Improving flavonoid production in Saccharomyces cerevisiae using synthetic biology tools

Flavonoids are plant secondary metabolites and represent one of the largest classes of natural products. Due to their health-beneficial properties, they have found potential applications in foods, beverages, cosmetics, and pharmaceuticals. Currently, their production is based on extraction from plant material. However, the low abundance of flavonoids in nature hinders efficient extraction and purification, thus inhibiting their market expansion. Chemical synthesis, although possible, relies on the use of harmful chemicals, harsh operating conditions, and high energy consumption.Metabolic engineering of microorganisms to develop so-called “microbial cell factories” has gained increasing attention as a more efficient and sustainable way to produce a variety of chemicals – including flavonoids. Saccharomyces cerevisiae (baker’s yeast) is one of the most well-studied and widely applied eukaryotic organisms for this endeavor. The fact that yeast shares cellular similarities to plants makes it a suitable host for the heterologous expression of flavonoid biosynthetic pathways. In this thesis, I present our efforts to improve the production of flavonoids in S. cerevisiae through the development and application of several synthetic biology tools. First, transcription factor-based biosensors for the isoflavonoid genistein and the flavonoid precursor p-coumaroyl-CoA were established. The latter sensor was used to devise a dynamic regulation strategy for the production of naringenin, a central flavanone and precursor for many flavanone derivatives. Cell growth was improved and naringenin titers were increased significantly. Next, a malonate assimilation pathway was implemented in yeast to enhance the supply of malonyl-CoA, an important precursor for all flavonoid compounds. By expressing a heterologous malonate transporter and malonyl-CoA synthetase, I constructed strains able to grow on externally supplied malonate. The malonate transporter was further evolved through targeted in vivo mutagenesis and beneficial mutations were identified through growth-based enrichment under selective conditions. Lastly, the production of the dihydrochalcone phloretin was explored. Its biosynthesis was accompanied by substantial byproduct formation and product degradation in the yeast cultivation medium. Different strategies, including enzyme scaffolding and antioxidant supplementation, were investigated to improve yeast-based production.Taken together, I addressed some significant challenges within microbial flavonoid production and showcased how synthetic biology tools may overcome these obstacles

A highly selective cell-based fluorescent biosensor for genistein detection

Genistein, an isoflavone found mainly in legumes, has been shown to have numerous health benefits for humans. Therefore, there is substantial interest in producing it using microbial cell factories. To aid in screening for high genistein producing microbial strains, a cell-based biosensor for genistein was developed by repurposing the Gal4DBD-ERα-VP16 (GEV) transcriptional activator in Saccharomyces cerevisiae. In the presence of genistein, the GEV sensor protein binds to the GAL1 promoter and activates transcription of a downstream GFP reporter. The performance of the biosensor, as measured by fold difference in GFP signal intensity after external genistein induction, was improved by engineering the sensor protein, its promoter and the reporter promoter. Biosensor performance increased when the weak promoter REV1p was used to drive GEV sensor gene expression and the VP16 transactivating domain on GEV was replaced with the tripartite VPR transactivator that had its NLS removed. The biosensor performance further improved when the binding sites for the inhibitor Mig1 were removed from and two additional Gal4p binding sites were added to the reporter promoter. After genistein induction, our improved biosensor output a GFP signal that was 20 times higher compared to the uninduced state. Out of the 8 flavonoids tested, the improved biosensor responded only to genistein and in a somewhat linear manner. The improved biosensor also responded to genistein produced in vivo, with the GFP reporter intensity directly proportional to intracellular genistein concentration. When combined with fluorescence-based cell sorting technology, this biosensor could facilitate high-throughput screening of a genistein-producing yeast cell factory

A p-Coumaroyl-CoA Biosensor for Dynamic Regulation of Naringenin Biosynthesis in Saccharomyces cerevisiae

In vivo biosensors that can convert metabolite concentrations into measurable output signals are valuable tools for high-throughput screening and dynamic pathway control in the field of metabolic engineering. Here, we present a novel biosensor in Saccharomyces cerevisiae that is responsive to p-coumaroyl-CoA, a central precursor of many flavonoids. The sensor is based on the transcriptional repressor CouR from Rhodopseudomonas palustris and was applied in combination with a previously developed malonyl-CoA biosensor for dual regulation of p-coumaroyl-CoA synthesis within the naringenin production pathway. Using this approach, we obtained a naringenin titer of 47.3 mg/L upon external precursor feeding, representing a 15-fold increase over the nonregulated system

UV-visible spectra character of larger diameter of gold nanoparticles (AuNPs)

The dependence of the optical properties of spherical gold nanoparticles (AuNPs) on particle size and wavelength were analyzed theoretically using Mie theory, where the complex refractive index of gold was corrected for the effect of a reduced mean free path of the conduction electrons in small particles. The simulation results indicated that larger diameter AuNPs have high sensitivity and the resolution compared to the small diameter AuNPs. Two different fabricated methods for synthesis larger diameter AuNPs have been proposed. One is the directly synthesis 50nm AuNPs, the other method is separated to two steps (prepared 13nm AuNPs seeds, then synthesis 50nm AuNPs). Comparing the UV-vis spectra and SEM image of the AuNPs synthesized by these two method, we find that the symmetry of the directly synthesis method is better than the other method. The experiment results show that the synthesis AuNPs are uniform. The major aim of this work is to provide a simple and fast method to synthesis larger diameter AuNPs and research the optical properties of the nanoparticles. © (2009) Trans Tech Publications

Engineered yeast tolerance enables efficient production from toxified lignocellulosic feedstocks

Lignocellulosic biomass remains unharnessed for the production of renewable fuels and chemicals due to challenges in deconstruction and the toxicity its hydrolysates pose to fermentation microorganisms. Here, we show in Saccharomyces cerevisiae that engineered aldehyde reduction and elevated extracellular potassium and pH are sufficient to enable near-parity production between inhibitor-laden and inhibitor-free feedstocks. By specifically targeting the universal hydrolysate inhibitors, a single strain is enhanced to tolerate a broad diversity of highly toxified genuine feedstocks and consistently achieve industrial-scale titers (cellulosic ethanol of >100 grams per liter when toxified). Furthermore, a functionally orthogonal, lightweight design enables seamless transferability to existing metabolically engineered chassis strains: We endow full, multifeedstock tolerance on a xylose-consuming strain and one producing the biodegradable plastics precursor lactic acid. The demonstration of "drop-in" hydrolysate competence enables the potential of cost-effective, at-scale biomass utilization for cellulosic fuel and nonfuel products alike

Wind power in China - Dream or reality?

After tremendous growth of wind power generation capacity in recent years, China now has 44.7 GW of wind-derived power. Despite the recent growth rates and promises of a bright future, two important issues - the capability of the grid infrastructure and the availability of backup systems - must be critically discussed and tackled in the medium term. The study shows that only a relatively small share of investment goes towards improving and extending the electricity infrastructure which is a precondition for transmitting clean wind energy to the end users. In addition, the backup systems are either geographically too remote from the potential wind power sites or currently financially infeasible. Finally, the introduction of wind power to the coal-dominated energy production system is not problem-free. Frequent ramp ups and downs of coal-fired plants lead to lower energy efficiency and higher emissions, which are likely to negate some of the emission savings from wind power. The current power system is heavily reliant on independently acting but state-owned energy companies optimizing their part of the system, and this is partly incompatible with building a robust system supporting renewable energy technologies. Hence, strategic, top-down co-ordination and incentives to improve the overall electricity infrastructure is recommended

13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation

Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL) for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using 13C-metabolic flux analysis (MFA). Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with 13C labeled glycerol or sodium bicarbonate. Through measurements of the 13C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate – oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO2 into biomass. As the human host is abundant in CO2 this finding requires further investigation in vivo as CO2 fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using 13C-MFA

Adapting to climate change by water management organisations: enablers and barriers

Climate change will be particularly experienced though the medium of water. Water organisations, that are managing societal and ecological needs for water, are therefore likely to experience the impact the most. This study reviews the current literature regarding adaptation to climate change by water management organisations and associated barriers. Literature on adaptive capacity is growing and a general consensus is emerging on the determinants of adaptive capacity, although variations exist regarding how it is to be evaluated, enhanced and applied to policy making due to its dynamic, contextual and latent nature. Since adaptive capacity is hard to measure and successful adaptation difficult to define, some studies focus on the existence of adaptation attributes of organisations. Studies reporting successful adaptation are minimal and barriers of adaptation are being discovered as adaptation research transitions into implementation. But the root causes of these barriers are often overlooked and the interconnectedness of the barriers is poorly addressed. Increasingly, combining top-down and bottom-up approaches to adaptation is being recommended due to the limitations of each. However, knowledge regarding how organisations operating at different scales can enhance adaptive capacity of other organisations operating at another scale is lacking due to the few studies of inter-organisational networks across scales. Social networks among actors are recognised as a key factor to enable adaptation. However, network studies generally focus on individual actors and rarely between public agencies/organisations. Moreover, the current literature is inadequate to understand the relationship between adaptation enabling characteristics, barriers and adaptation manifestation. The review demonstrates that research on understanding the emergence and sustenance of barriers is urgently required. Addressing these knowledge gaps will help to improve the design of adaptation strategies, thereby improving the ability of water management to address the ongoing challenges of climate change

The mid-Miocene Zhangpu biota reveals an outstandingly rich rainforest biome in East Asia

During the Mid-Miocene Climatic Optimum [MMCO, ~14 to 17 million years (Ma) ago], global temperatures were similar to predicted temperatures for the coming century. Limited megathermal paleoclimatic and fossil data are known from this period, despite its potential as an analog for future climate conditions. Here, we report a rich middle Miocene rainforest biome, the Zhangpu biota (~14.7 Ma ago), based on material preserved in amber and associated sedimentary rocks from southeastern China. The record shows that the mid-Miocene rainforest reached at least 24.2°N and was more widespread than previously estimated. Our results not only highlight the role of tropical rainforests acting as evolutionary museums for biodiversity at the generic level but also suggest that the MMCO probably strongly shaped the East Asian biota via the northern expansion of the megathermal rainforest biome. The Zhangpu biota provides an ideal snapshot for biodiversity redistribution during global warming