Methionine is a proteinogenic amino acid critical amino for the pharmaceutical, biotechnological and agricultural industries. The overall methionine biosynthetic pathway is as follows: 1. Acetylation of homoserine and its subsequent activation 2. Formation of homoserine through the replacement hydroxyl group by a thiol group, 3. methyl group transfer to the thiol forming methionine. In bacteria this process can occur in two ways: through trans-sulfurylation or direct sulfurylation. Direct sulfurylation is a more efficient process and has been proven to yield more methionine, however the enzymes catalysing this reaction are not extensively described. MetX and MetY are two of these enzymes. The aim of this experimental work is to improve the methionine production in E. coli bacteria through the genetic and structural manipulation of Met X and Met Y. Hence this laboratory work was divided into three major components: screening system calibration, enzymatic kinetic assays and mutagenesis. Screening system calibration aimed to geneticall modify E.coli in order to later test the most efficient metionine production strain in M9 minimal media. Enzymatic assays were performed to quantitavely characterise methionine binding to either Acetyl-CoA or Homoserine through a DTNB reaction at 420nm. Lastly, mutagenesis was perfomed through an array of computational biology techniques such as Pymol, AlphaFold2 and PROSS to produce a table of mutations that in the future will be implemented using error-prone PCR.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:his-21605 |
| Date | January 2022 |
| Creators | Rodrigues, Luísa |
| Publisher | Högskolan i Skövde, Institutionen för biovetenskap |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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