Application of Random Mutagenesis and Synthetic FadR Promoter for de novo Production of ω-Hydroxy Fatty Acid in Yarrowia lipolytica

As a means to develop oleaginous biorefinery, Yarrowia lipolytica was utilized to produce ω-hydroxy palmitic acid from glucose using evolutionary metabolic engineering and synthetic FadR promoters for cytochrome P450 (CYP) expression. First, a base strain was constructed to produce free fatty acids (FFAs) from glucose using metabolic engineering strategies. Subsequently, through ethyl methanesulfonate (EMS)-induced random mutagenesis and fluorescence-activated cell sorting (FACS) screening, improved FFA overproducers were screened. Additionally, synthetic promoters containing bacterial FadR binding sequences for CYP expression were designed to respond to the surge of the concentration of FFAs to activate the ω-hydroxylating pathway, resulting in increased transcriptional activity by 14 times from the third day of culture compared to the first day. Then, endogenous alk5 was screened and expressed using the synthetic FadR promoter in the developed strain for the production of ω-hydroxy palmitic acid. By implementing the synthetic FadR promoter, cell growth and production phases could be efficiently decoupled. Finally, in batch fermentation, we demonstrated de novo production of 160 mg/L of ω-hydroxy palmitic acid using FmeN3-TR1-alk5 in nitrogen-limited media. This study presents an excellent example of the production of ω-hydroxy fatty acids using synthetic promoters with bacterial transcriptional regulator (i.e., FadR) binding sequences in oleaginous yeasts.

Streamlining the Analysis of Dynamic 13C-Labeling Patterns for the Metabolic Engineering of Corynebacterium glutamicum as l-Histidine Production Host

Today’s possibilities of genome editing easily create plentitudes of strain mutants that need to be experimentally qualified for configuring the next steps of strain engineering. The application of design-build-test-learn cycles requires the identification of distinct metabolic engineering targets as design inputs for subsequent optimization rounds. Here, we present the pool influx kinetics (PIK) approach that identifies promising metabolic engineering targets by pairwise comparison of up- and downstream 13C labeling dynamics with respect to a metabolite of interest. Showcasing the complex l-histidine production with engineered Corynebacterium glutamicuml-histidine-on-glucose yields could be improved to 8.6 ± 0.1 mol% by PIK analysis, starting from a base strain. Amplification of purA, purB, purH, and formyl recycling was identified as key targets only analyzing the signal transduction kinetics mirrored in the PIK values.

MEASURING THE DYNAMIC POISSON'S RATIO OF WOOD WITH THE USE OF A RADIO INTERFEROMETER

A new method is proposed for determining the dynamic Poisson's ratio of both isotropic and anisotropic materials. The sample was loaded with a pulsed compressive load on a setup that implements the Kolsky technique using a split Hopkinson bar. The development in time of the longitudinal compressive deformation of the sample was determined by the signals recorded with the help of low-base strain gauges glued to the measuring bars. A millimeter-wave interferometer was used to measure the development of the radial components of the sample deformation in time. To assess the possible asymmetry of the radial expansion of the sample, measurements were carried out using two independent channels irradiating diametrically opposite zones of the lateral surface of the sample. The tests were carried out on a sample of pine with air humidity in the form of a cylinder 54 mm in diameter and 30 mm in height. A pulsed compressive load was loaded along the fibers. Using two channels of the radio interferometer, the separate displacement of the lateral surfaces of the sample was recorded both along and across the annual layers. It was determined that the displacements of the regions of the lateral surface of the sample during expansion along the annual layers are quite close, while during expansion across the annual layers they are very different. The relative transverse deformation of the sample in both cases was determined as the sum of lateral displacements divided by the sample diameter. As a result, two components of the dynamic Poisson's ratio were obtained, which amounted to ~0.2 (in the direction along the annual layers) and ~0.24 (in the direction across the annual layers).

Comparative evaluation of Aspergillus niger strains for endogenous pectin depolymerization capacity and suitability for D-galacturonic acid production

Pectinaceous agricultural residues rich in d-galacturonic acid (d-GalA), such as sugar beet pulp, are considered as promising feedstocks for waste-to-value conversions. Aspergillus niger is known for its strong pectinolytic activity. However, while specialized strains for production of citric acid or proteins are openly available, this is not the case for the production of pectinases. We therefore systematically compared the pectinolytic capabilities of six A. niger strains (ATCC 1015, ATCC 11414, NRRL 3122, CBS 513.88, NRRL 3, N402) using controlled batch cultivations in stirred-tank bioreactors. A. niger ATCC 11414 showed the highest polygalacturonase activity, specific protein secretion and a suitable morphology. Furthermore, d-GalA release from sugar beet pulp was 75% higher compared to the standard lab strain A. niger N402. Our study therefore presents a robust initial strain selection to guide future process improvement of d-GalA production from agricultural residues and identifies the most suitable base strain for further genetic optimizations.