Composition and production method of methionine
A technology of methionine and homoserine, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as unsuccessful production of methionine
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Embodiment 1
[0122] Example 1. One of the multiple pathways for the production of methionine, using an exogenously expressed nucleic acid sequence utilizing directed sulfhydrylation.
[0123] A. Construction of microorganisms with both metABC (transsulfuration) and metAZ (directed thiolation)
[0124] As mentioned previously, the endogenous product of methionine in E. coli is mainly produced by transsulfuration reactions. This example describes the engineering of E. coli to increase directed thiolation while also retaining the endogenous metAZ pathway.
[0125] Directed thiolation was increased by cloning O-succinylsulfhydrylase (EC 4.2.99.-), which converts O-succinyl homoserine to homocysteine by reaction with hydrogen sulfide. This enzyme is encoded by metZ and is found in some Pseudomonas species (Vermeij and Kertesz, J Bacteriol. 181:5833-5837, 1999 and Inoue et al., J. Bacteriol. 179:3956-3962 , 1997).
[0126] More specifically, metZ from Pseudomonas aeruginosa was cloned as a ...
Embodiment 3
[0142] Example 3 describes the determination of methionine production using the shake flask method. Briefly, cultures were grown for 50 hours at 30°C in medium supplemented with 150 mg / L methionine (to improve initial growth), and methionine was determined by HPLC. Table 2 shows that methionine production was higher in strains with both pathways compared to those with only transsulfuration or directed thiolation.
[0143] Table 2 Methionine production
[0144]
[0145] The synthesis of methionine in E. coli strains requires the genes metA and metB. When either gene was inactivated, E. coli lost the ability to produce methionine de novo. The data above indicate that addition of the metYX operon restores methionine production to levels similar to those obtained by methionine prototrophy. When cells gain access to both pathways, in some cases methionine production more than doubles. These results demonstrate that the two pathways are not mutually exclusive and that homoser...
Embodiment 2
[0151] Example 2. Homocysteine synthase utilizing O-acetyl-L-homoserine (OAHS) or O-succinyl-L-homoserine (OSHS)
[0152] This example describes the method used to isolate homocysteine synthase encoded by metY from Pseudomonas aeruginosa (ATCC 47085). This enzyme is inhibited by both L-methionine and S-adenosyl-L-methionine. Enzyme activity was analyzed according to Yamagata, Methods in Enzymology, 143:478,1987. A slight modification of the method used multiple sample points, guanidine was used to stop the reaction, and DTNB (5,5-dithiobis(2-nitrobenzoic acid) SigmaD8130) was used to detect homocysteine in the case of metA The generation of is described in Example 3.B. One enzyme unit (U) is defined as the production of one micromole of homocysteine per minute at room temperature.
[0153] MetY from Pseudomonas aeruginosa was expressed and analyzed using 17.5 μg of pure protein (N-labeled). In contrast to metY from Leptospira and most other published homocysteine ...
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