Team:NCTU Formosa/BP design

Butanol pathway

Motivation

Global energy and environmental problems have stimulated increasing efforts towards synthesizing biofuel from renewable resources. Compared to the traditional biofuel, ethanol, higher alcohols offer advantages as gasoline substitutes because of their higher energy density and lower hygroscopicity. ₍₁₎ The biggest benefits butanol offers as an alternative to ethanol in motor fuel derive directly from its chemical and physical properties. The vapor pressure of butanol is well below that of ethanol at room temperature. That means a gasoline-butanol blend would not suffer from the volatility concerns that plague gasoline-ethanol blends, thus reducing evaporative emissions from vehicles using it. Importantly, butanol is much less soluble in water than ethanol. That means it is much less prone to the kind of separation problems that have kept ethanol out of petroleum product pipelines. It also suggests it should be easier to separate from water after fermentation than ethanol, requiring less energy. Overall, butanol raises some fascinating possibilities for our alternate fuels strategy. ₍₂₎

Reference﹝1﹞: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. Reference﹝2﹞: Geoffrey Styles (2006) Higher Alcohols http://energyoutlook.blogspot.com/2006/08/higher-alcohols.html

Design

In traditional genetic engineering method(Figure 1), we use strong promoter to initiate our genes, however there are several disadvantages in this method. Firstly, E.coli will overexpress the target proteins which we need in synthetic pathway. However, this over-expression will cause E.coli wastes its limited growth resources. Then on the other hand, the activity and performance of the enzymes could be too low to produce enough product in the end. Therefore, it will unbalances the synthetic pathway, and the production of isobutanol will not be optimum. In addition, this is also a major 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. (the reaction pathway is shown in the picture of Overall pathway) 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.

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.) Following Figure 2 is about the strains where the alss, ilvC, ilvD and kivd genes are cloned from, along with their point mutation sites:

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). And we combine our low-temperature release device with butanol pathway because isobutanol will easily vaporize in high temperature, and the specific enzyme involved has better activity at 30℃.

In circuit A (BBa_K539691 ),Plac (BBa_R0010) is lacI regulated promoter to initiate the circuit to produce Alss, IlvC and IlvD. Gene alss, ilvC and ilvD will be translated into enzymes and catalyze pyruvate into 2-Ketoisovalerate. And the 37℃induced RBS we use is BBa_K115002 . The terminator is BBa_J61048. The host strain is DH5α. This circuit inhibits part BBa_K539742 (Figure 4) by producing TetR which is regulated by 37℃ RBS at 37℃ or higher.

In circuit B (BBa_K539742 ), Ptet BBa_R0040 is TetR repressible promoter, and the RBS we use is BBa_B0034 . Kivd (BBa_K539714 ) encodes 2-keto-acid decarboxylases which convert 2-Ketoisovalerate into aldehyde and then to isobutanol by alcohol dehydrogenases. Alcohol dehydrogenases are produced by E.coli itself naturally. The terminator is BBa_B0015.

By viewing our two circuits (Figure 3 and Figure 4) we could observe one circumstance that when the temperature reaches 37℃, tetR will be expressed and TetR will inhibit ptet (Figure 5). In this way, part BBa_K539742 will not be expressed, and if we keep the temperature over 37℃, we can accumulate the intermediate, 2-Ketoisovalerate, which is non-toxic for E.Coli. In the other hand, when we lower the temperature under 37℃, the 37℃ induced RBS (BBa_K115002) will not express so that part BBa_K539742 will not be inhibited by TetR (Figure 6). At the same time, KivD will be produced and start to catalyze 2-Ketoisovalerate into isobutyraldehyde and to isobutanol by alcohol dehydrogenases which is produced by E.coli naturally (the enzyme is not shown in Overall Reaction). The reason why we use low-temperature released device is because isobutanol is easy to evaporate and the enzyme which catalyze isobutyraldehyde to isobutanol has better activity in lower temperature, 30℃.