Team:UESTC Life/Project

From 2013.igem.org

Project Overview

Haloalkanes are widely used commercially. The majority of these compounds have been shown to be serious pollutants as they are toxic and quite persistent in the environment, such as a man-made industrial chemical 1,2,3-Trichloropropane (TCP) and an organic pesticide γ-Hexachlorocyclohex-ane (Lindane，γ-HCH). These halogenated compounds have been introduced into our environment as a consequence of industrial waste disposal and widespread open use in agriculture, which need to be removed to low levels from waste streams and during sanitation of polluted sites. For this reason, Microbial degradation of these compounds represents an important and efficient way to fulfill the target. In order to improve biodegradation efficiency, several powerful genetically engineered E. coli strains have been constructed by the co-expression of key enzymes involving in the biodegradation pathways of the two compounds. As putting the different selected enzymes together, it will have an ability to degrade more halogenated compounds besides the both. To construct the efficient co-expression system and achieve biodegradation of γ-HCH and TCP, these key enzymes, LinA, LinB, DhaA and HheC, are chosen, and foot and mouth disease virus 2A peptide and polycistronic co-expression strategies were adopted. Foot and mouth disease virus 2A peptide has been widely used for co-expression of multiple genes in eukaryote systems. However, the use of the 2A peptide in prokaryotes is limited, and so far only one paper described that F2A can work in E. coli as well.[1] To explore whether the 2A peptides can work in our co-expression system, several vectors were constructed by using all three 2A peptides, respectively. The results showed that all enzymes could co-express as a soluble protein with P2A peptide acting as a linker and F2A could work as the same as in eukaryotic system. Moreover, the resulting engineered E. coli exhibited an excellent capability for the degradation of TCP and γ-HCH.

Project Story

γ-isomer of hexachlorocyclohexane (γ-HCH)

HCH, formerly known as benzene hexachloride (BHC), is one of the now notorious organochlorine group of insecticides. About 600,000 tons were used throughout the world between the 1940s and the 1990s to control a wide range of agricultural, horticultural, and public health pests.[2,3] Mounting concerns about its nontarget toxicity and persistence have since caused it to be deregistered in most countries[2], but it is very stable in the environment[2,7] and it is still being manufactured in India for local and export uses, so residue problems will continue for many decades.[2] The majority of HCH waste has been discarded in the open or stored at various levels of containment near the production sites, HCH residues at many of the sites have percolated into the soil and then contaminated ground water.[2] Residues of HCH have now been reported for many countries in samples of air[8,9] water[10] soil[10,11,12]food commodities[13,14,15] and even from human blood samples.[16,17]

1,2,3-Trichloropropane (TCP)

1,2,3-Trichloropropane (TCP) is a toxic synthetic chlorinated hydrocarbon that is not known to occur naturally[20]. It is used as a chemical intermediate in organic synthesis, as a solvent, and as an extractive agent. In addition to these intentional uses, TCP is produced in considerable amounts as a by-product from the manufacture of epichlorohydrin. TCP does not noly contaminate soil, it also leaks down into groundwater and settles down at the bottom of the ground water reservoir because TCP is more dense than water. This makes TCP in its pure form a DNAPL (Dense Nonaqueous Phase Liquid) and it is therefore harder to remove from groundwater[19]. Groundwater and soils in various parts of the United States and in Europe are polluted with TCP as a result of improper disposal of TCP-contaminated effluents and due to the past use of the soil fumigant D-D, a mixture of 1,3-dichloropropene and 1,2-dichloropropane that contained TCP as a contaminant (U.S. Environmental Protection Agency, national priority site fact sheet and Tysons dump site). Due to its toxicity and persistence, TCP poses a serious risk to ecosystems and human health[18,19,21].

Four Key Enzymes

LinA and LinB

Selected LinA and LinB are cofactor-independent dehalogenase from Pseudomonas paucimobilis UT26, LinA mediates the first step of aerobic microbial degradation of γ-HCH to 1,3,4,6-tetrachloro-1,4-cyclohexadiene , which is further metabolized by the sequential activity of LinB.

DhaA and HheC

DhaA and HheC also are cofactor-free dehalohygenase. DhaA from Rhodococcus sp hydrolyzes carbon-halogen bonds in a wide range of haloalkanes, including TCP, to the corresponding haloalcohol, releasing halide ions. Haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1 is a potentially useful enzyme involved in the degradation of several important environmental pollutants, such as 1,3-dichloro-2-propanol, 2,3-dichloropropa-nol, 1-chloropropanol, epichlorohydrin and so on. Additionally, HheC has highly enantioselective dehalogenation of vicinal haloalcohols to epoxides, as well as the reverse reaction, the enantioselective and regioselective nucleophilic ring opening of epoxides by pseudo-halides such as azide and cyanide. In the synthesis of enantioselective medicine HheC being a important instrument puts it into a high gear. For the efficient degradation, we select two mutants DhaA31 and HheC/W239P, which have higher activity.[18,22]

　　　　　　　　　　　　　　　　Fig.2 Degradation pathway of 1,2,3-Trichloropropane (TCP)

Strategy

2A peptide Sequence

In foot-and-mouth disease virus (FMDV) and some other picornaviruses, the oligopeptide (≈20 amino acids) 2A region of the polyprotein mediates "cleavage" at its own C-terminus to release it from the 2B region. 2A is also active when placed between reporter proteins and in all eukaryotic systems tested - it acts as an autonomous element, making it an important tool for co-ordinated synthesis of multiple proteins from one open reading frame and making a ribosome jumping. (Fig.3)In recent years, there was little report about 2A peptide that is active in prokaryotic systems, even some papers stated 2A sequences can’t work in prokaryote. But Indian Scientists Dechamma et al. have found the F2A peptide could work in E.coli[1], which gives us creative advice.

　　　　
Fig.3 the picture comes from http://viralzone.expasy.org/all_by_protein/914.html.

In our project, at first step we constructed that F2A peptide sequence linked gene ''LinA'' and ''LinB'', and P2A linked gene ''DhaA'' and ''HheC''. (Fig.4)

Fig.4

Polycistroinc expression

Modular bacterial polycistronic expression system allows co-expression and co-purification of multiple polypeptides in E. coli from a single expression plasmid. The system is comprised of the polycistronic expression vector and a transfer vector which facilitates subcloning of component genes into the polycistronic expression vector. Restriction sites present in the polycistronic expression vector allow both affinity tagged and untagged complexes to be overexpressed. In this project, 2A peptide working in E.coli is just a exploration without guaranty from sufficient evident. Polycistroinc expression is the next way to achieve multi-enzyme co-expression. </br>

Fig.5.1 Fig.5.2

Reference(Click)