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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.

SUBMITTER: Pasoreck EK

PROVIDER: S-EPMC4980996 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Numerous wastewater treatment processes are designed by engineers to achieve specific treatment goals. However, the impact of these different process designs on bacterial community composition is poorly understood. In this study, 24 different municipal wastewater treatment facilities (37 bioreactors) with various system designs were analyzed by sequencing of PCR-amplified 16S rRNA gene fragments. Although a core microbiome was observed in all of the bioreactors, the overall microbial community composition (analysis of molecular variance; P = 0.001) as well as that of a specific population of Nitrosomonas spp. (P = 0.04) was significantly different between A/O (anaerobic/aerobic) systems and conventional activated sludge (CAS) systems. Community α-diversity (number of observed operational taxonomic units [OTUs] and Shannon diversity index) was also significantly higher in A/O systems than in CAS systems (Wilcoxon; P < 2 × 10-16). In addition, wastewater bioreactors with short mean cell residence time (<2 days) had very low community α-diversity and fewer nitrifying bacteria compared to those of other system designs. Nitrospira spp. (0.71%) and Nitrotoga spp. (0.41%) were the most prominent nitrite-oxidizing bacteria (NOB); because these two genera were rarely prominent at the same time, these populations appeared to be functionally redundant. Weak evidence (AOB:NOB « 2; substantial quantities of Nitrospira sublineage II) was also obtained suggesting that complete ammonia oxidation by a single organism was occurring in system designs known to impose stringent nutrient limitation. This research demonstrates that design decisions made by wastewater treatment engineers significantly affect the microbiome of wastewater treatment bioreactors. IMPORTANCE Municipal wastewater treatment facilities rely on the application of numerous "activated sludge" process designs to achieve site-specific treatment goals. A plethora of microbiome studies on municipal wastewater treatment bioreactors have been performed previously; however, the role of process design on the municipal wastewater treatment microbiome is poorly understood. In fact, wastewater treatment engineers have attempted to control the microbiome of wastewater bioreactors for decades without sufficient empirical evidence to support their design paradigms. Our research demonstrates that engineering decisions with respect to system design have a significant impact on the microbiome of wastewater treatment bioreactors.

Project description:Walnuts (Juglans of the Juglandaceae) are well-known economically important resource plants for the edible nuts, high-quality wood, and medicinal use, with a distribution from tropical to temperate zones and from Asia to Europe and Americas. There are about 21 species in Juglans. Classification of Juglans at section level is problematic, because the phylogenetic position of Juglans cinerea is disputable. Lacking morphological and DNA markers severely inhibited the development of related researches. In this study, the complete chloroplast genomes and two nuclear DNA regions (the internal transcribed spacer and ubiquitin ligase gene) of 10 representative taxa of Juglans were used for comparative genomic analyses in order to deepen the understanding on the application value of genetic information for inferring the phylogenetic relationship of the genus. The Juglans chloroplast genomes possessed the typical quadripartite structure of angiosperms, consisting of a pair of inverted repeat regions separated by a large single-copy region and a small single-copy region. All the 10 chloroplast genomes possessed 112 unique genes arranged in the same order, including 78 protein-coding, 30 tRNA, and 4 rRNA genes. A combined sequence data set from two nuclear DNA regions revealed that Juglans plants could be classified into three branches: (1) section Juglans, (2) section Cardiocaryon including J. cinerea which is closer to J. mandshurica, and (3) section Rhysocaryon. However, three branches with a different phylogenetic topology were recognized in Juglans using the complete chloroplast genome sequences: (1) section Juglans, (2) section Cardiocaryon, and (3) section Rhysocaryon plus J. cinerea. The molecular taxonomy of Juglans is almost compatible to the morphological taxonomy except J. cinerea (section Trachycaryon). Based on the complete chloroplast genome sequence data, the divergence time between section Juglans and section Cardiocaryon was 44.77 Mya, while section Rhysocaryon diverged from other sections in the genus Juglans was 47.61 Mya. Eleven of the 12 small inversions in the chloroplast genomes provided valuable phylogenetic information for classification of walnut plants at section and species levels. Our results are valuable for future studies on Juglans genetic diversity and will enhance the understanding on the phylogenetic evolution of Juglandaceae.

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Terpene metabolic engineering via nuclear or chloroplast genomes profoundly and globally impacts off-target pathways through metabolite signalling.

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