Biosynthesis of pyomelanin from methanol with engineered Komagataella phaffii and its characterizations.

Pyomelanin has been extensively applied in various fields. However, the yield of pyomelanin isolated from natural producers is low. Engineering microbial biosynthesis is considered a sustainable and economically feasible alternative. We utilize engineered Komagataella phaffii to increase the yield of homogentisic acid by 66-fold by balancing three different biosynthetic modules and further synthesizing pyomelanin by oxidative polymerization. During this process, we establish a pyomelanin color screening system and apply this system to perform directed evolution of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase and semirational design modification of hydroxyphenylpyruvate dioxygenase. We report the difference of pyomelanin between oxidative polymerization under alkaline conditions and that under laccase treatment. After high-density fermentation in a 5-L fermenter for 204 h, strain Pyo29 achieve the highest titer (70.5 ± 0.7 g/L). Finally, we demonstrate the potential application value of pyomelanin. This study provides a feasible solution for high-yield pyomelanin biosynthesis with substantial industrial production.