Overexpression Of Biosynthetic Genes Research Articles

Marker-free CRISPR-Cas9 based genetic engineering of the phytopathogenic fungus, Penicillium expansum

Filamentous fungi are prolific producers of secondary metabolites (SecMets), including compounds with antibiotic properties, like penicillin, that allows the producing fungus to combat competitors in a shared niche. However, the biological function of the majority of these small complex metabolites for the producing fungi remains unclear (Macheleidt et al., 2016). In an effort to address this lack of knowledge, we have chosen to study the microbial community of moldy apples in the hope of shedding more light on the role of SecMets for the dynamics of the microbial community. Penicillium expansum is one of the prevalent fungal species in this system, and in co-culture experiments with other apple fungal pathogens, we have observed up- and downregulation of several SecMets when compared to monocultures. However, molecular genetic dissection of the observed changes is challenging, and new methodologies for targeted genetic engineering in P. expansum are needed. In the current study, we have established a CRISPR-Cas9 dependent genetic engineering toolbox for the targeted genetic manipulation of P. expansum to allow for single-step construction of marker-free strains. The method and effect of different combinations of a Cas9-sgRNA expressing plasmids and repair template substrates in the NHEJ-proficient WT strain is tested by targeted deletion of melA, encoding a PKS responsible for pigment formation, which upon deletion resulted in white mutants. Co-transformation with a linear double-stranded DNA fragment consisting of two 2 kb homology arms flanking the PKS gene proved to be the most efficient strategy with 100% confirmed deletions by diagnostic PCR. Shorter homology arms (500–1000 bp) resulted in 20–30% deletion efficiency. Furthermore, we demonstrate the application of the CRISPR-Cas9 method for targeted deletion of biosynthetic genes without a visible phenotype, insertion of a visual reporter-encoding gene (mRFP), and overexpression of biosynthetic genes. Combined, these tools will advance in enabling the deciphering of SecMet biosynthetic pathways, provide in situ insight into when and where SecMets are produced, and provide an avenue to study the role of P. expansum SecMets in shaping the microbial community development on moldy apples via marker-free targeted genetic engineering of P. expansum.

Genetic background of sesquiterpene lactones biosynthesis in Asteraceae. A review

Asteraceae family is a rich source of many sesquiterpene lactones (STLs). These secondary metabolites exhibit multidirectional activity including anti-tumor, anti-inflammatory or antimicrobial, just to name a few. Promising approach of metabolic engineering offers a way of increasing the production of STLs by reconstruction of their biosynthetic pathway in a heterologous system. Moreover, their production in host plants might be increased through overexpression of biosynthetic genes and/or transcription factors (TFs) positively regulating the pathway. Either of the strategies requires extensive knowledge on the genetic background of STLs biosynthesis pathway. This review summarizes molecular investigations concerning biosynthesis of these medicinally essential metabolites.

Simultaneous over-expression and silencing of some benzylisoquinoline alkaloid biosynthetic genes in opium poppy

Opium poppy (Papaver somniferum L.) remains the only commercial source for several pharmaceutical alkaloids. In present study, opium poppy plants were genetically manipulated via VIGS technique using pTRV2-BBE (to silencing of BBE1 gene), pTRV2-COM (to simultaneous silencing of T6ODM and CODM genes) and pTRV2-BBE-COM (to simultaneous silencing of T6ODM, CODM and BBE1 genes) constructs. Also, via Agrobacterium-mediated transient expression technique, poppy plants were genetically manipulated using ACS (to simultaneous silencing of T6ODM, CODM and BBE1) and ACS-4ꞌOMT2 (to simultaneous silencing of T6ODM, CODM and BBE1 and over-expression of 4ꞌOMT2) constructs. HPLC analysis showed lower sanguinarine and noscapine levels and elevated levels of morphine in plants infiltrated with pTRV2-BBE construct. In plants infiltrated with pTRV2-COM construct, reduction in the morphine and codeine content and a substantial increase in accumulation of thebaine and papaverine were observed. A substantial increase in the accumulation of thebaine and papaverine and lower levels of morphine, codeine, sanguinarine and noscapine were observed in plants infiltrated with pTRV2-BBE-COM and ACS constructs. Plants infiltrated with ACS-4ꞌOMT2 showed lower levels of codeine, sanguinarine and noscapine and elevated levels of morphine, thebaine and papaverine. Although, previous studies reported the role of our selected genes in benzylisoquinoline alkaloids (BIAs) biosynthetic pathway, but we simultaneously engineered these genes to alter the levels of specific BIAs and take advantage of all genes at the same time. For the first time, we showed different pattern in BIAs accumulation in simultaneous gene manipulation compared with single gene manipulation in previous reports, especially for higher levels of thebaine (about 1550%) and papaverine (about 155%). We also determined potential role of BBE1 gene in noscapine biosynthesis. This is the first report of simultaneous silencing and over-expression of biosynthetic genes in opium poppy and our finding could establish vast potential for metabolic engineering in opium poppy.