Umeno Daisuke, Arnold Frances H
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
J Bacteriol. 2004 Mar;186(5):1531-6. doi: 10.1128/JB.186.5.1531-1536.2004.
Using methods of laboratory evolution to force the C(30) carotenoid synthase CrtM to function as a C(40) synthase, followed by further mutagenesis at functionally important amino acid residues, we have discovered that synthase specificity is controlled at the second (rearrangement) step of the two-step reaction. We used this information to engineer CrtM variants that can synthesize previously unknown C(45) and C(50) carotenoid backbones (mono- and diisopentenylphytoenes) from the appropriate isoprenyldiphosphate precursors. With this ability to produce new backbones in Escherichia coli comes the potential to generate whole series of novel carotenoids by using carotenoid-modifying enzymes, including desaturases, cyclases, hydroxylases, and dioxygenases, from naturally occurring pathways.
我们运用实验室进化方法促使C(30)类胡萝卜素合酶CrtM发挥C(40)合酶的功能，随后在功能重要的氨基酸残基处进一步诱变，发现合酶特异性在两步反应的第二步（重排）中受到控制。我们利用这一信息构建了CrtM变体，这些变体能够从合适的异戊烯基二磷酸前体合成此前未知的C(45)和C(50)类胡萝卜素骨架（单异戊烯基和二异戊烯基八氢番茄红素）。凭借在大肠杆菌中产生新骨架的这种能力，利用天然途径中的类胡萝卜素修饰酶（包括去饱和酶、环化酶、羟化酶和双加氧酶）就有潜力生成一整套新型类胡萝卜素。
