Biomaterials and Biocatalysts Group, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore, 117604, Singapore.
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore.
BMC Microbiol. 2018 Feb 21;18(1):14. doi: 10.1186/s12866-018-1151-6.
Rhodotorula toruloides is an outstanding producer of lipids and carotenoids. Currently, information on the key metabolic pathways and their molecular basis of regulation remains scarce, severely limiting efforts to engineer it as an industrial host.
We have adapted Agrobacterium tumefaciens-mediated transformation (ATMT) as a gene-tagging tool for the identification of novel genes in R. toruloides. Multiple factors affecting transformation efficiency in several species in the Pucciniomycotina subphylum were optimized. The Agrobacterium transfer DNA (T-DNA) showed predominantly single-copy chromosomal integrations in R. toruloides, which were trackable by high efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). To demonstrate the application of random T-DNA insertions for strain improvement and gene hunting, 3 T-DNA insertional libraries were screened against cerulenin, nile red and tetrazolium violet respectively, resulting in the identification of 22 mutants with obvious phenotypes in fatty acid or lipid metabolism. Similarly, 5 carotenoid biosynthetic mutants were obtained through visual screening of the transformants. To further validate the gene tagging strategy, one of the carotenoid production mutants, RAM5, was analyzed in detail. The mutant had a T-DNA inserted at the putative phytoene desaturase gene CAR1. Deletion of CAR1 by homologous recombination led to a phenotype similar to RAM5 and it could be genetically complemented by re-introduction of the wild-type CAR1 genome sequence.
T-DNA insertional mutagenesis is an efficient forward genetic tool for gene discovery in R. toruloides and related oleaginous yeast species. It is also valuable for metabolic engineering in these hosts. Further analysis of the 27 mutants identified in this study should augment our knowledge of the lipid and carotenoid biosynthesis, which may be exploited for oil and isoprenoid metabolic engineering.
罗氏酵母（Rhodotorula toruloides）是一种出色的脂类和类胡萝卜素生产菌。目前，关于关键代谢途径及其分子调控基础的信息仍然匮乏，这严重限制了将其作为工业宿主进行工程改造的努力。
我们已经将根癌农杆菌介导的转化（ATMT）作为一种基因标记工具，用于鉴定罗氏酵母中的新基因。优化了多个影响 Pucciniomycotina 亚门下几个物种转化效率的因素。根癌农杆菌转移 DNA（T-DNA）在罗氏酵母中主要表现为单拷贝染色体整合，可通过高效热不对称交错 PCR（hiTAIL-PCR）进行跟踪。为了证明随机 T-DNA 插入在菌株改良和基因挖掘中的应用，我们分别针对 cerulenin、nile red 和 tetrazolium violet 筛选了 3 个 T-DNA 插入文库，结果鉴定了 22 个在脂肪酸或脂质代谢中表现出明显表型的突变体。同样，通过转化体的目视筛选获得了 5 个类胡萝卜素生物合成突变体。为了进一步验证基因标记策略，我们详细分析了一个类胡萝卜素生产突变体 RAM5。该突变体在假定的八氢番茄红素脱氢酶基因 CAR1 处插入了一个 T-DNA。通过同源重组缺失 CAR1 导致与 RAM5 相似的表型，并且可以通过引入野生型 CAR1 基因组序列进行遗传互补。
T-DNA 插入诱变是罗氏酵母和相关油脂酵母物种中基因发现的有效正向遗传学工具。它对于这些宿主的代谢工程也很有价值。对本研究中鉴定的 27 个突变体的进一步分析应增加我们对脂类和类胡萝卜素生物合成的了解，这可能被用于油脂和异戊二烯代谢工程。
