Team:NAU-CHINA/Description

Our project is designed to degrade 3-phenoxybenzoateacid (3-PBA) thoroughly using the concept of Synthetic Biology and microbial biodegradation techniques. Also, we are eager to take the first step to make our project into reality. Microorganism were shown to acquire the ability to degrade various toxic compounds during their evolution. However, the efficiency of removal of these compounds by bacteria is actually low, because they evolve on the basis of environmental fitness rather than degradation efficiency. In our project, we found Sphingobiumwenxiniae JZ-1^T, which is resistant to 3-phenoxybenzoate acid and can utilize it as the sole carbon and energy sources for growth. In our lab, we aim to realize the biodegradation of 3-PBA using recombinant plasmid. The genes pbaC-pbaA1A2B-C23O-mhbDHIM, encoding enzymes of the 3-phenoxybenzoate degradation pathway were successfully built finally.

Background - Why we need to degrade 3-PBA thoroughly?

Our analysis:

Be harmful to the environment and human
Drosophila toxicological experiment
Cost-benefit analysis for engineered bacteria

Be harmful to the environment and human

The main metabolite in the degradation of insecticide pyrethroids is 3-phenoxybenzoate (3-PBA).Because of the wide use of pyrethroids and the stability of the diarylether compound itself, 3-PBA is typically detected as an important environmental contaminant. As a metabolite, 3-PBA is easy to be ignored by people. It’s time to arouse people’s attention on 3-PBA and pyrethroids because of the existed truth reported by paper and media. Through searching a large number of papers and analyzing data, we found that the harms of 3-PBA are as follows:

Long natural degradation half-life

The half life period of 3-PBA is about 180 days , longer than most of pyrethroids, making it difficult to be degraded in the environment.

Hindering the mineralization of pyrethroids，which cut off the biodegradation pathway that pesticide is transformed into non-toxic (poisonousless) small molecule.
Making secondary pollution to farm
Widespread occurrence in water, sediment, and soil
3-PBA is suspected of having reproductive toxicity
3-PBA is an important material in chemical industry.

Drosophila toxicological experiment

To analyze the toxicity of the 3-PBA, we used fruit fly to test. We tested 3-PBA’s influence on reproductive capacity of fruit fly, and found that 3-PBA can affect the normal secreting of sex hormone.

We counted the number of the new imagoes hatched by flies that were fed in medium with 3-PBA before.

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Cost-benefit analysis for engineered bacteria

Degradation comes at a price. Thorough biodegradation of 3-PBA involves a series of enzymatic reactions. Protein synthesis and enzyme reactions needs materials, ATP and NADH. Catechol , an intermediate metabolite of 3-PBA, is toxic to the Escherichia coli. 3-PBA can be degraded and absorbed entering the TCA circle to produce energy. Based on these facts we can get a formula Net proceeds(N)=Benefit(B)-Cost(C).

Method - How to degrade 3-PBA thoroughly?

Gene circuit design
Experimental test & previous parts improvement & analysis
Gene circuit redesign

Gene circuit design

Simplest genetic circuit ---our small step to degradation

Introduction:

We used constitutive promoter (BBa_J23101) to transcribe downstream genes including gene cluster pbaA1A2B and C23O. Constitutive promoter (BBa_J23100)was also used to transcribe gene pbaC. Gene cluster pbaA1A2B and gene pbaC encode the 3-phenoxybenzoate 1',2'-dioxygenase,which is an angular dioxygenase catalyzes the hydroxylation in the 1'and 2' positions of the benzene moiety of 3-phenoxybenzoate(3-PBA), yielding 3-hydroxybenzoate(3-HBA) and catechol. 3-HBA is further transformed by 3-hydroxybenzoate 6-hydroxylase, gentisate 1,2-dioxygenase, glutathione (GSH)-dependent maleylpyruvate isomerase and fumarylpyruvate hydrolase coding by gene cluster mhbDHIM. MhbM, MhbD, MhbI and MhbH convert 3-HBA to pyruvate and fumarate. Meanwhile, catechol is detoxified by catechol 2,3-dioygenase coding by gene C23O. The genetic circuit is shown in Fig1 and the whole metabolic pathway is shown in Fig2.

Fig1. Our device is designed to degrade 3-phenoxybenzoate completely

Fig2.The pathway of biodegradation of 3-pba in our engineering bacteria

Previous parts improvement

CopA promoter：

Overview

CopA, the principal copper efflux ATPase in Escherichia coli, is induced by elevated copper in the medium. CopA promoter is active in the presence of copper ion.

First experiment:

We test copA promoter in BL21（DE3）、DH5α. By measuring fluorescence intensity in cells by flow cytometer，we got data to analyze sensitivity and specificity of copA promoter.

Results:

In our experiment, copA promoter was induced by different concentration of copper ion (37.5umol/L、50umol/L、62.5umol/L、75umol/L) . It is shown in Fig1 that fluorescence intensity in cell increase firstly and decrease with small oscillations. At 4-5th hour fluorescence intensity in cell increases dramatically. Dose response curves was fitted to twice induction within 9 hours. CopA promoter has relative leaky basal expression(Fig 2)by comparing the negative control’s output and basal leakage of copA promoter in E. coli expression systems. In comparison of two graphs A、B, we can obviously find that the degradation of protein is much faster in DH5α than that in BL21（DE3），because BL21（DE3） has a deficiency of protease.. In the group of 0μmol/L Cu²⁺, the fluorescence shows a trend of falling firstly then rising. Actually, the fluorescence which produced by the leakage of copA will not change. The change quantity comes from the different growth periods of the E.coli. We added 40ul bacterial fluid into new medium with induction to start measuring. So bacteria will go through a period of growing from growth period to maturation period, so as to the change of the fluorescence. Maturation period is great period for the expression of protein.

Fig.3.A.Changes in fluorescence intensity induced by different concentration of copper ion in E.coli BL21 (DE3). B. Changes in fluorescence intensity induced by different concentration of copper ion in E.coli DH5α. C .Changes in fluorescence intensity in E.coli without induction comparing with two strains. D .Changes in fluorescence intensity in E.coli which do not contain copA promoter comparing with two strains.

Fig.4.CopA promoter has leaky basal expression

Second experiment:

We placed insulator RiboJ between copA promoter and RBS.

Fig.5.Two device used to detect copper in solution

Result:

We use 50uM copper ion to induce copA promoter (Fig 4).A device without RiboJ has an unstable Fluorescent quantity. At fourth hour, the fluorescence intensity in cells rose sharply. By contrast, a device with RiboJ response to copper ion and express GFP gradually. In addition, a device without RiboJ has high leakage with fluctuation. However, a device with RiboJ has low and stable leakage.

Fig .6 Changes in fluorescence intensity induced by copper ion in E.coli over time

Fig.7 leakage of copA promoter over time

Discussion:

We proposed that RiboJ help reduce and control leakage of copA promoter.

Reference:

[1]Outten F W, Outten C E, Hale J, et al. Transcriptional Activation of an Escherichia coli Copper Efflux Regulon by the Chromosomal MerR Homologue, CueR[J]. Journal of Biological Chemistry, 2000, 275(40): 31024-31029.

Gene circuit redesign(Click to see more.)

Practice - How to make our project into reality?

Social investigation
Applied design
Concept & achievement sharing

Reference:

[1]Cheng M, Chen K, Guo S, et al. PbaR, an IclR Family Transcriptional Activator for the Regulation of the 3-Phenoxybenzoate 1′, 2′-Dioxygenase Gene Cluster in Sphingobiumwenxiniae JZ-1T[J]. Applied and environmental microbiology, 2015, 81(23): 8084-8092.

[2] Characteristics of a 3-Phenoxybenzoic Acid Degrading-Dacterium and the Construction of a Engineering Bacterium DUAN Xiao-qin，ZHENG Jin-wei ，ZHANG Juan，HANG Bao-jian，HE Jian，LI Shun-peng (Key Laboratory of Microbiological Engineering of Agricultural Environment，Ministry of Agriculture，College of Life Sciences，Nanjing Agricultural University，Nanjing 210095，China)

[3] Microbial degradation of 3-phenoxybenzoic acid － A review Weiqin Deng1，Shuliang Liu1* ，Kai Yao2. 1College of Food Science，Sichuan Agricultural University，Ya’an 625014，Sichuan Province，China. 2 College of Light Industry and Food，Sichuan University，Chengdu 610065，Sichuan Province，China

[4] A Novel Angular Dioxygenase Gene Cluster Encoding 3-Phenoxybenzoate 1=,2=-Dioxygenase in Sphingobium wenxiniae JZ-1 Chenghong Wang, Qing Chen, Rui Wang, Chao Shi, Xin Yan, Jian He, Qing Hong, Shunpeng Li Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing, China

[5] MhbR, aLysR-type regulator involved in 3-hydroxybenzoatecatabolismviagentisate in Klebsiella pneumoniae M5a1Lu-Xia Lin, Hong Liu, Ning-Yi Zhou Key Laboratory of Virology, Wuhan Institute of Virology ,Chinese Academy of Sciences,Wuhan430071,China Graduate School, Chinese Academy of Sciences,Beijing100049,China