title: Terpene metabolic engineering via nuclear or chloroplast genomes profoundly and globally impacts off-target pathways through metabolite signalling.
abstract: The impact of metabolic engineering on nontarget pathways and outcomes of metabolic engineering from different genomes are poorly understood questions. Therefore, squalene biosynthesis genes FARNESYL DIPHOSPHATE SYNTHASE (FPS) and SQUALENE SYNTHASE (SQS) were engineered via the Nicotiana tabacum chloroplast (C), nuclear (N) or both (CN) genomes to promote squalene biosynthesis. SQS levels were ~4300-fold higher in C and CN lines than in N, but all accumulated ~150-fold higher squalene due to substrate or storage limitations. Abnormal leaf and flower phenotypes, including lower pollen production and reduced fertility, were observed regardless of the compartment or level of transgene expression. Substantial changes in metabolomes of all lines were observed: levels of 65-120 unrelated metabolites, including the toxic alkaloid nicotine, changed by as much as 32-fold. Profound effects of transgenesis on nontarget gene expression included changes in the abundance of 19 076 transcripts by up to 2000-fold in CN; 7784 transcripts by up to 1400-fold in N; and 5224 transcripts by as much as 2200-fold in C. Transporter-related transcripts were induced, and cell cycle-associated transcripts were disproportionally repressed in all three lines. Transcriptome changes were validated by qRT-PCR. The mechanism underlying these large changes likely involves metabolite-mediated anterograde and/or retrograde signalling irrespective of the level of transgene expression or end product, due to imbalance of metabolic pools, offering new insight into both anticipated and unanticipated consequences of metabolic engineering.
authors: Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors: Elise K
authors: EK
authors: Pasoreck
authors: Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors: Jin
authors: J
authors: Su
authors: Department of Biology, University of Pennsylvania, Philadelphia, PA, USA.
authors: Ian M
authors: IM
authors: Silverman
authors: Department of Biology, University of Pennsylvania, Philadelphia, PA, USA.
authors: Sager J
authors: SJ
authors: Gosai
authors: Department of Biology, University of Pennsylvania, Philadelphia, PA, USA.
authors: Brian D
authors: BD
authors: Gregory
authors: Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX, USA.
authors: Joshua S
authors: JS
authors: Yuan
authors: Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors: Henry
authors: H
authors: Daniell
mesh_terms: D018506:Gene Expression Regulation, Plant / Q000235:genetics / Q000502:physiology; D054628:Genome, Chloroplast / Q000235:genetics*; D060847:Metabolic Engineering; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*; D013185:Squalene / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: D013185:Squalene
keywords: Secondary metabolism; retrograde/anterograde signalling; squalene; transgenic/transplastomic plants