Combinatorial engineering for improved menaquinone-4 biosynthesis in Bacillus subtilis
Introduction
Menaquinones are a class of compounds found in various microorganisms and play important roles in sporulation, oxidative phosphorylation, and electron transport [1]. There are 14 types of menaquinones (MKs), which are named MK-n according to the number of isoprene units on the side chain [2]. The length of this tail varies depending on the microorganism [3]. In humans, the menaquinones are called vitamin K2 [4] and are the cofactors for the posttranslational conversion of glutamic acid residues of vitamin K dependent proteins. Menaquinones play important roles in blood coagulation, preventing osteoporosis [5], reducing cognitive diseases [6], preventing diabetes [7], mitigating cardiovascular calcification [8], and suppressing inflammation [9]. Therefore, vitamin K2 are widely used as drug treatments and dietary supplements in the food, health care, and pharmaceutical industries [10,11]. The chemical synthesis of MK-4 is challenging because of the need for stereoselectively synthesizing the all-trans configurations of biologically active molecules [12]. Therefore, biosynthetic MK-4 has attracted the attention of many researchers. Microbial production of MK-4 features the advantage of selectively producing all-trans isomer [13].
In previous studies, researchers have focused on the microorganisms such as B. subtilis natto [14], Flavobacterium sp. 238−7-K3−15 strain [15], and Pichia pastoris [16] to synthesize MK-4. The operating tools of B. subtilis natto and Flavobacterium sp. 238−7 were not mature, and there are few reports on metabolic engineering modification. Researchers have focused on culture conditions, medium composition, some additives, and random mutation screening techniques. Studies of P. pastoris were limited to expression of heterologous genes without metabolic modification. The quinone skeleton of MK-4 is synthesized by the variable side chain (C20) derived from isoprenoid pathway, and chorismate via the men gene cluster (menFDHBEC operon). Fig. 1 shows the MK-4 synthesis pathway, which includes shikimic acid (SA) pathway to supply the aromatic head [17], methylerythritol phosphate (MEP) pathway to assemble the C20 isoprenoid tail, and menaquinone pathway to make the final product MK-4 [18]. However, the MEP pathway was considered as rate-limiting for K2 production and has been intensively studied [19,20]. MEP module contains 8 genes including dxs (encoding 1-deoxyxylulose-5-phosphate synthase), dxr (encoding 1-deoxyxylulose-5-phosphate reductoisomerase), ispD (encoding 2-C-methylerythritol 4-phosphate cytidylyltransferase), ispE (encoding 4-diphosphocytidyl-2-C-methylerythritol kinase), ispF (encoding 2-C-methylerythritol 2,4-cyclodiphosphate synthase), ispG (encoding 4-hydroxy-3-methylbut-2-enyl diphosphate synthase), ispH (encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase), and idi (encoding isopentenyl-diphosphate delta-isomerase); Moreover, dxs and dxr encode the enzymes that have always been considered as rate-limiting enzymes in the MEP pathway [21]. Furthermore, menA encodes an enzyme that binds the aromatic head to the isoprenoid tail, and the reaction catalyzed by the enzyme MenA was also considered as rate-limiting for the biosynthesis of K2 [22].
B. subtilis is generally recognized as safe (GRAS), grows fast and has well-characterized genetics background, and many synthetic biology tools and specialized libraries are available for B. subtilis [23]. In this study, two key factors might affect the efficient synthesis of MK-4: 1) how to gradually convert IPP into GGPP to synthesize MK-4 is a challenge and 2) the supply of key precursor IPP is another challenge. In view of the above problems, we attempted to engineer B. subtilis 168 to effectively produce high value-added prenylated MK-4 through four pathways of metabolic engineering in this work, namely, reconstruction of MK-4 biosynthesis pathway, engineering MEP pathway, heterogeneous MVA isoprenoid pathway, and enhancing metabolic flux of menaquinone pathway. The resultant strain BY23 produced 120.1 ± 0.6 mg/L MK-4 in 250 mL shaker flasks and 145 ± 2.8 mg/L MK-4 in a 3-L bioreactor, and the results obtained here paves the way for the industrial production of MK-4 by microbial fermentation in the future.
Section snippets
Bacterial strains and medium
The strains with the genotypes of the constructed and engineered B. subtilis and primers used in this study were listed in Table 1, Table S1, respectively. The sequences of the heterologous encoding genes were listed in Table S2. B. subtilis 168 was used for host strain. The plasmids used in this study (p7Z6, p7Z6P43, p7C6P43, p7S6P43) replicate in E. coli, and antibiotic resistance were Zeor, Zeor, Cmr, and Spcr [24,25]. All strains were cultured in liquid Luria Bertani (LB) medium (per liter:
Reconstruction of MK-4 biosynthesis pathway in B. Subtilis
Though the biosynthesis pathway of MK-4 in the wild-type B. subtilis is complete, no MK-4 can be detected, maybe due to the low catalytic activities of key enzymes for MK-4 synthesis. Considering that menA gene encoding 1, 4-dihydroxy-2-naphtoic acid prenyltransferase and menG gene encoding demethylphylloquinol methyltransferase from Synechocystis sp. PCC 6803 are known to have catalytic activities for vitamin K synthesis [29], we introduced these two enzymes to strengthen MK-4 synthesis
Conclusions
In this study, Dxs, Dxr, and IspD-IspF from B. subtilis, CrtE and MenA from Synechocystis sp. PCC 6803 have been proved as rate-limiting enzymes in MEP pathway and menaquinone pathway of MK-4 synthesis. Moreover, the knockout of hepT gene could also significantly increase the production of MK-4. The final strain BY23, with the overexpression of the genes crtE, menA, menG, dxr, dxs, ispD-ispF, mvaK1, mvaK2, mvaD, mvaS, and mvaA, and knockout of hepT gene could produce 120.1 ± 0.6 mg/L of MK-4 in
Author agreement
All authors have seen and approved the final version of the manuscript being submitted. They warrant that the article is the authors' original work, hasn't received prior publication and isn't under consideration for publication elsewhere.
CRediT authorship contribution statement
Panhong Yuan: Conceptualization, Writing - original draft, Investigation, Visualization. Shixiu Cui: Methodology, Writing - review & editing. Yanfeng Liu: Methodology, Resources. Jianghua Li: Validation, Supervision. Xueqin Lv: Methodology, Writing - review & editing. Long Liu: Supervision, Writing - review & editing. Guocheng Du: Supervision, Project administration.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgement
This work was financially supported by the National Natural Science Foundation of China (31841484, 31840069, 21646119, and 31641845). Postgraduate Research & Practice Innovation Program of Jiangsu Provence (KYCX18_1486). The Fundamental Research Funds for the Central Universities (JUSRP51413B).
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