Terfehr Dominik, Dahlmann Tim A, Kück Ulrich
Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-University Bochum, Universitätsstr. 150, Bochum, 44780, Germany.
BMC Genomics. 2017 Mar 31;18(1):272. doi: 10.1186/s12864-017-3663-0.
Cephalosporins and penicillins are the most frequently used β-lactam antibiotics for the treatment of human infections worldwide. The main industrial producers of these antibiotics are Acremonium chrysogenum and Penicillium chrysogenum, two taxonomically unrelated fungi. Both were subjects of long-term strain development programs to reach economically relevant antibiotic titers. It is so far unknown, whether equivalent changes in gene expression lead to elevated antibiotic titers in production strains.
Using the sequence of PcbC, a key enzyme of β-lactam antibiotic biosynthesis, from eighteen different pro- and eukaryotic microorganisms, we have constructed a phylogenetic tree to demonstrate the distant relationship of both fungal producers. To address the question whether both fungi have undergone similar genetic adaptions, we have performed a comparative gene expression analysis of wild-type and production strains. We found that strain improvement is associated with the remodeling of the transcriptional landscape in both fungi. In P. chrysogenum, 748 genes showed differential expression, while 1572 genes from A. chrysogenum are differentially expressed in the industrial strain. Common in both fungi is the upregulation of genes belonging to primary and secondary metabolism, notably those involved in precursor supply for β-lactam production. Other genes not essential for β-lactam production are downregulated with a preference for those responsible for transport processes or biosynthesis of other secondary metabolites. Transcriptional regulation was shown to be an important parameter during strain improvement in different organisms. We therefore investigated deletion strains of the major transcriptional regulator velvet from both production strains. We identified 567 P. chrysogenum and 412 A. chrysogenum Velvet target genes. In both deletion strains, approximately 50% of all secondary metabolite cluster genes are differentially regulated, including β-lactam biosynthesis genes. Most importantly, 35-57% of Velvet target genes are among those that showed differential expression in both improved industrial strains.
The major finding of our comparative transcriptome analysis is that strain improvement programs in two unrelated fungal β-lactam antibiotic producers alter the expression of target genes of Velvet, a global regulator of secondary metabolism. From these results, we conclude that regulatory alterations are crucial contributing factors for improved β-lactam antibiotic titers during strain improvement in both fungi.
头孢菌素和青霉素是全球治疗人类感染最常用的β-内酰胺类抗生素。这些抗生素的主要工业生产菌是产黄顶头孢霉和产黄青霉，这两种真菌在分类学上并无关联。二者均是长期菌株开发项目的研究对象，目的是达到具有经济价值的抗生素效价。目前尚不清楚基因表达的等效变化是否会导致生产菌株中抗生素效价升高。
利用来自18种不同原核和真核微生物的β-内酰胺抗生素生物合成关键酶PcbC的序列，我们构建了系统发育树，以证明这两种真菌生产菌的远缘关系。为了解决这两种真菌是否经历了相似的遗传适应这一问题，我们对野生型和生产菌株进行了比较基因表达分析。我们发现菌株改良与两种真菌转录图谱的重塑有关。在产黄青霉中，748个基因表现出差异表达，而在工业菌株中，来自产黄顶头孢霉的1572个基因存在差异表达。两种真菌的共同之处在于，属于初级和次级代谢的基因上调，特别是那些参与β-内酰胺生产前体供应的基因。其他对β-内酰胺生产非必需的基因下调，尤其偏好那些负责运输过程或其他次级代谢物生物合成的基因。转录调控被证明是不同生物体菌株改良过程中的一个重要参数。因此，我们研究了两种生产菌株中主要转录调节因子Velvet的缺失菌株。我们鉴定出567个产黄青霉和412个产黄顶头孢霉的Velvet靶基因。在两种缺失菌株中，所有次级代谢物簇基因中约50%受到差异调节，包括β-内酰胺生物合成基因。最重要的是，35%-57%的Velvet靶基因位于两种改良工业菌株中表现出差异表达的基因之中。
我们比较转录组分析的主要发现是，两个不相关的真菌β-内酰胺抗生素生产菌的菌株改良计划改变了次级代谢全局调节因子Velvet的靶基因表达。从这些结果中，我们得出结论，调控改变是两种真菌菌株改良过程中β-内酰胺抗生素效价提高的关键促成因素。
