Metabolic Engineering of Microorganisms
Technological developments over the past century have made microorganisms a cornerstone in large-scale industrial production processes. Modern research focuses on metabolic engineering so that microbial strains produce high yields from desired end products. The arsenal of modern metabolic engineering contains tools that allow targeted rational interventions or global selection systems that combine classical approaches with omics technologies. It has recently included even more ambitious approaches to synthetic biology that radically reorganize the metabolism of organisms. show more show less
Numerous notable successes in yeast metabolic engineering have been achieved, such as the de novo production of opioids in yeast, vanillin, secondary metabolites of the flavonoid and stilbenoid family, and numerous terpenoids of plant and microbial origin such as artemisinin and carnosic acid.
Terpenoid production in S. cerevisiae is a characteristic example of modern biotechnology that incorporates all the variety of innovative approaches used in metabolic engineering. Terpenes attract keen interest as pharmaceuticals, flavorings and fragrances, and more recently as biofuels. More than 70,000 terpene compounds have been discovered in nature and new molecules are being added continuously.
This diversity stems from the enormous plasticity of the biosynthetic and terpene modification enzymes that result in the production of a large number of compounds as well as the rapid and dynamic change of their profile through small mutations and evolutionary pressure.
The activities of the Laboratory of Metabolic Engineering of INEB aims to improve the production platform of Saccharomyces for the increased yields of terpenes such as sclareol, miltiradiene, carnosic acid, salviol etc. At the same time, taking advantage of yeast advantages over other biosynthesis reconstitution systems (absence of endogenous terpene biosynthesis and modification enzymes, catalytic activity of heterologous enzymes), we undertake to characterize new enzymes and complex terpene biosynthesis pathways.
Yeast as “Cell factory” for the production of high added value chemicals
Microbial fermentation processes have been used by humans since ancient times for the processing of foods and production of beverages. Over the past century microbes became work-horses for large scale industrial production processes. However, since 1980s the advances in genetic engineering converted microbes to “cell factories” for the production of a diverse range of important chemical compounds. Manipulation of microbial metabolism holds major advantages since they can efficiently convert cheap raw materials like glucose, sucrose and biomass derived materials into high value chemicals and fuels, in an environmentally friendly manner. show more show less
S. cerevisiae is a preferred organism by industry as it can withstand high osmotic pressure and reduced pH compared to bacteria. However, despite the extensive synthetic and systems biological toolbox in yeast, it is recognized that further advances in tools and technologies are needed to expedite improvements in productivity of the cell platforms so as to reach sustainable, economically viable and environmentally friendly production processes for a multitude of compounds. This is expected to offer considerable benefits to society as drugs, industrial chemicals, food additives and fuels made inexpensively in an eco-friendly manner will enter the market.
In our recently published and unpublished work, we developed new molecular tools for the successive integration at desired loci of strong promoters and extra gene copies driven by strong promoters using recyclable cassettes. This enables successive modifications in tandem to take place on the yeast chromosomes, which is preferred by industry instead of plasmids. Additionally we proposed and successfully applied on small scale an approach to rewire yeast metabolism to desired end products by creating bottlenecks at critical nodes exploiting single allele deletion heterozygocity which suppresses proteins levels. Our approaches are simple, tap on different hubs not previously explored, which can be combined with existing and alternative approaches developed by other groups in the field, for additional improvements.
Elucidation of plant secondary metabolites biosynthesis
Chemical synthesis of industrially important plant secondary metabolites is often hampered by their complex stereochemistry and environmental concerns regarding the use of toxic catalysts and solvents. Production of the compounds of interest in heterologous microbial systems can provide a sustainable and environmentally friendly alternative. Several groundbreaking efforts have been reported, including the production of the antimalarial drug artemisinin, the fragrances sclareol and santalol, the flavoring agent vanillin, and the antioxidant resveratrol. Recently, the complete pathway of opioid biosynthesis comprising more than 20 enzymatic activities was reconstructed in yeast, demonstrating the potential of synthetic biology to contribute in the sustainable production of highly complex chemicals. In addition to the development of dedicated platform strains, such approaches also require detailed knowledge of the biosynthetic pathways and the genes involved. show more show less
Elucidation of plant secondary biosynthetic pathways can be a demanding process, involving the acquisition of transcriptomic or genomic sequence information and the analysis of a large number of candidate proteins for the identification of the activity responsible for a specific biosynthetic step. Frequently, this approach also requires the time-consuming chemical synthesis of precursors and the structure elucidation of pathway intermediates. Synthetic biology can expedite this process by enabling functional characterization of gene products and synthesis of substrates or intermediates on the same microbial platform.
We recently reported on the application of a modular yeast terpene production platform in the elucidation of the biosynthesis of carnosic acid and related diterpenes.
We conduct combined metabolome and transcriptome analysis of Salvia spp. and other aromatic and medicinal plants of interest in order to generate insights into isoprenoid pathway and identify key enzymes involved in terpene biosynthesis.
Metabolite analysis of S. pomifera leaf extracts led to the identification and isolation of several carnosic acid-related metabolites, suggested that transcriptomic analysis may yield important insights into the biosynthesis of these compounds and provided the basis for the successful pathway reconstruction in yeast cells.
Partners: A. Makris, A. Argiriou, S. Kampranis, V. Roussis, E. Ioannou, F. Trikka, C. Ignea, A.Athanasakoglou, A. Andreadelli
Systems biology and targeted genome engineering technology
Research focus on the identification of genetic variation that occurs naturally in organisms (e.g plants, fungi) and how genotype affects phenotype. The growing use of plants to produce food, fiber and biofuels represents a significant challenge for the agricultural sector in terms of breeding targets. To improve plants for food and energy in a climate smart way targeted horizontal technologies are used such as zinc finger nuclease (ZFN) technology. Climate change and global warming have negative impact on plant growth and productivity due to drought and more drought-tolerant plants are needed to offset the consequences of climate change. The research team of systems biology investigates how molecules interact within the cell during cell morphogenesis and how they work during formation of complex systems such as cell assemblages, structures and organisms. show more show less
The research links all levels of biological organization and evaluates metabolic and phenotypic changes as a result of genetic variation within the system. At another level, targeted genetic perturbation aims to create local genetic variation in a given genetic background to generate useful phenotypes for basic and applied research.
Sustainable and low input agriculture
Investigation of eco-friendly ways to recycle residual biomass into agricultural practices supporting a sustainable production system with positive effects on the environment (carbon footprint). Research focuses on the effect of biochars, generated by processing residual plant biomass, as soil amendments in plant development, nutrient availability, soil microbiology and water retention.