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Biotechnology and Biological Transformations

Efficient biosynthesis of low-molecular-weight poly-#-glutamic acid by stable overexpression of PgdS hydrolase in Bacillus amyloliquefaciens NB Yuanyuan Sha, Yatao Zhang, Yibin Qiu, Zongqi Xu, Sha Li, Xiaohai Feng, Mingxuan Wang, and Hong Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05485 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 16, 2018

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Journal of Agricultural and Food Chemistry

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Efficient biosynthesis of low-molecular-weight poly-γ-glutamic acid by stable

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overexpression of PgdS hydrolase in Bacillus amyloliquefaciens NB

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Yuanyuan Sha, †,§ Yatao Zhang, †,§ Yibin Qiu, †,§ Zongqi Xu, †,§ Sha Li, †,§ Xiaohai

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Feng, †,§ Mingxuan Wang, †,§ Hong Xu *,†,§

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†

8

China

9

§

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State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing 211816,

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

211816, China

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* Corresponding author at: Nanjing Tech University, Nanjing 211816, China

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Tel/Fax: +86-25-58139433

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E-mail address: [email protected] (Hong Xu)

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ACS Paragon Plus Environment


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ABSTRACT: Low-molecular-weight poly-γ-glutamic acid (LMW-γ-PGA) has

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attracted much attention owing to its great potential in food, agriculture, medicine and

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cosmetics. Current methods of LMW-γ-PGA production, including enzymatic

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hydrolysis, are associated with low operational stability. Here, an efficient method for

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stable biosynthesis of LMW-γ-PGA was conceived by overexpression of γ-PGA

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hydrolase in Bacillus amyloliquefaciens NB. To establish stable expression of γ-PGA

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hydrolase (PgdS) during fermentation, a novel plasmid pNX01 was constructed with a

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native replicon from endogenous plasmid p2Sip, showing a loss rate of 4% after 100

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consecutive passages. Subsequently, this plasmid was applied in a screen of high

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activity PgdS hydrolase, leading to substantial improvements to γ-PGA titer with

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concomitant decrease in the molecular weight. Finally, a satisfactory yield of 17.62 ±

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0.38 g/L LMW-γ-PGA with a weight-average molecular weight of 20–30 kDa was

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achieved by direct fermentation of Jerusalem artichoke tuber extract. Our study presents

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a potential method for commercial production of LMW-γ-PGA.

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KEYWORDS: Poly-γ-glutamic acid; Molecular weight; Bacillus amyloliquefaciens

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NB; PgdS hydrolase; Jerusalem artichoke

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INTRODUCTION

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Poly-γ-glutamic acid (γ-PGA), a natural high-molecular-weight polymer, is composed of

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D- and/or L-glutamic acid units linked via gamma amide linkages. γ-PGA is mainly

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synthesized by several Gram-positive bacteria (e.g. Bacillus subtilis, B. licheniformis),

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with a weight-average molecular weight (Mw) ranging from 100 to over 1000 kDa.1

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Interestingly, the biological functions and practical applications of γ-PGA are strongly

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dependent on its molecular weight especially in food and agriculture industry.2 Several

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studies have shown that low-molecular-weight γ-PGA (LMW-γ-PGA) (Mw1000

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kDa) for certain bioengineering applications as a crop cryoprotectant (2 kDa),3 fertilizer

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synergist (2 kDa),4 probiotic protectant (257 kDa),5 drug carrier (45–60 kDa),6 calcium

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absorption enhancer (11 kDa),7 bone tissue engineering nanocomposite (20–275 kDa),8

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and skin lightening agent (80–120 kDa).9 Compared with HMW-γ-PGA, LMW-γ-PGA

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can be easily absorbed by the body, owing to its moderate viscosity and manageable

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rheology, and it can be modified using chemical reagents as precursors for the synthesis

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of other substances, greatly broadening the applications of γ-PGA.10 Thus, the efficient

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production of LMW-γ-PGA is essential for further research and development of γ-PGA.

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Conventionally, LMW-γ-PGA is synthesized by the degradation of HMW-γ-PGA

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through physical and chemical methods.11 However, these methods are severely limited

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by high treatment costs and uncontrolled molecular weight reduction, which hinders the

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preparation and separation of specific LMW-γ-PGAs. In addition, the introduction of

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some chemical reagents seriously affects the biological activities of γ-PGA and even 3



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causes environmental pollution. In contrast, the enzymatic degradation using endo-type

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γ-PGA hydrolase (PgdS) for production of LMW-γ-PGA exhibits unique advantages,

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such as mild reaction conditions, high product specificity, and being pollution-free.12

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Until now, many PgdS hydrolases have been isolated and characterized from various

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strains. However, commercial PgdS hydrolases have not been obtained by scaled-up

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production of LMW-γ-PGA because of the complex conditions required and low

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hydrolytic activity.13 Furthermore, the separation processes in γ-PGA production and

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subsequent enzymatic hydrolysis are time-consuming and expensive, thus hampering the

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development of LMW-γ-PGA using in vitro enzymatic degradation method. In recent

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years, one-step direct fermentation has received greater attention because of its simplified

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process and decreased cost. According to Tian et al, an endogenous PgdS hydrolase was

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overexpressed in B. licheniformis WX-02, a glutamate-dependent γ-PGA producing

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strain, leading gradually to a decrease in γ-PGA Mw values from 1000–1200 kDa to 600–

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800 kDa. The results proved the feasibility of coupling PgdS hydrolase overexpression

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and synthesis of γ-PGA for one-step biosynthesis of LMW-γ-PGA.14 Although the

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molecular weight of the γ-PGA was reduced, the changes in molecular weight are yet to

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meet the requirements for LMW-γ-PGA in certain commercial applications. The most

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likely explanation is the lack of stable expression of the hydrolase system or low activity

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of endogenous PgdS hydrolase. Therefore, it is important to optimize the expression

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stability and screen out suitable PgdS hydrolase with high hydrolytic activity for efficient

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LMW-γ-PGA production.

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Recently, a novel glutamate-independent γ-PGA-producing strain was isolated and 4


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identified as Bacillus amyloliquefaciens NX-2S (CCTCC NO: M 2016346), which can

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directly make use of the raw inulin extract from Jerusalem artichoke tubers for efficient

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production of γ-PGA.15 By eliminating the endogenous plasmid in B. amyloliquefaciens

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NX-2S, the strain evolved into B. amyloliquefaciens NB (data not shown). For efficient

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production of LMW-γ-PGA, a stable expression system was necessary for continuous

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overexpression of PgdS hydrolase. However, in our previous work, a drastic plasmid

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elimination was observed in B. amyloliquefaciens NB (data not shown). Although many

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commercialized

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characteristics have been constructed and employed in large-scale production of

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industrial enzymes,16 their general applicability is still hampered by a severe instability of

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recombinants carrying those plasmids.17 Therefore, for high efficiency preparation of

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LMW-γ-PGA, it is necessary to construct a novel plasmid suitable for PgdS hydrolase

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expression in B. amyloliquefaciens NB. In this study, a novel expression-stable plasmid

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pNX01 was constructed and stably expressed in B. amyloliquefaciens NB. Then, a screen

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for γ-PGA hydrolase with high activity was performed by this plasmid and the best

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performing strain was selected for stable LMW-γ-PGA synthesis. Finally, batch

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fermentation using Jerusalem artichoke tuber extract was performed in a 7.5-L fermenter.

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This work greatly promotes the development of economical and sustainable production

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of LMW-γ-PGA, and the strategy used herein will be an attractive alternative for other

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high-value products.

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MATERIAL AND METHODS

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plasmids

with

different

transcriptional

patterns

and

genetic

Bacterial strains and plasmids. All bacterial strains and plasmids used in this work 5



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are listed in Table 1. B. amyloliquefaciens NB, a γ-PGA-producing strain, was isolated

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previously in our laboratory and served as the host strain for protein expression in this

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study. Escherichia coli DH5α was used for routine plasmid construction and

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maintenance. E. coli GM2163 was employed for plasmid demethylation and

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transformation.

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Media and culture conditions. For normal cloning and transformation

106

experiments, E. coli and B. amyloliquefaciens strains were grown at 37°C in Luria–

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Bertani (LB) medium (10 g/L tryptone, 10 g/L yeast extract, and 5 g/L NaCl) containing

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the appropriate antibiotic (100 μg/mL ampicillin for E. coli, 5 μg/mL chloramphenicol

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for B. amyloliquefaciens). For shake flask and batch fermentation, the seed medium and

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fermentation medium of the wild type and recombinant strains were consistent with

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previous reports.15 The cells were precultured in a 250 mL shake flask with 40 mL seed

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culture and incubated at 37°C with shaking at 200 rpm for 12 h. For flask cultures, 4.8

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mL seed culture was transferred into 500 mL flasks containing 80 mL of the modified

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medium, and cultured at 32°C and 200 rpm for 80 h (pH 7.5). Batch fermentation was

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performed in a BioFlo 115 7.5-L fermenter (New Brunswick Scientific) containing 4 L of

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medium. Cultivation was carried out at 32°C with stirring at 400 rpm for 92 h. The pH

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was maintained at 7.0 ± 0.1 by the addition of 2 M NaOH or 2 M HCl. Samples were

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collected periodically to determine the biomass, γ-PGA molecular weight, γ-PGA

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hydrolase activity, and γ-PGA production.

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DNA manipulation and plasmid construction. The primers used in this study are

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listed in Table 2. The novel protein expression vector pNX01 consisted of three 6


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fragments (One, Two, and Three). Fragment One, which includes an E. coli replication

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origin (ori-177) and an ampicillin-resistance marker (AmpR), was amplified from plasmid

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pHY300PLK using primers P01/P02. Fragment Two includes the replication origin

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amplified from plasmid p2Sip with primers P03/P04, and a chloramphenicol-resistance

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marker (CmR) amplified from plasmid pHT01 using primers P05/P06. Fragment Three

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includes P43 (K02174.1) amplified from B. subtilis 168 with primers P07/P08, unique

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restriction enzyme sites (SpeI-EcoRI-SalI-BglII-NotI), and α-amylase terminator TamyL

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(CP002634.1) amplified from B. amyloliquefaciens NB with primers P09/P10. All of the

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three DNA fragments were ligated with ClonExpress MultiS One Step Cloning Kit to

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generate pNX01. The green fluorescent protein GFP, amplified from plasmid

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EGFP-pBAD, was cloned into pNX01 with SpeI/NotI restriction sites, resulting in

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pNX01-gfp. To replace the plasmid replicon, the plasmid backbone was retained by

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reverse PCR, and then was connected with the replicons from pHT01 and pHY300PLK,

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generating pNX01-pHT01 and pNX01-pHY300PLK, respectively. The endo-type γ-PGA

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hydrolases PgdS1, PgdS2, PgdS3, and PgdS4 were amplified from B. subtilis NX-2, B.

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amyloliquefaciens NB, B. licheniformis 14580, and B. megaterium M, respectively. The

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signal peptide SPamyL from B. amyloliquefaciens NB was used as the secretory

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initiation signal for each gene, generating plasmids pNX01-pgdS1, pNX01-pgdS2,

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pNX01-pgdS3, and pNX01-pgdS4. All the constructed plasmids were confirmed by PCR

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and DNA sequencing. All the recombinant plasmids were transformed into B.

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amyloliquefaciens NB by high osmolarity electroporation.18

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Plasmid segregational stability assay. To determine the stability of the 7



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transformed plasmids, recombinants were passaged for 30, 50, and 100 consecutive

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generations in non-selective LB medium. Each generation was cultivated for 24 h and

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plated out on non-selective plates in triplicate. Then 100 single colonies from each plate

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were plated on selective plates. The number of colonies on plates with and without

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antibiotics was defined as Y and X, respectively. The plasmid loss rate was defined as

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R=(X−Y)/X.19

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GFP fluorescence assay. The fluorescence activity of GFP was determined as

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previously reported.20 Cells were grown in LB liquid medium for 60 h, during which the

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culture medium was sampled every 6 h. After cultivation, cells were harvested by

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centrifugation, washed 3x in PBS, and resuspended in dH2O. Then the cell suspension

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was transferred into 96-well plates. The GFP fluorescence was detected by the

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Synergy™ H4 multimode microplate reader (BioTek Instruments, Inc.). The relative

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fluorescence intensity of GFP expression was calculated as fluorescence intensity divided

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by cell growth.

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An endo-type γ-PGA hydrolase activity assay. Hydrolase activity of PgdS was

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evaluated by determining the amount of free amino groups released from γ-PGA.21 The

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hydrolyzed substrate γ-PGA from B. amyloliquefaciens NB was purified using a

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previously described method.22 Crude enzyme solution of γ-PGA hydrolase was obtained

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by removing the B. amyloliquefaciens cells via centrifugation (20 min, 12,000 × g).

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Enzyme activity was determined according to Tian et al.14 The enzymatic reaction,

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containing 200 μL of the crude enzyme solution and 800 μL of 10 g/L γ-PGA, was

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dissolved in 0.05 mM phosphate buffer, pH 7.4 and was incubated at 37°C for 4 h. A 8


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separate blank sample containing inactivated enzyme was set up for each sample to

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correct the results for the nonenzymatic release of amino groups. The reaction was

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stopped by immersion in boiling water for 5 min and then examined using the ninhydrine

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colorimetric spectrophotometric method. All the results were replicated at least 3 times.

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Analytical methods. Cellular biomass was determined according to Feng et al.23

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The γ-PGA concentration, molecular weight (weight-average molecular weight, Mw;

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number-average molecular weight, Mn) and the polydispersity index (Ip; Ip=Mw/Mn)

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were measured and calculated by using gel permeation chromatography (GPC) with an

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RI-10 refractive-index detector and a SuperposeTM 6 column (Shimadzu Corp.).24

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Viscosity of γ-PGA culture broth was measured by a rotational viscometer (NDJ-8S,

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Shanghai Hengping Scientific Instrument Co., Ltd.) with a No. 4 rotor at 60 rpm.25 The

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dissolved oxygen (DO) was measured by a probe in the bioreactor.

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RESULTS AND DISCUSSION

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Construction of a novel expression vector pNX01 for stable heterologous

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expression in B. amyloliquefaciens. The stability of expression plasmids in host cells is

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of great importance for the robustness and productivity of a production system, while

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instability might render the validation of an industrial process questionable or even

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impossible.26 In this study, to meet the demand for stable expression of PgdS hydrolase,

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construction of a stable plasmid was performed for efficient production of LMW-γ-PGA

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in B. amyloliquefaciens NB. In our previous work, a unique endogenous plasmid p2Sip

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was found in B. amyloliquefaciens NX-2S. A variety of traditional physical and chemical

9



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methods have been used for elimination of the native plasmid, but the results showed the

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ineffectiveness of these ways in B. amyloliquefaciens NX-2S, which indicates the high

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stability of the endogenous plasmid. Previous studies have shown that the replicon, a key

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element of a replicative plasmid, can affect the transformation efficiency, plasmid copy

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number, and segregational stability, which in turn have dramatic effects on heterologous

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gene expression, host metabolism, and final product formation.27 Therefore, in view of

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the high stability of endogenous plasmids, a method was proposed to construct a plasmid

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that contains the replicon from the endogenous plasmid p2Sip, which could serve as a

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potent tool for stability.

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In addition, the resistance of B. amyloliquefaciens NB to different antibiotics was

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analyzed. To isolate an antibiotic resistance screening marker suitable for B.

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amyloliquefaciens NB transformant screening, the growth of B. amyloliquefaciens NB on

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LB plates containing tetracycline, chloramphenicol, or kanamycin was observed. As

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shown in Table 3, 5 g/L chloramphenicol inhibited the growth of bacteria for the longest

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time. Therefore, 5 g/L chloramphenicol was chosen as the screening marker for NB

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transformants.

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Based on the analysis of replicons and antibiotic resistance in B. amyloliquefaciens

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NB, a novel expression plasmid pNX01 was constructed with the chloramphenicol gene,

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p2Sip replicon, ampicillin gene, E. coli replicon, P43 promoter, and amylase terminator

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TamyL. P43 is a constitutively expressed promoter that can drive strong transcription of

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heterologous proteins in B. amyloliquefaciens.28 In order to facilitate the later screening

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of different expression elements, both ends of the promoter and terminator sequences 10


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contain restriction sites. The plasmid encodes ampicillin resistance for E. coli and

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chloramphenicol resistance for B. amyloliquefaciens. The overall method used for vector

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construction is outlined in Figure 1A. As shown in Figure 1B, PCR analysis indicated

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that pNX01 was constructed successfully.

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pNX01 segregational stability in B. amyloliquefaciens NB. The gfp gene was

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chosen as a reporter to test the stability of pNX01 in B. amyloliquefaciens NB. GFP

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expression was confirmed by fluorescence intensity analyses and SDS-PAGE. As shown

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in Figure 2A, pNX01-gfp was capable of expressing GFP at different levels in B.

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amyloliquefaciens NB. The prominent bands in SDS-PAGE (Figure 2B) corresponding to

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25 kDa, the theoretical molecular weight of mature GFP, also confirmed GFP expression.

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These results suggest that pNX01 could support a functional expression system in B.

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amyloliquefaciens NB, providing a novel strategy for augmenting the stable

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overproduction of various heterologous proteins.

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Previous studies have revealed that the replication mechanism of the replicon plays

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an important role in plasmid stability.29 In order to identify the effects of different

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replicons on the maintenance of plasmids, 3 types of plasmids: pNX01 (rolling-circle

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type) (data not shown), pNX01-pHT01 (theta type),30 and pNX01-pHY300PLK

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(rolling-circle type),31 carrying replicons from plasmid p2Sip, pHT01, and pHY300PLK,

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respectively, were transformed into B. amyloliquefaciens NB by electroporation. The

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plasmid stability in different recombinants was investigated (Figure 3). For plasmid

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pNX01, the plasmid loss rate was the lowest throughout the cultivation process and only

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4% of cells lost the plasmid after 100 successive passages. Slightly less stable 11



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functioning of the pNX01-pHT01 was observed in NB; 52% of cells carried

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pNX01-pHT01 after 100 generations, indicating a slow loss of this plasmid. In

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comparison, plasmid pNX01-pHY300PLK was lost very rapidly by this strain when

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grown in the absence of selection; plasmid-containing cells comprised no more than 30%

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of the total population at the end of cultivation. This result suggests that endogenous

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replicon plays an important role in plasmid stability determination. It has been observed

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that artificial plasmid constructs containing the theta mode of replication are structurally

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much more stable than rolling-circle plasmids.32 This has been attributed to the fact that

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single-stranded DNA (ssDNA) is more prone to replication errors than double-stranded

240

DNA (dsDNA). Of particular interest is the observation that pNX01 showed a stronger

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replication stability in B. amyloliquefaciens NB. Previous studies have shown that the

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rolling-ring replicon contains 3 basic cassettes: replication initiation protein (Rep),

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double-strand origin (DSO), and single-strand origin (SSO).33 The sequences of Rep and

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DSO in different rolling-ring replicons are similar in functionally related families, while

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SSO sequences, which are important determinants of host range and host specificity, are

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quite different. A particular SSO can be more efficiently recognized by the RNA

247

polymerase of the origin host such that ssDNA can be efficiently transformed into

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dsDNA, thus ensuring stable replication of the plasmid.34 These results imply that

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pNX01 harboring the endogenous replicon would have the greatest stability and is

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suitable for the stable protein expression in B. amyloliquefaciens NB.

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Screening for γ-PGA hydrolase with high hydrolytic activity in B.

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amyloliquefaciens. To establish an efficient method for LMW-γ-PGA production, a 12


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screening of different endo-type γ-PGA hydrolases was performed based on the

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construction pNX01 in B. amyloliquefaciens NB. For the screening, 4 types of PgdS

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hydrolases, originating from B. subtilis NX-2, B. amyloliquefaciens NB, B. licheniformis

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14580, and B. megaterium M were inserted into pNX01, generating pNX01-pgdS1,

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pNX01-pgdS2, pNX01-pgdS3, and pNX01-pgdS4, respectively, and then were

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transformed into B. amyloliquefaciens NB to generate the recombinant NB

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(pNX01-pgdS1), NB (pNX01-pgdS2), NB (pNX01-pgdS3), and NB (pNX01-pgdS4),

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respectively. Fermentation of the strains was investigated in flask cultures.

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Figure 4A shows the effect of PgdS hydrolase on the growth of recombinant B.

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amyloliquefaciens NB. The final dry cell weight (DCW) of the 4 recombinants NB

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(pNX01-pgdS1), NB (pNX01-pgdS2), NB (pNX01-pgdS3), and NB (pNX01-pgdS4)

264

were 5.20 ± 0.15 g/L 、 4.91 ± 0.13 g/L 、 4.64 ± 0.13 g/L, and 4.43 ± 0.14 g/L,

265

respectively, which were all higher than that of the wild type strain (4.20 ± 0.12 g/L). As

266

shown in Figure 4B, enhancing the expression of PgdS hydrolase reduced the molecular

267

weight of γ-PGA. With prolonged fermentation, the molecular weight of γ-PGA

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gradually became smaller, which was consistent with the results reported by Tian et al.14

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In particular, at the end of fermentation, the γ-PGA Mw values for the recombinants NB

270

(pNX01-pgdS1), NB (pNX01-pgdS2), NB (pNX01-pgdS3), and NB (pNX01-pgdS4)

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were maintained at 20–30 kDa, 650–710 kDa, 690–750 kDa, and 785–830 kDa,

272

respectively, compared with the wild type strain (1300–1380 kDa). At the same time, the

273

hydrolase activities in the extracellular culture supernatant were determined (Figure 4C).

274

As expected, the recombinant strain NB (pNX01-pgdS1) generated the highest hydrolytic 13



275

activity (15.83 ± 0.42 U/mL). In contrast, NB (pNX01-pgdS2), NB (pNX01-pgdS3), and

276

NB (pNX01-pgdS4) exhibited comparatively lower activities, which were 8.82 ± 0.35

277

U/mL, 7.30 ± 0.34 U/mL, and 5.31 ± 0.30 U/mL, respectively. This result was consistent

278

with the result of the molecular weight assay. The wild type strain exhibited only weak

279

enzyme activity at the end of fermentation, which was similar to the results of a previous

280

study.35 Finally, the effect of PgdS hydrolase on γ-PGA production was investigated

281

(Figure 4D). Surprisingly, the PGA concentration of the recombinant strain NB

282

(pNX01-pgdS1) reached 15.92 ± 0.32 g/L, which was higher than that of any other

283

recombinants, indicating the efficiency of PgdS hydrolase from B. subtilis NX-2. The

284

viscosity profile of microbial biopolymers and their fermentation broths can be an

285

important factor in the design and operation of a successful bioprocess. As can be seen in

286

the Figure S1, the recombinant strain NB (pNX01-pgdS1) had lower value (20 ± 2

287

mPa·s) than that of the wild-type strain (750 ± 5 mPa·s) at the end of fermentation,

288

showing a 97.33% decrease. Low broth viscosity increased oxygen transfer and substrate

289

utilization,36 which explained the growth advantage and higher γ-PGA yield exhibited by

290

NB (pNX01-pgdS1). Additionally, the calculation of the polydispersity index (Mw/Mn)

291

reveals that four polymers present a uniform distribution of molecular weight, compared

292

with the control (Table S1), which demonstrates the advantage of these products in the

293

field of biomedical or pharmaceutical applications. In contrast with a previous report of

294

enhanced expression of endogenous PgdS hydrolase in B. licheniformis WX-02 with a

295

Mw of γ-PGA at 600–800 kDa,14 the advantage of this study is the production of γ-PGA

296

with a wider range of Mw (20–830 kDa) with heterologous expression of different γ-PGA

14


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hydrolases, which is more beneficial to broadening the industrial application of γ-PGA.

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Additionally, the ability to regulate the molecular weight of γ-PGA by various degrading

299

enzymes is unique. Previous studies have shown that the molecular weight of γ-PGA was

300

not only dependent on the activity of PgdS hydrolase, but also related to the substrate

301

specificity and stereoselectivity of enzyme.37 Different PgdS hydrolases may favor

302

cleaving different γ-glutamyl bonds between 2 glutamate residues such as DL, DD, or LL

303

in γ-PGA, thus producing γ-PGA acid with different molecular weights. Therefore,

304

screening of suitable PgdS hydrolase for the degradation of γ-PGA with a particular

305

stereochemical composition is of great significance for the preparation of a specific

306

LMW-γ-PGA. Based on these findings, it can be concluded that the PgdS hydrolase from

307

B. subtilis NX-2 was the most suitable for degradation of γ-PGA from B.

308

amyloliquefaciens NB.

309

Batch fermentation from raw inulin extract in a 7.5-L fermenter. Currently,

310

glucose, sucrose, and fructose are the dominant carbon sources used in γ-PGA

311

production.38 However, these nutrients are mainly derived from food biofuel crops, and

312

their excessive consumption can cause serious food shortages, causing many social

313

dilemmas.22 The Jerusalem artichoke (Helianthus tuberosus L.), a non-food material, is a

314

potential economically viable crop for bioenergy production from biomass because of its

315

fast growth, high production, and strong resistance to in hospitable growth

316

environments.15 The raw inulin extract made from Jerusalem artichoke tubers is rich in

317

carbohydrates and has been applied in the production of many important industrial

318

chemicals, such as ethanol, fructose, and lactic acid.39,40 In this study, upon construction

15



319

of stable plasmid and expression of high activity γ-PGA hydrolase, Jerusalem artichoke

320

was explored as an economical resource for the production of LMW-γ-PGA. The batch

321

fermentation of the recombinant NB (pNX01-pgdS1) was carried out in a 7.5-L

322

bioreactor. As shown in Figure 5, rapid growth of the recombinant strain with obvious

323

γ-PGA production occurred in the first 20 h. As the fermentation process progressed, the

324

molecular weight of γ-PGA decreased throughout the whole fermentation process,

325

resulting in higher DO values (45%), which in turn accelerated the growth of bacteria

326

(6.10 ± 0.33 g/L). Finally, the maximal titer of γ-PGA was markedly increased to 17.62 ±

327

0.38 g/L, with Mw value of 20–30 kDa. Previous studies have mainly focused on

328

increasing the production of HMW-γ-PGA, limiting the applications of γ-PGA in other

329

fields.41 In addition, the fermentation broths exhibited high viscosity, which seriously

330

weakens oxygen transfer in the process, and inhibits cell growth and γ-PGA production.42

331

In this study, the overexpression of PgdS hydrolase not only regulated the degree of

332

γ-PGA polymerization, but also promoted growth of the bacteria. In addition, the reduced

333

viscosity of the fermentation broth allowed for more convenient separation and

334

purification. Therefore, this method provides an environmentally friendly and

335

economical choice for LMW-γ-PGA production. Although 20–30 kDa LMW-γ-PGA has

336

been obtained by batch fermentation, the yield of LMW-γ-PGA was much lower than

337

that of HMW-γ-PGA. Further optimization strategies should be explored to improve the

338

fermentation process and enhance LMW-γ-PGA yield, including optimizing the

339

expression of γ-PGA hydrolase,43 combining inulinase expression to enhance the uptake

340

of substrates,44 or engineering efficient metabolic pathways for high yield of γ-PGA.45

16


Page 16 of 37

Page 17 of 37


341

Overall, B. amyloliquefaciens NB (pNX01-pgdS1) is a promising producer of

342

LMW-γ-PGA from Jerusalem artichoke tuber extract.

343

In this study, efficient production of LMW-γ-PGA was achieved by stable

344

overexpression of γ-PGA hydrolase in B. amyloliquefaciens NB for the first time. The

345

maximum production of LMW-γ-PGA (Mw, 20–30 kDa) reached 17.62 ± 0.38 g/L by

346

supplementing the raw inulin extract from Jerusalem artichoke tubers in batch

347

fermentation. This study provides the basis for an environmentally friendly, economical,

348

and effective process for specific LMW-γ-PGA production, which can be used for other

349

high value products. Additional optimization strategies are necessary to improve the

350

performance of the fermentation process and further enhance the production of

351

LMW-γ-PGA.

352

353

AUTHOR INFORMATION

354

Corresponding Author

355

*(H.X.) Present address: 30 South Puzhu Road, Nanjing 211816, China. Phone/fax:

356

+86-25-58139433. E-mail: xuh@ njtech.edu.cn.

357

ORCID ID

358

Hong Xu: 0000-0002-9085-9542

359

Funding

360

This work was funded by the National High Technology Research and Development

361

Program of China (863) (No. 2015AA020951), the National Nature Science Foundation 17



362

of China (No. 21878152), the Natural Science Foundation of the Jiangsu (No.

363

BK20150946), the Natural Science Research Project in Jiangsu Province (No.

364

15KJB530007) and the Jiangsu Synergetic Innovation Center for Advanced

365

Bio-Manufacture (XTB1804).

366

367

ASSOCIATED CONTENT

368

Supporting Information

369

The Supporting Information is available free of charge on the ACS Publications website.

370

Polydispersity index of purified γ-PGA samples from γ-PGA hydrolases overexpression

371

recombinants (Table S1); Effects of different γ-PGA hydrolases’ overexpression on the

372

broth viscosity (Figure S1) (PDF).

373

374

Notes

375

The authors declare no competing financial interest.

376

377

References

378

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furiosus

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acid synthesis for poly-gamma-glutamic acid production. Enzyme Microb. Technol. 2017,

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efficient signal peptide in Bacillus subtilis. N. Biotechnol. 2016, 33(3), 372-9.

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acid (γ‐PGA) overproduction. Microb. Biotechnol. 2014, 7(5), 446-455.

25



520

FIGURE CAPTIONS

521

Figure 1. Plasmid construction process (A) and confirmation of recombinant plasmid

522

(B). Line M: DL 5000 DNA marker; Line 1, 2, 3: fragment one (ori-177-AmpR) ， two

523

(replicon of p2Sip and CmR) and three (P43-MCS-Tamy) of plasmid pNX01.

524

Figure 2. Identification of the expression level and pattern of GFP in recombinant B.

525

amyloliquefaciens NB. The fluorescence intensity (A) and SDS-PAGE analysis (B) of

526

GFP expressed by pNX01 was monitored in recombinant B. amyloliquefaciens NB at

527

various time intervals.

528

Figure 3. Effect of different replicons on plasmid stability in B. amyloliquefaciens NB.

529

Figure 4. Comparison of biomass (A), weight-average molecular weight (B), γ-PGA

530

hydrolase activity (C), and γ-PGA production (D) in B. amyloliquefaciens NB by

531

over-expressing different γ-PGA hydrolases.

532

Figure 5. Production of low-molecular-weight γ-PGA in a 7.5-L fermenter. γ-PGA

533

weight-average molecular weight (Mw), γ-PGA hydrolase activity, γ-PGA production,

534

dissolved oxygen (DO), and biomass were measured at regular intervals.

26


Page 26 of 37

Page 27 of 37


TABLES Table 1. Strains and plasmids used in this study. Strains or plasmids

Relevant properties

Source

B. amyloliquefaciens NX-2S

wild-type strain, 2Sip+

This lab

B. amyloliquefaciens NB

cured 2Sip plasmid NB strain

This lab

B. subtilis NX-2

The strain with γ-PGA hydrolase enzyme

This lab

B. licheniformis 14580


This lab

B. megaterium M


This lab

Strains

E. coli DH5α

E. coli GM2163

F−,φ80dlacZΔM1, Δ(lacZYA-argF) U169, deoR, recA1, endA1, hsdR17(rk−, mk+), phoA, supE44, λ−thi-1, gyrA96, relA1 F−, ara-14 leuB6 thi-1 fhuA31 lacY1 tsx-78 galK2 galT22 supE44 hisG4 rpsL 136 (StrR) xyl-5 mtl-1 dam13::Tn9 (CamR) dcm-6 mcrB1 hsdR2 mcrA

This lab

This lab

B.subtilis 168

The strain with P43 promoter

This lab

NB (pNX01-gfp)

B. amyloliquefaciens NB with pNX01-gfp plasmid

This study

NB (pNX01)

B. amyloliquefaciens NB with pNX01 plasmid

This study

NB (pNX01-pHT01)

B. amyloliquefaciens NB with pNX01-pHT01plasmid

This study

NB (pNX01-pHY300PLK)

B. amyloliquefaciens NB with pNX01-pHY300PLK plasmid

This study

NB (pNX01-pgdS1)

B. amyloliquefaciens NB with pNX01-pgdS1 plasmid

This study

NB (pNX01-pgdS2)


This study

NB (pNX01-pgdS3)


This study

NB (pNX01-pgdS4)


This study

plasmids

27



Page 28 of 37

pHY300PLK

E. coli and B. subtilis shuttle vector; AmpR, TetR

TaKaRa, Dalian, China

pHT01

E. coli and B. subtilis shuttle vector; AmpR, CmR

MoBiTec, Goettingen, Germany

EGFP-pBAD

Source of egfp, 6xHis, AmpR

Addgene, Cambridge, MA

p2Sip

The endogenous plasmid of B. amyloliquefaciens NX-2S

This lab

pNX01

Replication origin (p15A ori) + AmpR (gram-negative) +

This study

replication origin (pAMa1) + CmR (gram-positive) + Promoter P43

(B.subtilis

168)

+

Terminator

TamyL

(B.

amyloliquefaciens NB) pNX01-gfp

pNX01 carrying gfp gene

This study

pNX01-pHT01

pNX01 carrying replicon of pHT01

This study

pNX01-pHY300PLK

pNX01 carrying replicon of pHY300PLK

This study

pNX01-pgdS1

pNX01 carrying pgdS gene (B. subtilis NX-2)

This study

pNX01-pgdS2

pNX01 carrying pgdS gene (B. amyloliquefaciens NB)

This study

pNX01-pgdS3

pNX01 carrying pgdS gene (B. licheniformis 14580)

This study

pNX01-pgdS4

pNX01 carrying pgdS gene (B. megaterium M)

This study

28


Page 29 of 37


Table 2. Primers and their sequences used for PCR in this study. Primers

sequences(5’-3’)

P01

TTCGCCTTGGAAAAATAATCTAGATTTCCATAGGCTCCGCCC

P02

TAAAAAAAAAAGAACCCTCACGCGGAACCCCTATTTGTTT

P03

AAACAAATAGGGGTTCCGCGTGAGGGTTCTTTTTTTTTTA

P04

AATAAAAGACCACATTAAAACTATTTGATTATGTAATTCT

P05

AGAATTACATAATCAAATAGTTTTAATGTGGTCTTTTATT

P06

ACCGTATGTTCAATGGCTCCCGGGTTTTGCATTCTACAAACT

P07

AGTTTGTAGAATGCAAAACCCGGGAGCCATTGAACATACGGT

P08

GCGGCCGCAGATCTGTCGACGAATTCACTAGTGTGTACATTCCTCTCTTACC

P09

ACTAGTGAATTCGTCGACAGATCTGCGGCCGCGGTAATAAAAAAACACCTCC

P10

GGGCGGAGCCTATGGAAATCTAGATTATTTTTCCAAGGCGAA

pNX01-gfp-F

TAAGAGAGGAATGTACACACTAGTATGGTGAGCAAGGGCGAGG

pNX01-gfp-R

AGGTGTTTTTTTATTACCGCGGCCGCTTACTTGTACAGCTCGTCC

pNX01-pHT01-F

AAACAAATAGGGGTTCCGCGATATTAGGAGCATTGAATAT

pNX01-pHT01-R

AATAAAAGACCACATTAAAATTATTGCACTTTTCTTAG

pNX01-pHY300PL

AAACAAATAGGGGTTCCGCGCTTAAGGAACGTACAGACGC

K-F pNX01-pHY300PL

AATAAAAGACCACATTAAAACTACTCTTTAATAAAATAAT

K-R pNX01-SPamyL-F

GGTAAGAGAGGAATGTACAACTAGTATGATTCAAAAACGAAAG

pNX01-SPamyL-R

GGCTGATGTTTTTGTAATCGGC

29



pNX01-pgdS1-F

GCCGATTACAAAAACATCAGCCGAGATTGCGGAAGCTGAT

pNX01-pgdS1-R

TGGAGGTGTTTTTTTATTACCGCGGCCGCTTATTGCACCCGTATACT

pNX01-pgdS2-F

GCCGATTACAAAAACATCAGCCACGGAAATTGCTGAAGCG

pNX01-pgdS2-R

TGGAGGTGTTTTTTTATTACCGCGGCCGCTTACGGGAGCCGGATGCT

pNX01-pgdS3-F

GCCGATTACAAAAACATCAGCCGATACGATCGGCGAGAAA

pNX01-pgdS3-R

TGGAGGTGTTTTTTTATTACCGCGGCCGCCTACTTAATTCTGACGCT

pNX01-pgdS4-F

GCCGATTACAAAAACATCAGCCGCTTTTCCTGCAGAAAAA

pNX01-pgdS4-R

TGGAGGTGTTTTTTTATTACCGCGGCCGCTTATGGCATGCGCTTTGC

30


Page 30 of 37

Page 31 of 37


Table 3. Analysis of the resistance of B. amyloliquaficiens NB to different antibiotics. Time

LB

LB+Tetracycline

LB+Chloramphenicol

LB+Kanamycin

5 ug/mL

10 ug/mL

5 ug/mL

10 ug/mL

5 ug/mL

10 ug/mL

12 h

+

-

-

-

-

-

-

24 h

+

+

-

-

-

-

-

36 h

+

+

+

-

-

-

-

48 h

+

+

+

-

-

+

-

+ represented the growth of bacteria, - represented the none-growth of bacteria.

31



Figure graphics Figure 1.

32


Page 32 of 37

Page 33 of 37


Figure 2.

33



Figure 3.

34


Page 34 of 37

Page 35 of 37


Figure 4.

35



Figure 5.

36


Page 36 of 37

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FOR TABLE OF CONTENTS ONLY

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Efficient Biosynthesis of Low-Molecular-Weight ... - ACS Publications

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