Homologous gene targeting of a carotenoids biosynthetic gene in Rhodosporidium toruloides by Agrobacterium-mediated transformation
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To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method.
The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11.
Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.
KeywordsAgrobacterium-mediated transformation Carotenoids Homologous gene targeting Rhodosporidium toruloides
This work was supported by the National Natural Science Foundation of China (21325627, 31370128).
Supplementary Table 1—Strains and plasmids used.
Supplementary Table 2—Primers used.
Sequence data—(a) The sequences of ADH2 (RHTO_03062) promoter region.
(b) The sequences of GPD (RHTO_3746) promoter region.
(c) The sequences of the RHTO_04602 locus.
(d) Homologous arm amplification sequences:CRTI-3225-R and CRTI-5695-R.
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