Expanding the CRISPR Toolbox for Engineering Lycopene Biosynthesis in Corynebacterium glutamicum

Abstract

Lycopene represents one of the central compounds in the carotenoid pathway and it exhibits a potent antioxidant ability with wide potential applications in medicine, food, and cosmetics. The microbial production of lycopene has received increasing concern in recent years. Corynebacterium glutamicum (C. glutamicum) is considered to be a safe and beneficial industrial production platform, naturally endowed with the ability to produce lycopene. However, the scarcity of efficient genetic tools and the challenge of identifying crucial metabolic genes impede further research on C. glutamicum for achieving high-yield lycopene production. To address these challenges, a novel genetic editing toolkit, CRISPR/MAD7 system, was established and developed. By optimizing the promoter, ORI and PAM sequences, the CRISPR/MAD7 system facilitated highly efficient gene deletion and exhibited a broad spectrum of PAM sites. Notably, 25 kb of DNA from the genome was successfully deleted. In addition, the CRISPR/MAD7 system was effectively utilized in the metabolic engineering of C. glutamicum, allowing for the simultaneous knockout of crtEb and crtR genes in one step to enhance the accumulation of lycopene by blocking the branching pathway. Through screening crucial genes such as crtE, crtB, crtI, idsA, idi, and cg0722, an optimal carotenogenic gene combination was obtained. Particularly, cg0722, a membrane protein gene, was found to play a vital role in lycopene production. Therefore, the CBIEbR strain was obtained by overexpressing cg0722, crtB, and crtI while strategically blocking the by-products of the lycopene pathway. As a result, the final engineered strain produced lycopene at 405.02 mg/L (9.52 mg/g dry cell weight, DCW) in fed-batch fermentation, representing the highest reported lycopene yield in C. glutamicum to date. In this study, a powerful and precise genetic tool was used to engineer C. glutamicum for lycopene production. Through the modifications between the host cell and the carotenogenic pathway, the lycopene yield was stepwise improved by 102-fold as compared to the starting strain. This study highlights the usefulness of the CRISPR/MAD7 toolbox, demonstrating its practical applications in the metabolic engineering of industrially robust C. glutamicum.

Keywords: CRISPR/MAD7; Corynebacterium glutamicum; lycopene production; metabolic engineering.

Figure 1

Establishment and optimization of CRISPR/MAD7 system in C. glutamicum. (a) The single plasmid-based CRISPR/MAD7 system is assisted by RecE/T for genome editing. The pEC series plasmid expressing MAD7, crRNA, and homologous arms with Kn^r. The RecE/T was carried by a pEC plasmid with Cm^r and under the control of an inducible promoter Ptrc. A total of 1 mmol/L of IPTG is used to induce RecE/T and MAD7 expression. (b) Transformants of C. glutamicum cells expressing the nuclease, with or without the combined expression of RecE/T, homology arm, and crRNA targeting crtI. (c) Transformants and editing efficiency of MAD7 nuclease under different promoters and expression plasmids targeting crtI. (d) The effect of the various PAM sequences on the efficiency and transformants of editing the crtI (red part represents the PAM sequence). (e) The effect of the various PAM sequences on the efficiency and transformants of editing the crtEb (red part represents the PAM sequence). (f) The effect of the various PAM sequences on the efficiency and transformants of editing the upp (red part represents the PAM sequence). Pj23119, a synthetic constitutive expression promoter; Ptrc and Ptac were inducible promoters; PlacM, a modified lac constitutive expression promoter; Ptuf, a strong constitutive promoter; lacIq, lac repressor of E. coli; Cm^r, chloramphenicol resistant; Kn^r, kanamycin resistant; IPTG, Isopropyl β-D-Thiogalactoside. CFUs, colony forming units. Data are analyzed using two-tailed t-test, ** p < 0.01.

Figure 2

Large DNA fragment deletion by CRISPR/MAD7 system in C. glutamicum. (a) Schematic representation of 5 kb, 20 kb, and 25 kb genome DNA fragment deletion in C. glutamicum. (b) Editing efficiency and transformants of 5 kb, 20 kb, and 25 kb genome large DNA fragment deletion by MAD7. (c) Comparison of the editing efficiency and transformants of 20 kb genome large DNA fragment deletion by MAD7 and FnCpf1, respectively. Data are analyzed using two-tailed t-test, * p < 0.05.

Figure 3

Scheme for site mutation via the CRISPR/MAD7-RecT system in C. glutamicum. (a) crRNA guiding MAD7 to cleave genomic DNA and produce DSBs that can be repaired by RecT and ssDNA. (b) Sequencing results of the crtE gene in the WT (C. glutamicum ATCC 13032) and MT (C. glutamicum ATCC 13032-crtE mutation) strains; nucleotides in red box represent the stop codons (TAA) introduced by ssDNA-directed recombineering. (c) Number of transformants and editing efficiency of the point mutation by CRISPR/MAD7-RecT system using dsDNA or various ssDNA.

Figure 4

Metabolic engineering of the lycopene pathway through single gene deletion and multiple gene overexpression. (a) Scheme of the lycopene biosynthesis pathway in C. glutamicum. Lycopene is distributed in lipid structures. (b) Lycopene yield of genes knockout using BHIS medium (2 g/L glucose was added). (c) A flow-chart for removing genome editing plasmids in C. glutamicum. (d) Lycopene production in C. glutamicum-∆crtEb with a combination of various genes using BHIS medium (2 g/L glucose was added).

Figure 5

Metabolic engineering of the lycopene pathway through dual gene deletion and multiple gene overexpression. (a) crRNA array guiding MAD7 to cleave genomic DNA and produce two DSBs that can be repaired by RecE/T and dsDNA. (b) The number of transformants and editing efficiency of the double gene knockout by CRISPR/MAD7 system using dsDNA. (c) Lycopene production of ∆crtEb∆crtR strain using BHIS medium (2 g/L glucose was added). (d) Lycopene production of WT strain and CBIEbR strain using BHIS medium (2 g/L glucose was added). Data are analyzed using two-tailed t-test, ** p < 0.01, *** p < 0.001.

Figure 6

High-Density fermentation for lycopene production. (a) Time courses showing changes for CBIEbR strain in lycopene production and cell growth (OD₆₀₀) during fed-batch fermentation. (b) The red liquid in the bottles was the lycopene fed-batch fermentation broth.