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.
Highlights
Carotenoids, which are natural pigments that provide the yellow to pink to red colors in various species and are widespread in plants, animals, and microorganisms, have a variety of biological functions (Alcaíno et al, 2016)
In E. coli, isoprenoid biosynthesis occurs through the methylerythritol phosphate (MEP) pathway, in which glyceraldehyde-3-phosphate and pyruvate are converted into two isoprenoid building blocks (viz., isopentenyl diphosphate (IPP) and dimethylallyl diphosphate) (Figure 1) with the help of cofactors cytidine triphosphate, adenosine triphosphate, and reduced nicotinamide adenine dinucleotide phosphate (NADPH)
Considering that lycopene is the direct precursor of β-carotene in E. coli, we deleted the zwf gene in E. coli strain ZF43 gdhA to explore the effect of its knockout on β-carotene production in the resultant strain ECW1 (ZF43 gdhA zwf )
Summary
Carotenoids, which are natural pigments that provide the yellow to pink to red colors in various species and are widespread in plants, animals, and microorganisms, have a variety of biological functions (Alcaíno et al, 2016). The overexpression of certain key enzymes in the MEP pathway, as well as strengthening of the precursor and cofactor supply, have proven to be effective strategies for increasing β-carotene production in E. coli (Sedkova et al, 2005; Yuan et al, 2006; Zhao et al, 2013; Li et al, 2015). Some strategies that have been useful for increasing β-carotene production in E. coli include the introduction of the heterologous pathway to increase IPP supply, combinatorial expression of the mevalonate and MEP pathways, and optimization of the fermentation process (Yoon et al, 2009; Nam et al, 2013; Yang and Guo, 2014; Ye et al, 2016b). With regard to energy balance and product-glucose conversion, MEP pathway is more energetically balanced and exhibits higher (about 19.8%) mass yield of glucose for terpenoids production (Ye et al, 2016a)
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