Metabolic Engineering of Bacillus Amyloliquefaciens as A Novel Cell Factory to Produce Spermidine

doi:10.21203/rs.3.rs-66197/v1

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Metabolic Engineering of Bacillus Amyloliquefaciens as A Novel Cell Factory to Produce Spermidine

https://doi.org/10.21203/rs.3.rs-66197/v1

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Background: Spermidine is a biologically active polyamine with extensive application potential in foods and pharmaceuticals. However, previously reported spermidine titers by biosynthesis methods are relatively low, which hinders the industrial fermentation of spermidine. To improve the spermidine titer, key genes affecting the spermidine production were mined to engineer the Bacillus amyloliquefaciens.

Results: Genes of S-adenosylmethionine decarboxylase (speD) and spermidine synthase (speE) from different microorganisms were expressed and compared in B. amyloliquefaciens. Therein, the speD from Escherichia coli and speE from Saccharomyces cerevisiae were confirmed to be optimal for spermidine synthesis, respectively. Then, these two genes were co-expressed to generate an engineering strain B. amyloliquefaciens HSAM2(PDspeD-SspeE) with a spermidine titer of 91.31 mg/L, improving by 10.90-fold compared with the control (HSAM2). Through further optimization of fermentation medium, the spermidine titer was increased to 227.35 mg/L, which was the highest titer among present reports. Moreover, the consumption of the substrate S-adenosylmethionine was consistent with the accumulation of spermidine, which contributed to understanding the synthetic pattern of spermidine.

Conclusions: Two critical genes for spermidine synthesis were obtained, and an B. amyloliquefaciens cell factory was constructed for enhanced spermidine production, which laid the foundation for further industrial production of spermidine.

Applied & Industrial Microbiology

Bacillus amyloliquefaciens

spermidine

metabolic engineering

fermentation optimization.

Spermidine is a multifunctional polyamine with the positive charge and low molecular weight [1, 2]. Generally, spermidine can electrostatically bind with negatively charged biomolecules such as DNA and RNA, which can stabilize the biomolecules and promote the cell growth [3, 4]. Moreover, spermidine has been reported to induce cell autophagy to extend life-span and reduce cardiovascular diseases-related morbidity [5–8]. In addition, spermidine also shows versatile functions such as reducing neurodegeneration, delaying senile dementia, preventing heart aging, and treating type 2 diabetes [9–12]. Therefore, spermidine has promising application potential in the fields of foods and pharmaceuticals, and development of spermidine-related products was extremely valuable.

At present, spermidine is mainly produced by chemical synthesis, while it has several disadvantages such as high energy consumption, mass toxic by-product, and heavy environmental pollution. In comparison, the microbial fermentation method is safe, efficient and environmentally friendly [13]. Currently, many microorganisms have been reported to synthesize trace spermidine [14–16]. The core metabolic pathway for spermidine synthesis is shown in Fig. 1. Putrescine and S-adenosylmethionine (SAM) are the precursors for spermidine formation. Firstly, the SAM is catalyzed to decarboxylated SAM (dcSAM) by SAM decarboxylase encoded by speD gene. Then, the aminopropyl group of the dcSAM is transfered to putrescine to produce the spermidine, which is mediated by the spermidine synthase encoded by speE gene [17–19]. In Saccharomyces cerevisiae, the spermidine production was increased by over-expressing genes of ornithine decarboxylase, SAM decarboxylase and spermidine synthase and deleting genes of anti-ornithine decarboxylase and polyamine transporter [20, 21]. In Synechocystis sp, overexpression of arginine decarboxylase genes Adc1 and Adc2 could enhance the intracellular spermidine content [22]. However, previously reported fermentation titers of spermidine are relatively low, and more work is needed to improve the spermidine production, such as explaining the metabolic mechanism, mining key genes and engineering the strain.

B. amyloliquefaciens has been an efficient platform workhorse for production of various bioproducts [23–25]. The complete spermidine synthesis pathway exists in B. amyloliquefaciens. Moreover, the precursors of spermidine including putrescine and SAM have been highly produced in B. amyloliquefaciens after metabolic engineering [26, 27]. Therefore, the B. amyloliquefaciens has the potential to be developed as an efficient spermidine cell factory. However, the genes responsible for spermidine synthesis from putrescine and SAM have not been investigated in B. amyloliquefaciens. Herein, the downstream genes of spermidine synthesis including SAM decarboxylase gene (speD) and spermidine synthase gene (speE) were selected from different microorganisms, and their effects on spermidine production were evaluated in B. amyloliquefaciens. Two efficient genes affecting the spermidine production were obtained, and the spermidine production was enhanced dramatically by combined expression and fermentation optimization.

Effects of different SAM decarboxylase genes on spermidine production

SAM can be catalyzed to decarboxylated SAM (dcSAM) by SAM decarboxylase, which is a key step for spermidine synthesis. Therefore, efficient SAM decarboxylase genes are believed to enhance the spermidine production. Previously, we constructed a high SAM-producing strain B. amyloliquefaciens HSAM2 [26], which might provide abundant SAM substrate for spermidine synthesis. Therefore, the HSAM2 was used as the host strain to express SAM decarboxylase genes (speD) from different microorganisms. The speD genes from B. amyloliquefaciens HZ-12, S. cerevisiae CICC 31001 and E. coli DH5α were selected to construct recombinant expression strains, named HSAM2(PHspeD), HSAM2(PSspeD) and HSAM2(PDspeD) respectively. After expressing these genes, the spermidine titers were improved significantly in all recombinant strains (Fig.2a). Among them, the maximum spermidine titer of 91.91 mg/L was obtained in HSAM2(PDspeD), increasing by 8.49-fold compared with the control strain HSAM2(pHY300PLK). These results indicated that the speD gene from E. coli DH5α was the optimal gene for enhanced spermidine production.

Subsequently, the speD gene of E. coli was integrated into the genome of HASM2 by homologous recombination, resulting in the integrated expression strain HSAM2ːːDspeD. After fermentation for 60 h, the spermidine titer reached 33.87 mg/L, which was 2.40-fold higher than that of the control strain HSAM2 (Fig.2b). In comparison, the spermidine titer of the integrated expression strain HSAM2ːːDspeD was much lower than that of plasmid-based expression strain HSAM2(PDspeD). This phenomenon was probably due to the low gene copy number during integration expression, while the pHY300PLK plasmid had the high copy number [28]. Therefore, recombinant plasmid expression was more suitable for the speD gene.

Effects of different spermidine synthase genes on spermidine production

Spermidine synthase catalyzes the transfer of the aminopropyl group from dcSAM to putrescine to form the spermidine [29], which is considered to be a rate-limiting step in biosynthesis of spermidine [30]. It is particularly essential to exploit efficient spermidine synthase genes (speE). Therefore, different speE genes were evaluated. According to the KEGG database, three spermidine synthase genes from E. coli DH5α, C. glutamicum ATCC13032 and S. cerevisiae CICC31001 were selected and expressed in B. amyloliquefaciens HZMD, resulting in recombinant strains HZMD(PDspeE), HZMD(PGspeE), and HZMD(PSspeE), respectively.

As shown in Fig.3, expressing speE genes from E. coli and S. cerevisiae significantly improved the spermidine production, while the gene from C. glutamicum showed no significant difference. Therein, the maximum spermidine titer reached 49.58 mg/L in HZMD(PSspeE), with a 23% increase than that of the control strain HZMD(pHY300PLK). Previously, overexpression of the native speE gene was confirmed to be efficient for spermidine synthesis in S. cerevisiae[31]. Herein, our results demonstrated that the speE gene from S. cerevisiae also enhanced the spermidine production in B. amyloliquefaciens. It can be inferred that overexpression of this speE gene presumably increased the enzymatic activity of spermidine synthase to promote the spermidine synthesis.

Effect of co-expressing speD and speE genes on spermidine production

Above results showed that genes of speD from E. coli DH5α and speE from S. cerevisiae CICC31001 were efficient to enhance the spermidine production. Therefore, these two genes were ligated into one pHY300PLK plasmid, co-expressed in HSAM2 to generate a recombinant strain HSAM2(PDspeD-SspeE). Then, the control strain HSAM2(pHY300PLK), single gene expression strain HSAM2(PDspeD), and co-expression strain HSAM2(PDspeD-SspeE) were compared after fermentation for 60 h. As shown in Figure 4, the spermidine titer of HSAM2(PDspeD-SspeE) reached 105.24 mg/L at 60 h, further improving by 15% compared with the HSAM2(PDspeD). It indicated that co-expression of speD and speE was effective to increase the spermidine titer, which was probably due to that more upstream substrates of SAM and putrescine were converted to form spermidine.

Optimize fermentation medium

To further improve the spermidine titer of the engineered HSAM2(PDspeD-SspeE), the key components of fermentation medium were optimized, including carbon sources, nitrogen sources and antibiotics. Carbon sources were important for cell growth and metabolites synthesis [32, 33]. Firstly, effects of carbon sources types on spermidine production were investigated. As shown in Fig. 5a, the maximum titer of spermidine was obtained using xylose as the carbon source. Furthermore, the concentration of xylose was optimized (Fig.5b). The maximum titer of spermidine reached 151.79 mg/L when the xylose concentration was 40 g/L, and no significant increase was observed at 60 g/L of xylose.

Corn pulp was a nutrition-rich nitrogen source, and effects of different corn pulp concentrations on spermidine production were investigated. As was indicated in Fig. 5c, the corn pulp concentration significantly affected the spermidine titer, and the maximum spermidine titer was obtained at 20 g/L. Several previous studies investigated the impact of antibiotics on the fermentation process [34-36]. Herein, different concentrations of tetracycline were added into medium. The increased tetracycline concentration resulted in the improved spermidine titer, and no further increase was noted when the tetracycline concentration reached 4 mg/mL (Fig.5d). These results indicated that adding tetracycline could improve spermidine production, which was probably due to that more plasmids could be maintained under the tetracycline stress.

The spermidine production process under the optimized fermentation midium

Under the optimized fermentation medium (40 g/L xylose, 10 g/L peptone, 20 g/L corn pulp, 2 g/L urea, 6.3 g/L (NH4)₂SO₄, 2.5 g/L NaCl, 3 g/L KH₂PO₄, and 4.2 g/L MgSO₄·7H₂O), the spermidine production process was investigated (Fig. 6). The spermidine was synthesized as the cell grew at the early stage of fermentation. The cell growth entered into the stationary phase at about 48 h, while the spermidine was further synthesized until 84 h, indicating that the spermidine synthesis was a partly growth-coupled process. At 84 h, the maximum spermidine titer reached 227.35 mg/L, which was currently the highest titer reported by microbial fermentation. SAM was the key precursor for spermidine synthesis [5, 37], and the SAM concentration was also measured to further understand the fermentation process. In the initial 24 h stage, the SAM was accumulated rapidly to reach the highest point, while the spermidine concentration did not have obvious improvement. When the fermentation time exceeded 24 h, the spermidine production showed a rapid increase accompanied by a sharp drop in the SAM concentration, indicating that the SAM was probably consumed to synthesize spermidine.

This study explored the possibility of synthesizing spermidine via engineering the B. amyloliquefaciens. Effects of different speD and speE genes on spermidine production were investigated in B. amyloliquefaciens. Two efficient genes of speD and speE were mined to enhance the spermidine production. Subsequently, these two genes were co-expressed to construct an engineering strain HSAM2(PDspeD-SspeE) with high spermidine production. After further optimization of the key medium components, the maximum spermidine titer of 227.35 mg/L was obtained, which was the highest titer among the microbial fermentation methods. Moreover, the detected SAM consumption also explained the accumulation of spermidine. This study excavated the key genes for spermidine synthesis and successfully acquired the highest spermidine titer, which provided the reference for breeding the industrial strain for spermidine production in the future.

Strains and plasmids

Table 1 listed all the strains and plasmids constructed in present study. All engineering B. amyloliquefaciens strains were modified from the wild-type strain B. amyloliquefaciens HZ-12 (M 2015234). Escherichia coli DH5α was used as the platform strain to construct expression and integration vectors based on plasmids of pHY300PLK and T2(2)-ori [38]. All designed primers in this study were showed in supplementary material (Table S1).

Table 1

Strains and plasmids used in this study.
Strains or plasmids	Characteristics	Source
B. amyloliquefaciens
HSAM2	HZ-12 integrated with SAM2, metA and metB, and deficient in mccA and sucC	Stored in lab
HSAM2(pHY300PLK)	HSAM2 harboring the plasmid pHY300PLK	This study
HSAM2(PHspeD)	HSAM2 harboring the plasmid PHspeD	This study
HSAM2(PSspeD)	HSAM2 harboring the plasmid PSspeD	This study
HSAM2(PDspeD)	HSAM2 harboring the plasmid PDspeD	This study
HSAM2ːːDspeD	HSAM2 integrated with speD from E. coli DH5α	This study
HZMD	HZ-12 integrated with SAM2 and DspeD	Stored in lab
HZMD(pHY300PLK)	HZMD harboring the plasmid pHY300PLK	This study
HZMD (PHspeE1)	HZMD harboring the plasmid PHspeE	This study
HZMD (PSspeE2)	HZMD harboring the plasmid PSspeE	This study
HZMD (PDspeE3)	HZMD harboring the plasmid PDspeE	This study
HSAM2(PDspeD-SspeE)	HSAM2 harboring the plasmid PDspeD-SspeE	This study
E.coli DH5α	F⁻ Φ80d/lacZΔM15, Δ(lacZYA-argF) U169, recA1, endA1, hsdR17 (r_K⁻, m_K⁺), phoA, supE44, λ⁻, thi-1, gyrA96, relA1	Stored in lab
S. cerevisiae CICC 31001	Wild type	Stored in lab
B. subtilis 168	Wild type	Stored in lab
B. licheniformis WX-02	Wild type	Stored in lab
plasmids
pHY300PLK	E. coli-Bacillus shuttle vector for gene expression, Ap^r, Tet^r	Stored in lab
PHspeD	pHY300PLK + P43 + TamyL + speD from HZ-12	This study
PSspeD	pHY300PLK + P43 + TamyL + speD from S. cerevisiae	This study
PDspeD	pHY300PLK + P43 + TamyL + speD from E. coli DH5α	This study
PDspeE	pHY300PLK + P43 + TamyL + speE from E. coli DH5α	This study
PGspeE	pHY300PLK + P43 + TamyL + speE from C. glutamicum	This study
PSspeE	pHY300PLK + P43 + TamyL + speE from S. cerevisiae	This study
PDspeD-SspeE	pHY300PLK + P43 + TamyL + speD from E. coli DH5α + P43 + TamyL + speE from S. cerevisiae	This study
T2(2)-ori	E. coli-Bacillus shuttle vector for gene knockout or integration; Kan^r	Stored in lab
T2-PDspeD	T2 (2) + A + B + P43 + TamyL + speD from E. coli DH5α	This study

Culture conditions

Cells were picked up from LB solid plates (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl and 15 g/L agar), and transferred into LB liquid medium to culture for 12 h at 37°C and 180 rpm to obtain seed cultures. Then, the inoculum was pipetted into the fermented medium. The initial fermentation medium contained 40 g/L sucrose, 3 g/L aspartate, 10 g/L peptone, 5 g/L corn pulp, 2 g/L urea, 6.3 g/L (NH₄)₂SO₄, 2.5 g/L NaCl, 3 g/L KH₂PO₄, 4.2 g/L MgSO₄·7H₂O, and pH 6.5. The inoculum size was 3% (v/v) and the tetracycline concentration was 8 µg/mL. The optimized fermentation medium was consisted of 40 g/L xylose, 10 g/L peptone, 20 g/L corn pulp, 2 g/L urea, 6.3 g/L (NH4)₂SO₄, 2.5 g/L NaCl, 3 g/L KH₂PO₄, and 4.2 g/L MgSO₄·7H₂O, 4 µg/mL tetracycline, and pH 6.5. Fermentation was carried out for 60 h at 37°C with shaking at 180 rpm.

Recombinant plasmid expression

Recombinant plasmid expression was carried out based on the procedures reported previously [39, 40]. The speD gene of B. amyloliquefaciens HZ-12 (named as BspeD) was amplified using primers BspeD-F and BspeD-R. By Splicing with Overlapping Extension PCR (SOE-PCR), the BspeD fragment was ligated with the P43 promoter from Bacillus subtilis 168 and the TamyL terminator from Bacillus licheniformis WX-02 to obtain gene expression module. Then, this module was inserted into the Bacillus-E. coli shuttle plasmid (pHY300PLK) at restriction sites of BamHI/XbaI to obtain the expression vector PBspeD1, which was subsequently electro-transformed into B. amyloliquefaciens HSAM2 competent cells, resulting in the recombination strain named as HSAM2(PBspeD). Other genes were expressed in pHY300PLK plasmids following the same method.

Homologous recombination

T2(2)-ori mediated homologous recombination was employed in gene integration expression [39, 40]. The upstream and downstream homology arms were amplified using primers A-F/A-R and B-F/B-R, respectively, which were further fused with the expression module of EspeD gene by SOE-PCR. Then, the fused fragment was ligated into the T2(2)-ori plasmid at BamHI/XbaI, resulting in the integrated vector T2(2)-EspeD. Subsequently, this plasmid was electro-transformed into B. amyloliquefaciens HSAM2 competent cells, which were cultured in LB plates containing 20 μg/mL of kanamycin. After verification by PCR, positive clones were inoculated into kanamycin-containing LB liquid medium (20 μg/mL), and cultured at 45°C for 8 h at 180 rpm. Single-crossover strains were selected by kanamycin resistance screening and PCR identification, subcultured in LB liquid medium for several times at 37°C (8h). The final cultures were diluted and incubated on LB plates to obtain individual colonies, which were transferred into LB and kanamycin-containing LB plates to screen kanamycin-sensitive colonies. The double-crossover strain was obtained by PCR verification.

Determination of spermidine

A volume of 1.5 mL HClO₄ aqueous solution (0.4 M) was added into 0.5 mL fermentative sample, extracted for 1 h. By centrifugation for 10 min at 12,000×g, 250 μL supernatant was collected, and mixed with 25 μL of 2 M NaOH, 75 μL of saturated NaHCO₃, and 500 μL of 5 mg/mL dansylchloride. Then, the mixture was reacted at 50°C for 45 min, added with 25 μL of 25% NH₄OH to remove the residual dansyl chloride by incubating for 15 min at 50°C. Finally, the mixture volume was added to 1.5 mL with acetonitrile. After centrifugation for 5 min at 2500×g, the supernatant was collected and filtered through a 0.22 μm membrane for HPLC (high-performance liquid chromatography) analysis, which was carried out using an Agilent 1260 HPLC system with an Agilent column Zorbax Eclipse XDB-C18 (4.6 mm×250 mm, 5 μm) at 30°C. The separation was achieved using a linear gradient of mobile phase A (acetonitrile) and B (H₂O) at a flow rate of 1 mL/min. The solvent gradient was as follows: 50% A (0–3 min), 50–90% A (3–20 min), 90% A (20–29 min), 90–50% A (29–32 min), and 50% A (32–35 min). The detection was carried out at 254 nm [41].

Determination of SAM

The SAM was detected by previously reported HPLC method [26] . For sample pretreatment, 1.5 mL of 0.4 M HClO₄was added into 500 μL fermentation broth to extract the total SAM for 1 h, vortexing for 10 s every 15 min. After centrifugation for 10 min at 12,000×g, 800 μL supernatant was collected and mixed with 95 μL 2 M NaOH and 15 μL saturated NaHCO₃. Then, the mixture was centrifuged for 5 min at 2500×g to obtain the supernatant, which was further filtered through a 0.22 μm membrane for HPLC analysis. The sample was analyzed on an Agilent 1260 HPLC system (Agilent, USA) with a Zorbax Eclipse XDB-C18 column (4.6 mm×250 mm, 5 μm). The ratio of mobile phases A (methanol) and B (40 mM NH₄H₂PO₂ solution containing 2 mM sodium heptane sulfonate) was set as 18:82, and the flow speed was controlled at 0.8 mL/min. The column temperature was set at 30°C, and the UV detection wavelength was controlled at 254 nm.

Statistical analysis

All fermentation experiments were conducted at least in triplicate, and data were calculated to obtain the mean value and standard deviation. The t test was carried out with Data Processing System (DPS) 7.05 to evaluate the significance of difference at the 95% confidence level.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by the Open Funds of Beijing Advanced Innovation Center for Food Nutrition and Human Health, and National Key Research and Development Program of China (No. 2018YFD0401200 and No. 2019YFA0906400).

Authors' contributions

X. Wei and Y. Liu designed this study, and contributed reagents and materials. D. Zou, L. Li, Y. Min and A. Ji conducted the experimental work. D. Zou analyzed the data. X. Wei, Y. Liu and D. Zou wrote and revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Haque SM, Ghosh B. Micropropagation of Kaempferia angustifolia Roscoe - An Aromatic, Essential Oil Yielding, Underutilized Medicinal Plant of Zingiberaceae Family. Journal of Crop Science & Biotechnology. 2018;21:147-153.
Jagu E, Pomel S, Pethe S, Loiseau PM, Labruère R. Polyamine-based analogs and conjugates as antikinetoplastid agents. European Journal of Medicinal Chemistry. 2017;139:982-1015.
Kusano T, Berberich T, Tateda C, Takahashi Y. Polyamines: essential factors for growth and survival. Planta. 2008;228:367-381.
Jie L, Christine F, Adrian P, Lionel H, Paul B, Katherine E, Fairhurst SA, Cathie M, Michael AJ. A novel polyamine acyltransferase responsible for the accumulation of spermidine conjugates in Arabidopsis seed. Plant Cell. 2009;21:318-333.
Madeo F, Eisenberg T, Pietrocola F, Kroemer G. Spermidine in health and disease. Science. 2018;359:6374.
Eisenberg T, Knauer H, Schauer A, Büttner S, Ruckenstuhl C, Carmona-Gutierrez D, Ring J, Schroeder S, Magnes C, Antonacci L. Induction of autophagy by spermidine promotes longevity. Nature cell biology. 2009;11:1305.
Soda K, Kano Y, Chiba F. Food Polyamine and Cardiovascular Disease -An Epidemiological Study. Glob J Health Sci. 2012;4:170-178.
Eisenberg T, Abdellatif M, Schroeder S, Primessnig U, Stekovic S, Pendl T, Harger A, Schipke J, Zimmermann A, Schmidt A. Cardioprotection and lifespan extension by the natural polyamine spermidine. Nat Med. 2016;22:1428.
Büttner S, Broeskamp F, Sommer C, Markaki M, Habernig L, Alavianghavanini A, Carmonagutierrez D, Eisenberg T, Michael E, Kroemer G. Spermidine protects against α-synuclein neurotoxicity. Cell Cycle. 2014;13:3903-3908.
Gupta VK, Lisa S, Tobias E, Sara M, Anuradha B, Koemans TS, Kramer JM, Liu KSY, Sabrina S, Stunnenberg HG. Restoring polyamines protects from age-induced memory impairment in an autophagy-dependent manner. Nature Neuroscience. 2013;16:1453.
Yue F, Li W, Zou J, Jiang X, Xu G, Huang H, Liu L. Spermidine prolongs lifespan and prevents liver fibrosis and hepatocellular carcinoma by activating MAP1S-mediated autophagy. Cancer Research. 2017;77:2938-2951.
Pichiah PB, Tirupathi, Suriyakalaa U, Kamalakkannan S, Kokilavani P, Kalaiselvi S, Sankarganesh D, Gowri J, Archunan G, Youn-Soo C, Achiraman S. Spermidine may decrease ER stress in pancreatic beta cells and may reduce apoptosis via activating AMPK dependent autophagy pathway. Medical Hypotheses. 2011;77:677-679.
Fan J, Ye H, Zhou ZJ, Zhao X. Research Progress on Production of Sugar Alcohols by Microbial Fermentation. Food & Fermentation Technology. 2013.
Qian ZG, Xia XX, Lee SY. Metabolic engineering of Escherichia coli for the production of putrescine: a four carbon diamine. Biotechnology and bioengineering. 2009;104:651-662.
Sekowska A, Bertin P, Danchin A. Characterization of polyamine synthesis pathway in Bacillus subtilis 168. Molecular microbiology. 1998;29:851-858.
Sekowska A, Coppée JY, Le Caer JP, Martin Verstraete I, Danchin A. S‐adenosylmethionine decarboxylase of Bacillus subtilis is closely related to archaebacterial counterparts. Molecular microbiology. 2000;36:1135-1147.
Torrigiani P, Bregoli AM, Ziosi V, Costa G. Molecular and biochemical aspects underlying polyamine modulation of fruit development and ripening. Stewart Postharvest Review. 2008;4:1-12.
Cona A, Rea G, R, Federico R, Tavladoraki P. Functions of amine oxidases in plant development and defence. Trends in Plant Science. 2006;11:80-88.
Imamura T, Fujita K, Tasaki K, Higuchi A, Takahashi H. Characterization of spermidine synthase and spermine synthase – The polyamine-synthetic enzymes that induce early flowering in Gentiana triflora. Biochemical & Biophysical Research Communications. 2015;463:781-786.
Kim SK, Jin YS, Choi IG, Park YC, Seo JH. Enhanced tolerance of Saccharomyces cerevisiae to multiple lignocellulose-derived inhibitors through modulation of spermidine contents. Metab Eng. 2015;29:46-55.
Kim SK, Jo JH, Park YC, Jin YS, Seo JH. Metabolic engineering of Saccharomyces cerevisiae for production of spermidine under optimal culture conditions. Enzyme & Microbial Technology. 2017;101:30-35.
Kera K, Nagayama T, Nanatani K, Saeki-Yamoto C, Tominaga A, Souma S, Miura N, Takeda K, Kayamori S, Ando E, et al. Reduction of Spermidine Content Resulting from Inactivation of Two Arginine Decarboxylases Increases Biofilm Formation in Synechocystis sp. Strain PCC 6803. J Bacteriol. 2018;200:9.
Feng J, Gu Y, Quan Y, Cao M, Gao W, Zhang W, Wang S, Yang C, Song C. Improved poly-γ-glutamic acid production in Bacillus amyloliquefaciens by modular pathway engineering. Metabolic Engineering. 2015;32:106-115.
Feng J, Gu Y, Quan Y, Zhang W, Cao M, Gao W, Song C, Yang C, Wang S. Recruiting a new strategy to improve levan production in Bacillus amyloliquefaciens. Scientific Reports. 2015;5:13814-13814.
Xu J, Yan W, Zhang W. Enhancing menaquinone-7 production in recombinant Bacillus amyloliquefaciens by metabolic pathway engineering. RSC Advances. 2017;7:28527-28534.
Ruan L, Li L, Zou D, Jiang C, Wen Z, Chen S, Deng Y, Wei X. Metabolic engineering of Bacillus amyloliquefaciens for enhanced production of S -adenosylmethionine by coupling of an engineered S -adenosylmethionine pathway and the tricarboxylic acid cycle. Biotechnology for Biofuels. 2019;12:1-12.
Li L, Zou D, Ji A, He Y, Liu Y, Deng Y, Chen S, Wei X. Multilevel Metabolic Engineering of Bacillus amyloliquefaciens for Production of the Platform Chemical Putrescine from Sustainable Biomass Hydrolysates. ACS Sustainable Chemistry & Engineering. 2020;8:2147-2157.
Uchida I, Makino Si, Sekizaki T, Terakado N. Cross‐talk to the genes for Bacillus anthracis capsule synthesis by atxA, the gene encoding the frans‐activator of anthrax toxin synthesis. Molecular microbiology. 1997;23:1229-1240.
Zou D, Min Y, Liu Y, Wei X, Wang J. Identification of a Spermidine Synthase Gene from Soybean by Recombinant Expression, Transcriptional Verification, and Sequence Analysis. Journal of Agricultural and Food Chemistry. 2020;68:2366-2372.
Imai A, Matsuyama T, Hanzawa Y, Akiyama T, Tamaoki M, Saji H, Shirano Y, Kato T, Hayashi H, Shibata D. Spermidine synthase genes are essential for survival of Arabidopsis. Plant Physiology. 2004;135:1565-1573.
Kim SK, Jin YS, Choi IG, Park YC, Seo JH. Enhanced tolerance of Saccharomyces cerevisiae to multiple lignocellulose-derived inhibitors through modulation of spermidine contents. Metabolic Engineering. 2015;29:46-55.
Mohammadkazemi F, Azin M, Ashori A. Production of bacterial cellulose using different carbon sources and culture media. Carbohydrate polymers. 2015;117:518-523.
Ruka DR, Simon GP, Dean KM. Altering the growth conditions of Gluconacetobacter xylinus to maximize the yield of bacterial cellulose. Carbohydrate Polymers. 2012;89:613-622.
Álvarez JA, Otero L, Lema J, Omil F. The effect and fate of antibiotics during the anaerobic digestion of pig manure. Bioresource technology. 2010;101:8581-8586.
Bauer A, Lizasoain J, Nettmann E, Bergmann I, Mundt K, Klocke M, Rincón M, Amon T, Piringer G. Effects of the antibiotics chlortetracycline and enrofloxacin on the anaerobic digestion in continuous experiments. BioEnergy Research. 2014;7:1244-1252.
Sara P, Michele P, Maurizio C, Luca C, Fabrizio A. Effect of veterinary antibiotics on biogas and bio-methane production. International Biodeterioration & Biodegradation. 2013;85:205-209.
Hamana K, Matsuzaki S. Polyamines as a chemotaxonomic marker in bacterial systematics. Critical reviews in microbiology. 1992;18:261-283.
Cai D, Wei X, Qiu Y, Chen Y, Chen J, Wen Z, Chen S. High-level Expression of nattokinase in Bacillus licheniformis by manipulating signal peptide and signal peptidase. Journal of Applied Microbiology. 2016;121:704.
Wei X, Zhou Y, Chen J, Cai D, Wang D, Qi G, Chen S. Efficient expression of nattokinase in Bacillus licheniformis : host strain construction and signal peptide optimization. Journal of Industrial Microbiology & Biotechnology. 2015;42:287-295.
Qiu Y, Zhang J, Li L, Wen Z, Nomura CT, Wu S, Chen S. Engineering Bacillus licheniformis for the production of meso -2,3-butanediol. Biotechnology for Biofuels. 2016;9:117.
Lu L, Liying R, Anying J, Zhiyou W, Shouwen C, Ling W, Xuetuan W. Biogenic amines analysis and microbial contribution in traditional fermented food of Douchi. Scientific Reports. 2018;8:1.

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Metabolic Engineering of Bacillus Amyloliquefaciens as A Novel Cell Factory to Produce Spermidine

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