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Liamocins, also known as heavy oils, are groups of extracellular lipids producing from Aureobasidium pullulans. In recent years, liamocins draw great attention due to their anticancer and antibacterial activities for applications in the pharmaceutical industry, as well as applications in the chemical industry as biosurfactants. For commercial applications, liamocins must be produced with high yield and high purity. However, the co-production of melanin, a black pigment, in liamocins fermentation presents a challenge in downstream product separation and purification. To elucidate the melanin biosynthesis in A. pullulans, we first studied the fermentation in shake-flasks under different light conditions, which resulted in different melanin production level. Then, the transcriptome of A. pullulans was sequenced and the expression levels of genes related to liamocins and melanin biosynthesis were analyzed to identify genes that were differentially expressed under light and dark conditions. Finally, CRISPR genome editing was used to knock out genes associated with melanin biosynthesis while genes associated with liamocins biosynthesis were overexpressed. The resulting mutant strains showed the ability to produce more liamocins with less melanin as indicated by the fermentation broth color as well as HPLC analysis. This study revealed the gene expression regulation in A. pullulans and provided the strategy of creating engineered A. pullulans for high-yield liamocins production with low melanin contamination.