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Metabolic engineering of Corynebacterium glutamicum for the high-level production of cadaverine that can be used for the synthesis of bio-polyamide 510 Hee Taek Kim, Kei-Anne Baritugo, Young Hoon Oh, Sung Min Hyun, Tae Uk Khang, Kyoung Hee Kang, Sol Hee Jung, Bong Keun Song, Kyungmoon Park, Il-Kwon Kim, Myung Ock Lee, Yeji Kam, Yong Taek Hwang, Si Jae Park, and Jeong Chan Joo ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00009 • Publication Date (Web): 28 Feb 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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ACS Sustainable Chemistry & Engineering

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Metabolic engineering of Corynebacterium glutamicum for the high-

2

level production of cadaverine that can be used for the synthesis of

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bio-polyamide 510

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Hee Taek Kim1,8, Kei-Anne Baritugo2,8, Young Hoon Oh1,8, Sung Min Hyun1,3, Tae Uk

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Khang1,4, Kyoung Hee Kang1, Sol Hee Jung2, Bong Keun Song1, Kyungmoon Park3,

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Il-Kwon Kim5, Myung Ock Lee6,7, Yeji Kam6, Yong Taek Hwang6, Si Jae Park2,*,

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Jeong Chan Joo1,*

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Institute of Chemical Technology, P.O. Box 107, 141 Gajeong-ro, Yuseong-gu,

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Daejeon 34114, Republic of Korea

Center for Bio-based Chemistry, Division of Convergence Chemistry, Korea Research

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52 Ewhayeodae-gil, Seodaemun-gu, Seoul 03760, Republic of Korea

Division of Chemical Engineering and Materials Science, Ewha Womans University,

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Sejong-ro, Jochiwon-eup, Sejong-si 30016, Republic of Korea

Department of Biological and Chemical Engineering, Hongik University, 2639

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Seongbuk-gu, Seoul 02841, Republic of Korea

Department of Chemical and Biological Engineering, Korea University, 145 Anam-ro,

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of Korea

Bioprocess R&D Center, DAESANG Corp., Icheon-si, Gyeonggi-do, 17384, Republic

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ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Lotte Chemical, 115 Gajeongbuk-ro, Yuseong-gu, Daejeon, 34110, Republic of Korea

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Department of Chemistry, KAIST, 291, Daehak-ro, Yuseong-gu, Daejeon 34141,

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Republic of Korea

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5 6

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work.

Hee Taek Kim, Kei-Anne Baritugo, and Young Hoon Oh contributed equally to this

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*Correspondence should be addressed to Si Jae Park ([email protected]) and Jeong Chan Joo ([email protected])

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1

Abstract

2

Fermentative production of cadaverine from renewable resources may support a

3

sustainable biorefinery process to produce carbon-neutral nylons such as bio-

4

polyamide 510 (PA510). Cost-competitive production of cadaverine is a key factor in

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the successful commercialization of PA510. In this study, an integrated biological and

6

chemical

7

polymerization with sebacic acid was developed to produce bio-PA510.

process

involving

cadaverine

biosynthesis,

purification,

and

its

8

To stably express ldcC from Escherichia coli in an engineered Corynebacterium

9

glutamicum PKC strain, an expired industrial L-lysine-producing strain, ldcC was

10

integrated into the chromosome of the C. glutamicum PKC strain by disrupting lysE

11

and controlling its expression via a strong synthetic H30 promoter. Cadaverine was

12

produced at a concentration of 103.78 g/L, the highest titer to date, from glucose by

13

fed-batch culture of this engineered C. glutamicum PKC strain. Fermentation-derived

14

cadaverine was purified to polymer-grade bio-cadaverine with high purity (99%) by

15

solvent extraction with chloroform and two-step distillation. Finally, bio-based PA510

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with good thermal properties (Tm 216°C and Tc 165°C) was produced by

17

polymerization of purified cadaverine with sebacic acid. The hybrid biorefinery

18

process combining biological and chemical processes demonstrated in this study is a

19

useful platform for producing bio-based chemicals and polymers.

20 21

Keywords:

22

Corynebacterium glutamicum, Lysine decarboxylase

Bio-polyamide,

PA510,

Cadaverine,

23

3


L-lysine,

Recombinant


1

Introduction

2

Increasing concerns regarding global climate change and depletion of fossil resources

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have drawn much attention for the development of sustainable processes as promising

4

alternatives to conventional petroleum-based chemical processes.1-4 Biorefinery

5

processes have been developed to promote sustainable chemical industries to produce

6

several platform chemicals, polymers, and fuels from renewable resources.

7

Fermentation processes employing metabolically engineered microbial host strains

8

have successfully been developed for production of platform chemicals such as organic

9

acids, alcohols, and amines, and polymers such as polyesters and polyamides from

10

biomass-derived sugars, including C6 glucose and C5 xylose.2-13

11

Polyamides (nylons) are polymers of repeating monomers of diamines and

12

dicarboxylic acids. Because polyamides have high thermal and mechanical strength

13

and abrasion resistance, they have extensively been used in the manufacture of textiles

14

such as carpet and sportswear and in the automotive industry for engineering

15

plastics.14-17 Moreover, polyamides can be used in clinical medicine to produce sutures,

16

catheters, and dental resin because of their high tensile strength and good elasticity and

17

flexibility.17,19 Global demands for representative petrochemical-based synthetic

18

polyamides, polyamide 6 (nylon 6) and polyamide 66 (nylon 66), were 4.2 and 2.1

19

million metric tons in 2010, respectively.18 Polyamides form diverse material

20

properties by employing monomers with different carbon atom numbers and functional

21

groups. Particularly, bio-based polyamides such as nylon 4, nylon 510, and nylon 56

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have extensively been developed for their possible applications in the apparel, food

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packaging, automobiles, and electronics industries.14-25

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L-Lysine, one of the most successful commercial products produced by microbial

2

fermentation processes, can be used as a versatile precursor to synthesize several

3

building-block chemicals such as cadaverine, 5-aminovaleric acid, glutaric acid, 1,5-

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pentanediol, adipic acid, and carprolactam.9,10,15-21 The annual production of L-lysine

5

by fermentation employing recombinant Escherichia coli and Corynebacterium

6

glutamicum as host strains exceeds 1.5 million tons.9 Cadaverine, a C5 diamine

7

biologically produced by decarboxylation of L-lysine using lysine decarboxylase as a

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biocatalyst, is a promising monomer for bio-based nylons such as nylon 56, nylon 510,

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and nylon 512. Microbial production of cadaverine has been examined in recombinant

10

E. coli and C. glutamicum strains by plasmid- or genome-based heterologous

11

expression of E. coli cadA and ldcC encoding inducible and constitutive lysine

12

decarboxylase, respectively.18-27

13

Plasmid-based heterologous expression of lysine decarboxylase gene has mainly been

14

examined for cadaverine production in recombinant C. glutamicum strains.18-27

15

Plasmid-based expression of E. coli cadA under a highly constitutive promoter in the

16

engineered C. glutamicum strain with cell-surface expression of amylase enabled

17

production of 2.34 g/L of cadaverine from 50 g/L of starch.26 However, plasmid-based

18

expression of heterologous genes has serious limitations such as a gradual reduction of

19

productivity and yield of target products in large-scale fermentation, as the metabolic

20

burden of host strains may be increased by the maintenance of plasmids and

21

segregational instability of plasmids, which may cause loss of plasmids during

22

cultivation.4,28,29 Chromosomal integration of heterologous genes is a good alternative

23

to plasmid-based expression of target genes.4 In a previous report, the highest

24

cadaverine concentration of 88 g/L was achieved by fed-batch fermentation of C.

5



1

glutamicum DAP-16. This strain was constructed by chromosome integration of a

2

codon-optimized variant of E. coli ldcC at the bioD locus encoding diothiobiotin

3

synthetase in an L-lysine hyper-producing strain C. glutamicum LYS-12, with

4

replacement of the native promoter by the tuf promoter (PtufldcCopt).24

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The selection of suitable promoters for efficient expression of heterologous genes is a

6

key factor for improving the production of target chemicals in host strains.30-33

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Previous studies showed that synthetic promoters have a favorable effect on tuning the

8

expression levels of heterologous genes in recombinant C. glutamicum, ultimately

9

supporting enhanced production of target products.31 For example, C. glutamicum

10

strains can be engineered to produce gamma-aminobutyrate (GABA), cadav¬!erine,

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and 5-aminovaleric acid from renewable resources by expressing glutamate

12

decarboxylase, lysine decarboxylase, and lysine 2-monooxygenase and delta-

13

aminovaleramidase, respectively, under synthetic promoters with different promoter

14

strengths.14-17,25,34,35 It was found that the final concentrations of GABA, cadaverine,

15

and 5-aminovaleric acid were highly dependent on the promoter strength and

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metabolic capacity of host strains to provide precursor metabolites such as L-glutamate

17

or L-lysine.14-17,25,34,35

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In this study, an integrated biological and chemical process for producing bio-PA510

19

was developed by combining microbial fermentation for the production of cadaverine,

20

purification of fermentation-derived cadaverine into polymer-grade cadaverine, and

21

polymerization of bio-cadaverine with sebacic acid into PA510 (Figure 1). Plasmid-

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and genome-based expression of E. coli ldcC was examined under the control of

23

synthetic promoters in a recombinant C. glutamicum PKC strain, an expired

24

commercial L-lysine-producing strain. Fed-batch fermentation of the C. glutamicum

6


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PKC strain expressing E. coli ldcC under the optimal promoter PH30, which is

2

integrated into lysE locus in the chromosome, resulted in the highest cadaverine

3

production. Polymer-grade cadaverine was obtained by sequential solvent extraction

4

and distillation of the fermentation broth. Finally, bio-based PA510 was produced by

5

poly-condensation of purified cadaverine with sebacic acid.

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1

Methods

2

Chemicals

3

Chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO,

4

USA) and used without further purification unless otherwise specified. Sulfuric acid

5

(Sigma-Aldrich, 95%) was employed as a dissolution solvent for the relative viscosity

6

analysis and m-cresol (Alfa Aesar, Haverhill, MA, USA, 99%) was used as a

7

dissolution solvent for molecular weight analysis.

8 9

Bacterial strains and plasmids

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All bacterial strains and plasmids used in this study are listed in Table 1. E. coli XL1-

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Blue (Stratagene, La Jolla, CA, USA) was used for general gene cloning studies and

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the C. glutamicum PKC strain was provided by DAESANG Corp (Gunsan, Korea).

13

Plasmids for the expression of E. coli ldcC under different synthetic promoters were

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constructed as previously described.25 Recombinant C. glutamicum PKC strains

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harboring plasmids for the expression of E. coli ldcC were constructed as described

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previously.25

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Genetic modifications of the C. glutamicum PKC genome were carried out by

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homologous recombination and two-step colony selection using kanamycin resistance

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and the SacB system.24 The chromosome integration plasmid (pK19mobsacB)

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containing the SacB system was introduced into the C. glutamicum PKC strain for

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homologous recombination of the gene expression cassette containing synthetic

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promoter, E. coli ldcC, and terminator into the lysE locus encoding an L-lysine

23

exporter.

24

8


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Plasmid construction

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All DNA manipulations were performed following standard procedures.36 Polymerase

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chain reaction (PCR) was performed in the C1000 Thermal Cycler (Bio-Rad, Hercules,

4

CA, USA). Primers used in this study (Table S1) were synthesized at Bioneer

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(Daejeon, Korea). Plasmid pK19mobsacB-lysEF was constructed by inserting the 5′-

6

region of lysE amplified from C. glutamicum PKC gDNA into pK19mobsacB at the

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HindIII and PstI sites. Plasmid pK19mobsacB-lysEFB was constructed by inserting the

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3′-region of lysE amplified from C. glutamicum PKC gDNA into pK19mobsacB-lysEF

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at the BamHI and EcoRI sites. Plasmids pK19mobsacB-lysEFB-L10ECLdcC,

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pK19mobsacB-lysEFB-L26ECLdcC,

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pK19mobsacB-lysEFB-I64ECLdcC,

12

pK19mobsacB-lysEFB-H36ECLdcC were constructed by inserting the gene expression

13

cassette containing synthetic promoter, E. coli ldcC, and terminator, which was

14

amplified

15

pCES208I16ECLdcC,

16

pCES208H36ECLdcC into plasmid pK19mobsacB-lysEFB at the PstI and BamHI

17

sites, respectively (Table 1).

from

plasmids

pK19mobsacB-lysEFB-I16ECLdcC, pK19mobsacB-lysEFB-H30ECLdcC,

pCES208L10ECLdcC,

pCES208I64ECLdcC,

and

pCES208L26ECLdcC,

pCES208H30ECLdcC,

and

18 19

Culture condition

20

E. coli XL1-Blue cells for gene cloning were cultured at 37°C in Luria-Bertani (LB)

21

medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl). Flask cultures of C.

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glutamicum were performed at 30°C and 250 rpm in a rotary shaker. The CG50

23

medium for flask cultivation contained (per liter): 50 g glucose, 30 g yeast extract, 30 g

24

(NH4)2SO4·7H2O, 0.5 g KH2PO4, 0.5 g MgSO4·7H2O, 0.01 g MnSO4·H2O, 0.01 g

9



1

FeSO4·7H2O, 0.5 mg biotin, and 0.3 mg thiamine-HCl. Kanamycin (Km) was added to

2

the culture medium at 20 mg/L for recombinant C. glutamicum strains. For

3

maintenance of pH around 7 during flask cultivation, 15 g/L of calcium carbonate was

4

added in culture medium.

5

Fermentation

6

Batch fermentations were carried out at 30°C and 600 rpm in a 2.5-L jar fermenter

7

(BioCNS, Daejeon, Korea) containing 500 mL of CG100 medium. CG100 medium

8

contained (per liter): 100 g glucose, 30 g yeast extract, 30 g (NH4)2SO4·7H2O, 0.5 g

9

KH2PO4, 0.5 g MgSO4·7H2O, 0.01 g MnSO4·H2O, 0.01 g FeSO4·7H2O, 0.5 mg biotin,

10

and 0.3 mg thiamine-HCl.s

11

Fed-batch fermentations were carried out at 30°C and 600 rpm in a 2.5-L jar

12

fermenter (BioCNS) initially containing 500 mL of CG100 medium. Glucose feeding

13

was started at a concentration of 10 g/L, and then the glucose concentration was

14

maintained at 10–40 g/L by continuously adding glucose solution. The feeding solution

15

contained (per liter): 700 g glucose, 270 g (NH4)2SO4·7H2O, 0.5 g MgSO4·7H2O, 0.01

16

g MnSO4·H2O, and 0.01 g FeSO4·7H2O. The culture pHs of batch and fed-batch

17

fermentations were maintained at pH 6.9 by automatic addition of 28% (v/v) NH4OH.

18

Foam formation was suppressed by adding Antifoam 204, and cell growth was

19

monitored by measuring the OD600.

20 21

Purification of cadaverine

22

Solvent extraction was conducted to extract cadaverine in the fermentation broth as

23

described by Kind et al. with some modifications.24 An appropriate amount of NaOH

24

was added to the supernatant in a glass reactor to adjust the pH to 14. The same

10


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1

volume of n-butanol or chloroform was added to the reactor and then mixed with the

2

pH-adjusted supernatant using a magnetic heat stirrer at 300 rpm and 55°C for 2 h. The

3

mixture was then transferred into separation funnels for phase separation. The organic

4

phase was collected in a round-bottom flask for further procedures and the aqueous

5

phase was treated again with the same extraction procedure as described above. The

6

collected organic phase from the 1st and 2nd extractions was concentrated using a rotary

7

evaporator at 60°C and 175 rpm. Fractional distillation was carried out using a

8

laboratory distillation apparatus at 25–120 mbar and 120–135°C to yield cadaverine

9

(diaminopentane) with a purity higher than 99.5%.

10 11

Polymerization of purified cadaverine for the synthesis of bio-based PA510

12

Purified cadaverine 65 g (636 mmol, 1.02 eq) and sebacic acid 126 g (623 mmol, 1.00

13

eq) were used as monomers for polymerization of bio-based PA510. Monomers and

14

deionized water 170 g (47 wt%) as a solvent were added to a 1-L autoclave reactor.

15

Next, the catalyst sodium hypophosphite monohydrate 13.2 mg (0.12 mmol, 79 ppm of

16

final polymer) was added. After the reactor was closed tightly, the reactor was flushed

17

with nitrogen gas at room temperature to remove the oxygen gas present in the reactor.

18

To obtain PA510 salt, the reaction temperature was raised to 140°C for 30 min. After

19

finishing salt formation, pre-polymerization was carried out. The reactor was heated

20

until 215°C, followed by increasing the pressure. After the pressure reached 17.2 bar,

21

the water vapor was slowly eliminated for 45 min to maintain a constant pressure.

22

Subsequently, the pressure in the reactor was gradually reduced to an atmospheric

23

pressure of 45 min to complete pre-polymerization. To perform polymerization, the

11



1

reaction temperature was increased to 270°C and the reactor was stirred for 80 min at 1

2

atm, followed by stirring for 10 min under full vacuum. PA510 polymerized in the

3

reactor was extruded under nitrogen pressure.

4

5

Analysis

6

The concentrations of organic acids and glucose were determined by high-

7

performance liquid chromatography using an Agilent Infinity 1260 System (Agilent

8

Technologies, Santa Clara, CA, USA) equipped with an Aminex HPX-76H column

9

(300 × 7.8 mm) (Bio-Rad). Concentrations of cadaverine and lysine were determined

10

using an Agilent Infinity 1260 System equipped with an Optimapak C18 column (150

11

× 4.6 mm) (Restek, Bellefonte, PA, USA) as reported previously.25 The purity of the

12

distillation product was measured with an Agilent 7890A system equipped with an

13

RTX 5-Amine column (30 m × 0.25 mm × 0.5 µm; Restek). Helium (purity 99.999%)

14

was used as carrier gas at a flow rate of 1.7 mL min-1. The inlet temperature was 250°C

15

and temperature at the detector was 300°C.

16

Bio-based PA510 was dissolved in hexafluoro-2-propanol (HFIP) and CDCl3 solvent

17

for proton nuclear magnetic resonance (1H-NMR, DD2 (500 MHz), Agilent) analysis.

18

Chemical shifts were expressed in parts per million (ppm) scale. Melting temperature

19

(Tm) and crystallization temperature (Tc) were measured by differential scanning

20

calorimetry (DSC, Q200, TA Instruments, New Castle, DE, USA). The PA510 sample

21

was melted gradually at 10°C/min and then cooled, and then reheated at the same rate

22

from 30 to 250°C. The degradation temperature (Td) was determined by Thermo-

23

Gravimetric Analysis (TGA, Q2950, TA Instruments). The PA510 sample was heated

12


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1

from 40°C to 900°C at 20°C/min under N2. Relative viscosity was measured with an

2

Ubbelohde viscometer at 25°C. To determine the viscosity, 0.3 g of prepared polymer

3

was dissolved in 30 mL 95% H2SO4 solvent. The RV value was calculated using a

4

formula. The RV value was dimensionless because the units of the numerator and

5

denominator canceled each other. RV = η/η0 (η: viscosity of the polymer solution, η0:

6

viscosity of the solvent). Molecular weight was determined by gel permeation

7

chromatography (PL220C, Agilent) with a Jordi column for polyamide analysis. The

8

gel permeation chromatography analysis solvent was m-cresol, and the sampling and

9

analysis temperature was 100°C with the PS standard. To validate the physical

10

properties as plastics, tensile strength was measured with a Universal testing machine

11

(5566, Instron, Norwood, MA, USA) following the ASTM D634 with test specimen

12

type1.

13

13



1

Results and discussion

2

Examination of the L-lysine production capacity of expired industrial C.

3

glutamicum strains

4

C. glutamicum has extensively been used for industrial production of L-amino acids,

5

particularly L-lysine, a direct precursor of cadaverine. Therefore, a cadaverine-

6

producing C. glutamicum strain can be relatively easily developed by heterologous

7

expression of lysine decarboxylase in C. glutamicum (Figure 1).9 However, it is

8

important to screen C. glutamicum strains with high L-lysine production capacity as

9

the host strain for the expression of lysine decarboxylase to achieve enhanced

10

production of cadaverine. Previously, we examined the L-lysine production ability of

11

expired industrial C. glutamicum strains to select an optimal host for 5-aminovalerate

12

production.35 C. glutamicum KCTC 1857 strain produced the largest amount of lysine

13

compared to other expired industrial strains that were mutated by ultraviolet light (UV)

14

or chemical analogues to attenuate feedback inhibition.35 The C. glutamicum KCTC

15

1857 strain showed the highest lysine production of 4.32 ± 0.45 g/L from 50 g/L of

16

glucose in shaking flask cultivation for 120 h, but also produced 1.17 ± 0.19 g/L of

17

lactic acid.35 A low concentration of dissolved oxygen in culture media frequently led

18

to the formation of by-products by the C. glutamicum strain, such as lactic acid and

19

acetic acid. This result indicates that metabolic flux of the C. glutamicum KCTC 1857

20

strain does not mainly use the tricarboxylic acid cycle, which maintains cell growth

21

and produces oxaloacetate, a precursor of lysine in this cycle.37 Thus, in this study, C.

22

glutamicum PKC strain, another expired industrial L-lysine producer kindly provided

14


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1

by Daesang Corp., was further examined to compare its L-lysine production capacity

2

with that of other strains examined previously.

3

C. glutamicum PKC strain produced 10.4 ± 1.56 g/L of L-lysine from 50 g/L of

4

glucose, which is more than two-fold higher than that produced by C. glutamicum

5

KCTC 1857 (Figure 2). Furthermore, no accumulation of lactic acid and acetic acid

6

was observed in flask cultivation of C. glutamicum PKC, suggesting that this strain can

7

be used as a practical biorefinery biocatalyst for L-lysine-derived products. Therefore,

8

the C. glutamicum PKC strain was examined as a host strain for enhanced production

9

of cadaverine by constructing an efficient expression cassette for the lysine

10

decarboxylase gene.

11 12

Plasmid-based expression of E. coli ldcC in recombinant C. glutamicum PKC

13

strain for cadaverine production

14

Synthetic promoter-based expression cassettes previously developed for the

15

heterologous expression of E. coli ldcC in recombinant C. glutamicum KCTC 185725

16

were transformed into the C. glutamicum PKC strain to investigate the effect of

17

promoter strength on cadaverine production (Table 1). The expression level of E. coli

18

ldcC in recombinant C. glutamicum PKC strains was controlled by three constitutive

19

promoter groups: high (H), intermediate (I), and low (L) strength (Figure 1). All

20

recombinant C. glutamicum PKC strains showed higher cadaverine production (8.50–

21

12.5 g/L) compared to previous results in recombinant C. glutamicum KCTC 1857

22

strains (0.64–1.01 g/L) (Figure 3).22 The highest cadaverine concentration of 12.5 ±

23

0.67 g/L was produced by recombinant C. glutamicum P-H30. In contrast, a relatively

24

lower amount of cadaverine (9.7 ± 0.71 g/L) was produced by the recombinant C.

15



1

glutamicum P-H36. Interestingly, C. glutamicum P-L10 (10.9 ± 0.93 g/L) and C.

2

glutamicum P-L26 (11.2 ± 2.61 g/L) strains with low strength promoters produced

3

higher concentration of cadaverine compared to the recombinant strains with

4

intermediate strength promoters, C. glutamicum P-I16 (8.50 ± 3.24 g/L) and C.

5

glutamicum P-I64 (11.0 ± 1.83 g/L). No significant correlation between promoter

6

strength and cadaverine production was observed in six recombinant C. glutamicum

7

PKC strains. However, it was found that L-lysine accumulated up to 0.47 ± 0.07 and

8

0.88 ± 0.16 g/L in the culture medium during flask cultures of recombinant C.

9

glutamicum P-L12 and C. glutamicum P-L26. In contrast, C. glutamicum P-H30 and C.

10

glutamicum P-H36 resulted in lower accumulation of L-lysine of approximately 0.05–

11

0.09 g/L in the culture medium. It was demonstrated that the deletion of L-lysine

12

exporter protein (LysE), which is mainly responsible for L-lysine secretion in C.

13

glutamicum, enhanced the production of L-lysine-derived platform chemical, 5-

14

aminovaleric acid by reducing much secretion of L-lysine into culture medium without

15

any deleterious effects on cell growth.

16

of cadaverine in C. glutamicum PKC strains may be enhanced if secretion of L-lysine

17

into cultivation medium is eliminated, which makes more L-lysine in the cells

18

available for cadaverine production.

34.,38

Therefore, it was reasoned that production

19 20

Examination of recombinant C. glutamicum PKC strains with genomic

21

integration of expression cassette for E. coli ldcC at lysE site for cadaverine

22

production

23

Because L-lysine accumulation in the culture medium may have resulted from

24

unstable expression of E. coli ldcC or metabolic burden caused by the plasmid-based

16


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1

expression system, the expression cassettes comprised of different combinations of six

2

synthetic promoters (L10, L26, I16, I24, H30, and H36) and E. coli ldcC were

3

integrated into the chromosome of a C. glutamicum PKC strain at the lysE locus via

4

homologous recombination (Table 1).21,39,40 Because the lysine exporter protein LysE

5

secretes L-lysine independently of cadaverine secretion, we predicted that

6

chromosomal integration of E. coli ldcC into the lysE locus of the C. glutamicum PKC

7

strain will lead to increased production of cadaverine by stable expression of lysine

8

decarboxylase and reduction of L-lysine secretion. The concentrations and yield of

9

cadaverine produced by recombinant C. glutamicum PKC strains expressing E. coli

10

ldcC integrated into the chromosome were 1.30–12.0 g/L and 0.05-0.42 mol

11

cadaverine/mol glucose. As shown in Figure 3, these were lower than those produced

12

by recombinant C. glutamicum PKC strains with plasmid-based expression of E. coli

13

ldcC (8.50–12.5 g/L, 0.30-0.44 mol cadaverine/mol glucose). Among the lysE deletion

14

mutants, only C. glutamicum G-H36 was able to produce relatively higher

15

concentrations of cadaverine (12.0 ± 0.76 g/L) compared to C. glutamicum P-H36

16

(9.70 ± 0.71g/L). Notably, the highest production and yield of cadaverine were

17

observed in C. glutamicum G-H36 (12.0 ± 0.76 g/L; 0.42 mol cadaverine/mol glucose)

18

and C. glutamicum P-H30 (12.5 ± 0.67 g/L; 0.44 mol cadaverine/mol glucose).

19

Interestingly, the C. glutamicum G-I64 strain showed significant reduction of

20

cadaverine production (1.30 ± 0.36 g/L) compared to C. glutamicum P-I64 (11.0 ± 1.83

21

g/L). Among all the lysE deletion mutants, only C. glutamicum G-L26 (2.36 ± 0.25

22

g/L) showed accumulation of L-lysine, suggesting that integration of E. coli ldcC into

23

the lysE locus successfully repressed secretion of L-lysine into the culture medium.

24

Because recombinant C. glutamcium PKC strains with genome-based expression of E.

17



1

coli ldcC can support stable expression of the lysine decarboxylase gene without

2

antibiotics along with low accumulation of L-lysine in the culture medium, these

3

strains can be examined for fermentative production of cadaverine to confirm their

4

production efficiency of cadaverine at a lower cost.24,28,29

5 6

Enhanced production of cadaverine in batch fermentations by recombinant C.

7

glutamicum PKC strains with chromosome-based E. coli ldcC expression

8

C. glutamicum is an obligate aerobic microorganism, wherein oxygen transfer is

9

known to be a crucial factor for balancing lysine production and cell growth.37,41-43 We

10

predicted that low production of cadaverine by recombinant C. glutamicum PKC

11

strains with chromosome-based E. coli ldcC expression in flask cultures was attributed

12

to the limited availability of oxygen in flask cultures. Thus, the cadaverine production

13

capability of six lysE deletion mutants with chromosome-based E. coli ldcC expression

14

was investigated in batch fermentations to support oxygen rich conditions. The time

15

profiles of cell growth, glucose consumption, extracellular L-lysine concentration, and

16

cadaverine concentration during batch fermentations of recombinant C. glutamicum

17

PKC strains are shown in Figure 4. The highest cadaverine concentration of 23.8 g/L

18

was produced by the C. glutamicum G-H30 strain, which was the second highest

19

producer in flask cultivation (5.26 ± 0.56 g/L) (Figure 3,4d). Recombinant C.

20

glutamicum G-L10 and C. glutamicum G-L26 produced larger amounts of cadaverine

21

(10.2 g/L and 8.83 g/L) than recombinant C. glutamicum G-I64 (8.40 g/L). Although

22

efficient glucose consumption was observed in C. glutamicum G-L10 and C.

23

glutamicum G-I64, relatively lower production of cadaverine (10.2 g/L and 8.40 g/L,

24

respectively) was observed compared to the other C. glutamicum strains. In contrast, C.

18


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1

glutamicum G-L26 showed retarded growth, slow glucose uptake capability, and

2

inefficient cadaverine production, although the culture condition in batch fermentation

3

was more favorable for maintaining cellular function compared to flask cultures

4

(Figure. 4). Interestingly, although C. glutamicum P-I16 resulted in the production of

5

8.50 ± 3.24 g/L cadaverine in flask culture, C. glutamicum G-I16 did not grow in batch

6

culture cultivation for unknown reasons (data not shown). Although six E. coli ldcC

7

expression cassettes under different synthetic promoters were integrated in the

8

chromosome of the C. glutamicum PKC strain by disrupting lysE, respectively, only

9

one strain, C. glutamicum G-I16, failed to grow in batch culture. The disruption of lysE

10

may result in the accumulation of L-lysine in the cytosol. Because toxic to the host

11

strain leading to cell rupture or death,44 this may have been caused by intracellular

12

accumulation of L-lysine which could not be rapidly converted into cadaverine in C.

13

glutamicum G-I16. Based on this observation in batch fermentation, prevention of

14

precursor secretion into the medium is not an ideal solution for maximizing the

15

production of target chemicals, although only a single enzyme reaction is involved in

16

the synthesis pathway of the target chemicals.45,46 Thus, metabolic engineering

17

strategies should be designed by carefully considering possible changes in metabolism

18

at the system level.47

19 20

High-level production of cadaverine by C. glutamicum G-H30 strain in fed-batch

21

culture

22

Because the C. glutamicum G-H30 strain produced the highest concentration of

23

cadaverine among the five recombinant C. glutamicum strains expressing E. coli ldcC

24

integrated into the chromosome examined in batch fermentations, C. glutamicum G-

19



1

H30 strain was selected as the host strain for high-level production of cadaverine by

2

fed-batch culture. Although the C. glutamicum G-H36 strain produced cadaverine in

3

batch culture at high concentrations for up to 18 h compared to the C. glutamicum G-

4

H30 strain (Figure 4), the cadaverine concentration with C. glutamicum G-H36 strain

5

reached a peak at 18 h and then decreased. Additionally, the final concentration of

6

cadaverine produced by the C. glutamicum G-H36 strain was lower than that of the C.

7

glutamicum G-H30 strain, and thus the C. glutamicum G-H30 was selected for fed-

8

batch fermentation.

9

The parental C. glutamicum PKC strain was also examined for its ability to provide

10

L-lysine as a direct precursor for cadaverine in fed-batch fermentation. As shown in

11

Figure S1, 140 g/L of L-lysine was produced in fed-batch culture of the C. glutamicum

12

PKC strain at 67 h. Based on this value, the efficiency of cadaverine production by the

13

C. glutamicum G-H30 strain developed was evaluated in this study. During fed-batch

14

fermentation of the C. glutamicum G-H30 strain, the highest L-lysine accumulation in

15

the culture medium up to 3.4 g/L was observed at 18 h accompanied by the production

16

of 10.6 g/L of cadaverine. The low concentration of L-lysine in the fermentation broth

17

indicates that disruption of the lysE locus was effective and L-lysine secretion was

18

successfully reduced. Through fed-batch fermentation of the C. glutamicum G-H30

19

strain, 103.8 g/L of cadaverine, the highest concentration to date to our knowledge,

20

was produced with a cadaverine productivity of 1.47 g/L/h at 65 h cultivation without

21

accumulation of L-lysine and by-products in the culture medium (Figure 5). The

22

cadaverine production achieved in fed-batch fermentation was 4.4-fold higher than that

23

of batch fermentation and conversion yield based on glucose was 53.5% (mol/mol).

24

Notably, deleterious effects such as growth inhibition and L-lysine accumulation by

20


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1

expression of E. coli ldcC integrated in the chromosome was not observed. Based on

2

these results, C. glutamicum G-H30 strain developed in this study is a good candidate

3

for high-level production of cadaverine from renewable resources.

4 5

Cadaverine purification from fermentation broth

6

Cadaverine was purified from fermentation broth by liquid-liquid extraction and

7

distillation. For efficient purification of cadaverine, the selection of appropriate

8

extraction solvent was conducted and the potential impurities that may impede

9

purification during concentration and distillation were analyzed. In a previous study, n-

10

butanol, 2-butanol, 2-octanol, and cyclohexanol were examined for cadaverine

11

extraction from the aqueous fermentation broth, and n-butanol was selected as

12

extraction solvent because of its low water solubility and high polarity to allow for

13

high solubility of cadaverine.24 In contrast, chloroform is non-reactive, immiscible

14

with, and denser than water and thus is a good extraction solvent for cadaverine

15

purification. Thus, in this study, the efficiency of chloroform and n-butanol as an

16

extraction solvent for cadaverine was compared. As shown in Figure 6a, the extraction

17

yields of both solvents were improved as extraction temperature increased. Chloroform

18

exhibited a slightly higher extraction yield (73.9% (w/w)) than n-butanol (70.6%

19

(w/w)) at 60°C (Figure 6a). Less water was present in the chloroform phase and more

20

than 90% of the chloroform remained after 10 cadaverine extractions (data not shown).

21

The boiling point of chloroform (61.2°C) is much lower than that of n-butanol

22

(117.7°C) and cadaverine (179°C), suggesting that chloroform can be easily removed

23

during the distillation process. Chloroform used in the extraction step was recovered

24

easily during the concentration and distillation steps, and the purity of the recovered

21



1

chloroform was higher than 99.5% (GC analysis), which is similar to that of unused

2

chloroform (data not shown). Considering the general characteristics of n-butanol as a

3

potential fuel, which may cause explosions during the distillation process and higher

4

yield of chloroform in the extraction of cadaverine, chloroform was selected as a

5

solvent to extract cadaverine from the fermentation broth. To improve the recovery

6

yield, cadaverine extraction using chloroform was conducted twice, and then the

7

extracted cadaverine solution was concentrated using a rotary evaporator at 60°C and

8

175 rpm. Finally, the concentrated cadaverine solution was further purified by

9

distillation at 25 mbar and 120°C. As a result, cadaverine recovery yields of 87.9%

10

(w/w) and 68.4% (w/w) were obtained after concentration and distillation with purities

11

of 72.0% and 99.7%, respectively (Figure 6b).

12 13

Investigation of potential impurities that impede purification yield of cadaverine

14

After solvent extraction, concentration, and distillation, bio-based cadaverine with

15

high purity was obtained, but a considerable amount of impurities was also detected

16

during purification. To identify potential impurities that may reduce purification yield,

17

GC analyses were conducted using the concentrated sample after liquid-liquid

18

extraction and distillated sample. As shown in Figure 7, three major peaks

19

corresponding to chloroform, cadaverine, and an unknown compound in the

20

concentrated sample were detected by GC analysis. All peaks except for cadaverine

21

were eliminated after distillation. However, a significant amount of the unknown

22

compound remained in the reservoir until the end of distillation, and the unknown

23

compound and cadaverine were distilled together above 135°C, reducing purification

24

yield (data not shown). Therefore, GC/MS analysis was conducted to identify the

22


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1

unknown compound (Figure S2). Unfortunately, the mass spectrum of this unknown

2

compound did not match to any compounds in the mass spectral database tested; 3-

3

amino-2-cyclohexene was the closest candidate with 0.43 similarity (Figure S2). The

4

unknown impurity in the concentrated sample could not be fully eliminated by

5

increasing the temperature and remained in the final distillation fraction, indicating that

6

the boiling point of the unknown compound is higher than that of cadaverine. Based on

7

this observation, the unknown impurity concentrated during the liquid-liquid extraction

8

process was likely because of its structural similarity to cadaverine. However, this

9

impurity has no deleterious effect on the polymerization of purified cadaverine and

10

sebacic acid as shown below, but further optimization of the purification process is

11

required to improve the yield and purity of cadaverine.

12 13

Polymerization of PA510 and its chemical properties

14

PA510 was synthesized from the purified cadaverine and sebacic acid. As shown in

15

Figure 8a, the following chemical shift values were detected as representatives; 4.56–

16

4.22 (multiplet, HFIP) and 7.27 (singlet, CDCl3) as a solvent peak used in analysis and

17

6.15 (2H, t), 3.27 (4H, q), 2.27 (4H, t), 1.62–1.55 (8H, m), 1.41–1.37 (10H, B) as

18

corresponding peaks to the backbone of PA510. Based on this result, the synthesized

19

polymer was identified as PA510. To characterize the chemical properties of the

20

synthesized PA510, DSC and TGA were conducted (Figure 8b, 8c). Melting

21

temperature (Tm) and crystallization temperature (Tc) were measured at 216°C and

22

165°C, respectively. The Tm value is similar to that of nylon 510 synthesized from

23

petroleum-based raw materials.24, 49 Degradation temperature (Td) was determined to

24

be 443°C by TGA, indicating that the synthsesized PA510 can be used as a heat-

23



1

resistant material. Based on thermal characteristic analyses, cadaverine is suitable for

2

use in the electronics and automobile industries, which demand high-performance

3

materials that are durable in harsh thermal environments. As shown in Table 2, the

4

relative viscosity was 4.6, average molecular weight (Mw) was 253,000 g/mol, and

5

number average molecular weight (Mn) was approximately 174,000 g/mol. The degree

6

of polymerization was much higher than that of chemical-based PA510. Thus, bio-

7

based cadaverine can be used as raw material for synthesizing nylon. The polymeric

8

characteristics of bio-based PA510 make it useful for extrusion applications, such as

9

films and filaments, which require comparatively high viscosity, while for injection

10

molding application such as electrical/electronic devices and automotive parts,

11

viscosity must be controlled to increase process ability by using a terminator such as

12

acetic acid.49 In addition, the tensile stress value at break was 39 MPa, which is

13

comparable to the value of chemical-based PA510.24,

14

based PA510 can be applied as a high-strength polyamide. Based on these results, the

15

polymerization product in this supports its application in diverse industrial areas

16

requiring polymers with high thermal and mechanical properties.

48

Therefore, synthesized bio-

17 18

Conclusion

19

In this study, the C. glutamicum PKC strain, an expired commercial L-lysine

20

producing host strain, was metabolically engineered for high-level production of

21

cadaverine from glucose by optimizing the expression of the E. coli lysine

22

decarboxylase gene. Fed-batch fermentation of the recombinant C. glutamicum G-H30,

23

expressing E. coli lysine decarboxylase integrated at the lysE locus of the chromosome

24

produced 103.78 g/L of cadaverine from glucose, which is the highest titer of

24


Page 24 of 49

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1

cadaverine reported to date to our knowledge. The purification yield of cadaverine was

2

enhanced by using chloroform as an extraction solvent, which resulted in the

3

production of polymer-grade bio-based cadaverine. Finally, polymerization of purified

4

cadaverine with sebacic acid resulted in bio-based PA510. Recombinant C. glutamicum

5

strains and fermentation and purification strategies of these strains, all of which were

6

developed to enhance the production of bio-based cadaverine in this study, are useful

7

for the development of a more economically feasible process for the sustainable

8

production of bio-based PA510.

9

25



1

Supporting Information

2

Supporting information for this study includes: table for list of primers, figure for

3

lysine production by C.glutamicum PKC and mass spectroscopy analysis of purified

4

cadaverine from fermentation broth. The related table and figures can be found via the

5

internet at https://pubs.acs.org.

6 7

Acknowledgements

8

This work was supported by the R& D Program of MOTIE/KEIT (10049674), Mid-

9

career Researcher Program from the Ministry of Science and ICT (MSIT) through the

10

National Research Foundation (NRF) of Korea (NRF-2016R1A2B4008707), and Ligin

11

Biorefinery from MSIT through the NRF of Korea (NRF-2017M1A2A2087135).

12 13 14

Competing interests The authors declare that they have no competing interests.

15 16 17

Availability of data and materials All data generated or analyzed during this study are included in this published article.

18 19 20

Consent for publication All authors consent for publication.

21 22 23

Funding Funding sources are declared in acknowledgement section.

24

26


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1

References:

2

(1)

Oh, Y.H., Eom, I.Y., Joo, J.C., Yu, J.H., Song. B.K., Lee, S.H., Hong, S.H., and

3

Park, S.J. (2015) Recent advances in development of biomass pretreatment

4

technologies used in biorefinery for the production of bio-based fuels,

5

chemicals and polymers Korean J. Chem. Eng. 32,1945.

6

(2)

Jang, Y.S., Kim, B.J., Shin, J.H., Choi, Y.J., Choi, S., Song, C.W., Lee, J.M.,

7

Park, H.G., and Lee, S.Y. (2012) Bio-based production of C2-C6 platform

8

chemicals Biotechnol. Bioeng. 109, 2437– 2459.

9

(3)

Joo, J.C., Khusnutdinova, A., Flick, R., Kim, T., Bornscheuer, U., Yakunin, A.

10

and Mahadevan, R. (2017) Alkene hydrogenation activity of enoate reductases

11

for an environmentally benign biosynthesis of adipic acid Chem. Sci. 8, 1406-

12

1413.

13

(4)

Wang, J., Niyompanich, S., Tai, Y.S., Wang, J., Bai, W., Mahida, P., Gao, T.,

14

Zhang, K (2016) Engineering of a highly efficient Escherichia coli strain for

15

mevalonate fermentation through chromosomal integration Appl Environ

16

Microbiol. 82,7176 –7184.

17

(5)

Chae, T.U., Ko, Y.S., Hwang, K.S., and Lee, S.Y. (2017) Metabolic Engineering

18

of Escherichia coli for the Production of Four-, Five- And Six-Carbon Lactams

19

Metab. Eng. 41, 82-91.

20

(6)

Chae, T.U., Won, J.K., Sol, C., Park, S.J., and Lee SY. (2015) Metabolic

21

engineering of Escherichia coli for the production of 1,3-diaminopropane, a

22

three carbon diamine Sci. Rep. 11(5):13040.

27



1

(7)

Oh, Y.H., Eom, G.T., Kang, K.H., Choi, J.W., Song, B.K., Lee, S.H., and Park,

2

S.J. (2015) Optimized Transformation of Newly Constructed Escherichia coli-

3

Clostridia Shuttle Vectors into Clostridium beijerinckii Appl. Biochem.

4

Biotechnol. 177,226-36.

5

(8)

Oh, Y.H., Eom, G.T., Kang, K.H., Joo, J.C., Jang, Y.A., Choi, J.W., Song, B.K.,

6

Lee, SH., and Park, S.J. (2016) Construction of heterologous gene expression

7

cassettes for the development of recombinant Clostridium beijerinckii

8

Bioprocess Biosyst. Eng. 39, 555-563.

9

(9) Becker, J., and Wittmann, C. (2015) Advanced Biotechnology: Metabolically

10

Engineered Cells for the Bio-Based Production of Chemicals and Fuels,

11

Materials, and Health-Care Products Angew Chem. Int. Ed. 54,2-25.

12

(10) Kim, T., Flick, R., Brunzelle, J., Singer, A., Evdokimova, E., Brown, G., Joo, J.C.,

13

Minasov, G.A., Anderson, W.F., Mahadevan, R., Savchenko, A., Yakunin, A.F.

14

(2017) Novel aldo-keto reductases for the biocatalytic conversion of 3-

15

hydroxybutanal to 1,3-butanediol: structural and biochemical studies Appl

16

Environ Microbiol. 83, e03172-16.

17

(11) Chae, C.G., Kim, Y.J., Lee, S.J., Oh, Y.H., Yang, J.E., Joo, J.C., Kang, K.H., Jang,

18

Y.A., Lee, H., Park, A.R., Song, B.K., Lee, S.Y., Park, S.J. (2016) Biosynthesis

19

of poly(2-hydroxybutyrate-co-lactate) in metabolically engineered Escherichia

20

coli Biotechnol Bioprocess Eng. 21, 169-174.

21

(12) David, Y., Baylon, M.G., Sudheer, P.D.V.N., Baritugo, K., Chae, C.G., Kim, Y.J.,

22

Kim, T.W., Kim, M.S., Na, J.G., Park, S.J. (2017) Screening of microorganisms

23

able to degrade low-rank coal in aerobic condition as potential coal

28


Page 28 of 49

Page 29 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

biosolubilization mediators from coal to biochemicals Biotechnol Bioprocess

2

Eng. 22, 178-185.

3

(13) Choi, S.Y., Park, S.J., Kim, W.J., Yang, J.E., Lee, H., Shin, J., Lee, S.Y. (2016)

4

One-step

5

carbohydrates in Escherichia coli Nat Biotechnol. 34, 435-440.

fermentative

production

of

poly(lactate-co-glycolate)

from

6

(14) Choi, J.W., Yim, S.S., Lee, S.H., Kang, T.J., and Park, S.J. (2015) Enhanced

7

production of gamma-aminobutyrate (GABA) in recombinant Corynebacterium

8

glutamicum expressing glutamate decarboxylase active in expanded pH range.

9

Microb. Cel.l Fact. 14, 21.

10 11

(15) Jiang, Y., Loos, K. (2016) Enzymatic synthesis of Biobased Polyesters and Polyamides. Polymers, 8, 243

12

(16) Park, S. J., Kim, E. Y., Noh,W., Park, H. M., Oh, Y. H., Lee, S. H., Song, B. K.,

13

Jegal, J. G., and Lee, S. Y. (2013) Metabolic engineering of Escherichia coli for

14

the production of 5-aminovalerate and glutarate as C5 platform chemicals

15

Metab. Eng. 16, 42–47.

16

(17) Park, S. J., Oh, Y. H., Noh,W., Kim, H. Y., Shin, J. H., Lee, E. G., Lee, S., David,

17

Y., Baylon, M. G., Song, B. K., Jegal, J., Lee, S. Y., and Lee, S. H. (2014)

18

High-level conversion of L-lysine into 5-aminovalerate that can be used for

19

nylon 6,5 synthesis. Biotechnol J. 9, 1322–1328.

20

(18) Qian, Z. G., Xia, X., and Lee, S. Y. (2011) Metabolic engineering of Escherichia

21

coli for the production of cadaverine: a five carbon diamine Biotechnol Bioeng.

22

108, 93–103.

29



1

(19) Mimitsuka, T., Sawai, H., Hatsu, M., and Yamada, K. (2007). Metabolic

2

engineering of Corynebacterium glutamicum for cadaverine fermentation

3

Biosci. Biotechnol. Biochem. 71, 2130–2135.

4

(20) Buschke, N., Schröder, H., and Wittmann, C. (2011). Metabolic engineering of

5

Corynebacterium glutamicum for production of 1,5-diaminopentane from

6

hemicellulose Biotechnol J. 6, 306–317.

7 8

(21) Kind, S., and Wittmann, C. (2011) Bio-based production of the platform chemical 1,5-diaminopentane Appl Microbiol Biotechnol. 91, 1287–1296.

9

(22) Kind, S., Jeong,W. K., Schröder, H., Zelder, O., and Wittmann, C. (2010)

10

Identification and elimination of the competing N-acetyldiaminopentane

11

pathway for improved production of diaminopentane by Corynebacterium

12

glutamicum Appl Environ Microbiol. 76, 5175–5180.

13

(23) Kind, S., Jeong, W. K., Schröder, H., and Wittmann, C. (2010). Systems-wide

14

metabolic pathway engineering in Corynebacterium glutamicum for bio-based

15

production of diaminopentane Metab. Eng. 12, 341–51.

16

(24) Kind, S., Neubauer, S., Becker, J., Yamamoto, M., Volkert, M., Abendroth, G.,

17

Zelder, O. and Wittmann, C (2014). From zero to hero—production of bio-

18

based nylon from renewable resources using engineered Corynebacterium

19

glutamicum Metab Eng. 25, 113–123.

20

(25) Oh, Y.H., Choi, J.W., Kim, E.Y., Song, B.K., Jeong, K.J., Park, K.M., Kim, I.K.,

21

Woo, H.M., Lee, S.H., and Park, S.J. (2015) Construction of Synthetic

22

Promoter-Based Expression Cassettes for the Production of Cadaverine in

23

Recombinant Corynebacterium glutamicum Appl Biochem Biotechnol.176 ,

24

2065-2075.

30


Page 30 of 49

Page 31 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

(26) Tateno, T., Okada, Y, Tsuchidate, T., Tanaka, T., Fukuda, H., and Kondo, A. (2009)

2

Direct production of cadaverine from soluble starch Corynebacterium

3

glutamicum coexpressing α-amylase and lysine decarboxylase Appl Microbiol

4

Biotechnol 82, 115-121.

5

(27) Li, M., Li, D., Huang, Y., Liu, M., Wang, H., Tang, Q., and Lu, F. (2014)

6

Improving the secretion of cadaverine in Corynebacterium glutamicum by

7

cadaverine-lysine antiporter J Ind Microbiol Biotechnol.41, 701-709.

8

(28) Kitade, Y., Okino, S., Gunji, W., Hiraga, K., Suda, M., Suzuki, N., Inui, M.,

9

Yukawa, H. (2013) Identification of a gene involved in plasmid structural

10

instability in Corynebacterium glutamicum Appl Microbiol Biotechnol 97:8219-

11

266.

12 13 14 15

(29) Friehs, K. (2004) Plasmid copy number and plasmid stability Adv Biochem Eng Biotechnol.2004; 86:47-82. (30) Tegel, H., Ottosson, J., and Hober, S. (2011) Enhancing the protein production levels in Escherichia coli with a strong promoter FEBS J 5, 729-739.

16

(31) Yim, S. S., An, S. J., Kang,M. S., Lee, J. H., and Jeong, K. J. (2013) Isolation of

17

fully synthetic promoters for high-level gene expression in Corynebacterium

18

glutamicum Biotechnol Bioeng.110, 2959– 2971.

19

(32) Barnard, G.C., Henderson, G.E., Srinivasan, S., and Gerngross, T.U. (2004) High

20

level recombinant protein expression in Ralstonia eutropha using T7 RNA

21

polymerase based amplification Protein Expr Purif. 38, 264-71.

22

(33) Patek, M., Holatko, J., Busche, T., Kalinowski, J., and Nesvera, J. (2013)

23

Corynebacterium glutamicum promoters: a practical approach Microb

24

Biotechnol.6, 103-117.

31



1

(34) Shin, J.H., Park, S.H., Oh, Y.H., Choi, J.W., Lee, M.H., Cho, J.S., Jeong, K.J., Joo,

2

J.C., Yu, J., Park, S.J., and Lee, S.Y. (2016) Metabolic engineering of

3

Corynebacterium glutamicum for enhanced production of 5 aminovaleric acid

4

Microb Cell Fact. 15,174.

5

(35) Joo, J.C., Oh, Y.H., Yu, J.H., Hyun, S.M., Khang, T.U., Kang, K.H., Song, B.K.,

6

Park, K., Oh, M.K., Lee, S.Y., and Park, S.J.(2017) Production of 5-

7

aminovaleric acid in recombinant C. glutamicum strains from miscanthus

8

hydrolysate solution prepared by a newly developed miscanthus hydrolysis

9

process. Bioresour Biotechnol. 245, 1692-1700.

10 11

(36) Sambrook, J., Russel, R.W. (2001) Molecular cloning: A laboratory manual, 3rd Ed. Cold spring harbor laboratory press, Cold spring harbor, N.Y.

12

(37) Sassi, A., Deschamps, A., Lebeault, J. (1996) Process analysis of l-lysine

13

fermentation with Corynebacterium glutamicum under different oxygen and

14

carbon dioxide supplies and redox potentials. Process Biochem. 31, 493-497.

15

(38) Rohles, C.M., Gieβelmann, G., Kohlstedt, M., Wittmann, C., Becker, J. (2016)

16

Systems metabolic engineering of Corynebacterium glutamicum for the production

17

of the carbon-5 platform chemicals 5-aminovalerate and glutarate. Microb Cell

18

Fact.15:154.

19

(39) Wu, G., Yan, Q., Jones, J., Tang, Y., Fong, S., Koffas, M.A. (2016) Metabolic

20

Burden: Cornerstones in Synthetic Biology and Metabolic Engineering

21

Applications Trends Biotechnol 64, 652-664.

22

(40) Adams, C., Hatfield, G. (1984) Effects of promoter strengths and growth

23

conditions on copy number of transcription-fusion vectors J. Biol. Chem. 259,

24

7399-403.

32


Page 32 of 49

Page 33 of 49 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

(41) Liu, Q., Zhang, J., Wei., X., Ouyang, S., Wu, Q., Chen, G.Q. (2008) Microbial

2

production of l-glutamate and l-glutamine by recombinant Corynebacterium

3

glutamicum harboring Vitreoscilla hemoglobin gene vgb Appl Microbiol

4

Biotechnol. 77, 1297-1304.

5

(42) Zimmermann, H., F., Anderlei, T., Buchs, J., Binder, M. (2006) Oxygen limitation

6

is a pitfall during screening for industrial strains. Appl Microbiol Biotechnol.

7

72, 1157-60.

8 9 10

(43) Rao, D., Razak, S., Praveena, B., Swamy, A. (2007) Dissolved Oxygen concentration

analysis

of

L-lysine

Fermentation

Production

by

Corynebacterium glutamicum. The Internet Journal of Pharmacology. 6(1).

11

(44) Stabler, N., Oikawa, T., Bott, M., Eggeling, L. (2011) Corynebacterium

12

glutamicum as a host for synthesis and export of D-Amino Acids. J Bacteriol.

13

193(7):1702-9.

14

(45) Chen, Z., Huang, J., Wu, Y., Liu, D. (2016) Metabolic engineering of

15

Corynebacterium glutamicum for the de novo production of ethylene glycol

16

from glucose. Metab Eng. 33:12-8.

17

(46) Jorge, J. M. P., Nguyen A. Q., Perez-Garcia, F., Kind, S., Wendisch, V. F. (2017)

18

Improved fermentative production of gamma aminobutyric acid via the

19

putrescine route: Systems metabolic engineering for production from glucose,

20

amino sugars, and xylose. Biotechnol Bioeng. 114(4):862-873.

21 22

(47) Lee, S.Y., Kim, H.U. (2015) Systems strategies for developing industrial microbial strains. Nature Biotechnol. 33(10): 1061–1072.

33



1

(48) Ma, W., Chen, K., Li, Y., Hao, N., Wang, X., Ouyang, P. (2017) Advances in

2

Cadaverine Bacterial Production and Its Applications. Engineering (Beijing).

3

3(3):308-317.

4

(49) Yan, Q., Zhao, T., Bai, Y., Zhang, F., Yang, W. (2009) Precipitation Polymerization

5

in Acetic Acid: Study of the Solvent Effect on the Morphology of

6

Poly(divinylbenzene). J Phys Chem B. 113(10): 3008-3014.

34


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1

Table 1. Lists of bacterial strains and plasmids used in this study Plasmid

Relevant characteristics

Reference

Strain

E. coli XL1-Blue

recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac Stratgene [Fe proAB lacIq Z∆M15 Tn10 (TetR)

C. glutamicum PKC

An expired industrial L-lysine-producing strain

C. glutamicum P-L10

C. glutamicum PKC with pCES208L10LdcC

This study

C. glutamicum P-L26

C. glutamicum PKC with pCES208L26LdcC

This study

C. glutamicum P-I16

C. glutamicum PKC with pCES208I16LdcC

This study

C. glutamicum P-I64

C. glutamicum PKC with pCES208I64LdcC

This study

C. glutamicum P-H30

C. glutamicum PKC with pCES208H30LdcC

This study

C. glutamicum P-H36

C. glutamicum PKC with pCES208H36LdcC

This study

C. glutamicum G-L10

C. glutamicum PKC derivative: LysE replaced with This study E. coli ldcC under L10 synthetic promoter.

C. glutamicum G-L26

C. glutamicum PKC derivative: LysE replaced with This study E. coli ldcC under L26 synthetic promoter

C. glutamicum G-I16

C. glutamicum PKC derivative: LysE replaced with This study E. coli ldcC under I16 synthetic promoter

C. glutamicum G-I64

C. glutamicum PKC derivative: LysE replaced with This study E. coli ldcC under I64 synthetic promoter

C. glutamicum G-H30

C. glutamicum PKC derivative: LysE replaced with This study E. coli ldcC under H30 synthetic promoter

C. glutamicum G-H36

C. glutamicum PKC derivative: LysE replaced with This study

35



Page 36 of 49

E. coli ldcC under H36 synthetic promoter Plasmids pCES208L10LdcC

pCES208 derivative; PL10, E. coli ldcC, Kmr

(25)

pCES208L26LdcC

pCES208 derivative; PL26, E. coli ldcC, Kmr

(25)

pCES208I16LdcC

pCES208 derivative; PI16, E. coli ldcC, Kmr

(25)

pCES208I64LdcC

pCES208 derivative; PI64, E. coli ldcC, Kmr

(25)

pCES208H30LdcC

pCES208 derivative; PH30, E. coli ldcC, Kmr

(25)

pCES208H36LdcC

pCES208 derivative; PH36, E. coli ldcC, Kmr

(25)

1

36


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1

Table 2 Comparison of material properties of synthesized bio-based polyamide

2

and commercially available polyamide.

Mechanical Property

Chemical-based

Biobased

Polyamide

Polyamide PA510

Melting temperature 214

215

167

158

Degradation temperature (Td, °C)

436

449 (Td5% 428)

Relative viscosity (g/mol)

3.6

4.6

128,655

174,294

199,477

25,000

48

39

(Tm, °C) Crystallization temperature (Tc, °C)

Number average molecular weight (Mn, g/mol) Weight average molecular weight (Mw, g/mol) Tensile test (stress at break) (Mpa) 3 4

37



Page 38 of 49

1

Figure legends

2

Figure 1. Schematic diagram of proposed production of bio-polyamide using

3

recombinant C. glutamicum strains containing chromosomal and plasmid expression

4

of E. coli LdcC using synthetic-promoter-expression cassettes used in this study.

5

Enzymes: LdcC, Lysine decarboxylase; LysE, lysine exporter. Abbreviations:

6

Glucose-6P, glucose 6-phosphate; PEP, phosphoenolpyruvate; CO2, carbon dioxide;

7

OAA, oxaloacetate; TCA cycle, tricarboxylic cycle; ASP, L-aspartate; ASP-P, L-

8

aspartyl-phosphate;

9

tetrahydrodipicolinate; DAP, D,L-Diaminopimelate.

DHDP,

L-2,3-dihydrodipicolinate;

THDP,

L-

10

Figure 2. Lysine production by different strains of C. glutamicum

11

Figure 3. Cadaverine production and lysine consumption of recombinant C.

12

glutamicum strains expressing either plasmid-based expression or chromosomal

13

integration of expression cassettes in shake-flask experiments

14

Figure 4. Comparison of cell growth (A), glucose consumption (B), lysine

15

concentration (C), and cadaverine production (D) of batch fermentation of

16

recombinant C. glutamicum strains with chromosomal integration of expression

17

cassettes with different strengths of promoters. Legends: H36-open circle, H30-

18

filled circle, L10-filled square, L26-open square, I64-open triangle

19

Figure 5. Lysine production of commercial L-lysine overproducing strain, C.

20

glutamicum PKC, used as a host strain (A) and cadaverine production and glucose

21

and lysine substrate consumption of recombinant strain C. glutamicum G-H30 with

22

chromosomal integration of lysine decarboxylase at the LysE site from fed-batch

23

fermentation from fed-batch experiments (B). Legends: cell growth – filled triangle,

38


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1

cadaverine concentration – open circle, glucose consumption – open triangle and

2

lysine concentration – filled circle

3

Figure 6. Cadaverine extraction yields obtained by n-butanol or chloroform extraction

4

of fermentation supernatant (a) and schematic illustration and mass balance of

5

cadaverine production process (b). All cadaverine concentrations were measured by

6

high-performance liquid chromatography, except that of the distillation product

7

measured by GC. Cadaverine amounts were calculated. (Symbols are: filled bar, n-

8

butanol extraction yield; open bar, chloroform extraction yield)

9

Figure 7. Gas chromatography analysis of purified cadaverine from fermentation broth

10

Figure 8. Results of nuclear magnetic resonance spectroscopy (1H NMR spectra) (a),

11

differential scanning calorimetry (DSC) (b), and thermogravimetric (TGA) analysis

12

(c) of biobased polyamide PA510

13

39



1

For Table of Contents Use Only.

2

3 4 5

Schematic diagram of sustainable production of bio-polyamide 510 from high level

6

production cadaverine using recombinant C. glutamicum PKC strains. Bio-polyamide

7

510 was produced by polymerization of sebacic acid with purified cadaverine.

40


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Figure 1 426x287mm (300 x 300 DPI)



Figure 2 143x118mm (300 x 300 DPI)


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Figure 3 143x107mm (300 x 300 DPI)



Figure 4 233x187mm (300 x 300 DPI)


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Figure 5 187x112mm (300 x 300 DPI)



Figure 6 115x26mm (300 x 300 DPI)


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Figure 7 92x67mm (300 x 300 DPI)



Figure 8 282x472mm (300 x 300 DPI)


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TOC 84x47mm (300 x 300 DPI)


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