León Rosa, Couso Inmaculada, Fernández Emilio
Departamento de Química y Ciencia de Materiales, Facultad de Ciencias Experimentales, Avda. Fuerzas Armadas s/n, Universidad de Huelva, 21007 Huelva, Spain.
J Biotechnol. 2007 Jun 15;130(2):143-52. doi: 10.1016/j.jbiotec.2007.03.005. Epub 2007 Mar 12.
Most higher plants and microalgae are not able to synthesize ketocarotenoids. In this study the unicellular chlorophyte Chlamydomonas reinhardtii has been genetically engineered with the beta-carotene ketolase cDNA from Haematococcus pluvialis, bkt1 (GeneBank accession no. X86782), involved in the synthesis of astaxanthin, to obtain a transgenic microalga able to synthesize ketocarotenoids. The expression of bkt1 was driven by the Chlamydomonas constitutive promoter of the rubisco small subunit (RbcS2) and the resulting protein was directed to the chloroplast by the Chlamydomonas transit peptide sequences of Rubisco small subunit (RbcS2) or Ferredoxin (Fd). In all transformants containing the bkt1 gene fused to the RbcS2 or the Fd transit peptides a new pigment with the typical ketocarotenoid spectrum was detected. Surprisingly this ketocarotenoid was not astaxanthin nor canthaxanthin. The ketocarotenoid was identified on the basis of its mass spectrum as 3,3'-dihydroxy-beta,epsilon-carotene-4-one (4-keto-lutein) or its isomer ketozeaxanthin.
大多数高等植物和微藻无法合成酮类胡萝卜素。在本研究中，单细胞绿藻莱茵衣藻已通过来自雨生红球藻的β-胡萝卜素酮酶cDNA（bkt1，基因库登录号：X86782）进行基因工程改造，该酶参与虾青素的合成，以获得一种能够合成酮类胡萝卜素的转基因微藻。bkt1的表达由莱茵衣藻核酮糖-1,5-二磷酸羧化酶小亚基（RbcS2）的组成型启动子驱动，所得蛋白质通过核酮糖-1,5-二磷酸羧化酶小亚基（RbcS2）或铁氧化还原蛋白（Fd）的莱茵衣藻转运肽序列定向到叶绿体。在所有含有与RbcS2或Fd转运肽融合的bkt1基因的转化体中，均检测到一种具有典型酮类胡萝卜素光谱的新色素。令人惊讶的是，这种酮类胡萝卜素既不是虾青素也不是角黄素。根据其质谱鉴定该酮类胡萝卜素为3,3'-二羟基-β,ε-胡萝卜素-4-酮（4-酮基叶黄素）或其异构体酮玉米黄质。
