Team:BUCT-China/Poster

Biodegradation of Plastics and Further Utilization of the Degradation Products for Biopolymer Synthesis

Presented by Team BUCT-China 2020

iGEM Team PI: Luo Liu (Primary), Kaili Nie (Secondary)
iGEM Team Instructor: Yongsheng Wang

iGEM Student Team Member: Shuming Jin (Leader), Jia Kenan, Shan Qi, Hanyi Zhang, Xiao Xiao , Sijia Xiao, Yushuo Li, Haiwei Quan, Shibo Hou, Zehui Han, Yuanzhi Chen, Min Li, Wenjing Li, Hao Qin, Qixing Wei, Yang Zhaoqi, Ran Zhao, and Yuxin Ning

College of Life Science and Technology, School of International Education

Beijing University of Chemical Technology, Beijing, China

Abstract

We observed a strain Microbulbifer hydrolyticus, which can degrade PE. This year, we made some improvements and conducted further exploration work based on last year's study. Our project is divided into two parts: the first part is degrading PE into alkanes. We build an artificial metabolic pathway and use surface display to express Laccase on the surface of spores, making the reaction more efficient and intuitive; In the second part, we constructed another engineered bacteria that can utilize alkanes to synthesize value-added products such as the new material PHFA (poly hydroxyl fatty acid). In a word, our research can not only solve the pollution of polyethylene, but also provide a low-cost raw material for the synthesis of PHFA. In our assumption, PHFA will be a new material with potential, and we will further study it in the future.

Introduction

In our project, we prepare to combine PE biodegradation and PHFA (poly hydroxyl fatty acid) biosynthesis process together and convert waste PE into brand new PHFA to reduce costs of PHFA production and meanwhile solve PE disposal problem. Specifically, our idea is to establish enzyme reaction system of PE degradation and fermentation system of PHFA synthesis.

Degradation:

We incorporated spore surface display technique in our enzyme expression system to observe the enzyme directly and to realize immobilization of enzyme. Therefore, we designed an engineered Bacillus Subtilis spore with the novel oxidase anchored at the outside spore. With the help of the spore, PE can be degraded within an enzyme reactor. Our design containing selection of anchor proteins and linker peptides, recombination of target gene, and utilization of a certain fluorescent protein to verify our plan.

Synthesis：

Considering the safety and operability of chassis organisms, we chose E. coli as our chassis bacteria. The bio-engineered E. coli synthesize PHFA within a fermentation system, having degraded PE as raw materials. We constructed the metabolic pathway for bio-synthesis by packing all the genes of required enzymes within a double plasmid system, which are further transferred to E. coli MG1655. And then, E. coli will absorb the PE degradation product---alkane by passive transport. Finally, through several steps of oxidation and polymerization, it will transform into PHFA.

Inspiration

The world has witnessed a great growth in the plastic productivity. From 98.92 million tons in 1990 to 300 million tons in 2019, it tripled over 30 years. Plastics is important and used widely, which leads to a huge number of plastic wastes. There are about 8 billion of waste plastic in the environment and its number is still growing. Besides, due to its corrosion resistance and durability, it can’t be degraded in even 100 years. The frequent reports of plastic pollution inspired us to biodegrade it using bacteria. After researching, we know that plastic can be classified into different groups based on their chemical structure and composition. Some of them can be biodegrade by various of bacteria, while others can’t, such as PE. We aim on finding a way to biodegrade PE and biosynthesis it into some environmentally-friendly material.

Last year, we found an oxidase that can degrade lignin in our team’s research. By studying the degradation mechanism, we believe that it can also degrade PE, and the idea is verified by experiments.

Based on last year’s work, we plan to optimize the degradation process of PE and make full use of the product of degradation this year. Besides, we expect to use synthetic biology method to build a synthetic pathway to realize the process of "waste plastic" to "treasure".

Preliminary study (with result)

Based on the study of a strain Microbulbifer hydroxyticus IRE-31, our team found that it has the potential to degrade PE, and we observed it by electron microscopy.

A. there is no Microbulbifer hydrolyticus, only a factor simulating the natural environment under UV conditions.

B.degradation of PE by Microbulbifer hydrolyticus (adding Cu+ to improve degradation efficiency)

Observing the changes of the surface of PE, it was found that the strain had a certain degradation effect on PE. We found that yifH is a key enzyme in the whole degradation process by conducting a genome-wide analysis of the strains and comparing with known oxidase gene sequence.

Through expressing the gene in E.coli, 14-16c alkanes were found by detecting the reaction product with GC-MS. So we can confirm that yifH is the oxidase gene we want.

Degrading design

Surface display technology
Since we get a novel laccase to degrade PE waste, we are trying to make most of it. We believe that Bacillus subtilis surface display technology can be applied to our project. The surface display plays the role of immobilized enzyme, which is reusable and convenient for the enzyme to contact with substrates directly. Besides, it is convenient to detect whether the enzyme is expressed successfully by GFP fusion protein.
We designed three main structures: anchor protein, linker peptide and target gene. Multiple review shows that the basic structure for the expression unit is as follow:
Promoter of anchor protein-CDS of anchor protein-linker-CDS of gene
At last, we combined target gene with a fluorescent protein GFP to verify the work.

1.Choosing anchor protein

（Anchor protein of CotG）

In the work of searching for suitable anchoring proteins, we found that cotG is widely used in catalysis. Although some researches also demonstrate that cotC, especially co-expression of cotG and cotC result in a high yield of enzyme reaction. We only choose to test cotG with its own promoter due to limited time.

2.Choosing linker
Linker plays a very important role in the whole system. We found that the performance of rigid linker is better than that of flexible linker and our former research reveals that rigid linker (EAAAK) is instrumental. However, a flexible linker (GGGGS) provides a higher enzyme activity compared with a stiff linker or no linker at all. Hence, we decided to try both linkers in two plasmids pCY1 and pCY2.

3.Recombinant chromosome
Recombinant chromosome After constructing the plasmids with DH5α, we linearized them with sca1. The linearized plasmids were electroporated into competent B. Subtilis WB800N cells. Since genome expression is more stable than episomal vectors, we insert target gene sequence between two amyE fragment, which is supposed to be introduced to amyE locus on genome of WB800N by a double crossover reaction. For integration–positive clones amyE gene is disrupted and WB800N can’t produce amylase. Therefore, we identified integration-positive clones by cultivating the bacteria on LB plates containing 1% starch. The integrated clones were expected to yield blue color after staining with iodine.

We further carried out PCR for integration-positive clones to verify the results. We have designed the primers listed below.

4.Verify test
We set up a control experiment to judge whether the surface display system is successful by observing the fluorescence intensity with the help of GFP. We constructed pCYG and pCG to test whether enzyme is anchored on spores’ surface.

I.One group of cells containing pCYG is expected to express a fused protein cotG-yifH-GFP. This protein will fix at the outer surface and emit green florescence when exited with ultra-violate light;

II.The other group of cells harboring pCG is expected to express only GFP within the cells. After cultivation of spores, we only need to compare the fluorescence intensity of the two groups to determine whether our anchoring protein is effective.

5. Enzymatic activity determination
Spores of WB800N harboring pCY1, pCY2, pCYG are cultivated, lyses and purified. We will incubate the spores with PE under 37℃ for different hours. The dry mass of remaining PE will be measured.

6.Plasmid
We constructed four plasmids as shown below.
Surface display plasmids：
I.pCY1 (cotG-flexible linker-yifH) II.pCY2 (cotG-rigid linker-yifH) Verify test plasmids：
III.pCYG (cotG-flexible linker-yifH-GFP) IV.pCG [promoter(cotG)-GFP]

Synthesis design

After degrading PE waste by surface display of novel laccase, we want to use synthetic biology to further utilize the bio-degraded products, which corresponds to our project idea----turning waste to wealth.

1.Artificial metabolic pathway
Previous research show that laccase turns PE into straight chain alkane with a length about 14 carbon and certain P450 enzyme is capable of oxidizing alkane. we decide to establish a new metabolic pathway in E. coli to convert degraded PE into PHFA, shown as follow：

2.Strain selection
We choose E. coli MG1655 as our chassis bacteria of synthetic metabolic pathway, which is a model strain with undoubted safety assurance and operability. Besides, to enrich the intermediates of our artificial pathway, we knocked out the fadE gene of the strain to inhibit consumption of β-oxidation.

3.Double plasmid system
There are specifically six enzymes are needed in the whole artificial metabolic pathway. To pack up all the six genes within one cell, we established a dual-plasmid system, as shown below:
I.Plasmid1

II.Plasmid2

4.Key part design
Due to COVID-19, we want to verify our synthesis design at limited time, so we build the key part design.

In this part, we used hydroxyhexadecanoic acid as the substrate, PPF or ACS2 as CoA ligase, WS or WS2 as Acyltransferase. We expressed pET-28a-WS2 / ACS2 and pET-28a-WS / PPF into E. coli MG1655. At the same time, the related plasmids were also expressed in E. coli MG1655 (fadE knockout to exclude the effect of β- oxidation).
The key part of the whole metabolic pathway is shown in the figure.

I.pET-28a-WS2/ACS2
II.pET-28a-WS/PPF

Results

Due to the COVID-19, we can’t enter the laboratory in time to complete the whole experiments. We established our experimental design in the early stage, even in the limited time we persisted to finish the decisive part of experiments to verify the feasibility of our design. With the result, we build a foundation for our follow-up work in the future.

The key part to the success of the whole metabolic pathway is shown in the figure. (PPF and ACS2-----CoA ligase, WS and WS2----- Acyltransferase)

In the verification experiment, we used hydroxyhexadecanoic acid as the substrate. We expressed pET-28a-WS2 / ACS2 and pET-28a-WS / PPF into E. coli MG1655. At the same time, the related plasmids were also expressed in E. coli MG1655 (fadE knockout to exclude the effect of β- oxidation).

1.SDS protein electrophoresis
Through SDS protein electrophoresis, we can confirm that our target genes are successfully expressed by corresponding molecular weight of target genes with protein marker.

2.Growth curve of E. coli
Knocking out the fadE gene has a certain impact on the metabolism of E. coli, like slowing down the growth, however the curve shows that E. coli still can grow normally.

The growth curve indicates that the new pathway we constructed is feasible. After knocking out, we can exclude the metabolism of fatty acids by the E. coli 's self-influence.

3.Results of GC-MS with hydroxyhexadecanoic acid as substrate
The GC-MS graph shows the substrate concentrations inside E. coli MG1655 (fadE knocked out with pET-28a-WS2/ACS2 or pET-28a-WS/PPF) are significantly reduced comparing to E. coli MG1655 (control group, pET-28a-WS2/ACS2, pET-28a-WS/PPF), which provides a strong support to the feasibility of our artificial pathway.

GC-MS diagram of substrate

4.GC-MS diagram of products
By GC-MS analysis, we detected a high content of cyclohexadecanolide, which means substrates reacted triumphantly. Although the polymerization didn’t take place, it still supports that our fatty acid CoA ligase and Acyltransferase metabolic pathway is successfully constructed to some extent.
We consider that low concentration of substrate inside E. coli inducing the happening of esterification instead of polymerization, which will be the key point of our future studies.

Future direction

For the PE degradation system:

1.Knock out the prerequisite genes for germination of bacillus subtilis spore to inhibit the unpredictable germination of engineered spore, by which enzyme reaction efficiency is enhanced. 2.Construct dedicated vector for surface displaying, such as inserting MCS (multiple cloning site) into both ends of target gene on current vector. For the PHFA synthesis system:
3.Enhance percent conversion of PHFA, by strengthening redox system, like overexpressing the oxidase of NADPH or NADH. 4.Introduce Golden Gate system to convenient the operation of multiple genes 5.Promote alkane intake efficiency of engineered E.coli. 6.Optimize reaction condition after analyzing experiments.

Proposed Implementation

we can apply our technique on everywhere which encounters with the Polythene waste. To make our design to be more applicable at various situation, we setup a unit system containing a biodegradation reactor, a filtration device, a biosynthesis reactor, a cell breaking device and a control part.

Taking Waste Hierarchy as reference, our technology and unit system are designed for waste recycling level. So that the unit system can apply into any organization’s factory, bureau of environmental protection in government, recycling company, nongovernmental organization. Anyone who needs to or wants to deal with PE waste and recycle it to make profits can take our unit system as one step.

When it comes to our unit system design, by certain pretreatment, the PE waste will become solid powder. The powder will be inserted into fluidized bed reactor with immobilized bacillus subtilis spores or bacillus subtilis spores only, in which PE degrades into alkane. We choose fluidized bed reactor (FBR) to be the reactor for the spores, which is appropriate to dispose substrate that has high viscosity or insoluble powder like PE power after pretreatment.

From FBR to synthesis reactor, the system needs a filtration. We decide to apply tubular filter having microfiltration film with a 0.04~20μm bore diameter. Although the filtering area of unit volume is relatively small for tubular filter, it has very simple structure which gives the convenience to clean and reuse immobilized spores.

After passing through tubular filter with microfiltration film, the alkane will be utilized by engineered E. coli to synthesize Polyhydroxy fatty acid in airlift filter fermenter. The airlift fermenter as a simple construction and costs less energy by blasting sterile air from nozzle in 250~300m/s flow velocity, then the air flow can cause circulation inside reactor. And mostly important, sterile air provides the engineered E. coli with plenty oxygen for fermentation.

Then, high pressure homogenizer will break the E. coli and PHFA (poly hydroxyl fatty acid) is gained. High pressure homogenizer has a high rate of breaking gram-negative bacteria and capability to operating at large scale.

Human practice

In this iGEM human practice, we set out to study the possible application prospects of our products. We've thought a lot about how we can use our end products, and we hope that our materials can be used as derivatives of PHA and have different characteristics.

At the same time, we recognize the remarkable achievements of 3D printing technology in the field of biomaterials. More and more people begin to pay attention to this technology, and we hope that we can get some inspiration from it. From these perspectives, we went to many related technology companies to learn about these materials and the application of 3D printing technology in biomaterials.

We have entered a number of biotechnology companies:
·3D printing operation

It was a very good experience for us in the process of this human practice. In addition to theoretical study, we also personally carried out the operation of 3D printing equipment, which made us very impressed.

·Expert advice

We learned from experts in the field of biomaterials and 3D printing. From this we have carried on the summary：
1. It is a long way from R & D to application of a material, with a long research cycle, which needs us to explore constantly.
2. The materials need to be tested strictly, including chemical property test, in vivo test and so on.
3. 3D printing technology has been widely used in the field of biomaterials, such as artificial tissues, artificial joints, repair materials, artificial meat and so on.
4. How to realize the aseptic operation of the whole production process is a difficult problem we must face.

At the same time, we have also reached the relevant cooperation intention. In the future, we will provide our products for them to do relevant testing and jointly study their application fields.
We are very grateful to these companies and experts who have helped us.

References and acknowledgement

Acknowledgement
Thanks to Yixun Jiang for Providing many advises on cloning and analysis make our experiment more feasible.

Thanks to Juntao Xu, Zhongyu Li, Ruipeng An and other lab members for providing tutoring while in the lab.

Thanks to Yongsheng Wang for support on the project.

Acknowledgement Langrun Wang and Zigi Ma, Provided technical support for video production.

Jane Liu, from team KEYSTONE, Provided technical support for the design of video.

Reference
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