背景:非常规酵母Rhodotorulatoruloides是生物技术中新兴的宿主生物体,因为其具有从多种碳源积累高水平的类胡萝卜素和细胞内储存脂质的天然能力。虽然使用R.toruloides的基因工程策略的数量正在增加,缺乏可用于修饰这种酵母的遗传工具仍然限制了菌株的发展。例如,几强,组成型R.toluloides启动子已经被表征,但迄今为止,仅鉴定了五个诱导型启动子。尽管氮限制培养条件通常用于诱导这种酵母中的脂质积累,对于R.toruloides,没有描述由氮饥饿调节的启动子。
结果:在这项研究中,我们使用基因组学和转录组学相结合的方法来鉴定可被氮饥饿诱导或抑制的新型R.toruloides启动子序列。RNA测序用于评估最近分离的R.toruloidesBOT-A2菌株在指数生长和氮饥饿期间的基因表达,当用葡萄糖或木糖作为碳源培养时。使用长和短读取测序的组合对BOT-A2的基因组进行测序,并在RNAseq数据的支持下进行注释。当比较氮消耗过程中的表达与指数生长过程中的表达时,差异表达分析用于鉴定具有|log2倍数变化|≥2的基因。评估来自这些基因中的16个的启动子区驱动荧光报告基因表达的能力。选择在氮饥饿下明显上调的三个启动子和下调的三个启动子并进一步表征。一个发起人,来自基因RTBOTA2_003877,被发现功能像一个开关,因为它仅在完全氮耗尽的情况下上调,而在氮源存在的情况下下调。
结论:确定了六个新的在氮饥饿下上调或下调的R.toluloides启动子。当工程化该生物体时,这些基本上有助于可用的启动子,并且被预见对于需要根据氮可用性特异性调节靶基因的未来工程化策略特别有用。
BACKGROUND: The non-conventional yeast Rhodotorula toruloides is an emerging host organism in biotechnology by merit of its natural capacity to accumulate high levels of carotenoids and intracellular storage lipids from a variety of carbon sources. While the number of genetic engineering strategies that employ R. toruloides is increasing, the lack of genetic tools available for modification of this yeast is still limiting strain development. For instance, several strong, constitutive R. toruloides promoters have been characterized, but to date, only five inducible promoters have been identified. Although nitrogen-limited cultivation conditions are commonly used to induce lipid accumulation in this yeast, no promoters regulated by nitrogen starvation have been described for R. toruloides.
RESULTS: In this study, we used a combination of genomics and transcriptomics methods to identify novel R. toruloides promoter sequences that are either inducible or repressible by nitrogen starvation. RNA sequencing was used to assess gene expression in the recently isolated strain R. toruloides BOT-A2 during exponential growth and during nitrogen starvation, when cultivated with either glucose or xylose as the carbon source. The genome of BOT-A2 was sequenced using a combination of long- and short-read sequencing and annotated with support of the RNAseq data. Differential expression analysis was used to identify genes with a |log2 fold change|≥ 2 when comparing their expression during nitrogen depletion to that during exponential growth. The promoter regions from 16 of these genes were evaluated for their ability to drive the expression of a fluorescent reporter gene. Three promoters that were clearly upregulated under nitrogen starvation and three that were downregulated were selected and further characterized. One promoter, derived from gene RTBOTA2_003877, was found to function like an on-off switch, as it was only upregulated under full nitrogen depletion and downregulated in the presence of the nitrogen source.
CONCLUSIONS: Six new R. toruloides promoters that were either upregulated or downregulated under nitrogen-starvation were identified. These substantially contribute to the available promoters when engineering this organism and are foreseen to be particularly useful for future engineering strategies requiring specific regulation of target genes in accordance with nitrogen availability.