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

Xiao Chao Xiong,Shan Qiang,Liang Liu,Yu Ling Qu,Jing Wang,Ching Yuan Hu,Yong Hong Meng

doi:10.3389/fmicb.2020.01346

Abstract

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.

Highlights

Β-carotene is a valuable terpenoid that has broad applications in the food industry as food additives, especially as colorants, as well as in the nutraceutical industry as nutritional supplements (Zhao et al, 2013; Ma et al, 2019). β-carotene is an antioxidant and a precursor for the formation of vitamin A
We found that Hxk (YALI0B22308g) was a useful gene that promotes the glucose utilization and generation of precursors for β-carotene biosynthesis in Y. lipolytica
The basal β-carotene producing strain Y.L-1 was constructed by integrating truncated hydroxymethylglutaryl-CoA reductase gene (tHMG) (YALI0E04807g), GGS1 (YALI0D17050g), carRA (KY971027), and carB (KY971026) genes into the Y. lipolytica PO1f genome

Summary

Introduction

Β-carotene is a valuable terpenoid that has broad applications in the food industry as food additives, especially as colorants, as well as in the nutraceutical industry as nutritional supplements (Zhao et al, 2013; Ma et al, 2019). β-carotene is an antioxidant and a precursor for the formation of vitamin A. On the contrary, introducing an additional copy of Hxk in Y. lipolytica resulted in the improvement of both biomass yield and lipid production (Lazar et al, 2014). Combinational overexpression of the key genes in the β-carotene biosynthesis pathway and engineering of lipid biosynthesis may improve the yield of β-carotene in Y. lipolytica. We found that Hxk (YALI0B22308g) was a useful gene that promotes the glucose utilization and generation of precursors for β-carotene biosynthesis in Y. lipolytica. The identification of the Hxk gene by increasing the glucose consumption provides a new target to engineer metabolically Y. lipolytica for β-carotene biosynthesis. The reconstitution of multiple related genes of the β-carotene biosynthesis pathway in Y. lipolytica represents the first step for large-scale fermentation to produce β-carotene

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