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The impact of model-assisted engineering was also tested for the production of long chain fatty alcohol, another commercially important molecule sharing the same pathway while differing only at the terminal reaction, and a titer of 1506 mg/L was achieved with a yield of 86.4% of the theoretical maximum.
Sequences were obtained through 3700 DNA Analyzer using Big Dye v3.1 terminal reaction (Applied Biosystem).
SO catalyses the oxidation of sulfite to sulfate, the terminal reaction in the catabolism of the sulfur-containing amino acids cysteine and methionine in mammals.
In addition, acyl-ACP thioesterase, catalyzing the terminal reaction to produce free FAs, is crucial in controlling metabolic flux towards FA.
Cystathionine γ-lyase (CGL) (EC4.4.1.1) is a pyridoxal-5′-phosphate-dependent enzyme that catalyzes the γ-cleavage of cystathionine [2-amino-4- 2-amino-2-carboxy-ethyl [2-amino-4- 2-amino-2-carboxy-ethyl [2-amino-4- 2-amino-2-carboxy-ethyl [2-amino-4- 2-amino-2-carboxy-ethyl [2-amino-4- 2-amino-2-carboxy-ethyl [2-amino-4- 2-amino-2-carboxy-ethyl [2-amino-4- 2-amino-2-carboxy-ethyl [2-amino-4- 2-amino-2-carboxy-ethyl
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Interestingly, two lines of observations suggest that these 5 terminal reactions are at a key position in the overall metabolism of the cell.
Surprisingly, rather than occurring in functions related to DNA metabolism, they fell into four metabolic genes (pgk, pgm, eno and pykA) ensuring the terminal reactions of glycolysis (Fig. 1A, the suppressed strains and metabolic mutations are listed Table 2).
In the case of metabolism, full viability is restored only by mutations in the 5 successive terminal reactions of the three-carbon part of glycolysis/gluconeogenesis out of 21 mutations leading to inactivity or absence of enzymes of the central carbon metabolism.
In other bacteria, ArgG and ArgH catalyze the two terminal reactions in the biosynthesis of arginine.
Two of the genes are HTGs: dapF and lysA, which complement the missing genes coding the two terminal reactions in lysine synthesis (fig. 1 A, table 1).
Only one product was present in each reaction as indicated by the SYBR Green dissociation curves of amplified products and by assay of terminal reactions by gel electrophoresis in 1% TBE agarose.
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