Application of adaptive laboratory evolution to improve the tolerance of Rhodotorula strain to methanol in crude glycerol and development of an effective method for cell lysis.

Rhodotorula toruloides can utilize crude glycerol as the low-cost carbon source for lipid production, but its growth is subjected to inhibition by methanol in crude glycerol. Here, transcriptome profiling demonstrated that 1004 genes were significantly regulated in the strain R. toruloides TO2 under methanol stress. Methanol impaired the function of membrane transport and subsequently weakened the utilization of glycerol, activities of the primary metabolism and functions of nucleus and ribosome. Afterwards the tolerance of TO2 to methanol was improved by using two-round adaptive laboratory evolution (ALE). The final strain M2-ale had tolerance up to 3.5% of methanol. 1 H NMR-based metabolome analysis indicated that ALE not only improved the tolerance of M2-ale to methanol but also tuned the carbon flux towards the biosynthesis of glycerolipid-related metabolites. The biomass and lipid titer of M2-ale reached 14.63 ± 0.45 g L-1 and 7.06 ± 0.44 g L-1 at 96 h in the crude glycerol medium, which increased up to 17.69% and 31.39%, respectively, comparing with TO2. Afterwards, an effective method for cell lysis was developed by combining sonication and enzymatic hydrolysis (So-EnH). The lytic effect of So-EnH was validated by using confocal imaging and flow cytometry. At last, lipid recovery rate reached 95.4 ± 2.7% at the optimized condition.

Attenuating the triacylglycerol catabolism enhanced lipid production of Rhodotorula strain U13N3.

Enhancing the lipid production of oleaginous yeasts is conducive to cutting the cost of feedstock for biodiesel. To increase the lipid productivity of Rhodotorula sp. U13N3, genes involving lipid degradation were knocked out and fermentation conditions were investigated. Results of transcription analysis demonstrated that genes encoding the ATG15-like lipase (ATG15) and peroxisomal acyl-CoA oxidase (ACOX2) were upregulated significantly at the lipogenesis stage. When ATG15 and ACOX2 were knocked out separately from the genome by the CRISPR/Cas9 method, both ΔATG15 and ΔACOX2 mutants showed better lipid production ability than the parent strain. Flow cytometry and confocal microscopic analyses indicated that simultaneous the knockout of ATG15 and ACOX2 did not impact the cell viability, whereas the lipid production was enhanced markedly as the lipid yield increased by 67.03% in shake flasks. Afterward, the ΔATG15ΔACOX2 transformant (TO2) was cultivated in shake flasks in the fed-batch mode; the highest biomass and lipid yield reached 45.76 g/L and 27.14 g/L at 216 h, respectively. Better performance was achieved when TO2 was cultivated in the 1-L bioreactor. At the end of fermentation (180 h), lipid content, yield, yield coefficient, and productivity reached 65.53%, 27.35 g/L, 0.277 g/g glycerol, and 0.152 g/L/h, respectively. These values were at the high level in comparison with Rhodotorula strains cultivated in glycerol media. Besides, fermentation modes did not affect the fatty acid composition of TO2 significantly. In conclusion, blocking the lipid degradation was an applicable strategy to increase the lipid production of Rhodotorula strains without compromising their cell viability. KEY POINTS: • ATG15-like lipase and acyl-CoA oxidase (ACOX2) participated in lipid degradation. • Knockout of ATG15 and ACOX2 increased lipid productivity, and lipid yield coefficient. • Cell viability maintained at high level in the knockout mutants during fermentation.

Characterization and bioactivity analysis of a heteropolysaccharide purified from Paenibacillus edaphicus strain UJ1.

Many polysaccharides produced by Paenibacillus spp. have attractive properties, such as rheological modification and immunomodulation. However, properties of P. edaphicus polysaccharides are not understood sufficiently. Here, the polysaccharide (PUM) was obtained from P. edaphicus strain UJ1 by batch fermentation, and the chemical characteristics, rheological and anti-inflammatory properties of PUM and its sulfate derivative (PUM-S) were investigated. The results indicated that PUM was a typical shear-thinning biopolymer with an estimated weight average molecular weight of 2.45 × 107 Da. PUM molecule consisted of D-Man, D-GlcA, D-Glc, D-Gal, and L-Fuc with the molar ratio of 3.00:1.07:3.21:0.81:0.76. It had the backbone â†’ 3)-ß-D-Man-(1 â†’ 3)-ß-D-Glc-(1 â†’ 3)-ß-D-Man-(1 â†’ 3)ß-D-Glc-(1 â†’ 4)-ß-D-GlcA-(1 â†’ 3)-ß-D-Man-(1 â†’ and two side chains, namely, pyruvoyl-Glc-(1→ and ß-L-Fuc-(1 â†’ 3)-ß-D-Gal-(1→. Moreover, PUM-S was prepared by SO3-pyridine method and had the weight average molecular weight of 1.42 × 105 Da. The bioactivity of PUM and PUM-S was analyzed in vitro in RAW 264.7 cells. The results indicated that both PUM and PUM-S facilitated cell proliferation at 50-500 µg/mL. Besides, PUM-S showed potential anti-inflammatory effect in the LPS-induced cells. According to transcription and molecular dynamics analyses, PUM-S expressed its activity probably by interacting with the Toll-like receptor 4. In general, P. edaphicus produced a polysaccharide with new chemical structure and promising rheological and bioactive properties.

The carbohydrate elicitor Riclinoctaose facilitates defense and growth of potato roots by inducing changes in transcriptional and metabolic profiles.

Promoting both root growth and defense is conducive to the production of potatoes (Solanum tuberosum L.), while the role of elicitors in this topic hasn't been fully understood. To investigate the effect of Riclinoctaose (RiOc) on root growth and defense, potato tissue cuttings were cultivated with different concentration of RiOc (0, 50, 200 mg/L) for 5 weeks and changes in root morphology, transcription, enzymatic and metabolomic profiles were monitored over time. The results indicated that RiOc triggered the salicylic acid (SA)-mediated defense response and facilitated the growth of adventitious and lateral roots in a dose- and time-dependent manner. MPK3/MPK6, SA- and auxin-signaling pathways and transcription factors such as WUS, SCR and GRAS4/GRAS9 participated in this process. Moreover, the 1H NMR based metabolome profiling demonstrated that potato roots altered the primary metabolism to respond to the RiOc elicitation and efficiency in production and allocation of defense and growth-related metabolites was improved. After 5-week treatment, the level of glucose, N-acetylglucosamine, glutamine, asparagine, isoleucine, valine, 3-hydroxyisovalerate and ferulate increased, while acetate, acetoacetate, fucose, and 2-hydroxyphenylacetate declined. In conclusion, RiOc played dual roles in activating the SA-mediated defense response and in promoting growth of potato roots by inducing changes in root transcription and metabolism.

Rhodotorula toruloides: an ideal microbial cell factory to produce oleochemicals, carotenoids, and other products.

Requirement of clean energy sources urges us to find substitutes for fossil fuels. Microorganisms provide an option to produce feedstock for biofuel production by utilizing inexpensive, renewable biomass. Rhodotorula toruloides (Rhodosporidium toruloides), a non-conventional oleaginous yeast, can accumulate intracellular lipids (single cell oil, SCO) more than 70% of its cell dry weight. At present, the SCO-based biodiesel is not a price-competitive fuel to the petroleum diesel. Many efforts are made to cut the cost of SCO by strengthening the performance of genetically modified R. toruloides strains and by valorization of low-cost biomass, including crude glycerol, lignocellulosic hydrolysates, food and agro waste, wastewater, and volatile fatty acids. Besides, optimization of fermentation and SCO recovery processes are carefully studied as well. Recently, new R. toruloides strains are developed via metabolic engineering and synthetic biology methods to produce value-added chemicals, such as sesquiterpenes, fatty acid esters, fatty alcohols, carotenoids, and building block chemicals. This review summarizes recent advances in the main aspects of R. toruloides studies, namely, construction of strains with new traits, valorization of low-cost biomass, process detection and optimization, and product recovery. In general, R. toruloides is a promising microbial cell factory for production of biochemicals.