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Production of ginsenoside F2 by using Lactococcus lactis with enhanced expression of #-glucosidase gene from Paenibacillus mucilaginosus Ling Li, So-Yeon Shin, Soo Jin Lee, Jin Seok Moon, Wan Taek Im, and Nam Soo Han J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04098 • Publication Date (Web): 23 Oct 2015 Downloaded from http://pubs.acs.org on October 27, 2015

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Journal of Agricultural and Food Chemistry

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Journal: Journal of Agricultural and Food Chemistry (ACS)

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Production of ginsenoside F2 by using Lactococcus lactis with enhanced

5

expression of β-glucosidase gene from Paenibacillus mucilaginosus

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7

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Ling Lia, So-Yeon Shina, Soo Jin Leea, Jin Seok Moona, Wan Taek Imb, and Nam Soo

9

Hana*

10 11

a

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and Food Sciences, Chungbuk National University, Cheongju 361-763, Korea

13

Brain Korea 21 Center for Bio-Resource Development, Division of Animal, Horticultural

b

Department of Biotechnology, Hankyong National University, Kyonggi-do 456-749, Korea

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Running title: Production of minor ginsenoside F2 using Lactococcus lactis

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Corresponding author: Nam Soo Han

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Phone: 82-43-261-2567; Fax: 82-43-271-4412

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E-mail: [email protected]

21 1

ACS Paragon Plus Environment


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Abstract

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This study aimed to produce a pharmacologically active, minor ginsenoside F2 from the

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major ginsenosides Rb1 and Rd by using a recombinant Lactococcus lactis strain expressing

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a heterologous β-glucosidase gene. The nucleotide sequence of the gene (BglPm) was derived

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from Paenibacillus mucilaginosus and synthesized after codon-optimization, and the two

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genes (unoptimized and optimized) were expressed in L. lactis NZ9000. Codon optimization

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resulted in reduction of unfavorable codons by 50% and a considerable increase in the

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expression levels (total activities) of β-glucosidases (0.002 units/mL, unoptimized; 0.022

30

units/mL, optimized). The molecular weight of the enzyme was 52 kDa, and the purified

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forms of the enzymes could successfully convert Rb1 and Rd into F2. The permeabilized L.

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lactis expressing BglPm resulted in a high conversion yield (74%) of F2 from the ginseng

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extract. Utilization of this microbial cell to produce F2 may provide an alternative method to

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increase the health benefits of Panax ginseng.

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Keywords: β-glucosidase, ginsenoside F2, Lactococcus lactis

37 38

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1. Introduction

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Ginseng, the root of Panax ginseng Meyer, is one of the most commonly consumed

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medicinal plants worldwide. According to the 2012 report, ginseng products topped the

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health functional foods produced in South Korea, with the production accounting to 49.2% of

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the total.1 Ginsenosides are the major active compounds of ginseng and these exhibit

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pharmacological activities such as anti-cancer, anti-inflammatory, anti-aging, and neuro-

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protective activities.2-4 Ginsenosides are triterpene saponins, and most of these compounds

46

consist of a dammarane skeleton with various sugar moieties at the C3 and C20 positions.5

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Most of the identified ginsenosides have been classified into three groups on the basis of the

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differences in the aglycone moiety: (1) 20(S)-protopananxadiol type (PPD) (Rb1, Rb2, Rb3,

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Rc, Rg3, Rh2, and Rd), (2) 20(S)-protopanaxatriol type (PPT) (Re, Rf, Rg1, Rg2, and Rh1),

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and (3) oleanolic acid.5 Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 are the major ginsenosides that

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constitute more than 80% of the total ginsenosides.6 In contrast, the minor ginsenosides (Rg3,

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Rh2, F2, C-K, Rg2, Rh1, and F1), which have significant pharmaceutical properties, are

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present in low concentrations in raw ginseng and are therefore difficult to extract. In

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particular, F2, which induces apoptosis of breast cancer cells,7 exists in raw ginseng and red

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ginseng at concentrations less than 0.01%.8

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Thus far, various methods have been developed for converting major ginsenosides into

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minor ginsenosides, such as acid or alkali treatment, heat treatment, and enzymatic or

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microbial bioconversion.9 The physical methods are not selective for the hydrolysis of

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glucose moieties; however, the enzymatic bioconversion method is highly selective and

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efficient in specifically hydrolyzing the glucose moieties of major ginsenosides and is

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therefore considered an optimal method. β-Glucosidases have been isolated and characterized

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from various organisms such as Paecilomyces sp.,10 Flavobacterium sp.,11 and 3



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Mucilaginibacter sp.12 and have been used to produce minor ginsenosides. However, the

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enzyme purification step and enzyme recycling system are critical to success of the

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enzymatic conversion method owing to the high cost of enzymes. Moreover, the microbial

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bioconversion methods employing β-glucosidase-producing microorganisms result in slow

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reaction rates due to low enzyme activities.13 Therefore, microbial cells with high β-

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glucosidase activity are necessary for use in the direct (in situ) production of minor

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ginsenosides from major ginsenosides via microbial fermentation.

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Lactic acid bacteria (LAB) are used as starter cultures for the production of fermented

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foods prepared from milk, vegetables, and cereals.14 LAB have a “Generally Recognized As

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Safe” (GRAS) status and are potential candidates for the production of minor ginsenosides.

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LAB exhibited excellent probiotic characteristics such as modulation of the intestinal

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microflora, resistance to acid and bile, and production of antimicrobial substances.15

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Furthermore, couples of useful gene expression systems have been developed.16

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Therefore, the aim of this study was to express β-glucosidase gene in Lactococcus lactis

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subsp. cremoris NZ9000 (hereafter, L. lactis NZ9000) and to produce ginsenoside F2 from

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major ginsenosides Rb1 and Rd using the recombinant cells. For this purpose, we used the β-

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glucosidase gene (BglPm) amplified from the chromosome of Paenibacillus mucilaginosus

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KCTC 3870T to construct a plasmid using the pNZ8008 vector. We isolated P. mucilaginosus

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from ginseng soil and the enzyme showed strong ginsenoside-transformation ability,

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especially against major ginsenosides Rb1 and Rd.17 In addition, to increase transcription of

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BglPm in L. lactis, its codon sequence was optimized. Furthermore, after plasmid

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transformation and gene expression, β-glucosidase (BGL) enzyme activity was analyzed, and

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recombinant cells were used to produce the minor ginsenoside F2.

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2. Materials and Methods

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2.1 Bacterial strains, media, and plasmids

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Bacterial strains, plasmids, and oligonucleotides used in this study are listed in Table 1. L.

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lactis NZ9000 and plasmid pNZ8008 were used for gene expression. L. lactis NZ9000 was

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grown in M17 medium (Difco, Detroit, MI, USA) with 0.5% (w/v) glucose (GM17) at 30°C.

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Escherichia coli MC1061 (MoBiTec) was used as the cloning host; it was grown in Luria-

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Bertani (LB) medium at 37°C under shaking conditions. For selection of Lactococcus and E.

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coli transformants, chloramphenicol (10 µg/mL) was added to the LB medium. Gene

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expression was induced in L. lactis by using nisin, which was prepared as follows: 2.5% (w/v)

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nisin powder (Sigma) dissolved in 0.05% (v/v) acetic acid, for obtaining a final concentration

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of 1 mg/mL.

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2.2 Codon usage analysis and optimization

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For high expression of the recombinant protein, BglPm was synthesized at Bioneer

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(Daejeon, Korea) after codon optimization by using proprietary algorithms on the basis of

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codon usage, repeat sequence, GC content, and messenger RNA structure without

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substitution in amino acid sequences. The 6× His-tag sequence and restriction sites of PstI

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and EcoRI were introduced into the optimized sequence. The frequency of each codon (count

106

per

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http://www.bioinformatics.org/sms2/codon_usage.html and http://www.kazusa.or.jp/codon/,

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respectively. The codon usage was defined as codon number in L. lactis divided by codon

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number in BglPm. A codon usage value below 20% is considered “unfavorable codon”.18

thousand)

of

BglPm

gene

and

L.

lactis

genome

110 5


were

analyzed

at


111

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2.3 Cloning of BglPm and transformation into L. lactis

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The unoptimized BglPm gene was amplified using Bgl-N and Bgl-C primers from pGEX-

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bglPm,17 whereas the optimized BglPm gene was digested with PstI and XhoI from pBHA-

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BglPm, which was synthesized by Bioneer (Daejeon, Korea). The two fragments were

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inserted into the corresponding sites of the expression plasmid pNZ8008, which was

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linearized by digesting with PstI and XhoI, resulting in pNZBgl-unopt and pNZBgl-opt,

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respectively (Table 1). The recombinant plasmids were transformed into E. coli MC1061 and

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transformants were selected on LB agar containing 10 µg/mL chloramphenicol. For

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transformation,

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electroporated into L. lactis NZ9000 as previously described.19 Transformation was

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performed using a Gene-Pulser unit combined with a Pulse Controller (Bio-Rad, Richmond,

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CA, USA). The electrocompetent cells (40 µL) were placed in a pre-chilled electroporation

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cuvette with 1 µL DNA and kept on ice for 5 min. A pulse was applied under the following

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conditions: 25 µF, 400 Ω, and 1300 V cm-1. Immediately thereafter, cells were resuspended

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in 1 mL GM17 broth containing 20 mM MgCl2 and 2 mM CaCl2, and incubated at 30°C for 1

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h. The transformed cells were selected using GM17 agar with chloramphenicol.

the

recombinant

plasmids

pNZBgl-unopt

and

pNZBgl-opt

were

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2.4 Expression and purification of recombinant pNZBgl-unopt and pNZBgl-opt

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The recombinant L. lactis harboring pNZBgl-unopt and pNZBgl-opt were grown in

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GM17 broth containing chloramphenicol until O.D600nm 0.4. Protein expression was induced

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by 1 ng/mL nisin and cells were further cultured for 3 h at 30°C. Cells were then collected by

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centrifugation at 10,000× g for 5 min, and the pellets were resuspended in 50 mM sodium

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phosphate buffer (pH 7.0). Cells were disrupted by sonication, and the supernatant fraction

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was used as the crude enzyme. The enzyme fraction was purified using Ni-NTA 6


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chromatography with a HisTrap-FF column (GE Healthcare, Uppsala, Sweden) and elution

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buffer (250 mM imidazole, 0.3 M sodium chloride, 50 mM sodium phosphate; pH 7.0).

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Expression and purification of β-glucosidase were analyzed by sodium dodecyl sulfate

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polyarylamide gel electrophoresis (SDS-PAGE). The relative intensity of SDS-PAGE bands

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was analyzed by Image Lab software (Bio-Rad).

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2.5 Enzyme activity assay

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Enzyme activities of crude and purified β-glucosidases were measured using ρ-

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nitrophenyl-β-D-glucopyranoside (PNPGlc) as substrate. Crude enzyme (50 µL) or purified

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enzyme (20 µL) was incubated in 300 µL 50 mM sodium phosphate buffer (pH 7.0)

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containing 2 mM PNPGlc at 37°C. One unit activity was defined as the amount of enzyme

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required to produce 1 µmol ρ-nitrophenyl (PNP) per minute, which was measured using the

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microplate reader at 405 nm.

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2.6 Biotransformation activity of purified enzyme

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To determine the enzymatic biotransformation activity on ginsenoside compounds, the 6-

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histidine-tag purified β-glucosidase was reacted with the ginsenosides Rb1, Rd, and Rg3. Rb1,

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Rd, Rg3, Rh2, and F2 (≥98.0% purity) were used as standard compounds (Biopurify,

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Chengdu, China). The purified enzyme (100 µL) was incubated in 50 mM sodium phosphate

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buffer (300 µL, pH 7.0) containing 0.2% (w/v) of each ginsenoside, at 37°C for 12 h. It was

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analyzed by thin-layer chromatography (TLC) using 60F254 silica gel plates (Merck, Germany)

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with CHCl3-CH3OH-H2O (65:35:10, lower phase) as the developing solvent. The spots on the

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TLC plate was detected by 10% (v/v) H2SO4 followed by heating at 110°C for 10 min. For

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analysis of transformation activity for PPD type ginsenosides mixture (PPDGM), the purified 7



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enzyme (150 µL) was incubated in 50 mM sodium phosphate buffer (300 µL, pH 7.0)

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containing 0.5% (w/v) PPDGM, at 37°C for 8 h. Then, the change in the concentration of

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substrates and products were measured using Agilent 1260 Infinity HPLC (high-performance

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liquid chromatography; Young In Scientific Co, Seoul, Korea) system equipped with a

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ZORBAX SB-C18 column (4.6 × 150 mm). Acetonitrile (solvent A) and water (solvent B)

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were used as the mobile phases. Gradient elution was started with 32% solvent A and 68%

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solvent B, and 65% solvent A for 8 min, 100% A for 12 min, holding 100% A from 12 to 15

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min, 32% A for 30 s, and holding 32% A from 15.1 to 25 min. The flow rate of the mobile

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phase was 1.0 mL/min and it was monitored at an absorbance of 203 nm using a UV

168

spectrophotometric detector.

169 170

2.7 Bioconversion of major ginsenosides to minor ginsenosides using recombinant L. lactis

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To produce the minor ginsenoside F2, the recombinant L. lactis harboring pNZBgl-opt

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that showed higher expression level and activity was used. After cultivation, whole cells were

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harvested by centrifugation at 7,000× g for 10 min at 4°C and suspended in 50 mM sodium

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phosphate buffer (pH 7.0). In case of cell lysates, the whole cells were disrupted via

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sonication and the supernatant fraction was recovered after centrifugation. In addition, the

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permeabilized cells were prepared by mixing whole cells with 0.5% (v/v) xylene in reaction

177

buffer.20 Subsequently, the three types of cells (whole cells, cell lysates, and permeabilized

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cells, at a final concentration of 50 mg/mL) were reacted in 50 mM sodium phosphate buffer

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(pH 7.0) with 1% (w/v) of PPDGM for 24 h. Samples were taken at intervals (0, 4, 8, 12, and

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24 h) and centrifuged (10,000× g, 2 min, 4°C) after boiling for 5 min. Both the supernatant

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and residual fractions were extracted with 50% (v/v) ethanol and the two fractions were

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pooled for analysis. Major ginsenosides (Rb1 and Rd) and minor ginsenoside (F2) were 8


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quantified using HPLC. The bioconversion yield of ginsenoside F2 (%) was calculated as

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follows: conversion yield (%) = ∆F2 / (∆Rb1 + ∆Rd) × 100

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2.8 Bioconversion of major ginsenosides to F2 during milk fermentation

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Three replicate experiments were performed using fermented milk as follows: skim milk

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(15 g) and PPDGM (1%) were added to 85 mL water. After inoculation with starter cultures

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(6.5 log colony-forming units [CFU]/mL) of recombinant L. lactis, the mixture was incubated

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at 37°C for 48 h. L. lactis population density was measured in terms of CFUs per milliliter by

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using the culture-pouring method. Samples were serially diluted (10-1, 10-3, 10-5, and 10-7)

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with sterile physiological saline (0.85% NaCl) and spiral-plated on MRS agar medium. MRS

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plates were then incubated anaerobically in gas pack jars at 37°C for 48 h. The pH of the

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samples was determined with a pH meter (IQ240; IQ Scientific Instruments, San Diego, CA,

195

USA). Finally, the concentrations of Rb1, Rd, and F2 were measured using the method

196

described in the previous section.

197 198

3. Results

199 200

3.1 Analysis of codon usage and construction of recombinant L. lactis

201

To increase the expression level of β-glucosidases in L. lactis, BglPm codons were

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optimized without amino acid substitution. Results of the comparison between two sequences

203

are presented as supporting information (Fig. S1). As shown, the optimized sequence had

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83.9% homology with the unoptimized (native) one. In addition, because L. lactis NZ9000

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has low GC content (35.8%), the GC content of BglPm was reduced from 57% to 47%.

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Furthermore, we compared the percentage of “unfavorable codons” per 50 bases (calculated 9



207

of the total unoptimized and optimized BglPm sequences), and it decreased by half in the

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optimized gene (Fig. 1).

209 210

3.2 Comparison of expression and purification levels of two recombinant enzymes

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The plasmids bearing the BglPm genes were expressed in L. lactis with C-terminal 6×

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His-tag and their products were confirmed by SDS-PAGE analysis. As shown in Fig. 2A, 52-

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kDa BGL recombinant proteins were expressed in L. lactis and they were purified to

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homogeneity using a Ni-NTA affinity procedure. The expression level of L. lactis harboring

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pNZBgl-opt was higher than that of pNZBgl-unopt and the relative intensity of the protein

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was increased by about 1.5 fold (Fig. 2B). These results suggested that the reduction of

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unfavorable codons in BglPm gene sequence affects its expression level.

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The activities of β-glucosidase in cell-free extracts of recombinant L. lactis harboring

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pNZBgl-unopt and pNZBgl-opt were found to be 0.002 units/mL and 0.022 units/mL,

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respectively (Table 2), which indicated a significant increase in protein translation via gene

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optimization; the optimization factor was 11.

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3.3 Biotransformation of Rb1, Rd, and Rg3 by purified enzyme

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The biotransformation activity of purified β-glucosidase was confirmed by TLC using the

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major ginsenosides Rb1 and Rd, and the minor ginsenoside Rg3 (Fig. 3(A)). The BGL

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enzyme hydrolyzed the outer glucose moiety of C3 and C20 positions of the major

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ginsenosides (Rb1 and Rd) and produced the minor ginsenoside F2: Rb1→ Rd → F2.

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Furthermore, the enzyme could transform the minor ginsenoside Rg3 to Rh2: Rg3 → Rh2.

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These bioconversion reactions and the relative structures of ginsenosides are shown in Fig. 4.

230

As shown by the results of HPLC, the ginsenosides Rb1, Rd, and F2 were detected at 3.7 min, 10


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4.8 min, and 6.9 min retention times, respectively. Using the PPDGM as the substrate, the

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enzyme could transform Rb1 and Rd into F2 (Fig. 3(B)). In 0.5% (w/v) PPDGM solution,

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Rb1 and Rd were present at concentrations of 1.97 mM and 1.61 mM, respectively. After 8

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hrs reaction, 2.11 mM F2 was synthesized from the major ginsenosides Rb1 and Rd, and 1.45

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mM Rd still remained in the reaction solution. These results are consistent with a previous

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report that showed expression of the same gene in E. coli.17

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3.4 Production of the minor ginsenoside F2 using various forms of recombinant cells

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To increase the bioconversion rate of PPDGM to F2, we tested three forms of

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recombinant cells (L. lactis harboring pNZBgl-opt), including whole cells, cell lysates, and

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permeabilized cells. The whole cells of L. lactis harboring pNZ8008 were used as the control.

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Rb1 and Rd were present at concentrations of approximately 3.3 mM and 2.9 mM,

243

respectively, in 1% of PPDGM solution (Table 3). All three forms of recombinant cells

244

completely converted the major ginsenoside Rb1 into Rd or F2 within 24 h, whereas the

245

control fraction did not. On the other hand, small amounts of Rd, which is also a major

246

component of PPDGM, remained after the 24 h reaction (Fig. 5). The bioconversion yields of

247

whole cells, cell lysates, and permeabilized cells were 50%, 91%, and 74%, respectively

248

(Table 3).

249 250 251

3.5 Bioconversion of major ginsenosides to F2 during milk fermentation To explore whether F2 can be produced by the starter culture during milk fermentation, 1%

252

(w/v) of PPDGM dissolved in skim milk was fermented after inoculation of recombinant L.

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lactis. The inoculated cell densities (6.5 log CFU/mL) of L. lactis reached a maximum level

254

(8.8 log CFU/mL) after 24 h and decreased slowly thereafter, suggesting that the 11



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transformant grew well in skim milk including PPDGM. The initial pH of medium was

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approximately 7.3, and it was dropped consistently during the fermentation process. The pH

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at the end of the fermentation was 4.8 ± 0.15, which was higher than that of other forms of

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yogurt reported previousy.21,22 During the fermentation period, the concentration of

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ginsenosides (Rb1, Rd, and F2) and the bioconversion rate was measured. In all, 0.5 mM of

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F2 was synthesized from the major ginsenosides Rb1 and Rd (initial concentrations were 2.0

261

mM and 1.3 mM, respectively). The conversion ratio was 15%, which is lower than that

262

observed in the cellular conversions under buffer conditions. This low ration may be

263

attributed to the decrease in β-glucosidase activity at low pH conditions generated during the

264

milk fermentation. In summary, this experiment provides a possible application of L. lactis

265

developed in this study for manufacturing a ginseng-based yogurt, which serves as a health

266

functional food.

267 268

4. Discussion

269

Recent studies have focused on the various pharmacological effects of the minor

270

ginsenoside F2. For instance, Mai et al. reported that F2 suppresses the proliferation of breast

271

cancer stem cells by modulating apoptotic and autophagic fluxes.7 Shin et al. reported that F2

272

represses glioblastoma multiforme, a common malignant brain tumor, by inducing apoptosis

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of golima cells and inhibiting angiogenesis.23 In addition, F2 promoted hair growth and

274

anagen induction24 and reduced obesity via the inhibition of adipogenesis in the 3T3-L1 cell

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line.25 Furthermore, application of F2 to the skin could improve skin conditions, skin

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moisture content, skin complexion, and result in skin whitening.26 Therefore, F2 has immense

277

potential for use as a chemotherapeutic agent, cosmetic ingredient, and a functional food

12


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additive. Despite these potential applications, the production of the ginsenoside F2 has not

279

been intensely studied.

280

E. coli is the most widely used host microbe for heterologous enzyme production,

281

because of its well-characterized expression system, fast growth-rate, well-known genetic

282

information, and various high cell-density culture techniques.27 On the other hand, LAB

283

system will likely compete with E. coli in the food industry because it is suitable for food-

284

grade expression of enzymes. LAB are useful for the production of a variety of raw materials

285

for foods and feeds, and these are added as starter or adjunct cultures in different food

286

products.28,29 These cultures are known to influenced the texture, flavor, and shelf life of food.

287

Furthermore, the growth media required for LAB (e.g., MRS used for LAB culture costs ~9

288

€/L) is only three times costlier than media used for E. coli.30 Although further improvements

289

are needed, such as food grade selection makers, utilizing broad host microbes, and high cell-

290

density culture techniques, the LAB system has tremendous potential for protein production

291

in the food industry.31

292

In this study, to increase the expression of BglPm gene in L. lactis, we optimized the

293

codon sequence of the gene, and the expression level increased by about 11 fold. Teng et al.

294

also reported that differences in codon usage between the sequence and the expression host

295

would considerably affect the expression level of recombinant protein.32 In addition, the GC

296

content of BglPm was reduced from 57% to 47% because L. lactis NZ9000 has low GC

297

content (35.8%). This change might affect the increase of BglPm expression level in L. lactis.

298

Sinclair and Choy reported a consistent result that reduction of GC content would lead to 7.5%

299

increase in the expression of human glucocerebrosidase in Pichia pastoris cells.33 Therefore,

300

enhanced expression of BglPm can be interpreted to occur due to (i) improved translational

301

efficiency by codon optimization and (ii) increased mRNA transcription by reduced GC 13



302

content. The bioconversion yield of cell lysates was the highest among the three types of

303

recombinant cells used, which indicated the highest accumulation of β-glucosidase in the

304

cytoplasm. Therefore, secretion of β-glucosidase via the cell membrane would be helpful in

305

various aspects, such as to increase the target enzyme’s production yield, simplify enzyme

306

purification process, and convert ginsenosides directly during microbial fermentation.

307

Furthermore, the secretion system of L. lactis would also provide merit because of the

308

absence of lipopolysaccharide and protease, compared to the well-known protein secretion

309

hosts such as Bacillus subtilis and E. coli.

310

In conclusion, we produced the minor ginsenoside F2 from the major ginsenosides Rb1

311

and Rd, by cloning BglPm from P. mucilaginosus and expressing it in L. lactis. After codon

312

optimization, the percentage of unfavorable codons decreased by half and the expression

313

levels of β-glucosidases significantly increased. SDS-PAGE analysis of the purified protein

314

resulted in a single band with a molecular weight comparable to that of β-glucosidase (52

315

kDa). The whole cells of L. lactis harboring pNZBgl-opt were reacted with PPDGM for 24 h,

316

and Rb1 and Rd were converted into F2, resulting in a conversion yield of 50%. Moreover,

317

when the harvested cells were permeabilized with xylene, the conversion yield sharply

318

increased up to 74%. Thus, our study findings demonstrate that the permeabilized L. lactis

319

expressing the β-glucosidase gene can be used to produce F2 in ginseng extract. To our

320

knowledge, this is the first report of heterologous expression of β-glucosidase in an LAB

321

system. We believe that the application of this cell factory system to produce F2 will provide

322

an alternative approach to increase the health functions of Panax ginseng.

323 324

Acknowledgement

325

This study was supported by the Intelligent Synthetic Biology Center of Global Frontier 14


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326

Project, funded by the Korean Ministry of Science, ICT and Future Planning

327

(2013M3A6A8073553) and the National Research Foundation of Korea (NRF) grant

328

(2015R1A2A2A01007156) funded by the Korea Government (MEST).

329 330

Supporting information

331

Comparision of sequences of unoptimized and optimized BglPm gene. The alignment of two

332

sequences was performed using Vector NTI and displayed using GeneDoc software.

333

The supporting information is available free of charge on the ACS Publications website at

334

http://pubs.acs.org.

335

15



336 337 338

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model in SD rats. J. Ginseng Res. 2012, 36, 86-92.

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436 437

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438


Figure legends

439 440

Fig. 1 Percentage of “unfavorable” codons per 50 bases calculated over the entire genes for

441

unoptimized and optimized beta-glycosidase from Paenibacillus sp.

442 443

Fig. 2 SDS-PAGE analysis of the recombinant L. lactis harboring pNZBgl-unopt and

444

pNZBgl-opt (A) and the relative band intensity (B) analyzed using Image Lab software.

445

Control, no induction; M, molecular markers; T, total fraction; S, soluble fraction; I, insoluble

446

fraction; E, elusion fraction.

447 448

Fig. 3 Biotransformation of standard compounds (Rb1, Rd, and Rg3(S)) and PPDGM using

449

purified enzyme of recombinant L. lactis harboring pNZBgl-opt, they were analyzed using

450

TLC (A) and HPLC (B), respectively. St, ginsenoside standards; 1, Rb1; 2, reaction mixture

451

of Rb1; 3, Rd; 4, reaction mixture of Rd; 5, Rg3(S); 6, reaction mixture of Rg3(S); (a), before

452

enzyme reaction; (b), after enzyme reaction.

453 454

Fig. 4 Schematic view of transformation pathways for F2 production and the relative

455

structures of ginsenosides.

456 457

Fig. 5 Production of minor ginsenoside F2 using various forms of recombinant L. lactis

458

(pNZBgl-opt) cells. Whole cells without nisin induction (A) used as control; whole cells (B),

459

whole cell lysates (C), and permeabilized cells (D) with nisin induction. Rb1 (◆); Rd (▲);

460

F2 (●).

21



Page 22 of 30

Table 1 Strains, plasmids, and primers used in this study Strains, plasmids,

Relevant characteristics

Source

MG1363 derivated, pepN::nisRK, expression host

MoBiTec

araD139, ∆(ara, leu)7697, ∆lacX74, galU-, galK-, hsr-,

MoBiTec

and primers Strains Lactococcus lactis NZ9000 Escherichia MC1061

coli

+

hsm , strA, cloning host

Plasmids pNZ8008

PnisA, gus A, CmR, replicon of rolling circle plasmid

MoBiTec

pSH71 pNZBgl-unopt

pNZ8008 carrying unoptimized Bgl gene

This study

pNZBgl-opt

pNZ8008 carrying optimized Bgl gene

This study

Bgl-N

5′-TACTGCAGATGGAATATATTTTTCCACAG-3′

This study

Bgl-C

5′-TACTCGAGTTAGTGGTGATGATGGTGATGCA

This study

Primers

GCACTTTCGTGGATGC-3′

22


Page 23 of 30


Table 2 Total activities of β-glucosidase in cell-free extracts of induced and uninduced culture of recombinant L. lactis harboring pNZBgl-unopt and pNZBgl-opt

L. lactis (pNZBgl)

Step

Total Volume Activity activity (mL) (units/mL) (units)

Yield Induction Optimization (%) factor factora

unoptimized Crude enzyme + uninduced

100

0.001

0.1

Crude enzyme

100

0.002

0.2

100

Ni-NTA purification

100

0.001

0.1

50

Crude enzyme

100

0.022

2.2

100

Ni-NTA purification

100

0.017

1.7

77

unoptimized + induced

optimized + induced a

1 2

1

11

Optimization factor = total activity of optimized BGL/total activity of unoptimized BGL

23



Page 24 of 30

Table 3 Concentration of ginsenosides Rb1, Rd, and F2 and bioconversion yields in each sample using various recombinant cells

Ginsenoside Rb1 (mM)

Ginsenoside Rd (mM)

Ginsenoside F2 (mM) Conversion yield (%)a

Samples 0h

24 h

0h

24 h

0h

24 h

Control

3.37 ± 0.07

3.35 ± 0.01

2.89 ± 0.05

2.89 ± 0.11

0.05 ± 0.00

0.05 ± 0.00

0

Whole cells

3.25 ± 0.07

0.09 ± 0.00

3.00 ± 0.06

1.42 ± 0.04

0.09 ± 0.00

2.46 ± 0.06

50

Cell lysates

3.27 ± 0.01

0.05 ± 0.00

2.88 ± 0.01

1.93 ± 0.00

0.09 ± 0.00

3.90 ± 0.03

91

Permeabilized cells

3.36 ± 0.03

0.02 ± 0.00

2.94 ± 0.01

0.95 ± 0.03

0.06 ± 0.00

4.01 ± 0.18

74

a

Conversion yield (%) = ∆F2/(∆Rb1 + ∆Rd)×100 The values in the table are average determined from three independent experiments and standard errors are shown.

24



30 pNZBgl-unopt pNZBgl-opt

25

20

15

10

5

Codon number Fig. 1

25


42 0 40 0-

40 0 35 0-

35 0 30 0-

30 0 25 0-

25 0 20 0-

20 0 15 0-

15 0 10 0-

50 -

10 0

0 150

Percentage "unfavorable" codons (%)

Page 25 of 30


Page 26 of 30

(B) 2500

Relative intensity

2000

1500

1000

500

0

T

S 10*I control

Fig. 2

26


T S 10*I pNZBgl-unopt

T

S 10*I pNZBgl-opt

Page 27 of 30


(B) 2000

Rb1

(a)

Peak Area (mAU*s)

1500

1000

Rd

500

0 2000

(b) F2

1500

1000

500

0 0

2

4

6

8

Retention time (min)

Fig. 3

27


10


Glc

Page 28 of 30

Glc

Rb1

Rd

F2

Glc

Rg3

Rh2

Fig. 4

28


Page 29 of 30


(B) 0.5

0.4

0.4

Concentration (%)

Concentration (%)

(A) 0.5

0.3

0.2

0.3

0.2

0.1

0.1

0.0

0.0 0

5

10

15

20

0

25

5

10

(C)

20

25

20

25

(D)

0.5

0.5

0.4

0.4

Concentration (%)

Concentration (%)

15

Time (h)

Time (h)

0.3

0.2

0.1

0.3

0.2

0.1

0.0

0.0

0

5

10

15

20

25

0

5

Time (h)

10

15

Time (h)

Fig. 5

29



Table of contents PPD type ginsenoside mixture xylene Lactococcus lactis

Glc Β-glucosidase

Rb1

Rd Β-glucosidase

Glc

F2 F2

30


Page 30 of 30

Production of Ginsenoside F2 by Using ... - ACS Publications

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