Terreic acid is a potential anticancer drug as it inhibits Bruton’s tyrosine kinase; however, its biosynthetic molecular steps remain unclear. In this work, the individual reactions of terreic acid biosynthesis were determined by stepwise pathway assembly in a heterologous host, Pichia pastoris, on the basis of previous knockout studies in a native host, Aspergillus terreus. Polyketide synthase AtX was found to catalyze the formation of partially reduced polyketide 6-methylsalicylic acid, followed by 3-methylcatechol synthesis by salicylate 1-monooxygenase AtA-mediated decarboxylative hydroxylation of 6-methylsalicylic acid. Our results show that cytochrome P450 monooxygenase AtE hydroxylates 3-methylcatechol, thus producing the next product, 3-methyl-1,2,4-benzenetriol. A smaller putative cytochrome P450 monooxygenase, AtG, assists with this step. Then, AtD causes epoxidation and hydroxyl oxidation of 3-methyl-1,2,4-benzenetriol and produces a compound terremutin, via which the previously unknown function of AtD was identified as cyclooxygenation. The final step involves an oxidation reaction of a hydroxyl group by a glucose-methanol-choline oxidoreductase, AtC, which leads to the final product: terreic acid. Functions of AtD and AtG were determined for the first time. All the genes were reanalyzed and all intermediates and final products were isolated and identified. Our model fully defines the molecular steps and corrects previous results from the literature.

Natural red pigments have been widely used as food and cosmetics additives. However, due to toxic byproducts or allergen issues, it is still necessary to look for some other red pigment products. This study proposed combinatorial strategies to improve production of a new kind of red pigments from the fungus Geomyces WNF-15A, isolated from Antarctica. A high-production medium was developed by statistical experimental design, which was further simplified for industrial use by single-factor experiments. Strain breeding by atmospheric room temperature plasma mutagenesis generated a mutant, Geomyces sp. WNF-15A-M210, which increased production of red pigments by 24.4% and shortened culture phase by 33.3% comparing with the wild-type. The production of red pigments by this mutant favored a weak alkaline condition but required only mild dissolved oxygen tension. Control of initial pH 8.5 (process pH around 7.5) increased red pigments production by 19% comparing with natural condition. Precursor and inhibitor addition experiments indicated that the red pigments were synthesized by polyketide pathway, and feeding 6 mmol/L precursor of sodium acetate by three aliquots at days 3 to 5 improved biosynthesis of red pigments by 27%. Finally, the developed culture process was verified in a 5-L stirred tank bioreactor. The red pigments production of the pH regulation group reached 1.11-fold of the control and 1.95-fold of the precursor regulation group, respectively. This study provides high-production strain, optimized medium, and bioprocess for the possible industrial production of Antarctic Geomyces red pigments in future.

Polar regions are rich in microbial and product resources. Geomyces sp. WNF-15A is an Antarctic psychrotrophic filamentous fungus producing high quality red pigment with potential for industrial use. However, efficient biosynthesis of red pigment can only realize at low temperature, which brings difficult control and high cost for the large-scale fermentation. This study aims to develop transposon insertion mutation method to improve cell growth and red pigment production adaptive to normal temperature. Genetic manipulation system of this fungus was firstly developed by antibiotic marker screening, protoplast preparation and transformation optimization, by which transformation efficiency of ∼50% was finally achieved. Then transposable insertion systems were established using Helitron, Fot1 and Impala transposons. The transposition efficiency reached 11.9%, 9.4% and 4.6%, respectively. Mutant MP1 achieved the highest red pigment production (OD520 of 39) at 14°C, which was 40% higher than the wild-type strain. Mutant MP14 reached a maximum red pigment production (OD520 of 14.8) at 20°C, which was about 2-fold of the wild-type strain. Mutants MP2 and MP10 broke the repression mechanism of red pigment biosynthesis in the wild-type and allowed production at 25°C. For cell growth, 8 mutants grew remarkably better (12%∼30% biomass higher) than the wild-type at 25°C. This study established an efficient genetic manipulation and transposon insertion mutation platform for polar filamentous fungus. It provides reference for genetic breeding of psychrotrophic fungi from polar and other regions.

The collection of photogenerated electrons is an essential part of the photoelectrochemical process on a semiconductor photoanode, which is often a bottleneck in a particulate photoanode. Herein, by ingeniously inserting...