Laboratory of Systems and Synthetic Biology, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands.
International Research Center in Critical Raw Materials-ICCRAM, Advanced Materials, Nuclear Technology and Applied Bio/Nanotechnology, University of Burgos, Plaza Misael Bañuelos s/n, 09001 Burgos, Spain.
FEMS Microbiol Lett. 2019 Apr 1;366(7). doi: 10.1093/femsle/fnz071.
Currently, there is no consensus regarding the mechanism underlying Aspergillus niger citrate biosynthesis and secretion. We hypothesise that depending on the experimental setup, extracellular citrate accumulation can have fundamentally different underlying transcriptomic landscapes. We show that varying the amount and type of supplement of an arginine auxotrophic A. niger strain results in transcriptional down-regulation of citrate metabolising enzymes in the condition in which more citrate is accumulated extracellularly. This contrasts with the transcriptional adaptations when increased citrate production is triggered by iron limitation. By combining gene expression data obtained from these two very distinct experimental setups with hidden Markov models and transporter homology approaches, we were able to compile a shortlist of the most likely citrate transporter candidates. Two candidates (An17g01710 and An09g06720m.01) were heterologously expressed in the yeast Saccharomyces cerevisiae, and one of the resultant mutants showed the ability to secrete citrate. Our findings provide steps in untangling the complex interplay of different mechanisms underlying A. niger citrate accumulation, and we demonstrate how a comparative transcriptomics approach complemented with further bioinformatics analyses can be used to pinpoint a fungal citrate exporter.
目前，对于黑曲霉柠檬酸生物合成和分泌的机制尚未达成共识。我们假设，根据实验设置的不同，细胞外柠檬酸的积累可能具有根本不同的潜在转录组景观。我们表明，改变精氨酸营养缺陷型黑曲霉菌株的补充量和类型会导致在细胞外积累更多柠檬酸的条件下，柠檬酸代谢酶的转录下调。这与铁限制触发柠檬酸产量增加时的转录适应形成对比。通过将这两种非常不同的实验设置中获得的基因表达数据与隐马尔可夫模型和转运蛋白同源性方法相结合，我们能够编制出最有可能的柠檬酸转运蛋白候选名单。两个候选物（An17g01710 和 An09g06720m.01）在酵母酿酒酵母中异源表达，其中一个突变体显示出分泌柠檬酸的能力。我们的研究结果为阐明黑曲霉柠檬酸积累的不同机制之间的复杂相互作用提供了步骤，我们展示了如何使用比较转录组学方法辅以进一步的生物信息学分析来确定真菌柠檬酸外排泵。
