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Biosynthesis of L-Erythrose by Assembly of Two Key Enzymes in Gluconobacter oxydans Xingxing Zou, Yuhong Ren, Jinping Lin, Xinlei Mao, and Shengyun Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02201 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 14, 2017

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Journal of Agricultural and Food Chemistry

Biosynthesis of L-Erythrose by Assembly of Two Key Enzymes in Gluconobacter oxydans Xingxing Zou‡,†, Jinping Lin‡,†, Xinlei Mao†, Shengyun Zhao§ and Yuhong Ren *,† †

State Key Laboratory of Bioreactor Engineering, New World Institute of

Biotechnology, East China University of Science and Technology, Shanghai 200237, China. §

Fujian Key Laboratory of Eco-Industrial Green Technology, Wuyi University, Wuyishan, 354300, China

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ACS Paragon Plus Environment


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ABSTRACT: L-erythrose, a rare aldotetrose, possesses various pharmacological activities.

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However, efficient L-erythrose production is challenging. Currently, L-erythrose is

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produced by a two-step fermentation process from erythritol. Here, we describe a novel

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strategy for the production of L-erythrose in Gluconobacter oxydans (G. oxydans) by

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localizing the assembly of L-ribose isomerase (L-RI) to membrane-bound sorbitol

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dehydrogenase (SDH) via the protein-peptide interactions of the PDZ domain and PDZ

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ligand. To demonstrate this self-assembly, green fluorescent protein (GFP) replaced L-RI

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and its movement to membrane-bound SDH was observed by fluorescence microscopy.

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The final L-erythrose production was improved to 23.5 g/L with the stepwise metabolic

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engineering of G. oxydans, which was 1.4-fold higher than that obtained using

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co-expression of SDH and L-RI in G. oxydans. This self-assembly strategy shows

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remarkable potential for further improvement of L-erythrose production.

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KEYWORDS: L-erythrose; self-assembly; G. oxydans; protein–peptide interactions

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INTRODUCTION

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Erythrose, a rare aldotetrose, contains two adjacent chiral carbon atoms.1 Several reports

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show that erythrose possesses hypoglycemic effect. It also exhibits inhibitory effect on

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cancer cells and may be used in treating cancer.2 2-Deoxy-L-erythrose, a derivative of

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L-erythrose, possesses anti-HIV activity.3

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Previous reports show that transketolases can produce L-erythrose from glyceraldehyde

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and a combination of β-hydroxypyruvate and glyceraldehyde.4,5 However, this method is

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not suitable for the production of L-erythrose because of its cost and low yield. Currently, a

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report described a two-step reaction for producing L-erythrose.6 In the first step, erythritol

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is completely oxidized to L-erythrulose using Gluconobacter frateurii IF0 3254.7 Next,

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L-erythritol was isomerized to L-erythrose using the constitutive L-ribose isomerase (L-RI)

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from a mutant strain of Acinetobacter sp. DL-28.8 However, the yield of L-erythrose from

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L-erythrulose was only 18%.

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Currently, methods involving single step synthesis of erythritol are limited. G. oxydans is

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extensively used in synthetic biology methods for carbohydrate production because of the

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efficiency of its membrane-bound dehydrogenase.9 The membrane-bound sorbitol

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dehydrogenase (SDH) of G. oxydans converts erythritol to L-erythrulose, and a strain of G.

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oxydans with high SDH activity, DSM2003, was obtained upon regulation of growth

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conditions.10,11 SDH and L-RI are the key enzymes required for the synthesis of L-erythrose.

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However, the active site of SDH is oriented towards the periplasm, and therefore,

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substances that can be used as energy sources have to cross only the outer membrane to 3



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access SDH.12 Moreover, L-RI is located within the cytoplasm. Therefore, heterologous

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expression of L-RI alone is insufficient to achieve satisfactory yields of L-erythrose. To

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shorten the spatial distance between SDH and L-RI in G. oxydans, strategies involving the

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assembly of multienzyme cascades should be considered.

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The substrate was delivered more conveniently when the spatial distance between the

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enzymes was reduced.13 Strategies that involve enzyme fusion and modular scaffolds have

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shortened the distance between proteins in many instances. However, loss of enzymatic

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activity is a disadvantage of these strategies.14 Therefore, self-assembly using

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protein-peptide interactions was developed as an alternative strategy.15

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In this study, we developed a novel strategy for the production of L-erythrose by

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positioning the assembly of L-RI to membrane-bound SDH in G. oxydans DSM2003. This

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method significantly increased the production of L-erythrose, the details of which would be

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discussed.

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MATERIALS AND METHODS Construction of Fusion Genes

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The target gene encoding L-RI was synthesized using codon optimization (Genscript

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Biotechnology co., Ltd. Nanjing, China), while SDH was amplified from the cDNA derived

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from the genome of G. oxydans DSM2003. Positioning was achieved using a normal

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enzymatic assembly method.16 Oligonucleotide sequences of the PDZ domain17 were

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previously constructed in our laboratory. The PDZ-ligand was fused to the reverse primer of

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L-RI to generate the fragment L-RI-PDZlig. ER/K, a α-helical linker segment, was inserted 4


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between the SDH and PDZ fragments. Then, the sequences encoding PDZ-SDH or

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L-RI-PDZlig were ligated to the PtufB promoter to produce fragments PtufB-PDZ-SDH and

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PtufB-L-RI-PDZlig. Next, both the expression vector pBBR1MCS-5 and the fragment

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PtufB-L-RI-PDZlig were double-digested with SacI and XbaI (Thermo Fisher Scientific,

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Rockford, IL, USA) and ligated with T4 DNA ligase, yielding the plasmid

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PtufB-L-RI-PDZlig (Figure S1A). The co-expression plasmid containing PtufB-PDZ-SDH

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and PtufB-L-RI-PDZlig were constructed similarly (Figure S1B).

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Bacterial Strain Construction

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The two control plasmids were constructed to express L-RI with or without the PDZ ligand

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fusion, and these were co-expressed with the plasmid expressing SDH-PDZ domain fusion

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protein in G. oxydans DSM2003. All plasmids were transformed into G. oxydans DSM2003

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using triparental conjugation18 as previously reported for heterologous protein expression.

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Cells were collected by centrifuge, washed twice using pure water and stored at -18 °C.

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Enzymatic Activity Assays

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The activity of SDH and PDZ-SDH were evaluated by the L-erythrulose concentration. The

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standard assay mixture contained 20 g/L freshly prepared erythritol (99%, Aladdin Chemistry

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Co. Ltd., Shanghai, China) in 100 mM sodium phosphate buffer (pH 7.0) and 20 g/L resting

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cells (WCW). The reaction was performed at 30 °C for sampling per hour. The L-erythrulose

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content was analyzed using high-pressure liquid chromatography (Agilent, 1100 series) and

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detected using an ultraviolet-visible (UV-vis) detector (Spectrasystem UV1000, λ = 210 nm).

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To verify the effect of PDZ ligand on L-RI enzyme activity, vectors encoding L-RI 5



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with/without the PDZ ligand were constructed. The standard assay mixture contained 8.5 g/L

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freshly prepared L-erythrulose (85%, Aladdin Chemistry Co. Ltd., Shanghai, China) in 100

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mM sodium phosphate buffer (pH 7.0) and 20 g/L resting cells (WCW). The reaction was

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performed at 30 °C and samples were withdrawn every hour. The L-erythrose content was

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analyzed using high-pressure liquid chromatography (HPLC) and detected using an UV-vis

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detector (Spectrasystem UV1000, λ = 210 nm).

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Substrate Inhibition Assays

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To determine the effect of substrate on the cells, different concentrations of erythritol were

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used and the molar concentration of the solution was adjusted with NaCl to achieve the same

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concentration of NaCl as in the control. Resting cells (50 g/L) were suspended in different

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concentrations of erythritol solution for 20 h in a 4 °C refrigerator. 20 g/L erythritol was

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catalyzed by the treated cells and the remaining catalytic activity of the cells was measured.

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Fluorescence Localization Assays

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Vectors expressing SDH and fusions of the green fluorescent protein (GFP) with the PDZ

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domain or the PDZ ligand were co-expressed in G. oxydans DSM2003 to determine whether

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the GFP-tagged protein localized to the membrane-bound SDH. The control strain expressed

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GFP alone. Recombinant strains were cultured in sorbitol medium at 30 °C and 200 rpm for

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20 h. The harvested cells were washed twice with phosphate-buffered saline (PBS; pH 7.0)

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and resuspended in 20 mM PBS. The resuspended cells were disrupted by sonication in the

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presence of phenyl methane sulfonyl fluoride (PMSF), a protease inhibitor. After

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centrifugation at 12,000 g for 30 min, the supernatant was subjected to ultracentrifugation at 6


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40,000 g for 1 h (Himac CP-WX, Hitachi, Japan) to obtain the membrane fraction, which

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were subsequently resuspended and visualized by epifluorescence (excitation, 469 nm;

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emission, 525 nm). The fluorescence intensity of the membrane fractions were measured

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using an automatic cellular imaging multifunctional detection system (Cytation3, Bio-Tek

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Instruments, Inc., Winooski, VT, USA).

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L-RI Localization Assays

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SDH with PDZ domain and L-RI with PDZ ligand were co-expressed in G. oxydans

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DSM2003 to determine whether the L-RI was localized to the membrane-bound SDH; vector

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expressing L-RI was used as control.

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resting cells (WCW) in the same way as described above, and then used to catalyze 8.5 g/L

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freshly prepared L-erythrulose. The reaction conditions and the detection method were as the

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same as L-RI enzyme activity assays.

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RESULTS AND DISCUSSION

Membrane components were extracted from 20 g/L

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SDH is consisted of two hetero subunits (sldB, 13.7 KDa; and sldA, 77 KDa). The sldB is

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located upstream of sldA gene.19 Hydrophobicity analysis of the sldB amino acid sequence

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suggested that it has four transmembrane regions and the N-terminal of sldB is exposed to

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cytoplasm. The self-assembly strategy was based on fusing a PDZ domain and its

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corresponding ligand (PDZlig) to the N-termini of sldB and C-termini of L-RI, respectively,

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yielding PDZ domain-SDH (SPd) and L-RI-PDZlig (LPl). The strategy for positioning the

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assembly of SDH and L-RI is outlined in Figure 1.

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Effect of pH on SDH and L-RI Activity 7



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To investigate the effect of pH on erythritol oxidation, same concentration of G. oxydans

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resting cells was used to transform 20 g/L erythritol. The initial pH of the reaction system

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was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 with 0.1 M NaOH and HCl solutions, as

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required. The concentration of L-erythrulose in the reaction solution after one hour was

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measured and the initial reaction rate was calculated. The results are shown in Figure S2A. At

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initial reaction pH of 3.0 and 8.0, the initial reaction rate was approximately zero and the

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reaction did not proceed. SDH was catalytically active in the pH range of 4.0-7.0 and

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oxidized erythritol most rapidly at pH 5.0.

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L-RI activity was measured at various pH in the range of 6.0 to 8.0, using the same

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concentration of resting cells (WCW) that independently expressed L-RI to transform 20 g/L

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L-erythrulose. The concentration of L-erythrose in the reaction solution was measured after

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every three hours and the results are show in Figure S2B. The enzyme activity of LRI

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increased with increase in pH from 6.0 to 8.0.

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PDZ Domain or Ligand Fusion Alter the Enzymatic Activities

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To examine the enzymatic activities of SPd and LPl, strains expressing SPd and LPl were

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used; strains independently expressing SDH and L-RI were used as controls. The reactions

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were performed at 30 °C for several hours with the same concentration of resting cells, and

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the specific activities obtained are summarized in Figure 2. The specific activities of SPd and

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LPl decreased by 36.1% and 21.2% compared to those of SDH and L-RI under the same

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conditions. These results showed that fusion of SDH and L-RI with PDZ domain and ligand,

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respectively, still remained most of their enzymatic activities. 8


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Verifying Positioning Assembly Using GFP-tagged PDZ Ligand and By Measuring the

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Activity of L-RI in Membranes

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To confirm the self-assembly, GFP was fused with PDZ ligand instead of LPl in the

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co-expression strain (SGS); a strain expressing GFP alone (uSG) was used as a control. The

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membrane fractions were in the sediment after ultracentrifugation. The sediment in which

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GFP were located in the membrane fraction fluoresced green. To confirm assembly due to

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GFP positioning, the membrane compositions were resuspended and visualized by

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epifluorescence, (Figure 3A). To further delineate the changes in membrane compositions

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between SGS and uSG, fluorescence intensity was measured using a microplate reader using

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the same concentration of cells. As shown in Figure 3B, membrane composition of SGS

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showed approximately 2-fold greater fluorescence intensity than uSG. The results indicated

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that GFP could be localized to the membrane protein via protein-peptide interactions.

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To further verify L-RI located on the membranes via the self-assembly of SDH and L-RI,

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SPd and LPl were co-expressed in G. oxydans DSM2003 (SPP); vector expressing L-RI (uSL)

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was used as control. The membrane fractions of SPP and uSL were extracted and the activity

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of L-RI was measured. As shown in Figure 3C, the membranes of SPP showed significant

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activity of L-RI, whereas the activity of L-RI in the control’s membranes was undetectable.

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Although some L-RI was remained in the supernatant of the assembled cells, the activity of

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L-RI in the supernatant (data not shown) was lower than that in the membranes of the

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assembled cells. The result suggested that the most of L-RI was located on the membranes

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via self-assembly with SDH.

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Self-assembly of SPd and LPl Enhanced L-Erythrose Production

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For this cascade reaction catalyzed by SDH and L-RI, the activity of L-RI is much less than

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SDH’s in G. oxydans DSM2003. Moreover, the expression level of L-RI was so low that its

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band almost could not be identified by protein electrophoresis meaning that the amount of

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L-RI was very low in the cytoplasm of cells. Therefore, it is important for improving the

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catalytic efficiency by shortening the spatial distance between the SDH located in the

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cytoplasmic membrane and the L-RI distributed in the cytoplasm.

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Prior to the self-assembly experiments, SDH fused directly with L-RI via different linkers

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was constructed to co-locate SDH and L-RI. However, the activity of these fusion proteins

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could not be detected in G. oxydans DSM2003, suggesting that this strategy was not suitable

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for the assembly of these two enzymes. Co-expression of SPd and LPl resulted in the

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self-assembly of SDH and L-RI in G. oxydans DSM2003. The resting cells of SPP were used

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to produce L-erythrose from L-erythritol, uSL was used as control. 10 g/L resting cells of

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SPP and uSL were used to catalyze 10 g/L erythritol to L-erythrose, respectively (Figure 4).

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The results showed that the self-assembly of SPd and LPl significantly increased L-erythrose

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production during resting cell catalysis, resulting in higher L-erythrose production (220.34

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mg/L) for SPP, whereas the production titer of uSL was 166.52 mg/L under the same

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condition. The increase in yield in SPP may be benefit from the membrane located L-RI,

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because, compared with uSL, L-erythrulose would arrive in the L-RI located on the

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membrane more quickly than in the L-RI random distribution in cytoplasm once

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L-erythrulose penetrated the cytoplasmic membrane. The results suggested that the reduction 10


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in intermediate product diffusion in the assembled cells lead to more efficient conversion of

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the substrate to L-erythrose compared to the unassembled cells.

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Optimizing the Catalytic Conditions

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The L-erythrose yield at various temperatures using SPP and uSL were monitored and the

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results are shown in Figure S3a. The L-erythrose production of SPP was more than that of

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uSL at the same temperature. SPP produced the maximum amount of L-erythrose at 30°C,

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which was selected as the final catalytic temperature for all future in vitro whole-cell

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catalysis experiments. G. oxydans DSM2003 was exposed to different concentrations of

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erythritol to test substrate inhibition. The relative activity of cells (compared to untreated

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cells) decreased with increasing concentrations of erythritol (Figure S3b). The catalytic

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activity was less than 30% when erythritol concentration was 150 g/L. To determine the

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optimal cell concentration, different concentrations of G. oxydans DSM2003 were used for

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catalyzing the conversion of 100 g/L erythritol under the same condition. The results are

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shown in Figure S3c. The initial reaction rate increased with cell concentrations and peaked

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at 50 g/L cell concentration (WCW).

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After optimizing the catalytic conditions, SPP and uSL were used to catalyze reactions with

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50 g/L WCW and 100 g/L erythritol at pH 7.0 and 30 °C. As shown in Figure 5, the substrate

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was rapidly converted to L-erythrulose in the initial stages of the reaction, and the

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consumption rate of SPP was slower than that of uSL between 0 and 12 h; however, the

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substrate was completely transformed at 36 h. Finally, the reaction catalyzed by SPP resulted

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in a titer of 23.5 g/L L-erythrose, which was 1.4-fold higher than that of uSL (17.2 g/L). 11



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These results suggested that the reduction in intermediate product diffusion in SPP lead to

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more efficient conversion of the substrate to L-erythrose by LPl compared to that in uSL.

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Self-assembly of SPd and LPl reduces the spatial distance between these two enzymes of the

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L-erythrose biosynthetic cascade, which enhances the reaction yield by decreasing the

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intermediate diffusion.

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In this work, we used a protein pair to set a target on SDH, the L-RI was successfully

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positioned on the membrane protein. By this assembly approach, the position in space of

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these two enzymes was closed in the cells leading to increasing 1.4-fold the yield of

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L-erythrose compared with the unassembled cells. The best titer of L-erythrose reached 23.5

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g/L. This study shows the positioning assembly strategy in a heterologous production

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pathway for L-erythrose synthesis in G. oxydans.

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ABBREVIATIONS SDH，sorbitol dehydrogenase; L-RI, L-ribose isomerase; GFP, green fluorescent protein; PDZ, PDZ domain; PDZlig, ligand for PDZ domain; HPLC, high -performance liquid chromatography; WCW, wet cell weight.

ASSOCIATED CONTENT

SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website. Additional figures and tables

AUTHOR INFORMATION Corresponding Author * (Y. H. Ren) E-mail: [email protected]. Phone: +86 21 6425 2163. Fax: +86 21 6425 0068.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

‡These authors contributed equally to this article. X. X. Zou and J. P. Lin designed and preformed most of experiments, participated in all data analysis, and drafted much of the manuscript. S. Y. Zhao provided advice on metabolic pathway analysis 13



and molecular biology experiments. X. L. Mao provided advice on cultivation of G. oxydans. Y. H. Ren conceived the experiments, provided regular advice as the study progressed and revised the manuscript. All authors read and approved the final manuscript.

Funding sources This work was funded by the National Special Fund for the State Key Laboratory of Bioreactor Engineering (2060204).

Notes The authors declare no competing financial interest.

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REFERENCES

(1) Académie de Pharmacie (Paris). Journal de pharmacie et de chimie. Imprimeurs de l'Université royale de France: Paris, France, 1845. (2) Sener, A.; Devis, G.; Somers, G.; et al. The insulinotropic action of D-erythrose. Diabetologia, 1977, 13, 125-130. (3) Kruger, N.J.; von Schaewen, A. The oxidative pentose phosphate pathway: structure and organization. Curr. Opin. Plant Biol. 2003, 6, 236-246. (4) Dickens, F.; Williamson, D.H. Formation of erythulose from hydroxypyruvate in the presence of yeast carboxylase. Nature, 1956, 178, 1349-1350. (5) Bongs, J.; Hahn, D.; Schörken, U.; et al. Continuous production of erythrulose using transketolase in a membrane reactor. Biotechnol. Lett. 1997, 19, 213-216. (6) Mizanur, R.M.D.; Takeshita, K.; Moshino, H.; et al. Production of L-erythrose via L-erythrulose from erythritol using microbial and enzymatic reactions. J. Biosci. Bioeng. 2001, 92, 237-241. (7) Takeshita, K.; Shimonishi, T.; Izumori, K. Production of L-psicose from allitol by Gluconobacter frateurii IFO 3254. J. Ferment. Bioeng. 1996, 81, 212-215. (8) Shimonishi, T.; Izumori, K. A new enzyme, L-ribose isomerase from Acinetobacter sp. strain DL-28. J. Ferment. Bioeng. 1996, 81, 493-497. (9) Prust, C.; Hoffmeister, M.; Liesegang, H.; et al. Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans. Nat. Biotechnol., 2005, 23,

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195-200. (10) Moonmangmee, D.; Adachi, O.; Shinagawa, E.; et al. L-Erythrulose production by oxidative fermentation is catalyzed by PQQ-containing membrane-bound dehydrogenase. Biosci. Biotechnol. Biochem. 2002, 66, 307-318. (11) Yang, X.P.; Wei, L.J.; Ye, J.B.; et al. A pyrroloquinoline quinine-dependent membrane-bound d-sorbitol dehydrogenase from Gluconobacter oxydans exhibits an ordered Bi Bi reaction mechanism. Arch. Biochem. Biophys. 2008, 477, 206-210. (12)

Deppenmeier,

U.;

Hoffmeister,

M.;

Prust,

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Biochemistry

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biotechnological applications of Gluconobacter strains. Appl. Microbiol. Biotechnol. 2002, 60, 233-242. (13) Wheeldon, I.; Minteer, S.D.; Banta, S.; et al. Substrate channeling as an approach to cascade reactions. Nat. Chem. 2016, 8, 299-309. (14) Lee, H.; DeLoache, W.C.; Dueber, J.E. Spatial organization of enzymes for metabolic engineering. Metab. Eng. 2012, 14, 242-251. (15) Liu, F.; Banta, S.; Chen, W. Functional assembly of a multi-enzyme methanol oxidation cascade on a surface-displayed trifunctional scaffold for enhanced NADH production. Chem. Commun. 2013, 49, 3766-3768. (16) Gao, X.; Yang, S.; Zhao, C.; et al. Artificial multienzyme supramolecular device: highly ordered self‐assembly of oligomeric enzymes in vitro and in vivo. Angew. Chem. Int. Ed. Engl. 2014, 53, 14027-14030.

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(17) Lee, H.; DeLoache, W.C.; Dueber, J.E. Spatial organization of enzymes for metabolic engineering. Metab. Eng. 2012, 14, 242-251. (18) Hölscher, T.; Görisch, H. Knockout and overexpression of pyrroloquinoline quinone biosynthetic genes in Gluconobacter oxydans 621H. J. Bacteriol. 2006, 188, 7668-7676. (19) Hoshino T,; Sugisawa T,; Shinjoh M, et al. Membrane-bound D-sorbitol dehydrogenase of Gluconobacter suboxydans IFO 3255—enzymatic and genetic characterization. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics. 2003, 1647(1): 278-288.

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FIGURE LEGENDS

Figure 1. Schematic view of the positioning assembly of L-RI and SDH due to the interaction between PDZ and its ligand, PDZlig.

Figure 2. The specific activities of SDH, SPd, L-RI, and LPl. (A) Gray and red shadows show the enzyme activity of SDH and SPd, respectively. (B) Gray and red shadows show the enzyme activity of L-RI and LPl, respectively. Activity values are the arithmetic mean of at least three different measurements. Error bars show the standard deviation of three measurements, ** denotes p < 0.01.

Figure 3. Visualization of membrane composition of SGS (left) and uSG (right) by inverted fluorescence microscopy (A). Fluorescence intensity of membrane compositions of SGS and uSG (B). The L-RI activities of membrane compositions of uSL and SPP (C). Each data point represents the mean ± standard deviation of three measurements, *** denotes p < 0.001.

Figure 4. Long-term catalysis reactions of strains SPP and uSL with 10 g/L substrate. Each data point represents the mean ± standard deviation of three measurements, ** denotes p < 0.01.

Figure 5. Long-term catalysis reactions of strains SPP and uSL at pH 7.0 (0.1 M sodium phosphate) and a substrate concentration of 100 g/L. The erythrose production of SPP (hollow squares) and uSL (solid squares), substrate consumption of SPP

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(hollow circles) and uSL (solid circles), and intermediate product of SPP (hollow triangles) and uSL (solid triangles) are indicated. Each data point represents the mean ± standard deviation of three measurements.

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FIGURE GRAPHICS Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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TABLE OF CONTENTS

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Biosynthesis of L-Erythrose by Assembly of Two Key Enzymes in

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