Mortimer Cara L, Misawa Norihiko, Ducreux Laurence, Campbell Raymond, Bramley Peter M, Taylor Mark, Fraser Paul D
Centre for Systems and Synthetic Biology, School of Biological Sciences Royal Holloway University of London, Egham, Surrey, UK.
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi-machi, Ishikawa, Japan.
Plant Biotechnol J. 2016 Jan;14(1):140-52. doi: 10.1111/pbi.12365. Epub 2015 Apr 2.
To produce commercially valuable ketocarotenoids in Solanum tuberosum, the 4, 4' β-oxygenase (crtW) and 3, 3' β-hydroxylase (crtZ) genes from Brevundimonas spp. have been expressed in the plant host under constitutive transcriptional control. The CRTW and CRTZ enzymes are capable of modifying endogenous plant carotenoids to form a range of hydroxylated and ketolated derivatives. The host (cv. Désirée) produced significant levels of nonendogenous carotenoid products in all tissues, but at the apparent expense of the economically critical metabolite, starch. Carotenoid levels increased in both wild-type and transgenic tubers following cold storage; however, stability during heat processing varied between compounds. Subcellular fractionation of leaf tissues revealed the presence of ketocarotenoids in thylakoid membranes, but not predominantly in the photosynthetic complexes. A dramatic increase in the carotenoid content of plastoglobuli was determined. These findings were corroborated by microscopic analysis of chloroplasts. In tuber tissues, esterified carotenoids, representing 13% of the total pigment found in wild-type extracts, were sequestered in plastoglobuli. In the transgenic tubers, this proportion increased to 45%, with esterified nonendogenous carotenoids in place of endogenous compounds. Conversely, nonesterified carotenoids in both wild-type and transgenic tuber tissues were associated with amyloplast membranes and starch granules.
为了在马铃薯中生产具有商业价值的酮类胡萝卜素，来自短波单胞菌属的4,4'-β-加氧酶（crtW）和3,3'-β-羟化酶（crtZ）基因已在组成型转录控制下在植物宿主中表达。CRTW和CRTZ酶能够修饰植物内源性类胡萝卜素，形成一系列羟基化和酮化衍生物。宿主（品种Desirée）在所有组织中都产生了大量非内源性类胡萝卜素产物，但显然是以经济上至关重要的代谢物淀粉为代价的。冷藏后，野生型和转基因块茎中的类胡萝卜素水平均有所增加；然而，不同化合物在热加工过程中的稳定性有所不同。叶片组织的亚细胞分级分离显示，类胡萝卜素存在于类囊体膜中，但主要不存在于光合复合物中。确定了质体小球的类胡萝卜素含量急剧增加。这些发现通过叶绿体的显微镜分析得到了证实。在块茎组织中，酯化类胡萝卜素占野生型提取物中总色素的13%，被隔离在质体小球中。在转基因块茎中，这一比例增加到45%，酯化非内源性类胡萝卜素取代了内源性化合物。相反，野生型和转基因块茎组织中的非酯化类胡萝卜素与造粉体膜和淀粉颗粒有关。
