Discovery of a Short-Chain Îµ-Poly-l

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Biotechnology and Biological Transformations

Discovery of a short-chain #-poly-L-lysine and its highly efficient production via synthetase swap strategy Delei Xu, Rui Wang, Zhaoxian Xu, Zheng Xu, Sha Li, Mingxuan Wang, Xiaohai Feng, and Hong Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06019 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019

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Journal of Agricultural and Food Chemistry

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Discovery of a short-chain ε-poly-L-lysine and its highly efficient production via

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synthetase swap strategy

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Delei Xu1,2,3, Rui Wang1,2,3, Zhaoxian Xu4, Zheng Xu1,2,3*, Sha Li1,2,3, Mingxuan

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Wang1,2,3, Xiaohai Feng1,2,3, Hong Xu1,2,3*

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1State

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University, Nanjing 211816, China

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2College

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211816, China

Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech

of Food Science and Light Industry, Nanjing Tech University, Nanjing

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3The

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Tech University, Nanjing 211816, China

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4School

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and Technology, Nanjing 210094, China

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*Corresponding author

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Hong Xu; Nanjing Tech University; Tel/Fax:+86-25-58139433;E-mail address:

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[email protected];

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Zheng Xu; Nanjing Tech University; Tel/Fax:+86-25-58139433;E-mail address:

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[email protected];

Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture, Nanjing

of Environmental and Biological Engineering, Nanjing University of Science

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ACS Paragon Plus Environment


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ABSTRACT

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ε-Poly-L-lysine (ε-PL) is a natural antimicrobial cationic peptide, which is

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generally recognized as safe for use as a food preservative. To date, the production

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capacity of strains that produce low-molecular weight ε-PL remains very low and thus

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unsuitable for industrial production. Here, we report a new low-molecular weight

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ε-PL-producing Kitasatospora aureofaciens strain. The ε-PL synthase gene of this

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strain was cloned into a high ε-PL-producing Streptomyces albulus strain. The

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resulting recombinant strain efficiently produced ε-PL with a molecular weight of

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1.3-2.3 kDa and yielded of 23.6 g/L following fed-batch fermentation in a 5 L

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bioreactor. In addition, circular dichroism spectra showed that this ε-PL takes on a

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confoirmation similar to an antiparallel pleated-sheet. Moreover, it demonstrated

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better antimicrobial activity against yeast compared to the 3.2-4.5 kDa ε-PL. This

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study provides a highly efficient strategy for production of the low-molecular weight

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ε-PL, which helps to expand its potential applications.

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KEYWORDS: ε-Poly-L-lysine, low molecular weight, synthetase swap, efficient

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production, natural preservative.

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INTRODUCTION

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ε-Poly-L-lysine (ε-PL) is an antimicrobial compound consisting of 25–35 L-lysine

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residues that are connected by unique linkages between the ε-amino groups and

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α-carboxyl groups.1,2 To date, Streptomyces and Kitasatospora are the main

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organisms known to produce ε-PL.3 Because of its unique structure, ε-PL exhibits

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advantageous characteristics, such as, water-solubility, thermostability, and

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biodegradability, and is a cationic homobiopolymer, which is non-toxic to humans

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and the environment.4,5 In particular, because of its broad antimicrobial effect against

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bacteria, filamentous fungi, yeast, and viruses, it has been approved for used as a

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natural food preservative in Japan, the USA, South Korea, China and other countries.6,

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7

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applications in the medical field because of its ability to selectively remove

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endotoxins (which reduces cell toxicity), improve cell adhesion, inhibit pancreatic

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lipase activity, and inhibit the production of oral bacterial toxins.8, 9

In addition to its applications in the food industry, ε-PL also has potential

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Similar to many other biopolymers, the biological functions of ε-PL are strongly

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dependent on its molecular weight (Mw).1 For example, Shima et al. investigated the

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antibacterial activity of ε-PLs with different Mw on Escherichia coli and found that,

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ε-PLs with less than 9 degree of polymerization had no obvious antibacterial activity,

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whereas ε-PLs with more than 9 degrees of polymerization had significant

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antibacterial activity. Furthermo ure, no apparent increase in antibacterial activity was

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observed for ε-PLs beyond 15 L-lysine residues.10 Recent studies have shown that

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low-Mw ε-PLs have a stronger inhibitory effect on yeast than high-Mw ε-PLs.11,12



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Moreover, high L-lysine residues of ε-PLs lead to an unpleasant bitter taste.13,14

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Therefore, efficient production of different Mw ε-PLs is very important for an

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in-depth study of their properties and exploring their broad applications.

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To date, several ε-PL-producing strains able to secrete low-Mw ε-PL have been

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screened. However, these strains exhibit a lower production capacity compared to the

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high-Mw ε-PL-producing strains. For example, the ε-PLs commonly used currently

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have a Mw ranging from 3.2 to 4.5 kDa (corresponding to 25-35 L-lysine residues),

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and are produced by S. albulus NBRC14147 (previously known as S. albulus

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IFO14147), S. lydicus USE-11, S. albulus PD-1, and Streptomyces. sp M-Z18. These

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strains can produce 1.236-4.0 g/L ε-PL in shake-flask fermentation using a two-stage

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culture method.1,15-17 In contrast, researchers have identified six low-Mw ε-PL

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producing strains from 1,300 Actinomycete colonies.17 ε-PL production in most of

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these strains was only 0.4-0.8 g/L, lower than in high-Mw ε-PL-producing strains

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using the same culture method. Therefore, it is difficult to identify strains producing

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high yields of low-Mw ε-PL, indicating that further research is necessary before these

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strains can be used in the industry. In addition to natural production of ε-PL by these

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strains, low-Mw ε-PL can also be obtained by adding chemically modified

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cyclodextrins and glycerol or randomly mutating ε-PL synthetase (Pls) to reduce the

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degree of polymerization in high-Mw ε-PLs. For example, Nishikawa et al. found that

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the Mw of ɛ-PL could be reduced from 3.2-4.5 kDa to less than 2.5 kDa by adding a

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mixture of chemically modified cyclodextrins and glycerol,13 while Chen et al.

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showed that the Mw of ɛ-PL could be reduced from 3.2-4.5 kDa to less than 1.1-4.1


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kDa by using glycerol as the carbon source.12 However, the addition of polyol and

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cyclodextrins could hinder ɛ-PL separation and purification and glycerol alone may be

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insufficient to reduce ε-PL length. Recently, Hamano et al. demonstrated that mutated

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Pls could produce short-chain ε-PLs.11 However, as the ɛ-PL synthesis mechanism

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remains unclear, the Mw of ɛ-PL obtained by random mutation of Pls cannot be

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controlled. In addition to low-Mw ɛ-PL production by microorganisms, Tao et al.

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reported a new chemical strategy based on ring-opening polymerization for obtaining

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ε-PLs of varying Mw from lysine18. Unfortunately, this chemically synthesized ε-PL

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has some safety risks for the food industry and there is no evidence to suggest that the

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chemical synthesis of ɛ-PL has a higher production efficiency than microbial

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fermentation. In summary, although these strategies are promising, a simple, efficient,

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and safe method for the production of low-Mw ɛ-PL is still needed. Recently, the

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unravelling of the molecular mechanisms underlying ɛ-PL biosynthesis has confirmed

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that the Mw of ɛ-PL is directly affected by the Pls.11 This raised the question of

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whether we could produce low-Mw ɛ-PL by introducing the corresponding pls gene

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into the high-yield ɛ-PL-producing strains.

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In this study, a novel low-Mw ɛ-PL-producing strain was identified and its pls

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gene was cloned and analyzed. This pls gene was introduced into the high-yield

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ɛ-PL-producing S. albulus PD-1 strain by knocking out its endogenous pls gene.

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Furthermore, by optimizing different pls promoters, an efficient ɛ-PL-producing strain,

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capable of producing low-Mw ɛ-PL, was constructed. We hope this work will provide

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a reference for the production of other biopolymers.



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MATERIALS AND METHODS

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Strains, Plasmids, and Cultivation Conditions. S. albulus PD-1 was isolated

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from soils and deposited in China Center for Type Culture Collection (Accession No.

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M2011043). The Bacterial strains and plasmids used in this study are listed in Table

111

1.

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The mannitol soya flour (MS) medium containing (g/L): mannitol (20), soya flour

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(20), agar (20) was used to grow spores. Medium 3G (M3G) containing (g/L): glucose

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(50), (NH4)2SO4 (10), yeast extract (5), KH2PO4 (1.36), K2HPO4 (0.8), MgSO4·7H2O

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(0.5), ZnSO4·7H2O (0.04), and FeSO4·7H2O (0.03) was used for submerged

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cultivation. The initial pH of M3G was adjusted to 6.8 with ammonia. Seed cultures

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were prepared in 500 mL flasks containing 100 mL of M3G medium and grown at 30

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°C and 200 rpm shaking condition for 24 hrs. Shake flask fermentation was conducted

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at 30 ºC and 200 rpm shaking condition for 72 hrs. Batch and fed-batch fermentation

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were carried out in a 5 L bioreactor (KF-5L, KoBio Tech Co., Ltd., Korea). The seed

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culture (300 mL), was transferred into a bioreactor containing 2.7-L sterile medium.

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For the batch fermentation, the pH was reduced from an initial 6.8 and maintained at

123

4.0. For the fed-batch fermentation, a classical two-stage fermentation strategy was

124

applied for ɛ-PL production. The pH was maintained at 6.0 during the bacterial

125

growth phase and the changed to 4.0 with 30% ammonia during the ɛ-PL production

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phase. When the glucose concentration in the culture broth decreased to

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approximately 10 g/L, a feeding solution (glucose, 500 g/L; (NH4)2SO4, 50 g/L) was

128

pumped into the broth to maintain the glucose concentration at approximately 10 g/L.


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In order to avoid foaming during the fermentation process, the anti-foam agent

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KM-70 was used with an automatic control system.

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The microbial strains used for the antibacterial experiments were obtained from

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the General Microbiological Culture Collection Center (CGMCC), Beijing, China. LB

133

medium (10 g/L NaCl, 10 g/L tryptone, and 5 g/L yeast extract) was used for the

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cultivation of bacteria. YPD medium (10 g/L yeast extract, 10 g/L peptone, and 20

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g/L glucose) was used for yeasts. PDA medium (20 g/L glucose and 200 g/L potato

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juice ) was used for molds cultivation..

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General Molecular Biological Techniques. Plasmid preparation, restriction

138

endonuclease digestions, and other DNA manipulations were carried out according to

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standard procedures for Streptomyces and E. coli.19 The primers used in this study are

140

listed in Table S1, included in the Supporting Information. Intergeneric conjugation

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from E. coli ET12567 (pUB307) to Streptomyces was performed as described by Xu

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et al.20

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Isolation and Identification of Strain PL-1. Soil samples were collected from the

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suburbs of Nanjing, China, and were mixed with CaCO3 and air-dried in the shade for

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10 days. One gram of soil was suspended in 9 mL of sterile double distilled water and

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dilutions of up to 10-5 were carried out. Aliquots (0.1 mL) of the 10-2, 10-3, 10-4, and

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10-5 dilutions were spread on starch casein agar medium containing (g/L): soluble

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starch (10), casein (0.3), KNO3 (2), NaCl (2), K2HPO4 (2), MgSO4·7H2O (0.05),

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CaCO3 (0.02), FeSO4·7H2O (0.01), and agar (15). The pH was adjusted to 7.0 prior to

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sterilization.21 To suppress fungal and bacterial growth, actidione (20 mg/L) and



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nalidixic acid (100 mg/L) were added to the medium. The plates were incubated at 30

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°C for 7 days. Based on the colony morphology, the isolated strains were cultivated in

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tryptic soy broth (Becton Dickinson) at 30 °C and with 200 rpm shaking to prepare

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the mycelium for genomic DNA (gDNA) extraction. gDNA was isolated using the

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salting-out protocol22 and stored at -80 °C. To identify strains harboring the pls gene,

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conserved regions of the known Pls and its homologous amino acid sequences were

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chosen as the screening target. Degenerate primers P1-F and P1-R, which amplify a

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3000 bp fragment of the target region, were designed to screen different strains. The

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gDNA of S. albulus PD-1 (ɛ-PL producer, containing the pls gene [plsI gene]) was

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used as the positive control, while a blank sample was used as the negative control. A

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partial 16S rDNA region was amplified for species identification using the 27F and

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1492R universal primers (27F, 5′-GAGAGTTTGATCCTGGCTCAG-3′) and (1492R,

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5′-ACGGCTACCTTGTTACGACTT-3′). The amplified DNA fragments were

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sequenced and their homologies were analyzed using BLAST on the National Center

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for Biotechnology Information (NCBI) website. MEGA version 4.0 was used for

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additional phylogenetic analysis.

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Cloning of the pls Gene in Strain PL-1 and its Bioinformatics Analysis. Based

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on the intact pls gene of strain PL-1 (the plsII gene), the sequences upstream and

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downstream of the plsII gene were obtained by genome walking according to the

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manufacturer’s instructions (Genome Walker Kit, Takara Biotech Co., Ltd., Dalian,

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China). All primers used are shown in Table S1. Ttransmembrane topology was

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predicted using the Constrained Consensus TOPology prediction server (CCTP;


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http://cctop.enzim.ttk.mta.hu/?_=/jobs/submit).23 Sequences were searched using

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InterProScan (http://www.ebi.ac.uk/interpro/search/sequence-search) for conserved

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domain analysis.24 Substrate specificities of the non-ribosomal peptide synthetase

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adenylation domains (A domains) were predicted by using NRPSpredictor2

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(http://nrps.informatik. uni-tuebingen.de/Controller? cmd = SubmitJob).25

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Purification of the PlsII A Domain Protein. The nucleotide sequence of the A

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domain gene was amplified from strain PL-1 with primers A-domain-F/A-domain-R

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listed in Table S1. The resulting PCR fragment was inserted into the pET28a vector

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backbone digested with EcoRI and HindIII to create plasmid pET28a-plsII by

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seamless cloning.26 The resulting recombinant plasmid was introduced into E. coli

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BL21 (DE3) for protein expression. The recombinant protein was further purified

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using Ni-chelating affinity chromatography.

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Analysis of the Substrate Specificity of the PlsII A Domain. As the A domains

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mediate

the

ATP-dependent

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aminoacyl-O-AMP with pyrophosphate (PPi) release, a colorimetric assay for PPi

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based on the formation of molybdopyrophosphate, was used for the evaluation of

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adenylation, as described by Katano and Hajime et al.27 Briefly, the PPi can be

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spectrophotometrically

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[(P2O7)Mo18O54]4− anions in an acidic acetonitrile-water solution containing MoO42-

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anions. These [(P2O7)Mo18O54]4− anions are further reduced by ascorbic acid, resulting

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in a more readily distinguishable blue coloration, which was read at an absorbance of

194

620 nm with a microplate reader.

detected

activation

based

of

on

amino-acid

the

yellow


substrates

color

such

produced

as

by


195

Construction of the plsI Gene Knock-out Mutant. The plsI gene of S. albulus

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PD-1 was disrupted by a double-crossover event. To construct the disruption plasmid,

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the 1.02 kb gene fragment carrying a thiostrepton resistance gene (tsr) was amplified

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from pXY200 using primers Tsr-F and Tsr-R. Two DNA fragments (3.24 kb and 3.15

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kb) carrying the upstream and downstream regions of the target genes, respectively,

200

were also amplified from S. albulus PD-1 using the primers listed in Table S1. Next,

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these three amplified fragments were mixed with the 6.5 kb EcoRI/XbaI fragment

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from pKC1139 and ligated using the ClonExpress MultiS One Step Cloning Kit

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(Vazyme, China) according to the manufacturer’s protocol, to obtain the plsI gene

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disruption plasmid pKC1139-tsr-plsI. This gene disruption plasmid was then

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transferred by conjugation from E. coli ET12567 (pUB307) to S. albulus PD-1 and the

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thiostrepton-resistant double crossover mutant phenotypes were isolated after five

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rounds of nonselective growth. The disruption mutation was confirmed by PCR using

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primers check-F and check-R.

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Plasmid Construction for PL-1 Pls Expression. To evaluate the effect of

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different promoters on ɛ-PL production, the plsII gene controlled by its native

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promoter, a strong promoter ermE*, and the promoter from plsI were constructed

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using an integrated plasmid pSET152. The plsII gene expression cassette was

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amplified from the strain PL-1 gDNA using primer pair PlsII-F/PlsII-R and then

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inserted between the XbaI and EcoRI sites of pSET152, yielding recombinant plasmid

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pSET152-PplsII-plsII. The ermE* promoter and plsII gene open reading frame (ORF)

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were amplified from plasmid pIB13928 and strain PL-1, respectively. The resulting


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PCR products were inserted into the XbaI-EcoRI sites of pSET152, generating

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pSET152-PermEp*-plsII. The plsI promoter and plsII gene ORF were amplified from

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the gDNA of strain PD-1 and strain PL-1, respectively. The resulting PCR products

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were inserted into the XbaI-EcoRI sites of pSET152, generating pSET152-PplsI-plsII.

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All constructs were confirmed by DNA sequencing. These constructs were then

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introduced into the corresponding strains by intergeneric conjugation and the

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transformed strains were selected by screening for apramycin resistance.

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Determination of ɛ-PL Antimicrobial Activity. The minimum inhibitory

225

concentration (MIC), the lowest concentration of ɛ-PL that inhibits microbial growth ,

226

was determined using the two-fold micro-dilution technique according to the

227

guidelines of the Clinical and Laboratory Standards.29 The maximum concentration of

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ɛ-PL tested in this assay was 1280 μg/mL. Briefly, the ɛ-PL was two-fold serially

229

diluted with LB, YPD or PDA in 10 mL test tubes. Next, 100 μl of the diluted volume

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of inoculum was added to each tube, resulting in a final density of 5 × 105 cfu/mL for

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bacteria, 5 × 105 cfu/mL for yeasts and 5 × 105 cfu/mL for molds, followed by

232

incubation at 37 °C for 12-18 hrs with shaking at 200 rpm.

233

Determination of Cell Growth, Glucose Concentration, and Fermentation

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Culture Purification. Cell concentration was measured in terms of dry cell weight

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(DCW). The harvested culture samples were filtered and the mycelia were washed

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and dried to a constant weight at 65 °C. The glucose concentration in the culture broth

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was determined using an SBA biosensor analyzer (Shandong Science Academy, Jinan,

238

China). ε-PL was extracted from the culture filtrate as a white powder of (ε-PL·HCl)



239

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using previously described purification procedures.30

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High Performance Liquid Chromatography (HPLC) Analysis. ε-PL yield was

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determined by HPLC, according to a previously reported method.30 A TSKgel

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ODS-120T column (4.6 × 250 mm; Tosoh, Tokyo) connected to an Agilent 1200

243

HPLC system was equilibrated with 95% (v/v) solvent A (water, 0.05% trifluoroacetic

244

acid) and 5% (v/v) solvent B (acetonitrile). The gradient program was run as follows:

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95% (v/v) solvent A and 5% (v/v) solvent B for 10 min, 15% solvent B for 5 min,

246

then maintained at 15% of solvent B for 20 min. ɛ-PL concentration was determined

247

using calibration curves (R2 > 0.99) (data not shown). ɛ-PL degree of polymerization

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was evaluated using HPLC as previously reported.31 A TSKgel ODS-80Ts column

249

(4.6 × 250 mm; Tosoh, Tokyo) was equilibrated with 45% (v/v) solvent C (10 mM

250

NaH2PO4, 100 mM NaClO4·H2O, 10 mM sodium octane sulfonate, pH 2.6) and 55%

251

(v/v) solvent D (20 mM NaH2PO4, 200 mM NaClO4·H2O, 20 mM sodium octane

252

sulfonate [pH 2.6], 50% (v/v) acetonitrile) using a gradient program of 55–78%

253

solvent D over 70 min at 50 °C.

254

Matrix Assisted Laser Desorption Ionization-Time of Flight Mass

255

Pectrometry

(MALDI-TOF/MS)

256

MALDI-TOF/MS

257

2,5-dihydroxybenzoic acid was used as a the matrix.

(Autoflex

2,

Analysis. Bruker

ε-PL

Mw

Daltonics

was Inc.,

verified USA)

by with

258

Circular Dichroism (CD) Spectra Analysis. CD was scanned at a far-UV range

259

(185–260 nm) using Chirascan Circular Dichroism spectrometer (Applied

260

Photophysics, UK) with a 1 mm path-length quartz cuvette at 20 °C. The sample


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261

concentration used for circular dichroism analysis was 1.0 mg/mL of 5 mM sodium

262

phosphate buffer (pH=7.0), in the absence and presence of 30 mM SDS micelles. The

263

average of three scans was used to obtain the circular dichroism data, which was

264

expressed in terms of ellipticity (θ), in mdeg.

265

Statistical Analysis. At least three replicates of each experiment were performed.

266

Data are expressed as the mean±standard deviation. Statistical analysis was conducted

267

by one-way ANOVA followed by Duncan’s test (p < 0.05) using SPSS 17.0 software.

268

RESULTS AND DISCUSSION

269

Discovery of a New ɛ-PL-producing Strain. The conventional method for the

270

screening for ε-PL-producing strains mainly relies on its polycationic characteristics,

271

which can produce transparent zones with acidic dyes3; however, this process is

272

laborious. Currently, degenerate primers based on conserved domains in gene clusters

273

are enabling rapid screening of new secondary metabolites from nature.32,33 In this

274

study, as shown in Figure S1, two highly conserved regions, FDASCEEMW and

275

QTHLFHDR, were identified based on the alignment of the known Pls and its

276

homologous amino acid sequences. Degenerate primers were designed to amplify

277

these conserved regions to aid in the rapid discovery of potential ɛ-PL

278

producing-strains. Of the 19 Actinomycetes strains that were screened, a target

279

specific-PCR product was rapidly identified from one strain (PL-1; Figure S2) and

280

confirmed by DNA sequencing. In addition, the amino acid sequence of this newly

281

identified strain shared 55.08% identity (data not shown) with similar Pls regions in S.

282

albulus PD-1, suggesting that strain PL-1 might be a ε-PL producer. Subsequently, a



283

nearly complete 16S rDNA sequence (1389 bp) was amplified from strain PL-1 by

284

PCR (accession number MK073012). Based on the 16S rDNA sequence analysis,

285

strain PL-1 was identified as Kitasatospora aureofaciens (Figure 1).

286

Using partial nucleotide sequences encoding PlsII, multiple primers were

287

designed to amplify the 5′-upstream region and the 3′-downstream regions of the

288

known plsII genes via Genome Walking. Using these primers, a 3963 bp nucleotide

289

sequence containing the intact pls gene of K. aureofaciens PL-1 was identified; this

290

sequence was deposited with the GenBank database under accession no. MK090575.

291

In addition, the amino acid sequence of PlsII showed 55.04 % homology with PlsI of

292

S. albulus PD-1 (Figure S3) and transmembrane structure prediction revealed that

293

both the proteins have six transmembrane helices. To verify the substrate specificity

294

of the adenylation domain, we used colorimetric determination of PPi anion formation,

295

as previously explained. The A domain of PlsII was overproduced as N-His6–tagged

296

fusion proteins and purified with Ni-sepharose (Figure 2). This purified protein was

297

tested against an array of 23 different amino acids for substrate specificity. The A

298

domain of PlsII exhibited absolute specificity for L-lysine (Figure 2), consistent with

299

the software’s prediction (data not shown). These results indicate that the synthase

300

identified in strain PL-1 is a Pls, rather than another amino acid synthases such as

301

poly-diaminopropionic acid synthetase or poly-diaminobutyric acid synthetase. In

302

addition, the purified ɛ-PL from strain PL-1 was subjected to 1H NMR, the resulting

303

signals corresponded to the main ɛ-PL signals (Figure S4). Based on the above

304

mentioned results, the degree of ɛ-PL polymerization and the production capacity of K.


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305

aureofaciens PL-1 were further tested. Ion-pair chromatography (Figure 3)

306

demonstrated that the ɛ-PL from K. aureofaciens PL-1 has a polymerization degree

307

of 10-18, compared to the ɛ-PL from the S. albulus PD-1, which has a polymerization

308

degree of 25-33. However, the ɛ-PL production capacity and biomass concentration of

309

K. aureofaciens PL-1 however were only 25% (1.37 g/L) and 50% (7.56 g/L) of that

310

of S. albulus PD-1 in a batch fermentation process, respectively (Table 2). Because an

311

oligomeric ɛ-PL with ten or more lysine residues is desirable, as it does not have a

312

bitter taste and exhibits stronger inhibitory activity against yeasts than high-Mw

313

ɛ-PL,11,14 we attempted to increase low-Mw ɛ-PL production in K. aureofaciens PL-1.

314

However, despite the many strategies used, no significant increase in ɛ-PL production

315

was achieved (data not shown). Thus, based on an an in-depth analysis of the Pls

316

mechanism,34 we attempted to introduce the plsII gene into a high-ɛ-PL yielding strain

317

of S. albulus PD-1, by knocking out its endogenous plsI gene, in order to obtain a

318

high-yield low-Mw ɛ-PL producing-strain.

319

The Knock-out of the plsI Gene of S. albulus PD-1. Since 1977, S. albulus has

320

been used to produce ɛ-PL35and as a food preservative. In addition, this strain is

321

known to produce ɛ-PL in extremely high quantities (> 20 g/L), because of its ability

322

to produce the high levels of ATP and biomass required for the synthesis of ɛ-PL.36

323

We hypothesized that a S. albulus PD-1 strain in which the endogenous pls gene had

324

been knocked-out might serve as a potent host for heterologous production of the

325

low-Mw ɛ-PL. Therefore, the plsI gene in S. albulus PD-1 was knocked-out using the

326

homologous double exchange method as follows. First, an internal 2.26 kb region of



327

the plsI gene was replaced by the tsr gene, resulting in the plasmid pKC1139-tsr-plsI,

328

with 3.24 kb left-flanking and 3.15 kb right-flanking sequences (Figure 4A).

329

Subsequently, this temperature-sensitive plasmid was introduced into S. albulus PD-1

330

by conjugation, resulting in a thiostrepton-resistant, apramycin-sensitive marked

331

mutant. The △plsI mutation was confirmed by PCR amplification using primers

332

flanking the deleted regions of the chromosome (Figure 4B), followed by DNA

333

sequencing, and the mutant strain was designated as S. albulus PD-4. HPLC analysis

334

of the fermentation broth showed that S. albulus PD-4 could not secrete ɛ-PL (Figure

335

4C), thus constituting an ideal host for heterologous expression of the plsII gene. In

336

addition, except for the loss of its ɛ-PL producing ability, S. albulus PD-4 spores were

337

indistinguishable from those of the parental S. albulus PD-1 strain (Figure 4D). As

338

this strain also had a powerful metabolic pathway for the biosynthesis of L-lysine and

339

ATP, which are two key factors of ɛ-PL biosynthesis,37,38 we concluded that S.

340

albulus PD-4 would be an excellent host for low-Mw ɛ-PL production.

341

Validation of S. albulus PD-4 as a Host for Low-Mw ɛ-PL Production. To

342

validate the feasibility of using S. albulus PD-4 as a host for low-Mw ɛ-PL production,

343

plasmid pSET152-PPlsII-plsII plasmid (Figure 5A), in which the plsII gene is under the

344

control of its native promoter, was introduced into S. albulus PD-4 by conjugation.

345

The ɛ-PL concentration in the culture broth was determined on the third day of

346

cultivation. As shown in Figure 5B, S. albulus PD-4-pSET152-PPlsII-plsII only

347

produced 0.85 g/L ɛ-PL, compared to S. albulus PD-1 (1.23 g/L), indicating that it has

348

some differences in the ɛ-PL yield compared to the original S. albulus PD-1 strain.


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349

Thus, we attempted to improve ɛ-PL production using promoters derived from

350

different sources, as the promoter is one of the important elements in Pls expression

351

and ɛ-PL production.37 When plsII gene was expressed under the control of the

352

constitutive ermE* promoter, ε-PL production was not detected. Previous studies have

353

shown that the Pls is tightly regulated; ɛ-PL is only synthesized during the mid-log

354

and stationary phases of growth.39 This suggests that a constitutive promoter is not

355

suitable for pls gene expression. When psII gene was under the controlled of the plsI

356

gene promoter from S. albulus PD-1, the ɛ-PL production was improved from 0.85 to

357

1.14 g/L, a 34.1% increase (Figure 5B). This result indicates that S. albulus PD-1

358

could produce a high quantities of ɛ-PL not just because of the sufficient precursor

359

and ATP supply, as well as the excellent performance of its pls gene promoter. In

360

either case, the biomass of the recombinant strain was not significantly altered relative

361

to the wild-type strain S. albulus PD-1 strain. In addition, the Mw of the ɛ-PL was

362

confirmed by MALDI-TOF/MS after purification. The Mw of the ɛ-PL produced by S.

363

albulus PD-4-pSET152-PplsI -plsII was 1.3-2.3 KDa, which is the same as the Mw of

364

the ɛ-PL from K. aureofaciens PL-1, and dissimilar to the 3.2-4.5 kDa ɛ-PL produced

365

by wild type S. albulus PD-1 (Figure 6). This result is consistent with a number of

366

previous studies, which showed that the Mw of ɛ-PL is controlled by the Pls itself

367

instead and not by the ɛ-PL degrading enzyme.36,40 Based on these results, S. albulus

368

PD-4-pSET152-PPlsI-plsII was selected for further experimentation and renamed S.

369

albulus PD-5 for the sake of simplicity.

370

Performance of Fed-batch Operation Using Strain S. albulus PD-5. To verify



371

the feasibility and stability of recombinant plasmid expression under large-scale

372

production conditions, S. albulus PD-5 was cultured in a 5 L bioreactor; a final

373

biomass concentration of 26.8 g/L and ε-PL concentration of 23.6 g/L were achieved

374

using S. albulus PD-5 (Figure 7), compared to 25.8 g/L of ε-PL produced by S.

375

albulus PD-1 after 168 hrs fermentation.41 This result demonstrated that our Pls

376

replacement strategy had no negative effect on ε-PL production. In addition, a number

377

of microorganisms producing low-MW ε-Pls, such as Streptomyces sp. USE-33, S.

378

celluloflavus USE-31, S. celluloflavus subsp. USE-32, and Streptomyces sp. USE-51,

379

have also been identified in nature, and their ability to produce ε-PL was also shown

380

to be low.17 Thus, the Pls replacement strategy is a highly efficient strategy for

381

obtaining low-Mw ε-PL. Moreover, given the rapid development of microbial genome

382

sequencing approaches, a large number of pls genes will be annotated in the database,

383

the S. albulus PD-4 host could be engineered to produce ε-PL with different Mw,

384

greatly accelerating the traditionally time-consuming strain improvement process.

385

Antibacterial Activity of ε-PLs with Different Mw. The MIC method was

386

employed to examine the antimicrobial activity of the low-Mw ε-PL against

387

microorganisms causing food spoilage compared with the high-Mw ε-PL. The results

388

are shown in Table 3.

389

The low-Mw ε-PL from S. albulus PD-5 exhibited a slightly weaker inhibitory

390

activity against bacteria compared to the high Mw ε-PL from S. albulus PD-1.

391

However, it exhibited better inhibitory activity against yeasts compared to the high

392

Mw ε-PL. Previously, we showed that poly L-diaminopropionic acid, a novel


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393

non-proteinic amino acid oligomer (Mw ranging from 0.55 to 1.5 kDa), also exhibited

394

a stronger inhibitory activity against yeasts.30 These results are likely to be related to

395

the yeast cell membrane, which contains a higher ratio of lipids than bacteria; thus,

396

the low-Mw polyamino acids might have a better adsorption effect on yeasts.30

397

Unfortunately, the ε-PL from S. albulus PD-5 exhibited poor antimicrobial activity

398

against molds, similar to the ε-PL from S. albulus PD-1. Previous reports have

399

suggested that the protease in mold has the typical ε-PL endo-type degradative

400

activity, which might contribute to the high MIC values of ε-PL on molds.42 In

401

summary, although the low-Mw ε-PL had a reduced inhibitory effect on bacteria, it

402

had higher antimicrobial activity towards yeasts, compared to the commonly used

403

ε-PL. In addition, as the long-chain length ε-PL gives a bitter taste if used in high

404

quantitates, the use of the low-Mw ε-PL in food susceptible to yeast contamination,

405

such as food with a low pH, low water activity, low humidity, high salt content, or

406

high sugar content,43,44could not only improve their taste but also expand the

407

applications of ε-PL.

408

ɛ-PL mainly acts by adsorbing to the surface of the microbial membrane, which

409

causes physiological damage to the cell, and thus, the microorganisms cannot easily

410

develop resistance to it.45 Recently, researchers have shown that the antimicrobial

411

mechanism of ε-PL against E.coli and yeast involves a “carpet model”, which can

412

cause micelle formation, phospholipid bilayer bending, and perforations on the cell

413

membrane, eventually leading to cell death.46, 47 In order to study the difference in the

414

antimicrobial activity of ε-PLs with different Mw, we investigated the secondary



Page 20 of 46

415

structure of these ε-Pls using CD. The CD spectra results showed that these two types

416

of ε-PL take on a confoirmation similar to an antiparallel pleated-sheet (Figure 8),

417

which is consistent with a previous report.48 In addition, in a water environment or a

418

solvent that mimics the membrane environment, the strength of this conformation

419

increases with increasing ε-PL Mw. This indicates that the dominant secondary

420

structure is one of the factors that influence ε-PL activity, along with the positive net

421

charge, flexibility, size, and hydrophobicity.49

422

Abbreviations Used. ε-PL, ε-Poly-L-lysine; Mw, molecular weight; Pls, ε-PL

423

synthetase; M3G, Medium 3G; gDNA, genomic DNA; PPi, pyrophosphate; MIC, the

424

minimum inhibitory concentration; DCW, dry cell weight; HPLC, high-performance

425

liquid

426

ionization-time of flight mass spectrometry; CD, circular dichroism spectra.

427

ORCID

428

Hong Xu: 0000-0002-9085-9542

429

Funding

chromatography;

MALDI-TOF/MS,

matrix-assisted

laser

desorption

430

This work was supported by the National Key R&D Program of China (No.

431

2017YFD0400403), the National Nature Science Foundation of China (Nos.

432

21878152 and 51703095), the Jiangsu Synergetic Innovation Center for Advanced

433

Bio-Manufacture (No. XTB1804), the State Key Laboratory of Materials-Oriented

434

Chemical Engineering (ZK201606), and the Postgraduate Research & Practice

435

Innovation Program of Jiangsu Province (KYCX18_1102).

436

Supporting Information description


Page 21 of 46


437

The sequences of all the primers used in this study are detailed in Table S1. Two

438

highly conserved regions, FDASCEEMW and QTHLFHDR, were identified based on

439

an alignment of the known Pls and its homologous amino acid sequences (an overall

440

similarity ranging from 99.7 % [between EXU90606.1 and WP_038523657.1] to

441

1280

>1280

Penicillium

>1280

>1280

chrysogenum

CGMCC

3.3890 aMICs

were determined by the broth dilution method.

bHigh-Mw

cLow-Mw

ε-PL·HCl with 3.2-4.5 kDa. ε-PL·HCl with the 1.3-2.3 kDa.



Figure graphics Figure 1


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Figure 2



Figure 3


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Figure 4



Figure 5


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Figure 6



Figure 7


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Figure 8



Graphic for table of contents


Page 46 of 46

Discovery of a Short-Chain Îµ-Poly-l

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