFP and mCherry proteins and succeeded in identifying the most potent SP-protein combination for each of them. This thoroughly evaluated measurement tool can determine the optimal combination of SP with any protein of interest. Almost all yeasts have extracellular amylase or cellulase activity, the activities of amylase and cellulase were higher in most of the yeasts at 30 ℃, and the highest amylase and cellulase activities were observed in Tetracladium sp. and M. gelida respectively[10].
2) Previous Projects on Biodiesel
Biofuels produced by algae have proved to be a potential alternative to fossil fuels. Linkoping Sweden (2016) have been working with the model algae C. reinhardtii. These sequences combined result in a knock-out of a gene coding for starch synthesis, when the starch production is cut off, the fatty acid synthesis is naturally favored. Large scale production of the biodiesel from C. reinhardtii could be the answer to a sustainable environment in the future. Washing (2011) put AAR and ADC genes downstream of a strong constitutive promoter. They found that alkanes could not be detected when ADC was expressed only, C14 alcohols could be detected when AAR was expressed alone, and alkanes of different lengths (C13, C15, C17) could be detected only when AAR and ADC were expressed, which provides a direction for the subsequent optimization. The researchers reported that they used noodle manufacturing waste as raw materials and converted it into bioethanol and biodiesel[11].
References
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- 8.Raul D, Biswas T, Mukhopadhyay S, Kumar Das S, Gupta S. (2014). Production and partial purification of alpha amylase from bacillus subtilis (MTCC 121) using solid state fermentation. Biochemistry Research International. 2014, 568141.
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- 11.Yang X, Lee JH, Yoo HY, Shin HY, Thapa LP, Park C, Kim SW. Production of bioethanol and biodiesel using instant noodle waste. Bioprocess Biosyst Eng. 2014, 37(8):1627-35.