Comparative genomics analysis and transposon mutagenesis provides new insights into high menaquinone-7 biosynthetic potential of Bacillus subtilis natto.

This research combined Whole-Genome sequencing, intraspecific comparative genomics and transposon mutagenesis to investigate the menaquinone-7 (MK-7) synthesis potential in Bacillus subtilis natto. First, Whole-Genome sequencing showed that Bacillus subtilis natto BN-P15-11-1 contains one single circular chromosome in size of 3,982,436 bp with a GC content of 43.85 %, harboring 4,053 predicted coding genes. Next, the comparative genomics analysis among strain BN-P15-11-1 with model Bacillus subtilis 168 and four typical Bacillus subtilis natto strains proves that the closer evolutionary relationship Bacillus subtilis natto BN-P15-11-1 and Bacillus subtilis 168 both exhibit strong biosynthetic potential. To further dig for MK-7 biosynthesis latent capacity of BN-P15-11-1, we constructed a mutant library using transposons and a high throughput screening method using microplates. We obtained a YqgQ deficient high MK-7 yield strain F4 with a yield 3.02 times that of the parent strain. Experiments also showed that the high yield mutants had defects in different transcription and translation regulatory factor genes, indicating that regulatory factor defects may affect the biosynthesis and accumulation of MK-7 by altering the overall metabolic level. The findings of this study will provide more novel insights on the precise identification and rational utilization of the Bacillus subtilis subspecies for biosynthesis latent capacity.

Adaptive laboratory evolution for improved tolerance of vitamin K in Bacillus subtilis.

Menaquinone-7 (MK-7), a subtype of vitamin K2 (VK2), assumes crucial roles in coagulation function, calcium homeostasis, and respiratory chain transmission. The production of MK-7 via microbial fermentation boasts mild technological conditions and high biocompatibility. Nevertheless, the redox activity of MK-7 imposes constraints on its excessive accumulation in microorganisms. To address this predicament, an adaptive laboratory evolution (ALE) protocol was implemented in Bacillus subtilis BS011, utilizing vitamin K3 (VK3) as a structural analog of MK-7. The resulting strain, BS012, exhibited heightened tolerance to high VK3 concentrations and demonstrated substantial enhancements in biofilm formation and total antioxidant capacity (T-AOC) when compared to BS011. Furthermore, MK-7 production in BS012 exceeded that of BS011 by 76% and 22% under static and shaking cultivation conditions, respectively. The molecular basis underlying the superior performance of BS012 was elucidated through genome and transcriptome analyses, encompassing observations of alterations in cell morphology, variations in central carbon and nitrogen metabolism, spore formation, and antioxidant systems. In summation, ALE technology can notably enhance the tolerance of B. subtilis to VK and increase MK-7 production, thus offering a theoretical framework for the microbial fermentation production of other VK2 subtypes. Additionally, the evolved strain BS012 can be developed for integration into probiotic formulations within the food industry to maintain intestinal flora homeostasis, mitigate osteoporosis risk, and reduce the incidence of cardiovascular disease. KEY POINTS: • Bacillus subtilis was evolved for improved vitamin K tolerance and menaquinone-7 (MK-7) production • Evolved strains formed wrinkled biofilms and elongated almost twofold in length • Evolved strains induced sporulation to improve tolerance when carbon was limited.

Correction: Tang et al. Vitamin K2 Modulates Mitochondrial Dysfunction Induced by 6-Hydroxydopamine in SH-SY5Y Cells via Mitochondrial Quality-Control Loop. Nutrients 2022, 14, 1504.

In the original publication [...].

Insights into Regulating Mechanism of Mutagenesis Strains of Elizabethkingia meningoseptica sp. F2 by Omics Analysis.

Vitamin K2 plays an important role in electron transport, blood coagulation, and calcium homeostasis, and researchers have been trying to use microbes to produce it. Although our previous studies have shown that gradient radiation, breeding, and culture acclimation can improve vitamin K2 production in Elizabethkingia meningoseptica, the mechanism is still unclear. This study is the first which performs genome sequencing of E. meningoseptica sp. F2 as a basis for subsequent experiments and further comparative analyses with other strains. Comparative metabolic pathway analysis of E. meningoseptica sp. F2, E. coli, Bacillus subtilis, and other vitamin K2 product strains revealed that the mevalonate pathway of E. meningoseptica sp. F2 is different in bacteria at the system level. The expressions of menA, menD, menH, and menI in the menaquinone pathway and idi, hmgR, and ggpps in the mevalonate pathway were higher than those in the original strain. A total of 67 differentially expressed proteins involved in the oxidative phosphorylation metabolic pathway and citric acid cycle (TCA cycle) were identified. Our results reveal that combined gradient radiation breeding and culture acclimation can promote vitamin K2 accumulation probably by regulating the vitamin K2 pathway, oxidative phosphorylation metabolism pathway, and the citrate cycle (TCA cycle).

Identification of chitin synthase activator in Aspergillus niger and its application in citric acid fermentation.

The biosynthesis of citric acid (CA) using Aspergillus niger as a carrier is influenced by mycelium morphology, which is determined by the expression level of morphology-related genes. As a key component of the fungal cell wall, chitin content has an important effect on morphogenesis, and to investigate the effects of this on fermentation performance, we used RNA interference to knockdown chitin synthase C (CHSC) and chitin synthase activator (CHS3) to obtain the single-gene mutant strains A. niger chs3 and chsC and the double mutant A. niger chs3C. We found that the CA fermentation performance of the two single mutants was significantly better than that of the double mutant. The mutant A. niger chs3-4 exhibited CA production potential compared to that of the parent strain in scale-up fermentation; we determined certain characteristics of CA high-yielding strain fermentation pellets. In addition, when chsC alone was silenced, there was very little change in chs3 mRNA levels, whereas those of chsC were significantly reduced when only chs3 was silenced. As this may be because of a synergistic effect between chsC and chs3, and we speculated that the latent activation target of CHS3 is CHSC, our results confirmed this hypothesis. This study is the first application of a separation and combination silence strategy of chitin synthase and chitin synthase activator in the morphology of A. niger CA fermentation. Furthermore, it provides new insights into the method for the morphological study of A. niger fermentation and the interaction of homologous genes. KEY POINTS: • The function of chitin synthase C (chsC) and chitin synthase activator (chs3) is tightly interrelated. • Mycelial morphology was optimized by knockdown of CHS3, resulting in the overproduction of citric acid. • The separation and combination silence strategies are promising tools for the interaction of homologous housekeeping genes.

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis.

BACKGROUND: Menaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis. Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation. RESULTS: We used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01 mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion. CONCLUSIONS: In summary, a "high-efficiency, low-carbon, cofactor-recycling" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids.

Rational Design by Structural Biology of Industrializable, Long-Acting Antihyperglycemic GLP-1 Receptor Agonists.

Glucagon-like peptide-1 (GLP-1) is easily degraded by dipeptidyl peptidase-4 (DPP-4) in the human body, limiting its therapeutic effect on type II diabetes. Therefore, improving GLP-1 receptor agonist (GLP-1RA) stability is a major obstacle for drug development. We analyzed human GLP-1, DPP-4, and GLP-1 receptor structures and designed three GLP-1RAs, which were introduced into fusion protein fragments and changed in the overall conformation. This modification effectively prevented GLP-1RAs from entering the DPP-4 active center without affecting GLP-1RAs' ability to bind to GLP-1R, the new GLP-1RA hypoglycemic effect lasting for >24 h. Through molecular modeling, molecular dynamics calculation, and simulation, possible tertiary structure models of GLP-1RAs were obtained; molecular docking with DPP-4 and GLP-1R showed access to the fusion protein. The overall conformational change of GLP-1RAs prevented DPP-4 binding, without affecting GLP-1RAs' affinity to GLP-1R. This study provides important drug design ideas for GLP-1RA development and a new example for application of structural biology-based protein design in drug development.

Preparation of Cellulose-Based Flocculant and Its Application in the Enrichment of Vitamin K2 in Fermentation Supernatant.

Nutritional food supplements and pharmaceutical products produced with vitamin K2 as raw materials a very promising market in the global scope. The main production method of vitamin K2 is microbial fermentation, but approximately 50% of vitamin K2 synthesized by the main production strain Bacillus subtilis natto exists in extracellular form, which is not easy to separate and extract. In order to solve this problem, in this study, we synthesized a novel cellulose flocculant, MCC-g-LMA, by grafting reaction using microcrystalline cellulose (MCC) and lauryl methacrylate (LMA) as monomers, and ammonium persulfate as an initiator to flocculate VK2 from the fermentation supernatant. The flocculant was characterized by Fourier transform infrared spectroscopy (FTIR), elemental analysis, and scanning electron microscopy (SEM), and the grafting reaction was successful. When the flocculant dosage was 48.0 mg/L and pH was 5.0, the flocculation rate of the MCC-g-LMA on the fermentation supernatant reached 85.3%, and the enrichment rate of VK2 reached 90.0%. Furthermore, we explored the flocculation mechanism of VK2 by the MCC-g-LMA and speculated that the flocculation mechanism mainly included adsorption bridging, hydrophobic association and net trapping and sweep effect. In this study, the extraction method for trace high-value biological products in the fermentation supernatant was improved, which provided a method and theoretical basis for the efficient separation and purification of VK2 and other terpenoids.

Vitamin K2 Modulates Mitochondrial Dysfunction Induced by 6-Hydroxydopamine in SH-SY5Y Cells via Mitochondrial Quality-Control Loop.

Vitamin K2, a natural fat-soluble vitamin, is a potent neuroprotective molecule, owing to its antioxidant effect, but its mechanism has not been fully elucidated. Therefore, we stimulated SH-SY5Y cells with 6-hydroxydopamine (6-OHDA) in a proper dose-dependent manner, followed by a treatment of vitamin K2. In the presence of 6-OHDA, cell viability was reduced, the mitochondrial membrane potential was decreased, and the accumulation of reactive oxygen species (ROS) was increased. Moreover, the treatment of 6-OHDA promoted mitochondria-mediated apoptosis and abnormal mitochondrial fission and fusion. However, vitamin K2 significantly suppressed 6-OHDA-induced changes. Vitamin K2 played a significant part in apoptosis by upregulating and downregulating Bcl-2 and Bax protein expressions, respectively, which inhibited mitochondrial depolarization, and ROS accumulation to maintain mitochondrial structure and functional stabilities. Additionally, vitamin K2 significantly inhibited the 6-OHDA-induced downregulation of the MFN1/2 level and upregulation of the DRP1 level, respectively, and this enabled cells to maintain the dynamic balance of mitochondrial fusion and fission. Furthermore, vitamin K2 treatments downregulated the expression level of p62 and upregulated the expression level of LC3A in 6-OHDA-treated cells via the PINK1/Parkin signaling pathway, thereby promoting mitophagy. Moreover, it induced mitochondrial biogenesis in 6-OHDA damaged cells by promoting the expression of PGC-1α, NRF1, and TFAM. These indicated that vitamin K2 can release mitochondrial damage, and that this effect is related to the participation of vitamin K2 in the regulation of the mitochondrial quality-control loop, through the maintenance of the mitochondrial quality-control system, and repair mitochondrial dysfunction, thereby alleviating neuronal cell death mediated by mitochondrial damage.

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

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.

Cloning and functional characterization of the geranylgeranyl diphosphate synthase(GGPPS)from Elizabethkingia meningoseptica sp.F2.

To date, there is no functional characterization of EmGGPPS (from Elizabethkingia meningoseptica sp.F2) as enzymes catalyzing GGPP. In this research, maltose-binding protein (MBP), disulfide bond A (DbsA), disulfide bond C (DbsC), and two other small protein tags, GB1 (Protein G B1 domain) and ZZ (Protein A IgG ZZ repeat domain), were used as fusion partners to construct an EmGGPPS fusion expression system. The results indicated that the expression of MBP-EmGGPPS was higher than that of the other four fusion proteins in E. coli BL21 (DE3). Additionally, using EmGGPPS as a catalyst for the production of GGPP was verified using a color complementation assay in Escherichia coli. In parallel with it, the enzyme activity experiment in vitro showed that the EmGGPPS protein could produce GGPP, GPP and FPP. Finally, we successfully demonstrated MK-4 production in engineered E. coli by overexpression of EmGGPPS.

A study of hydrophobins-modified menaquinone-7 on osteoblastic cells differentiation.

Menaquinone-7 is involved in bone metabolism and can be used to prevent and treat osteoporosis. However, as a fat-soluble vitamin, menaquinone-7 has poor water solubility. As a surfactant, hydrophobins can change the affinity/hydrophobicity of the covered interface. In this study, menaquinone-7 was modified by hydrophobins, and the different addition ratios were explored. Moreover, Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and water contact angle (WCA) measurements indicated that hydrophobins effectively bind to menaquinone-7 and greatly increase the hydrophilicity of the surface of menaquinone-7. Studies on the metabolism of MC3T3-E1 cells showed that compared with native menaquinone-7, HGFI-modified menaquinone-7 can significantly promote osteoblast differentiation but inhibit osteoclast differentiation. Besides, the Mito-Tracker Green experiments show that HGFI-modified menaquinone-7 can significantly promote the activity of mitochondria in cells. These findings indicate that hydrophobins can be used as an effective biomaterial to modify menaquinone-7, promote the formation of osteoblasts, and better to bone balance.

Correction to: Combining mutagenesis on Glu281 of prenyltransferase NovQ and metabolic engineering strategies for the increased prenylated activity towards menadione.

The published online version contains some errors.

Combining mutagenesis on Glu281 of prenyltransferase NovQ and metabolic engineering strategies for the increased prenylated activity towards menadione.

Prenyltransferase NovQ is a vital class involved in the biosynthesis of secondary metabolites such as clorobiocin and novobiocin. To investigate the relationship between structure and catalytic properties of NovQ, here, we have analyzed the substrate-binding site, namely PT barrel, and revealed that menadione hydroquinol formed intermolecular interactions with the residue Glu281 near the center of the active pocket. In this study, Glu281 was substituted with 9 diverse amino acids and catalytic properties of mutants were observed in vitro. Among them, E281Q showed 2.05-fold activities towards the aromatic substrate and prenyl donor, while others obtained catalytic efficiency between 8.4 and 88.6% of that of wild-type NovQ. Furthermore, the effects of catalytic conditions and substrate status on the activity of NovQ and its mutants were considered to obtain the optimized prenylated reaction. When the evolutionary NovQ variant E281Q was overexpressed in the host constructed to synthesize dimethylallyl diphosphate through the engineered mevalonate (MVA) pathway, we harvested up to 4.7 mg/L prenylated menadione at C-3 position by exogenously supplying the aromatic substrate. The construction of the microbial platform based on NovQ opens a new orientation to further biosynthesize various vitamin K2 with other ABBA prenyltransferases in E. coli.

Synthesis of the carboxymethyl cellulose magnetic nanoparticles for efficient immobilization of prenyltransferase NovQ.

Prenyltransferase NovQ immobilized carboxymethyl cellulose magnetic nanoparticles (NCMNs) were successfully synthesized via a valuable approach integrated from nanocomposite preparation, and applied for the production of vitamin K2 using menadione hydroquinol and dimethylallyl diphosphate (DMAPP) as substrates. To investigate the interaction between nanoparticles and NovQ, we characterized the nanocomposite, and revealed that carboxymethyl cellulose (CMC) and Fe3O4 formed a core-shell structure to absorb NovQ in the reaction systems, resulting from the high affinity of immobilized materials. Meanwhile, NCMNs with excellent pH and temperature tolerance, enhanced prenylated activity, and improved stability were found. Molecular docking analysis was also conducted to justify the contribution of multiple amino acids and effect of nanoparticles on catalytic properties of NovQ. Taken together, our study introduces a promising strategy to prepare magnetic nanoparticles and improve the performance of catalyst, which aims for opening new orientations for synthesis of magnetic nanoparticles used for prenyltransferase immobilization.

Construction of a novel MK-4 biosynthetic pathway in Pichia pastoris through heterologous expression of HsUBIAD1.

BACKGROUND: With a variety of physiological and pharmacological functions, menaquinone is an essential prenylated product that can be endogenously converted from phylloquinone (VK1) or menadione (VK3) via the expression of Homo sapiens UBIAD1 (HsUBIAD1). The methylotrophic yeast, Pichia pastoris, is an attractive expression system that has been successfully applied to the efficient expression of heterologous proteins. However, the menaquinone biosynthetic pathway has not been discovered in P. pastoris. RESULTS: Firstly, we constructed a novel synthetic pathway in P. pastoris for the production of menaquinone-4 (MK-4) via heterologous expression of HsUBIAD1. Then, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (PGAP) appeared to be mostsuitable for the expression of HsUBIAD1 for various reasons. By optimizing the expression conditions of HsUBIAD1, its yield increased by 4.37 times after incubation at pH 7.0 and 24 °C for 36 h, when compared with that under the initial conditions. We found HsUBIAD1 expressed in recombinant GGU-23 has the ability to catalyze the biosynthesis of MK-4 when using VK1 and VK3 as the isopentenyl acceptor. In addition, we constructed a ribosomal DNA (rDNA)-mediated multi-copy expression vector for the fusion expression of SaGGPPS and PpIDI, and the recombinant GGU-GrIG afforded higher MK-4 production, so that it was selected as the high-yield strain. Finally, the yield of MK-4 was maximized at 0.24 mg/g DCW by improving the GGPP supply when VK3 was the isopentenyl acceptor. CONCLUSIONS: In this study, we constructed a novel synthetic pathway in P. pastoris for the biosynthesis of the high value-added prenylated product MK-4 through heterologous expression of HsUBIAD1 and strengthened accumulation of GGPP. This approach could be further developed and accomplished for the biosynthesis of other prenylated products, which has great significance for theoretical research and industrial application.

Effect of static magnetic field on morphology and growth metabolism of Flavobacterium sp. m1-14.

Increasing evidence shows that static magnetic fields (SMFs) can affect microbial growth metabolism, but the specific mechanism is still unclear. In this study, we have investigated the effect of moderate-strength SMFs on growth and vitamin K2 biosynthesis of Flavobacterium sp. m1-14. First, we designed a series of different moderate-strength magnetic field intensities (0, 50, 100, 150, 190 mT) and exposure times (0, 24, 48, 72, 120 h). With the optimization of static magnetic field intensity and exposure time, biomass and vitamin K2 production significantly increased compared to control. The maximum vitamin K2 concentration and biomass were achieved when exposed to 100 mT SMF for 48 h; compared with the control group, they increased by 71.3% and 86.8%, respectively. Interestingly, it was found that both the cell viability and morphology changed significantly after SMF treatment. Second, the adenosine triphosphate (ATP) and glucose-6-phosphate dehydrogenase (G6PDH) metabolism is more vigorous after exposed to 100 mT SMF. This change affects the cell energy metabolism and fermentation behavior, and may partially explain the changes in bacterial biomass and vitamin K2 production. The results show that moderate-strength SMFs may be a promising method to promote bacterial growth and secondary metabolite synthesis.

Improvement of menaquinone-7 production by Bacillus subtilis natto in a novel residue-free medium by increasing the redox potential.

Bacillus subtilis natto is a GRAS bacterium. Nattokinase, with fibrinolytic and antithrombotic activities, is one of the major products of this organism. It is being gradually recognized that B. subtilis natto can also be used as a biosynthetic strain for vitamin K2, which has phenomenal benefits, such as effects in the prevention of cardiovascular diseases and osteoporosis along with antitumor effects. Knocking out of the aprN gene by homologous recombination could improve the redox potential and slightly increase the concentration of MK-7. By detecting the change in redox potential during the growth of B. subtilis natto, a good oxygen supply and state of the cell membrane were found to be beneficial to vitamin K2 synthesis. A two-step RSM was used to optimize the operation parameters and substrate concentration in the new residue-free fermentation culture. The optimal conditions for the residue-free medium and control were determined. The optimum concentrations of soybean flour, corn flour, and peptone were 78.9, 72.4, and 24.8 g/L, respectively. The optimum rotational speed and volume of the culture medium using a shaking flask were 117 rpm and 10%, respectively. The state and composition of the cell membranes were more stable when engineered bacteria were cultured in this residue-free fermentation medium. Finally, the concentration of MK-7 increased by 37% to 18.9 mg/L, and the fermentation time was shortened by 24 h.

Evaluation of multiple fused partners on enhancing soluble level of prenyltransferase NovQ in Escherichia coli.

To obtain the soluble production of recombinant NovQ, it has been constructed into the pET28a system. Unfortunately, NovQ was mostly accumulated as inclusion bodies and existed in insoluble fractions of E. coli cell lysate. Four partners, namely His6, TrxA, GST and MBP, were investigated in fusion expression and co-expression to achieve soluble expression in E. coli strains BL21 (DE3) and Rosetta™ (DE3). MBP fusion expression revealed a forceful function in enhancing solubility compared with others, in which the soluble protein was approximately 70% of the total cellular proteins in E. coli. Improvement of rare tRNA abundance promoted the yield of total recombinant protein and the expression level of soluble protein. Besides, one-step purification method was applied and the purity of recombinant protein obtained using Ni-NTA resin was over 90%, where soluble recombinant MBP-NovQ was cleaved using TEV protease in vitro. This method could be an ideal method for soluble expression of ABBA prenyltransferases in E. coli.