An Engineered Aro1 Protein Degradation Approach for Increased cis,cis-Muconic Acid Biosynthesis in Saccharomyces cerevisiae.

Muconic acid (MA) is a chemical building block and precursor to adipic and terephthalic acids used in the production of nylon and polyethylene terephthalate polymer families. Global demand for these important materials, coupled to their dependence on petrochemical resources, provides substantial motivation for the microbial synthesis of MA and its derivatives. In this context, the Saccharomyces cerevisiae yeast shikimate pathway can be sourced as a precursor for the formation of MA. Here we report a novel strategy to balance MA pathway performance with aromatic amino acid prototrophy by destabilizing Aro1 through C-terminal degron tagging. Coupling of a composite MA production pathway to degron-tagged Aro1 in an aro3Δ aro4Δ mutant background led to the accumulation of 5.6 g/liter protocatechuic acid (PCA). However, metabolites downstream of PCA were not detected, despite the inclusion of genes mediating their biosynthesis. Because CEN.PK family strains of S. cerevisiae lack the activity of Pad1, a key enzyme supporting PCA decarboxylase activity, chromosomal expression of intact PAD1 alleviated this bottleneck, resulting in nearly stoichiometric conversion (95%) of PCA to downstream products. In a fed-batch bioreactor, the resulting strain produced 1.2 g/liter MA under prototrophic conditions and 5.1 g/liter MA when supplemented with amino acids, corresponding to a yield of 58 mg/g sugar.IMPORTANCE Previous efforts to engineer a heterologous MA pathway in Saccharomyces cerevisiae have been hindered by a bottleneck at the PCA decarboxylation step and the creation of aromatic amino acid auxotrophy through deleterious manipulation of the pentafunctional Aro1 protein. In light of these studies, this work was undertaken with the central objective of preserving amino acid prototrophy, which we achieved by employing an Aro1 degradation strategy. Moreover, resolution of the key PCA decarboxylase bottleneck, as detailed herein, advances our understanding of yeast MA biosynthesis and will guide future strain engineering efforts. These strategies resulted in the highest titer reported to date for muconic acid produced in yeast. Overall, our study showcases the effectiveness of careful tuning of yeast Aro1 activity and the importance of host-pathway dynamics.

Addressing the challenges for sustainable production of algal biofuels: I. Algal strains and nutrient supply.

Microalgae hold promise for the production of sustainable replacement of fossil fuels due to their high growth rates, ability to grow on non-arable land and their high content, under the proper conditions, of high energy compounds that can be relatively easily chemically converted to fuels using existing technology. However, projected large-scale algal production raises a number of sustainability concerns concerning land use, net energy return, water use and nutrient supply. The state-of-the-art of algal production of biofuels is presented with emphasis on some possible avenues to provide answers to the sustainability questions that have been raised. Here, issues concerning algal strains and supply of nutrients for large-scale production are discussed. Since sustainability concerns necessitate the use of wastewaters for supply of bulk nutrients, emphasis is placed on the composition and suitability of different wastewater streams. At the same time, algal cultivation has proven useful in waste treatment processes, and thus this aspect is also treated in some detail.

Addressing the challenges for sustainable production of algal biofuels: II. Harvesting and conversion to biofuels.

In order to ensure the sustainability of algal biofuel production, a number of issues need to be addressed. Previously, we reviewed some of the questions in this area involving algal species and the important challenges of nutrient supply and how these might be met. Here, we take up issues involving harvesting and the conversion ofbiomass to biofuels. Advances in both these areas are required if these third-generation fuels are to have a sufficiently high net energy ratio and a sustainable footprint. A variety of harvesting technologies are under investigation and recent studies in this area are presented and discussed. A number of different energy uses are available for algal biomass, each with their own advantages as well as challenges in terms of efficiencies and yields. Recent advances in these areas are presented and some of the especially promising conversion processes are highlighted.

Strain variation in microalgal lipid production during mixotrophic growth with glycerol.

Algal cultivation at high latitudes is challenged by the relatively low annual solar flux. One possible scenario to overcome this limitation is the use of mixotrophic growth to potentially boost biomass and lipid production. Here the effect of glycerol addition on the growth and lipid production by twelve indigenous microalgae was examined. The results show that there is considerable strain dependent variation in the maximum growth rate under mixotrophic conditions with the addition of glycerol causing in some cases up to a 2.4-fold increase in growth rate and a up to a 1.9-fold increase in biomass. In addition, glycerol increased total lipid production 40-60% in some strains. These results also show the value in screening culture collections for desired traits independent of strain identification since here one (PCH02) of the five Chlorella strains showed a large increase in lipid with glycerol.

Effect of nitrogen regime on microalgal lipid production during mixotrophic growth with glycerol.

Mixotrophic growth of microalgae to boost lipid production is currently under active investigation. Such a process could be of practical importance if a cheap source of organic carbon, such as waste glycerol from biodiesel production, could be used. Several previous studies have already demonstrated that this carbon source can be used by different indigenous strains of microalgae. In this study it is shown that different nitrogen limitation strategies can be applied to further increase lipid production during growth with glycerol. In one strategy, cultures were grown in nitrogen replete medium and then resuspended in nitrogen free medium. In a second strategy, cultures were grown with different initial concentrations of nitrate. Lipid production by the two microalgal strains used, Chlorella sorokiniana (PCH02) and Chlorella vulgaris (PCH05), was shown to be boosted by strategies of nitrogen limitation, but they responded differently to how nitrogen limitation was imposed.

Utilization of biodiesel-derived glycerol or xylose for increased growth and lipid production by indigenous microalgae.

Microalgae are a promising alternative for sustainable biofuel production, but production yields and costs present a significant bottleneck. Here, the use of glycerol and xylose to boost the lipid yield was evaluated using ten strains from the Université de Montréal collection of microalgae. This report shows that some microalgal strains are capable of mixotrophic and heterotrophic growth on xylose, the major carbon source found in wastewater streams from pulp and paper industries, with an increase in growth rate of 2.8-fold in comparison to photoautotrophic growth, reaching up to µ=1.1/d. On glycerol, growth rates reached as high as µ=1.52/d. Lipid productivity increased up to 370% on glycerol and 180% on xylose for the strain LB1H10, showing the suitability of this strain for further development of biofuels production through mixotrophic cultivation.

Screening microalgae native to Quebec for wastewater treatment and biodiesel production.

Biodiesel production from microalgae lipids is being considered as a potential source of renewable energy. However, practical production processes will probably require the use of local strains adapted to prevailing climatic conditions. This report describes the isolation of 100 microalgal strains from freshwater lakes and rivers located in the vicinity of Montreal, Quebec, Canada. Strains were identified and surveyed for their growth on secondary effluent from a municipal wastewater treatment plant (La Prairie, QC, Canada) using a simple and high throughput microalgal screening method employing 12 well plates. The biomass and lipid productivity of these strains on wastewater were compared to a synthetic medium under different temperatures (10±2°C and 22±2°C) and a number identified that showed good growth at 10°C, gave a high lipid content (ranging from 20% to 45% of dry weight) or a high capacity for nutrient removal.

Algal biofuels: challenges and opportunities.

Biodiesel production using microalgae is attractive in a number of respects. Here a number of pros and cons to using microalgae for biofuels production are reviewed. Algal cultivation can be carried out using non-arable land and non-potable water with simple nutrient supply. In addition, algal biomass productivities are much higher than those of vascular plants and the extractable content of lipids that can be usefully converted to biodiesel, triacylglycerols (TAGs) can be much higher than that of the oil seeds now used for first generation biodiesel. On the other hand, practical, cost-effective production of biofuels from microalgae requires that a number of obstacles be overcome. These include the development of low-cost, effective growth systems, efficient and energy saving harvesting techniques, and methods for oil extraction and conversion that are environmentally benign and cost-effective. Promising recent advances in these areas are highlighted.