Enhancing β-Carotene Production in Escherichia coli by Perturbing Central Carbon Metabolism and Improving the NADPH Supply

Abstract

Beta (β)-carotene (C₄₀H₅₆; a provitamin) is a particularly important carotenoid for human health. Many studies have focused on engineering Escherichia coli as an efficient heterologous producer of β-carotene. Moreover, several strains with potential for use in the industrial production of this provitamin have already been constructed via different metabolic engineering strategies. In this study, we aimed to improve the β-carotene-producing capacity of our previously engineered E. coli strain ZF43ΔgdhA through further gene deletion and metabolic pathway manipulations. Deletion of the zwf gene increased the resultant strain's β-carotene production and content by 5.1 and 32.5%, respectively, relative to the values of strain ZF43ΔgdhA, but decreased the biomass by 26.2%. Deletion of the ptsHIcrr operon further increased the β-carotene production titer from 122.0 to 197.4 mg/L, but the provitamin content was decreased. Subsequently, comparative transcriptomic analysis was used to explore the dynamic transcriptional responses of the strains to the blockade of the pentose phosphate pathway and inactivation of the phosphotransferase system. Lastly, based on the analyses of comparative transcriptome and reduction cofactor, several strategies to increase the NADPH supply were evaluated for enhancement of the β-carotene content. The combination of yjgB gene deletion and nadK overexpression led to increased β-carotene production and content. The best strain, ECW4/p5C-nadK, produced 266.4 mg/L of β-carotene in flask culture and 2,579.1 mg/L in a 5-L bioreactor. The latter value is the highest reported from production via the methylerythritol phosphate pathway in E. coli. Although the strategies applied is routine in this study, the combinations reported were first implemented, are simple but efficient and will be helpful for the production of many other natural products, especially isoprenoids. Importantly, we demonstrated that the use of the methylerythritol phosphate pathway alone for efficient β-carotene biosynthesis could be achieved via appropriate modifications of the cell metabolic functions.

Keywords: Escherichia coli; NADPH supply; metabolic engineering; phosphotransferase system inactivation; β-carotene.

Figure 1

Simplified carbon metabolic profile of the producing Escherichia coli strain. The heterologous genes gps and crtEBIY were integrated into the E. coli genome at the ldhA locus to introduce the β-carotene pathway (Li et al., 2015). The genetic manipulations in this study are shown in colored fonts, where red represents gene deletion and blue represents gene overexpression. The multiple arrows represent multiple reactions. G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F1,6P, fructose-1,6-bisphosphate; G-l-6P, gluconolactone-6-phosphate; G3P, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; 6PG, 6-phosphogluconate; Ru5P, ribose-5-phosphate; 2-KG, 2-ketoglutaric acid; Glu, glutamic acid; DXP, 1-deoxy-d-xylulose-5-phosphate; MEP, 2C-methyl-d-erythritol-4-phosphate; CDP-ME, 4-diphosphocytidyl-2C-methyl-d-erythritol; CDP-MEP, 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate; MEC, 2C-methyl-d-erythritol-2,4-cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate.

Figure 2

Effects of zwf deletion and phosphotransferase system inactivation on β-carotene production (A) and biomass generation (B) in Escherichia coli strain ZF43ΔgdhA. The results represent the means from four independent experiments, and the error bars represent standard deviations.

Figure 3

Comparative transcriptomic analysis of Escherichia coli strains ZF43ΔgdhA, ECW1, and ECW2. (A) Fraction of correlated genes in KEGG pathways of strain ECW1 relative to those of strain ZF43ΔgdhA. The x-axis indicates the ratio of the number of genes differentially expressed in the pathway relative to the total number of genes in the pathway. The y-axis numbers in parentheses are the number of genes differentially expressed in the pathway. Red indicates an upregulated pathway, blue a downregulated pathway, and green a pathway containing both upregulated and downregulated genes. (B) Box plot of the log₂FoldChange of the differentially expressed genes participating in the TCA cycle, EMP/gluconeogenesis pathway, pentose phosphate pathway, and oxidative phosphorylation in strains ECW1 and ECW2 relative to those in strain ZF43ΔgdhA. The bold line in the box indicates the median and the yellow dot indicates the mean. The lower and upper bounds of the box indicate the first and third quartiles, respectively, and the whiskers show ±1.5× the interquartile range. (C) Transcriptional levels of genes related to the anaplerotic pathways, acetate pathways, NADPH pathways, and methylerythritol phosphate (MEP) pathways in strains ECW1 and ECW2 relative to those in strain ZF43ΔgdhA. (D) Fraction of correlated genes in KEGG pathways of strain ECW2 relative to those of strain ECW1. The meaning of the colors is the same as in (A).

Figure 4

NADPH supply to increase β-carotene production by different engineered Escherichia coli strains. Production of β-carotene (A) and biomass (B) by the different strains. The results represent the means from four independent experiments, and the error bars represent standard deviations. (C) Fed-batch fermentation of strain ECW4/p5C-nadK. Ultraviolet spectrophotometer was used to quantify the β-carotene production quickly during the fed-batch fermentation, and HPLC was used to re-quantify the production of some samples to confirm the quantitative accuracy of the absorption method after fermentation. Results showed that the quantifications obtained from the two methods were consistent.