Difference between revisions of "Part:BBa K1972005"

Latest revision as of 01:42, 27 October 2016

dszB (a desulfinase gene), E.coli-optimized

DszB is a a desulfinase which can convert 2-(2-hydroxybiphenyl) sulfinate (HBPS) to 2-hydroxybiphenyl (2HBP) during the 4S pathway, in which dibenzothiophene (DBT) undergoes three successive oxidation steps and one a hydrolytic step leading to the formation of 2-hydroxybiphenyl (2HBP)

This single monooxygenase gene was synthesized whith E.coli codon optimized by Generay and the sequence was found in NCBI (GenBank Accession number L37363.1)

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 267
Illegal AgeI site found at 1104
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 864

We constructed this single enzyme with other three dsz enzymes which are also E.coli codon optimized. 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.

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-We constructed this single enzyme with other three dsz enzymes.
+We constructed this single enzyme with other three dsz enzymes which are also E.coli codon optimized.
 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.