Physiological characterization of the high malic acid-producing Aspergillus oryzae strain 2103a-68.

Malic acid is a C4 dicarboxylic acid that is currently mainly used in the food and beverages industry as an acidulant. Because of the versatility of the group of C4 dicarboxylic acids, the chemical industry has a growing interest in this chemical compound. As malic acid will be considered as a bulk chemical, microbial production requires organisms that sustain high rates, yields, and titers. Aspergillus oryzae is mainly known as an industrial enzyme producer, but it was also shown that it has a very competitive natural production capacity for malic acid. Recently, an engineered A. oryzae strain, 2103a-68, was presented which overexpressed pyruvate carboxylase, malate dehydrogenase, and a malic acid transporter. In this work, we report a detailed characterization of this strain including detailed rates and yields under malic acid production conditions. Furthermore, transcript levels of the genes of interest and corresponding enzyme activities were measured. On glucose as carbon source, 2103a-68 was able to secrete malic acid at a maximum specific production rate during stationary phase of 1.87 mmol (g dry weight (DW))⁻¹ h⁻¹ and with a yield of 1.49 mol mol⁻¹. Intracellular fluxes were obtained using ¹³C flux analysis during exponential growth, supporting the success of the metabolic engineering strategy of increasing flux through the reductive cytosolic tricarboxylic acid (rTCA) branch. Additional cultivations using xylose and a glucose/xylose mixture demonstrated that A. oryzae is able to efficiently metabolize pentoses and hexoses to produce malic acid at high titers, rates, and yields.

Can We Approach Theoretical Lipid Yields in Microalgae?

Can we approach theoretical lipid yields in microalgae? Yes: we can substantially reduce the gap between current and theoretical maximum yield. A realistic maximum is approximately 0.5g triacylglycerol (TAG) per mol photons, about five times higher than what is currently achieved in outdoor cultivation. Achieving this realistic maximum will require several breakthroughs. First, outdoor operation typically has low yields, mainly caused by fluctuating insolation. Future adaptive control models will help increase these yields. Additionally, the lipid production capacity of currently used strains needs to increase. Powerful strain-specific molecular toolboxes are being developed, shifting the bottleneck towards understanding metabolism and identifying target genes. Finally, strains and processes should be improved concurrently to fully exploit the potential lipid production from microalgae.

Dynamics of triacylglycerol and EPA production in Phaeodactylum tricornutum under nitrogen starvation at different light intensities.

Lipid production in microalgae is highly dependent on the applied light intensity. However, for the EPA producing model-diatom Phaeodactylum tricornutum, clear consensus on the impact of incident light intensity on lipid productivity is still lacking. This study quantifies the impact of different incident light intensities on the biomass, TAG and EPA yield on light in nitrogen starved batch cultures of P. tricornutum. The maximum biomass concentration and maximum TAG and EPA contents were found to be independent of the applied light intensity. The lipid yield on light was reduced at elevated light intensities (>100 µmol m-2 s-1). The highest TAG yield on light (112 mg TAG molph-1) was found at the lowest light intensity tested (60 µmol m-2 s-1), which is still relatively low to values reported in literature for other algae. Furthermore, mass balance analysis showed that the EPA fraction in TAG may originate from photosynthetic membrane lipids.