LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, F-31077 Toulouse, France; TWB, 3 rue Ariane, 31520 Ramonville-St. Agne, France.
LISBP, Université de Toulouse, CNRS, INRA, INSA, 135 Avenue de Rangueil, F-31077 Toulouse, France; TWB, 3 rue Ariane, 31520 Ramonville-St. Agne, France.
Metab Eng. 2018 Jan;45:237-245. doi: 10.1016/j.ymben.2017.12.005. Epub 2017 Dec 14.
2,4-dihydroxybutyrate (DHB) is a precursor for the chemical synthesis of the methionine analogue 2-hydroxy-4-(methylthio)butyrate. Since no annotated metabolic pathway exists for its microbial production from sugar, we have conceived a two-step synthetic metabolic pathway which converts the natural amino acid homoserine to DHB. The pathway proceeds through the homoserine transaminase-catalyzed deamination of homoserine to obtain 2-oxo-4-hydroxybutyrate (OHB), and continues with the reduction of OHB to DHB, which is catalyzed by an OHB reductase enzyme. We identified homoserine transaminase and OHB reductase activity in several candidate enzymes which act on sterically cognate substrates, and improved OHB reductase activity of lactate dehydrogenase A of Lactococcus lactis by structure-based enzyme engineering. Fed-batch cultivation of a homoserine-overproducing Escherichia coli strain which expressed homoserine transaminase and OHB reductase enzymes resulted in the production of 5.3g/L DHB at a yield of 0.1g/g.
2,4-二羟基丁酸（DHB）是一种用于化学合成蛋氨酸类似物 2-羟基-4-（甲硫基）丁酸的前体。由于微生物从糖中生产 DHB 没有注释的代谢途径，我们设想了一种两步合成代谢途径，该途径将天然氨基酸高丝氨酸转化为 DHB。该途径通过高丝氨酸转氨酶催化的高丝氨酸脱氨作用获得 2-氧代-4-羟基丁酸（OHB），然后通过 OHB 还原酶催化 OHB 还原为 DHB。我们在几种候选酶中鉴定出了高丝氨酸转氨酶和 OHB 还原酶活性，这些酶作用于空间匹配的底物上，并通过基于结构的酶工程提高了乳酸乳球菌乳酸脱氢酶 A 的 OHB 还原酶活性。表达高丝氨酸转氨酶和 OHB 还原酶的高丝氨酸过量生产大肠杆菌菌株的分批补料培养导致 5.3g/L DHB 的产量为 0.1g/g。
