Team:Worldshaper-Nanjing/Poster

Poster: Worldshaper-Nanjing

Team Achievement

This is the poster of Team Worldshaper-Nanjing 2020. This year, our project is focusing on turning substandard grains into biodiesel using engineered Y. lipolytica. As followed is our achievements in this iGEM season.

Turning Waste into Treasure:
Reuse of Substandard Grains as Fermentation Substrate to Produce Biodiesel

Presented by: Team Worldshaper-Nanjing 2020

Team Roster:

• PIs: Shan Dong, Ruyi Shi, Xixi Song
• Instructors: Xiaoman Sun, Tianqiong Shi, Chao Ye, Wenzheng Liu
• Team members: Xinhe Shen, Jianpu Gao, Zihan Tian, Zhenhao Feng, Yu Dai, Yiheng Yao, Guanwen Ding, Yiling Dai, Tingting Da, Yiran Xu, Ruifan Cao, Xiyu Huang, Xinyuan Fang, Yang Shi, Ziyou Yang, Xinyao Xu, Yingtong Zhou, Tengzhong Zhang, Taixiang Yang

Abstract

Substandard grains, such as long-term stored grains, cannot be eaten due to the existed toxin and contaminants. Therefore, these grains attracted much attention as the fermentation substrate. Meanwhile, the biodiesel attracted increasing attention as a promising biofuel to replace fossil diesel for the last two decades. In this work, Yarrowia lipolytica has been selected as the model host for biodiesel production. Taking into account the disability to degrade the raw starch of Y. lipolytica, we separately express heterologous alpha‑amylase and glucoamylase enzymes in it. The results showed that the strains expressing glucoamylase were able to grow on starch as sole carbon source. By optimizing the C/N=100, the total lipid content increases up to 21 % of DCW, which sets up the basis for further studies.In the future, we aim to provide a practical, economical, and environment-friendly method to treat substandard grains and produce biodiesel.

Introduction & Inspiration

Our team initially wanted to find a way to use bio-synthetics to turn kitchen waste into something usable. However, the composition of kitchen waste is so complex that there is no suitable way to treat it. We finally narrowed down our topic to substandard grain based on background survey and experts advises.

Substandard grains refer to raw grains, including corn, rice, wheat, etc. that contain several toxic and harmful substances that do not meet the limit requirements of the national food safety standards and may be fatal in several cases (see below).

Biodiesel has attracted more and more attention recently because of much less Green House Gas (GHG) emissions than that of fossil fuel counterparts on a per unit energy basis (See below). Therefore, bioenergy production is a key development project worldwide.

The goal of our project is to turn starch from substandard grains into biodiesel using genetically engineered Y. Lipolytica, a type of oleaginous yeast.

Figure Life-cycle GHG emissions from gasoline, diesel and biofuels in the U.S. (Source: Renew. Sustain. Energy Rev. 2018, 82 (March), 2387–2400.)

Project Modeling

We firstly constructed the enzyme-constrained model of Y. Lipolytica under the lead of our teacher to predict whether our idea is practicable, and provide theoretical evidence for our further modification within the experiments. The new generation of the model is based on the traditional metabolic network model, iYLI647.

Predication:

Y. Lipolytica can utilize 36 kinds of carbon sources.
The rate of glucose is the maximum, while that of starch is only 6.34% lower. Consequently, Y. Lipolytica is able to effectively utilize starch as carbon source.

Figure The predict results of the carbon source utilization capacity of Y. lipolytica of ec_iYLI647 model.

Design

In order to meet the requirement for the complete hydrolysis of raw starch, the α-amylase and glucoamylase genes from Aspergillus niger were introduced into Y. lipolytica to further improve the ability and efficiency of degrading starch. The main functional biobricks we constructed include AnGlu Expression Unit (BBa_K3578010), AnAmyA Expression Unit (BBa_K3578011), Combined Expression system (Planning).

Figure Schematic diagram of the starch decomposition system design.

Results and Conclusion

Starch Degradation Test

The α-amylase and glucoamylase expression units were separately constructed to test their ability to degrade starch. After electrophoresis verification (Figure 1-A), two engineered Y. lipolytica expressing the AnGlu and AnAmyA individually were cultured in the starch (sole carton source) medium to further verify the starch degradation ability.

Figure 1 (A) Agarose gel electrophoresis for plasmid identification; (B) Growth curve in starch medium as sole carbon source; (C) Photos of Y. lipolytica solution after culturing 96 hours

As shown in Figure 1-B, only Po1g-AnGlu strain can grow well in the starch medium. The result can also be directly observed as shown in Figure 1-C, in which Polg-AnGlu possess a much higher cell density.

To sum up, the expression of AnGlu in Y. lipolytica is sufficient to allow growth on starch.

Lipid Production Test

Since the Polg-AnGlu strain can growth well on the starch medium, we then measured the lipid titer and content. The Polg-AnGlu can produce up to 0.76 ± 0.04 g/L lipid and 14.66 ± 0.43% of DCW as fatty acids in the starch media and a C/N ratio of 30.

Meanwhile, it shows that improving C/N ratio is an effective strategy to strengthen lipid accumulation (Figure 2-A). And this conclusion was also verified by lipid bodies visualization carried out by addition of 1 mg/mL Nile red to the cell suspension (Figure 2-B).

Figure 2 (A) Lipid production and content in the starch medium with different C/N ratio; (B) Fluorescence micrograph of Y. lipolytica cell.

Human Practices

To confirm the operability of our project, we launched the human practice research carefully, including a general background investigation, public survey, and professional interview.

Public Survey

Low public awareness for stale grains problems. Still, a respectable amount of 24% of participants considered stale grains edible.

These statistics revealed that it is necessary to improve people’s awareness of stale food, thus public science activities scheduled.

Improvement from Expert Interviews

• narrowing down our research goals
• expand the scope of raw materials from stale grain to substandard grains
• inspiration for implementation design
• inspiration for further expanding alternative raw material

Proposed Implementation

The whole project, from raw materials collection to supplying biodiesel to fuel station, was designed with 11 steps.

Figure 1 The whole process of biodiesel production.

Some steps worth pointing out are as follows:

1. Collect raw materials
We can cooperate with government departments to collect the stored materials, and transport these grains to biodiesel manufacture factory and store them in factory’s storage.

2. Fermentation Process
The mechanical agitating fermentation tank we selected is conducive to solid and nutrients fully contact, easy to absorb nutrients; On the other hand, it can break bubbles (Figure 2A).

3. Liquid extraction device
Three-phase centrifugal separator will be used to separate lipid and wastewater from residues respectively (Figure 2B).

Figure 2 Schematic diagram of (A) The mechanical agitating fermentation tank (B) Three-phase centrifugal separator.

Future Work

1. Further increase AnGlu gene copy number to accelerate starch degradation rate.

2. The strategies for improving the lipid titer, yield and rate

3. Further expand the scope of our usable waste substrates

Acknowledgements

Thanks and acknowledgements for our families and all other people involved in helping make us a successful iGEM team.

General Support:
Hangzhou Sipu Education and Technology, Co., Ltd.

Lab Support:
Nanjing Normal University, School of Food Science and Pharmaceutical Engineering

Wiki Support:
Mr. Dong Zhiheng, Senior IT engineer
Jiangnan-China 2020 iGEM team

Human Practices Support:
Dr. Sun, School of Food Science and Pharmaceutical Engineering of NNU
Prof. Shen Xinchun, College of Food Science and Engineering of NUFE
Director of the Granary, Yuyang, Hunan
Prof. Zhao Zongbao, Dalian Institute of Chemical Physics, Chinese Academy of Sciences

Partnership:
Jiangnan-China 2020 iGEM team