De Novo Biosynthesis of Î²-Valienamine in Engineered Streptomyces

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Letter

De novo biosynthesis of #-valienamine in engineered Streptomyces hygroscopicus 5008 Li Cui, Ying Zhu, Xiaoqing Guan, Zixin Deng, Linquan Bai, and Yan Feng ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00138 • Publication Date (Web): 05 Oct 2015 Downloaded from http://pubs.acs.org on October 6, 2015

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ACS Synthetic Biology

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De novo biosynthesis of β-valienamine in engineered

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Streptomyces hygroscopicus 5008

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Li Cui, Ying Zhu, Xiaoqing Guan, Zixin Deng, Linquan Bai,* and Yan Feng*

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State Key Laboratory of Microbial Metabolism, School of Life Science &

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Biotechnology, and Joint International Research Laboratory of Metabolic &

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Developmental Sciences, Shanghai Jiao Tong University, Shanghai 200240,

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China

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ABSTRACT: The C7N aminocyclitol β-valienamine is a lead compound for the

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development of new biologically active β-glycosidase inhibitors as chemical

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chaperone therapeutic agents for lysosomal storage diseases. Its chemical

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synthesis is challenging due to the presence of multi-chiral centers in the

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structure. Herein, we took advantage of a heterogeneous aminotransferase

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with stereospecificity and designed a novel pathway for producing

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β-valienamine in Streptomyces hygroscopicus 5008, a validamycin producer.

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The aminotransferase BtrR from Bacillus circulans was able to convert

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valienone to β-valienamine with an optical purity of up to > 99.9% enantiomeric

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excess value in vitro. When the aminotransferase gene was introduced into a

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mutant of S. hygroscopicus 5008 accumulating valienone, 20 mg/L of

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β-valienamine was produced after 96 hours cultivation in shaking flasks. This

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work provides a powerful alternative for preparing the chiral intermediates for

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pharmaceutical development.

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KEYWORDS: β-valienamine, valienone, aminotransferase, validamycin,

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biosynthesis

ACS Paragon Plus Environment


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C7N aminocyclitols remain increasing interest in the biochemistry of

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glycosidase inhibitors for their significant bioactivities.1, 2 For instance,

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β-valienamine, (1S, 2S, 3R, 6R)-6-amino-4-(hydroxymethyl) cyclohex-4-ene-1,

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2, 3-triol is an optical building block for synthesizing the inhibitors of

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β-glycosidases.2-4 Its two derivatives, N-octyl-β-valienamine and

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N-octyl-4-epi-β-valienamine, have been proven to be promising chemical

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chaperone therapeutic agents for lysosomal storage diseases including

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Gaucher, GM1-gangliosidosis, and Morquio B disease caused by the disorder

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of β-glycosidase.5-8 The chemical synthetic routes of this compound have been

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investigated since 1980s.9-15 However the presence of multiple chiral centers

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in β-valienamine is quite challenging due to an insufficient stereospecificity of

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the chemical catalysts. Moreover, the multi-steps, harsh reactions (lower than

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-70°C), and chemical pollutions also cause synthetic and economic hurdles.

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Biosynthesis has emerged as a powerful alternative,16,17 especially for chiral

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amines in asymmetric synthesis.18 Recently, instead of using traditional

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rhodium, an aminotransferase was found to be remarkably able to convert

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prositagliptin ketone into sitagliptin with enantiomeric excess (e.e.) value >

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99.9%.19 However, there is no similar report using aminotransferase for the

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biosynthesis of β-valienamine. It is also valuable to construct a biosynthetic

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pathway for producing β-valienamine in vivo.

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In our previous work, 2-epi-5-epi-valiolone, 5-epi-valiolone and valienone

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were found to be the intermediates in the biosynthetic pathway of validamycin

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A in Streptomyces hygroscopicus 5008 (Figure 1, 1-3).20,21 It is noteworthy that

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valienone has a similar structure with β-valienamine except that the former has

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a carbonyl group, while the latter has an amino group on C6. Such similarity



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suggests that valienone would be an ideal precursor for certain

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aminotransferase to produce β-valienamine directly by a transamination

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reaction (Figure 1). Therefore, we focused on screening aminotransferases

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that can catalyze the conversion of valienone into β-valienamine in a desired

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stereospecific manner, and we designed an artificial biosynthetic pathway in S.

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hygroscopicus 5008 for microbial synthesis of β-valienamine.

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The key challenge in designing this biosynthetic pathway is the selection of

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an aminotransferase for the bioconversion of valienone into β-valienamine. To

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identify suitable enzyme candidates, we used an integrative platform (RxnIP)22

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to collect enzyme information, especially for enzymes recognizing cyclitol

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substrates, which is similar to valienone in structure. The sugar

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aminotransferases (SATs) convert keto-sugars into amino-sugars in the

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biosynthesis of macrolide antibiotics and belong to the subgroup

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DegT_DnrJ_EryC1 in the Pfam database.23 We chose SATs that typically use

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scyllo-inosose and NDP-4-keto-sugar as substrates for further analysis. As

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shown in Figure 2, four main evolutionary branches existed in a maximum

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likelihood phylogenetic tree, and one aminotransferase was therefore selected

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from each branch. These aminotransferases were BtrR24 from Bacillus

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circulans catalyzing the transamination of 2-deoxy-scyllo-inosose and

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2-deoxy-3-amino-scyllo-inosose in butirosin A biosynthesis, DesI25 from

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Streptomyces venezuelae in the biosynthesis of

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TDP-4-amino-4,6-dideoxyglucose, Per26 from Caulobacter crescentus for

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GDP-4-amino-6-deoxy-mannose biosynthesis, and ArnB27 from Escherichia

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coli for UDP-4-amino-arabinose biosynthesis. The four candidate genes were

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amplified by PCR or chemically synthesized, cloned into plasmid pET28a, and


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overexpressed in E. coli BL21 (DE3) to yield soluble proteins. The recombinant

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proteins with the N-terminal His-tag were purified by Ni-NAT columns and

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confirmed to be a homogeneous band by SDS-PAGE (Figure S1).

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To assess the feasibility of these aminotransferases to convert the

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non-natural substrate valienone into β-valienamine, we checked both the

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catalytic activity and enantio-preference of these enzymes in vitro. The

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aminotransferase activity assay was performed at 37°C in sodium phosphate

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buffer (pH 7.5) with valienone and cofactor pyridoxal 5′-phosphate (PLP). To

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identify the appropriate amino donors, L-glutamine and L-glutamate were

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tested. As shown in Figure S2a, L-glutamine showed a higher activity for all of

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the selected enzymes and was therefore used for further enzyme kinetic

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assays. Quantitative analysis and the stereo-purity of β-valienamine were

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detected through pre-column derivatization with ortho-phthaldialdehyde (OPA)

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using high-performance liquid chromatography (HPLC) with fluorescence

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detection. The derivatives of standards valienamine (S-configuration) and

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β-valienamine (R-configuration) were separated through HPLC by an Eclipse

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XDB-C18 column with retention times of 5.1 and 5.7 min, respectively (Figure

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3a).

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The mixture of the reaction was analyzed with the same condition. The

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results indicated that BtrR, Per, and ArnB showed detectable activity after 3

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hours of incubation (Figure 3c-e). More interestingly, BtrR and Per

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demonstrated a stringent stereospecificity with >99.9% e.e. value, while ArnB

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had low stereospecificity with 61% e.e. value for its β-valienamine product.

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Moreover, BtrR showed the highest activity among these three candidates and

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converted 5 mM valienone into β-valienamine with 76% conversion after 12



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hours at the enzyme concentration of 5 mg/mL for sample preparation. The molecular weight and the absolute configuration of the purified amine

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product were further verified by high-resolution mass spectrometry (HR-MS)

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with positive mode, 1H NMR, NOE, NOESY, 13C NMR spectra. The product

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gave an ion peak at m/z 176.0936 [M+H]+ (Table S1 and Figure S3), and its

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OPA derivative gave an ion peak at m/z 374.1041 [M+Na]+ (Table S2 and

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Figure S6a), matching the molecular formula of β-valienamine and its OPA

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derivative well. 1H NMR analysis of the substrate (valienone) and the product

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(β-valienamine) showed that the chemical shifts of all of the protons moved to

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a higher magnetic field, especially the adjacent protons (δ of H-5 from 6.20 to

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5.50 ppm) and (δ of H-1 from 4.16 ppm to 3.27 ppm ), and a new signal

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appeared at δ3.35 (s, 1H,H-6), which is consistent with the chemical shift of a

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methylene that is attached to an amino group. The signal of H-1 was changed

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from a doublet to a triplet with a coupling constant of 9.8 Hz, indicating that

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both C-2 and C-6 have the same relative configuration (Figure S4a, b). Further

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NOE and NOESY analysis showed that there was a correlation between H-2

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(δ3.50 ppm) and H-6, confirming the configuration of the aminated C-6 as the

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R-configuration (Figure S4c, d). Meanwhile, the transamination also can be

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observed through the chemical shifts in 13C NMR spectra (Figure S4e, f).

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Further comparison of the catalytic efficiency of BtrR and Per towards

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valienone (Figure 4) was performed by Michaelis-Menten kinetic analysis with

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L-glutamate dehydrogenase (L-GDH) as a coupling enzyme (Figure S2b).28

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The Km value of BtrR is only one-sixth that of Per toward valienone, indicating

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its stronger substrate-binding capacity. Considering the native substrates of all

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candidates, the BtrR prefers small scyllo-inosose substrates


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(2-deoxy-scyllo-inosose and 2-deoxy-3-amino-scyllo-inosose), whereas others

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prefer large NDP-4-keto-sugars. Possibly, this is the reason why BtrR shows a

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higher binding affinity for valienone. Additionally, the kcat value of BtrR was 3.6

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folds of that of Per, resulting in an overall 13-fold higher catalytic efficiency (kcat

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/Km) than Per. Combining e.e. values and kinetic parameters summarized in

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Figure 4, BtrR was proven to be the best catalytic candidate to transfer an

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amino group from L-glutamine to valienone for the production of chiral

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β-valienamine in vitro.

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The biosynthetic pathway of validamycin A has been thoroughly

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studied,20,21,29,30 and its producer, S. hygroscopicus 5008, gives a stable yield

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of validamycin A. To achieve the acquisition of β-valienamine in the microbe

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via a fermentation process, we designed a novel pathway by introducing the

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btrR gene into an engineered S. hygroscopicus 5008 to generate a new

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pathway for the biosynthesis of β-valienamine. We have identified that the C7

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cyclitol kinase ValC in the validamycin A biosynthetic pathway is responsible

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for the phosphorylation of valienone.29 Obviously, the presence of ValC is

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supposed to consume valienone and form a competitive reaction with BtrR.

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Therefore, the gene valC was knocked out using REDIRECT Technology.29

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The mutant ∆valC and wild-type 5008 were cultured in the fermentation media

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for 72 hours, and the production of valienone and validamycin A were analyzed

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by HPLC. The results showed that the production of validamycin A was

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abolished (Figure S5a) and valienone was accumulated in the ∆valC mutant

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(Figure 5b, S5b). Subsequently, the btrR gene was cloned on the integrative

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vector pPM927 under the control of the strong PvalA promoter30 and

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integrated into the ∆valC mutant. Thus, a ValA-ValD-ValK-BtrR pathway was



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constructed for the biosynthesis of β-valienamine in the mutant ∆valC::btrR.

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Mutant ∆valC::btrR and ∆valC were cultured with 50 mL fermentation medium

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for 5 days in shaking flasks, and the productivity of β-valienamine was

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monitored by HPLC with OPA-derivatization for every 12 hours. As shown in

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Figure 5a, the characteristic peak of β-valienamine with a retention time of 5.7

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min was observed in the fermentation broth of the mutant ∆valC::btrR but

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absent in mutant ∆valC, which was confirmed by further HR-MS analysis (data

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not shown). The accumulation of β-valienamine was increased with prolonged

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incubation time and reached the highest of 20 mg/L at 96 hours (Figure 5b).

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The fermented β-valienamine product was also verified by HR-MS with

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pre-column derivatization using OPA. The spectrum matched well with that of

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the standard β-valienamine and showed the characteristic ion peak at m/z

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374.1038 [M+Na]+ (Table S2 and Figure S6b). The above data showed that the

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designed ValA-ValD-ValK-BtrR pathway for β-valienamine biosynthesis is

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functional, and the engineered strain is able to produce the unnatural product

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β-valienamine.

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In summary, we successfully designed and constructed an artificial

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pathway for generating highly stereospecific β-valienamine both in vitro and in

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vivo. Compared with the current chemical methods for β-valienamine

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synthesis, our biological strategy represents a promising alternative by

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integrating a heterogeneous aminotransferase gene and engineering host

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metabolic flux due to its sustainable and efficient process. To further increase

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β-valienamine production, we are investigating the engineering of BtrR for

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improved catalytic activity on valienone, by in silico design based on the

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protein structure analysis and systematically optimizing the pathway flux of the


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host microbe. Our research not only builds a new and efficient route to

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synthesize β-valienamine, but also facilitates further synthesis of its active

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derivatives with glycosidase inhibitory activity for treating related diseases.

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MATERIALS AND METHODS

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Chemicals. Valienone was synthesized by WuXi AppTec Company (Wu Xi,

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China). β-Valienamine was prepared and identified after bioconversion with

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BtrR in vitro in our lab. Valienamine was kindly provided by professor Yuguo

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Zheng (Zhejiang University of Technology). Other chemicals were purchased

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from Sigma-Aldrich, Inc. (St. Louis, MO).

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Molecular phylogenetic analysis by Maximum Likelihood. For

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phylogenetic analysis, full-length amino acid sequences were aligned by

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Clustal X (Des Higgins et.al., Conway Institute UCD Dublin) with the following

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parameters: protein weight matrix = Gonnet; gap open = 10; gap extension =

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0.2. Maximum likelihood analysis was employed by MEGA6 software with the

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JTT matrix-based model.

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Expression and purification of SATs. Candidate SAT genes were amplified

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and ligated into the pET28a plasmid. Expression plasmids were transformed

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into E.coli BL21 (DE3). Transformants were grown in LB medium with 100

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µg/mL kanamycin at 37°C until the cell density at 600 nm reached 0.3-0.5

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followed by induction with 0.8 mM isopropyl-β-D-thiogalactopyranoside for 10

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hours to produce N-terminal His6-tagged recombinant proteins. The cells were

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harvested by centrifugation, resuspended in 20 mM potassium phosphate

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buffer (pH 7.5), and disrupted by sonication. The fusion proteins were purified

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with Ni-NTA resin column with 20 mM potassium phosphate buffer (pH 7.5)

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containing different concentrations of imidazole. The purified proteins were



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detected by SDS- PAGE.

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Enzymatic activity and the kinetic parameter assays. To measure the

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activity of sugar aminotransferases (SATs), 1 mg/mL purified SAT was

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incubated with 5 mM valienone as amino acceptor and 5 mM L-glutamine as

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an amino donor in 20 mM potassium phosphate (pH 7.5) containing 0.3 mM

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pyridoxal 5′-phosphate as a cofactor at 37°C for 3 hours followed by

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pre-column ortho-phthaldialdehyde derivatization and fluorescence detection.

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The kinetic parameters were determined at 37°C using NADH-dependent

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L-glutamate dehydrogenase (L-GDH) as a coupling enzyme with a reaction

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system involving 0.5 U L-GDH, 5 mM ammonium chloride and 0.5 mM NADH

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in addition to the normal reaction system. The reduction in the absorbance of

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NADH (ε340=6220 M-1 cm-1) was monitored continuously at 340 nm by a

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multi-scan spectrum microplate spectrophotometer (Spectra Max, Molecular

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Devices). One unit of enzyme activity was defined as 1 µmol of NADH

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consumed per min.

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Stereopurity determination of β-valienamine by HPLC with pre-column

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derivatization using OPA. Stereometric purity of β-valienamine was

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determined by online fluorescence derivatization of ortho-phthaldialdehyde on

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Agilent 1200 Infinity LC System. β-Valienamine and its (S)-enantiomer

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standards were derivatized by 0.15 M OPA in the 0.40 M (pH 9.0) borate buffer.

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The separation of the derivative mixture was performed using an Eclipse

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XDB-C18 (5 µm, 4.6×150 mm) column eluted with 22% acetonitrile at 30°C for

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18 min under the detection with the fluorescence detector at 445 nm emission

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and 340 nm excitation wavelengths with medium sensitivity. β-valienamine and

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valienamine showed the retention times at 5.7 and 5.1 min, respectively.


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β-Valienamine purification from the reaction mixture and the

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fermentation broth of ∆valC::btrR. A mixture of the reaction was filtered

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through a Microcon YM-10 (Millipore) to remove protein (>10,000 MW) and

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then purified by Dowex 1×2 (OH- form) (0.5×15 cm) eluted with water. Isolation

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of β-valienamine from fermentation broth of ∆valC::btrR through two-column

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chromatography system was performed including Dowex 50×8 (H+ form)

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eluted with 0.5 M ammonia water and Dowex 1×2 (OH- form) eluted with water.

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High resolution-mass spectroscopy (HR-MS) and nuclear magnetic

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resonance spectroscopy (NMR). High-resolution mass spectral analysis was

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performed using an Agilent 1290-MS 6230 TOF-MS system with positive

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model. For NMR identification, the purified samples were dried and

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redissolved in D2O solution, and one-dimensional 1H, 13C and two-dimensional

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1

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MHz spectrometer.

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Construction of ∆valC::btrR mutant. To construct the ∆valC::btrR mutant, a

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0.9 kb XhoI/NdeI PvalA promoter fragment and a 1.3 kb NdeI/XbaI fragment

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containing the btrR gene were cloned into pBluescript II SK(+) plasmid

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digested by XhoI and XbaI. Subsequently, the fragment containing PvalA and

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btrR was cleaved with EcoRI from the recombinant plasmid and cloned into the

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integrative vector pPM927. The resulting plasmid was introduced into the

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∆valC mutant by intergeneric conjugation mediated by E. coli ET12567

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(pUZ8002) and confirmed by PCR amplification.

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Fermentation of S. hygroscopicus 5008 and its mutants. S. hygroscopicus

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5008 and its mutants were cultivated for 72-120 hours at 37°C at 220 rpm in

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the fermentation medium (gram per liter: corn powder 100, soybean flour 25,

H-1H COSY, NOESY NMR spectra were obtained using Bruker Avance III 400



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yeast extract 5, NaCl 1, and KH2PO4 1.5). Samples were collected every 12

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hours. The supernatant of the fermentation broth was extracted with

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chloroform and centrifuged at 12,000 rpm for 10 min before HPLC analysis.

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Validamycin A analysis by HPLC. The fermentation broth of S.

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hygroscopicus 5008 and ∆valC mutant were separated through Eclipse

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XDB-C18 (5 µm, 4.6×150 mm) column, eluted with CH3CN/H2O (3: 97) as

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mobile phase at 30°C for 18 min and monitored at 210 nm by diode array

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detector.

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Valienone accumulation analysis by HPLC with DNPH pre-column

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derivatization. The accumulation of valienone in the fermentation broth of the

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∆valC mutant was evaluated by HPLC with a pre-column derivatization using

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2,4-dinitrophenylhydrazine. The reaction was performed with 7.57 mM DNPH

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in 5% H3PO4 solution. Analysis was performed using an Eclipse XDB-C18 (5

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µm, 4.6×150 mm) column, eluted with 50% acetonitrile at 30°C for 18 min, and

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detected at 380 nm by diode array detector.

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ASSOCIATED CONTENT

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Supporting Information

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Additional figures as described in the text. This material is available free of

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charge via the Internet at http://pubs.acs.org

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AUTHOR INFORMATION

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Corresponding Authors

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* Yan Feng Tel: +86-21-34207189

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* Linquan Bai Tel: +86-21-34206722 E-mail: [email protected]

E-mail: [email protected]


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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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This work was supported by National 973 Program (2012CB721003), National

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Science Foundation of China (31200061), and Research Fund for the Doctoral

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Program of Higher Education (13Z102090066). We are grateful to Prof. Yuguo

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Zheng for the generous gift of valienamine standard, Prof. Shuangjun Lin for

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the help on NMR analysis, and Prof. Meifeng Tao and Dr. Guangyu Yang for

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the help on plasmid construction.

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REFERENCES

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(1) Mahmud, T. (2003) The C7N aminocyclitol family of natural products.

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application of unsaturated carbaglycosylamine glycosidase Inhibitors.

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neuronopathic lysosomal diseases: competitive inhibitors as chemical

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For Table of Contents Use Only De novo biosynthesis of β-valienamine in engineered Streptomyces hygroscopicus 5008 Li Cui, Ying Zhu, Xiaoqing Guan, Zixin Deng, Linquan Bai,* and Yan Feng* 196x160mm (300 x 300 DPI)


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Figure 1. Biosynthetic pathway of validamycin A (dashed frame) and de novo aminotransferase-catalyzed route for β-valienamine biosynthesis (solid frame) in S. hygroscopicus 5008. 117x52mm (300 x 300 DPI)



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Figure 2. Phylogenetic analysis of SATs and the reactions catalyzed by the selected (red) candidates. The multiple alignments were performed by Clustal X, and the phylogenetic tree was generated using MEGA6. BtrR_Bci (CAD41947), SpcS2_Ssp (ABW87804), StsC_Sgl (AIR96275), GtmB_Mec (CAE06512), StrS_Sgr (CAA68523), WecE_Eco (AAC76796), ArnB_Eco (AAM92146), ArnB_Sty (NP_461239), Per_Eco (AAG57096), Per_Ccr (AAK22996), LmbS_Sli (CAA55764), DesI_Sve (AAC68684), PseC_Hpy (AAD07433), PglE_Cje (AAD09304). 574x210mm (300 x 300 DPI)


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Figure 3. HPLC analysis of the reaction mixture via pre-column derivatization using OPA. (a) Valienamine and β-valienamine standards; (b) Reaction mixture without enzyme as the blank control; (c, d, e) Reactions catalyzed by BtrR, Per, and ArnB, respectively. 200x139mm (300 x 300 DPI)



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Figure 4. Enzymatic conversion and kinetic properties of selected SATs. (Column) Enzymatic conversion of valienone to β-valienamine by selected SATs. Data were collected from three measurements with 1 mg/mL enzymes incubated with 5 mM valienone, 5 mM L-glutamine, and 0.3 mM PLP at 37°C in sodium phosphate buffer (pH 7.5); (Table) Kinetic parameters of different SATs; 0.5 U L-GDH was added into the reaction system as a coupling enzyme with 5 mM ammonium chloride and 0.5 mM NADH. 210x77mm (300 x 300 DPI)


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Figure 5. Production of β-valienamine in the ∆valC mutant integrated with the heterogeneous aminotransferase gene btrR. (a) HPLC analysis of 96-hour fermentation broths with pre-column derivatization using OPA; (b) Time course of β-valienamine and valienone titer in ∆valC and ∆valC::btrR mutant. 297x420mm (300 x 300 DPI)


De Novo Biosynthesis of Î²-Valienamine in Engineered Streptomyces

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