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As a valuable carotenoid, β-carotene is commercially used in food, cosmetics, animal feeds, and other industries. Metabolic engineering of microorganisms has been widely explored to improve the production of β-carotene. Compared with the traditional genetic modifications mainly focused on the pathways of mevalonate (MVA) and β-carotene biosynthesis, this study aims to increase the β-carotene production through promoting the synthesis of precursor substances by overexpressing hexokinase and hydroxymethylglutaryl-CoA synthase in an engineered  Yarrowia lipolytica . In this study, we investigated the effect of the unique hexokinase gene ( Hxk ) overexpression on β-carotene accumulation and glucose consumption. The  Hxk  gene was introduced into a β-carotene producing strain Y.L-1 to generate strain Y.L-2, and this increased the β-carotene content by 98%. Overexpression of the  Hxk  gene led to increasing in hexokinase activity (329% higher), glucose-6-phosphate content (92% higher), and improvement of the transcriptional level of  Hxk  (315% higher) compared to the control Y.L-1 strain. Moreover,  Hxk  overexpression accelerated the utilization rate of glucose. The gene  erg13  encoding hydroxymethylglutaryl-CoA synthase was also overexpressed to increase the precursor supply for β-carotene biosynthesis. Recombinant Y.L-4 harboring two copies of  erg13  produced 8.41 mg/g dry cell weight (DCW) of β-carotene, which was 259% higher than Y.L-1. The β-carotene content of 9.56 mg/g DCW was achieved in strain Y.L-6 by integrating  erg13  into the chromosome and  Hxk  overexpression. The 3-Hydroxy-3-Methylglutaryl-CoA content in the cells was increased by overexpressing two copies of the  erg13  gene. Finally, the titer of β-carotene reached 2.4 g/L using a 50 L bioreactor by the engineered strain, and the fermentation cycle was shortened from 144 to 120 h. Overall, overexpression of  Hxk  and  erg13  could improve β-carotene production and successfully overcoming the bottleneck of precursor generation to support a more efficient pathway for the production of the target product. Our results revealed a novel strategy to engineer the pathway of β-carotene synthesis.

frontiers in microbiology

Promoting the Synthesis of Precursor Substances by Overexpressing Hexokinase (Hxk) and Hydroxymethylglutaryl-CoA Synthase (Erg13) to Elevate β-Carotene Production in Engineered Yarrowia lipolytica