Part:BBa_K1696011
Riboflavin producing parts
This part is based on the BBa_K1172303 in Part Registry constructed by 2013 Team Bielefelf-Germany which was also aimed at producing riboflavins.
Riboflavin (vitamin B2), which belongs to vitamin B group, is the universal precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). It is synthesized from one molecule of GTP and two molecules of ribulose 5-phosphate (Ru-5-P) through a seven-step synthetic pathway, which is similar in all organisms. [1] Riboflavin was synthesized and produced in a great amount for its remarkable applications in medicine, food and industrial process. Besides, the macroscopic yellow coloration and fluorescent property make it a perfect substrate for convenient detection.
Fig1.Pathway for engineered riboflavin and FMN production in E.coli.
As we learn from metabolic flux ( Fig.1), it reveals the relevant pathways of riboflavin production and engineering strategies for riboflavin production in E.coli. In their previous research(Tao, et al) [1] ,they construct a high-yield E.coli strain with a yield of 229.1 mg/L.
Based on their study, we constructed a flavin producing part (ribABDEC cluster) named EC10.(Figure 2).
Fig2. The design of this part.
The riboflavin biosynthesis genes ribABDEC from E. coli are overexpressed under the control of the inducible trc promoter. Enzymes encoded by the genes shown are: ribA, GTP cyclohydrolase II; ribB, 3,4-dihydroxy-2-butanone 4-phosphate synthase; ribD, fused diaminohydroxyphosphoribosylaminopyrimidine deaminase/ 5-amino-6-(5-phosphoribosylamino)uracil reductase; ribE, 6,7-dimethyl-8-ribityllumazine synthase; ribC, riboflavin synthase gene.
The RBS here is a weak syn-RBS to make the metabolic flux accumulate in riboflavin instead of FMN.
Fig3. The production of EC10 in tubes.
The part we designed, compared with BBa_K117230, has been well optimized and the yield of that reached 17 mg/L( as shown in Fig.4). We can see from the results, the functionality of their parts has successfully improved.
Fig4. The yield of riboflavin in different strains :EC10, Rf02S (Δpgi+EC10), EC10*(BBa_K1696010)
For riboflavin measurements, culture samples were diluted with 0.05 M NaOH to the linear range of the spectrophotometer and the A444 was immediately measured, according to Chen’s method[1].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3410
Illegal BamHI site found at 1370
Illegal XhoI site found at 64 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 262
Illegal AgeI site found at 992
Illegal AgeI site found at 1034
Illegal AgeI site found at 2752
Illegal AgeI site found at 3163 - 1000COMPATIBLE WITH RFC[1000]
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