Part:BBa_K1696003
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 3. Lactate metabolic pathway. The pflB and poxB knocking out and ldhE insertion will result in the accumulation of lactate.
pflB knockout
PFL is a crucial enzyme in the glucose metabolism under anaerobic condition, which can catalyze one molecule of pyruvate to one molecule of formicate and one molecule of acetylcoenzyme A (AcCoA). According to metabolic pathway, pyruvate is consumed both in the PFL and LDH reactions. In the wild type E. coli, LDH reaction is not as competitive as the reaction through PFL, and therefore, knockout of the PFL related genes will contribute to redistribute the metabolic flux. When the PFL pathway is blocked, E. coli will conceivably alter the distribution of these products to overcome the imbalanced reducing equivalents caused by the pathway knockout. Therefore, knockout of pflB not only decreased the carbon flow to acetyl-CoA under anaerobic condition but also reduced the anaerobic consumption of NADH through reductive TCA.
After the knockout of pflB, though the growth rate was slightly slowed down, the yield of lactate of engineered strain was improved greatly and showed a significant difference from the wild type MG1655. From the result, we can see our part functioned as expected.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 36
Illegal BsaI.rc site found at 1058
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