Difference between revisions of "Part:BBa K1696003"

Revision as of 14:47, 16 September 2015

L-lactate producing part1

L-Lactic acid, one of the most important chiral molecules and organic acids, is produced via pyruvate from carbohydrates in diverse microorganisms catalyzed by an NAD+-dependent L-lactate dehydrogenase (ldhA).[1]LDH is an enzyme found in nearly all living cells (animals, plants, and prokaryotes), which catalyzes the conversion of pyruvate to lactate with NADH serving as the coenzyme. In our project, we intend to introduce high-yield exogenous L-(+)-lactate dehydrogenase gene (ldhA) from Lactobacillus, which can produce a relatively larger amount of L-lactate. Similar metabolic pathway has been found in a variety of organisms, to distinguish the differences, we renamed the heterogenous L-lactate dehydrogenase ldhE while homogenous one keeps original.

We choose a strong promoter+ strong RBS to construct L-Lactate part. The NAD+-dependent L-lactate dehydrogenase gene is from Lactobacillus casei.

Fig.1 Design of the part.

But we found the yield was no different between BL21(blank) and BL21+ldhE(experientmental). Shown in Fig2.

Fig2. The lactate yield of BL21 and BL21+ldhE in flask-shaking fermentation condition (LB medium).

In order to enhance the transformation efficiency from glucose to lactate, as well as improve the characterization of our ldhE part, we decide to knockout the the pflB and poxB.

Figure 6. Lactate metabolic pathway. The pflB and poxB knocking out and ldhE insertion will result in the accumulation of lactate.

Sequence and Features