Part:BBa_K539742

TetR repressible promoter (BBa_R0040)+ kivd (alpha-ketoisovalerate decarboxylase)+Ter (BBa_B0015)

Please refer to the wiki for our overall concept:[http://2011.igem.org/Team:NCTU_Formosa/BP_design 2011 NCTU_FORMOSA]

In traditional genetic engineering method(Figure 1), we use strong promoter to initiate our genes, but this way E.coli will overexpress the proteins we need in synthetic pathway. However, this overexpression of target proteins will cause E.coli wastes its limited growth resources, or the activity and performance of the enzymes may be too low. In this situation, it unbalances the synthetic pathway, and the production of isobutanol will not be optimum. This is also a problem in the production of isobutanol which is poisonous to E.coli.

To solve the problem, we need to adjust the expression of the genes, and make sure every intermediate can be catalyzed by the very next enzyme. The intermediates of isobutanol can be catalyzed step by step till they become the target products we want. In the new method we design, we control the pathway by stopping the mechanism when it reaches the step to produce non-toxic intermediate (2-Ketoisovalerate ) which we want to accumulate, then under specific thermal control, the mechanism would continue to express. The advantage of our new method is that the precursors are much less toxic for E.coli than our target product (isobutanol) is. We then Apply this new method to our project. We first accumulate lots of the non-toxic intermediate as the precursor, 2-Ketoisovalerate, to a certain amount, and then convert the entire non-toxic precursor into the product, isobutanol, all at once.

In order to achieve the goal of our experimental design, we apply carbohydrate fermentation to gain our product. First, we choose glucose as the resource to get through biosynthetic pathway, and we can harvest isobutanol which is the derivative of butanol. Glucose can be catalyzed into isobutanol through afterward enzymes- Alss, Ilvc, Ilvd,and Kivd step by step. We can also stop any step to accumulate the intermediates we want (see picture Overall Reaction).

Figure.1 Overall conception of isobutanol synthesis pathway

We clone the genes which will be translated into enzymes such as AlsS, IlvC, IlvD ,KivD, and assemble the genes into two circuits as following. The enzymes are crucial for producing butanol.(Reference: Atsumi, S.; T. Hanai and J.C. Liao (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels, Nature, 451:86-89.)

Circuit 2

The part is built in kivd and TetR repressible promoter that constitutively ON and repressed by tetR. And in the end, terminatorPart:BBa_B0015 is included. The coding sequence of kivd is from Lactococcus lactis subsp. It encodes alpha-ketoisovalerate decarboxylase which is responsible for the decarboxylation into aldehydes of α-keto acids derived from amino acid transamination. This is part of the isobutanol biosynthesis pathway.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 658
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]