Difference between revisions of "Part:BBa K3036001"

Revision as of 02:36, 12 October 2019

Acetokinase

This part encodes encodes acetokinase (ACK) of E. coli. The natural function of ACK is to convert acetate into acetyl phosphate. However, the reversibility of the reaction confers ACK a potential to produce acetic acid using acetyl phosphate as a precursor. Our team made use of this part, together with phosphotransacetylase (PTA), to overproduce acetic acid.

Sequence and Features

Characterized by BNU-China 2019

Biology and Usage

Derived from E. coli DH5alpha genome, the gene ack encodes acetokinase (ACK), of which the natural function is to reversibly convert acetate into acetyl phosphate.

However, the reversibility of the reaction confers ACK a potential to produce acetic acid using acetyl phosphate as a precursor. Together with phosphotransacetylase (PTA), which produces acetyl phosphate out of acetyl-CoA, ACK notably enhances yield of acetate through a two-step enzymatic reaction[1]. In our project, we take advantage of the pathway to overexpress acetate using an intestinal microbe inside human intestine, where acetate signals consumption of human white fat tissue.

Properties

The function of this part is validated in an acetate-overproducing system, where ACK and PTA are coexpressed in a polycistron system under control of a Plac promoter. Using Megazyme acetic acid assay kit, we verified that the yield of acetate is enhanced by a remarkable 9 fold, as is shown in Fig. 1.

This overproduction can be further enhanced by coexpressing enzymes involved in acetyl-CoA-yielding processes, such as fatty acyl-CoA synthetase (FadD), key enzyme in beta-oxidation of higher fatty acids (Fig. 2).

Experimental approach

1.Transfer the plasmid into E. coli competent cells.
2.Culture the strain in LB-ampicillin (50 ng/μL) at 37℃ for 5 hours, use a strain containing pUC19 as control group.
3.Induce both groups by addition of IPTG to a final concentration of 5 mM.
4.Keep culturing at 37℃ and take samples at 0 hr, 2 hr and 4 hr after induction.
5.Measure acetate content using Megazyme acetic acid assay kit.
6.Three replicas are tested in each group.

Reference

[1] Kakuda H, Shiroishi K, Hosono K, Ichihara S. Construction of Pta-Ack Pathway Deletion Mutants of Escherichia coli and Characteristic Growth Profiles of the Mutants in a Rich Medium. Biotech. Biochem., 58 (12), 2232~2235, 1994.

Sequence and Features