Identification and Functional Analysis of Two Novel Genes—Geranylgeranyl Pyrophosphate Synthase Gene (AlGGPPS) and Isopentenyl Pyrophosphate Isomerase Gene (AlIDI)—from Aurantiochytrium limacinum Significantly Enhance De Novo β-Carotene Biosynthesis in Escherichia coli

Abstract

Precursor regulation has been an effective strategy to improve carotenoid production and the availability of novel precursor synthases facilitates engineering improvements. In this work, the putative geranylgeranyl pyrophosphate synthase encoding gene (AlGGPPS) and isopentenyl pyrophosphate isomerase encoding gene (AlIDI) from Aurantiochytrium limacinum MYA-1381 were isolated. We applied the excavated AlGGPPS and AlIDI to the de novo β-carotene biosynthetic pathway in Escherichia coli for functional identification and engineering application. Results showed that the two novel genes both functioned in the synthesis of β-carotene. Furthermore, AlGGPPS and AlIDI performed better than the original or endogenous one, with 39.7% and 80.9% increases in β-carotene production, respectively. Due to the coordinated expression of the 2 functional genes, β-carotene content of the modified carotenoid-producing E. coli accumulated a 2.99-fold yield of the initial EBIY strain in 12 h, reaching 10.99 mg/L in flask culture. This study helped to broaden current understanding of the carotenoid biosynthetic pathway in Aurantiochytrium and provided novel functional elements for carotenoid engineering improvements.

Recent Advances on Key Enzymes of Microbial Origin in the Lycopene Biosynthesis Pathway

“…coli. The recombinant strain showed a β-carotene content of 5.14 mg/L, which was 39.7% higher than that with the original crtE gene of Pantoea ananatis . Furthermore, improving the catalytic activity through enzyme engineering is also an effective strategy .…”

“…Specifically, the strain expressing the idi gene from Aurantiochytrium limacinum produced 10.99 mg/L of βcarotene, the next transformation product of lycopene, in 12 h, which was increased by 80.9% and 113.9% compared with the control strain expressing the endogenous idi gene of E. coli and the control strain not carrying the idi gene, respectively. 64 In addition to the overexpression strategy, a directed evolution technique based on error-prone PCR was also used to improve the catalytic activity of IDI from Saccharomyces cerevisiae. The mutant IDI with higher specific activity and catalytic efficiency was screened via three rounds of error-prone PCR, resulting in an 80% increase of the lycopene content compared to wildtype IDI.…”

“…The recombinant strain showed a βcarotene content of 5.14 mg/L, which was 39.7% higher than that with the original crtE gene of Pantoea ananatis. 64 Furthermore, improving the catalytic activity through enzyme engineering is also an effective strategy. 69 The directed evolution of GGPPS led to a 4-fold increase of lycopene production, while the directed evolution of PSY only increased lycopene production by 2-fold.…”

Marine thraustochytrid: exploration from taxonomic challenges to biotechnological applications

“…This results in the reduced accumulation of fatty acids by increasing the consumption of malonyl-CoA through the MVA pathway of carotenoid synthesis (Song et al, 2022). The geranylgeranyl pyrophosphate synthase encoding gene (AlGGPPS) and isopentenyl pyrophosphate isomerase encoding gene (AlIDI) from A. limacinum MYA-1381 were integrated into the de novo carotene biosynthetic pathway in Escherichia coli, which increased the carotenoid by 2.99-fold compared with the initial strain (Shi et al, 2023). This indicated the participation of the two novel genes functioned coordinately in the carotene biosynthesis and provided novel functional elements for carotenoid engineering improvements (Shi et al, 2023).…”

Transcending membrane barriers: advances in membrane engineering to enhance the production capacity of microbial cell factories

“…Advanced technologies such as gene editing [ 5 – 8 ], protein engineering [ 9 – 11 ], and dynamic genetic circuits [ 10 – 12 ] have been widely applied in the construction and performance optimization of microbial cell factories. Although traditional synthetic biology strategies focus on the construction and optimization of metabolic pathways, such as strengthening central and major metabolic pathways [ 13 – 15 ], improving the supply and turnover of cofactors [ 16 , 17 ], removing competitive metabolic bypaths, and enhancing metabolic flow [ 18 , 19 ], these methods have shown significant limitations in increasing the yield of many hydrophobic natural products (e.g., steroids, terpenoids, alkaloids, and liposoluble vitamins). In particular, many hydrophobic natural products tend to accumulate in biological membrane structures rather than being secreted into the culture medium, a tendency that, due to the limited space of the cell membrane structure, restricts the effective accumulation of target chemicals.…”