Enhancing carotenoid production in Rhodotorula mucilaginosa KC8 by combining mutation and metabolic engineering

Abstract

Rhodotorula mucilaginosa has been considered as a potential industrial yeast due to its unicellular and fast-growing characteristics, and its ability to produce carotenoids, including torularhodin. However, its low total carotenoid production limits its commercial application. In this study, mutation breeding and metabolic engineering were employed to enhance carotenoid production in the R. mucilaginosa strain KC8. After chemical–physical mutagenesis, R. mucilaginosa K4 with a 67% greater concentration of carotenoids (14.47 ± 0.06 mg L⁻¹) than R. mucilaginosa KC8 (8.67 ± 0.07 mg L⁻¹) was obtained. To further enhance carotenoid production, gene HMG1 encoding the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was introduced from another yeast, Saccharomyces cerevisiae, and overexpressed in R. mucilaginosa K4. The carotenoid production of HMG1-gene-overexpression transformant G1 reached 16.98 mg L⁻¹. To relieve the feedback inhibition of ergosterol, and to down-regulate ergosterol synthesis, ketoconazole, an ergosterol synthesis inhibitor, was added at a concentration of 28 mg L⁻¹. The carotenoid production of the transformant G1 reached 19.14 ± 0.09 mg L⁻¹, which was 121% higher than in R. mucilaginosa KC8. This suggests that a combination of chemical–physical mutagenesis, overexpression of the HMG1 gene, and adding ketoconazole is an effective strategy to improve carotenoid production.

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