Engineering microbial consortia of Elizabethkingia meningoseptica and Escherichia coli strains for the biosynthesis of vitamin K2

Abstract

Background: The study and application of microbial consortia are topics of interest in the fields of metabolic engineering and synthetic biology. In this study, we report the design and optimisation of Elizabethkingia meningoseptica and Escherichia coli co-culture, which bypass certain limitations found during the molecular modification of E. meningoseptica, such as resistance to many antibiotics and fewer available molecular tools.

Results: The octaprenyl pyrophosphate synthase from E. meningoseptica sp. F2 (EmOPPS) was expressed, purified, and identified in the present study. Then, owing to the low vitamin K2 production by E. coli or E. meningoseptica sp. F2 monoculture, we introduced the E. meningoseptica and E. coli co-culture strategy to improve vitamin K2 biosynthesis. We achieved production titres of 32 mg/L by introducing vitamin K2 synthesis-related genes from E. meningoseptica sp. F2 into E. coli, which were approximately three-fold more than the titre achieved with E. meningoseptica sp. F2 monoculture. This study establishes a foundation for further engineering of MK-n (n = 4, 5, 6, 7, 8) in a co-cultivation system of E. meningoseptica and E. coli. Finally, we analysed the surface morphology, esterase activity, and membrane permeability of these microbial consortia using scanning electron microscopy, confocal laser scanning microscopy, and flow cytometry, respectively. The results showed that the co-cultured bacteria were closely linked and that lipase activity and membrane permeability improved, which may be conducive to the exchange of substances between bacteria.

Conclusions: Our results demonstrated that co-culture engineering can be a useful method in the broad field of metabolic engineering of strains with restricted molecular modifications.

Keywords: Elizabethkingia meningoseptica; Metabolic engineering; Microbial consortia; Vitamin K2.

Fig. 1

Production of vitamin K2 by the monoculture and co-culture systems of Escherichia coli and Elizabethkingia meningoseptica sp. F2. The error bars represent the standard error of at least three biological replicates. *Statistical significance (p < 0.05) compared to the monoculture system of E. coli and E. meningoseptica sp. F2

Fig. 2

Bioinformatic and SDS-PAGE analysis of EmOPPS. A Alignment of amino acid sequences of prenyltransferases. The black box on a colour background indicates similar amino acid residues. The two conserved DDXXD motifs are indicated. B SDS-PAGE analysis of recombinant protein levels in Escherichia coli BL21 (DE3) cells at 30 °C for 4 h. Lanes 1, 3, 5, 7: supernatant. Lanes 2, 4, 6, 8: precipitate

Fig. 3

Menaquinone-n (n = 4, 5, 6, 7, 8) production in the engineered Escherichia coli (J02 strains). LC–MS analysis of the production of J02 strains (A–C). A HPLC chromatograms of different MK-n B, C mass spectra of MK-n, MK-4 (m/z = 445), MK-5 (m/z = 513), MK-6 (m/z = 581), MK-7 (m/z = 649), and MK-8 (m/z = 717)

Fig. 3

Menaquinone-n (n = 4, 5, 6, 7, 8) production in the engineered Escherichia coli (J02 strains). LC–MS analysis of the production of J02 strains (A–C). A HPLC chromatograms of different MK-n B, C mass spectra of MK-n, MK-4 (m/z = 445), MK-5 (m/z = 513), MK-6 (m/z = 581), MK-7 (m/z = 649), and MK-8 (m/z = 717)

Fig. 4

Co-culture compatibility determination and upstream strain optimisation. A Optimisation of the inoculation ratios for the CO-1 cultivation system. Elizabethkingia meningoseptica and Escherichia coli were inoculated into the co-culture system at a ratio of 3:1, 3:2, 3:3, and 3:4 v/v. Statistical significance (p < 0.05) was determined by comparing with the initial inoculation ratio (3:1). B Different types of co-cultivation systems. The co-culture strains were inoculated at a ratio of 3:3. The error bars represent the standard error of at least three biological replicates. Statistical significance (p < 0.01) compared to the CO-1 cultivation system

Fig. 5

Design of Elizabethkingia meningoseptica and Escherichia coli co-culture system for vitamin K2 biosynthesis. (I) Mevalonate pathway: atoB acetoacetyl-CoA acetyltransferase; HMGS HMG-CoA synthase; HMGR HMG-CoA reductase; MK mevalonate kinase; MVD1 mevalonate pyrophosphate decarboxylase; idi IPP isomerase. MenA (1,4-dihydroxy-2-naphthoate octaprenyltransferase), MenG, and UbiE (Q-8/MK-8 biosynthesis methyltransferase) encoded the head structure biosynthesis enzymes that are involved in the vitamin K2 biosynthetic pathway starting from isochorismate. Gene names in red represent the overexpressed genes. (II) HPLC analysis of vitamin K2 products in E. meningoseptica and E. coli co-culture system

Fig. 6

Visualisation of different inoculates using scanning electron microscopy images. A Escherichia coli. Blue circles surround the connected E. coli cells in view. B Elizabethkingia meningoseptica sp. F2. Green circles surround the connected E. meningoseptica sp. F2 cells in view. C E. meningoseptica and E. coli co-culture. Red circles surround the connected E. meningoseptica and E. coli cells in view. D E. meningoseptica and E. coli co-culture treated with 1% POE. Yellow circles surround the connected E. meningoseptica and E. coli cells in view

Fig. 7

Flow cytometry dot plots of monoculture system of Escherichia coli (A), B and Elizabethkingia meningoseptica sp. F2 as a reference and C co-culture strains of E. meningoseptica and E. coli cells treated without or D with 1% POE