Thioesterase from Bacteroides thetaiotaomicron
Acyl-ACP thioesterase BT_2075
Usage and Biology
This part contains a promoter BBa_J23119, the RBS* BBa_K2924009, the coding region of the Acyl-[acyl-carrier-protein] hydrolase from Bacteroides thetaiotaomicron BBa_K2924003 and a double terminator BBa_B0015.
Fatty acids are long aliphatic chained carboxylic acids, which can be saturated or unsaturated. They have mostly an even number of carbon atoms from 4 to 28. Butyric acid (Fig. 1) is a straight-chained saturated 4:0 fatty acid. It is a oily and colorless liquid and has a unpleasant, rancid odor 1. It has a molecular mass of 88.11 g/mol.
Butyric acid is found in animal fat and plant oils 1, bovine milk, butter, cheese, and human breast milk 2.
Fatty acids are synthesized and elongated by fatty acid synthases, which is a complex containing multiple enzymes. The determination of its length is controlled by thioesterases, a subgroup of hydrolases. They hydrolyze acetyl-CoA esters or acyl carrier protein esters to the corresponding free fatty acid and the Coenzyme A or the acyl carrier protein.
The Acyl-[acyl-carrier-protein] thioesterase of Bacteroides thetaiotaomicron has been tested to show catalytic activities in producing a high amount of 4:0 fatty acid 3, 4. B. thetaiotaomicron is a naturally gut bacteria of the human intestine and colon 5 and is using undigested dietary polysaccharides as main fermentable source for the short-chain fatty acids 6.
Use of butyric acid
Butyric acid has a broad range of usage in chemical and fuel industries 4, 7. In manufacture, it is used as artificial flavoring ingredient for candies, certain liquors or syrups 1.
By engineering metabolic pathways to produce designer fatty acids with the correct amount of carbons in the chain, such fatty acids could be used directly for chemicals or fuels with less processing 8 and less usage of fossil resources. Therefore, it is a step towards a environmental friendly alternative 9.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
The part was cloned via Gibson Cloning into the pSHDY plasmid. The pSHDY plasmid is an RSF1010-based, low-copy self-replicating vector derived from pVZ321 and has a broad host range, which can ensure the conjugation from Escherichia coli to cyanobacteria and other microorganisms.
Unfortunately, it was not possible to test E. coli transformants or the Synechocystis conjugants with gas chromatography-mass spectrometry (GC-MS) due to the unavailability of a method for short-chain fatty acid detection. This experiments can be conducted as soon as a suitable method is created.
: National Center for Biotechnology Information. PubChem Database. Butyric acid, CID=264, https://pubchem.ncbi.nlm.nih.gov/compound/Butyric-acid (accessed on Sept. 20, 2019)
: McNabney, Sean M., and Tara M. Henagan. "Short chain fatty acids in the colon and peripheral tissues: a focus on butyrate, colon cancer, obesity and insulin resistance." Nutrients 9.12 (2017): 1348.
:Jing, Fuyuan, et al. "Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity." BMC biochemistry 12.1 (2011): 44.
: Liu, Xiping, et al. "Biosynthesis of butenoic acid through fatty acid biosynthesis pathway in Escherichia coli." Applied microbiology and biotechnology 99.4 (2015): 1795-1804.
: Xu, Jian, et al. "A genomic view of the human-Bacteroides thetaiotaomicron symbiosis." Science 299.5615 (2003): 2074-2076.
: Rios-Covian, David, et al. "Shaping the metabolism of intestinal Bacteroides population through diet to improve human health." Frontiers in microbiology 8 (2017): 376.
: Jawed, Kamran, et al. "Engineered production of short chain fatty acid in Escherichia coli using fatty acid synthesis pathway." PloS one 11.7 (2016): e0160035.
: Ziesack, Marika, et al. "Chimeric fatty acyl-acyl carrier protein thioesterases provide mechanistic insight into enzyme specificity and expression." Appl. Environ. Microbiol. 84.10 (2018): e02868-17.
: Baroi, G. N., et al. "Butyric acid fermentation from pretreated and hydrolysed wheat straw by an adapted Clostridium tyrobutyricum strain." Microbial biotechnology 8.5 (2015): 874-882.