Difference between revisions of "Part:BBa K1972013"

Latest revision as of 03:16, 29 October 2016

dszBAC with tac promoter and dszD with lac promoter

We constructed dszBCA genes with tac promoter and dszD gene with lac promoter choose E.coli BL21 as our host cell. When IPTG is added to our system, DszABCD enzymes will be successful expressed and these four enzymes convert dibenzothiophene (DBT) undergoes through three successive oxidation steps and one a hydrolytic step to 2-hydroxybiphenyl (2HBP) by the 4s pathway.

Each single enzyme gene was synthesized by Generay with E.coli optimized and we constructed them together and suquencing comformed. the sequences can be found in NCBI (GenBank Accession number L37363.1)

Sequence and Features

The DszB desulfurase catalyzes the rate-limiting step of the 4S-pathway and the Y63F amino acid substitution was previously reported to enhance its activity and stability. Therefore, the Y63F amino acid substitution of the DszB desulfurase was performed.

Figure 1. Bio-circuit of BBa_K1972013 after our third optimization

Furthermore, the desulfurization experiment showed that the activities of enzyme C and A were still stronger than B, which caused undesirable accumulation of intermediate products (DBTO2/HBPS), seriously affecting the activity of enzyme B. The promoters of dsz genes were further adjusted. Gene dszB, dszC and dszA were controlled by the tac promoter, which was strong enough and endogenous for E.coli. At the same time, dszD was under the weak lac promoter as an independent operon, making the expression of enzyme D relatively weak, with the purpose of indirectly attenuating the effect of the enzyme A and C (as shown in Figure 1).

Figure 2. The desulfurization results of Recombinant strain BL21-dszBACD (optimized) tested by HPLC

The desulfurization efficiency of the recombinant strain BL21-dszBACD (optimized) is greatly improved.

Reference

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