Endophytic bacteria as contributors to theanine production in Camellia

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Biotechnology and Biological Transformations

Endophytic bacteria as contributors to theanine production in Camellia sinensis Jun Sun, Manman Chang, Haijing Li, Zhaoliang Zhang, Qi Chen, Ying Chen, Yu Yao, An Pan, Chengying Shi, Chunling Wang, Jian Zhao, and Xiaochun Wan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03946 • Publication Date (Web): 03 Sep 2019 Downloaded from pubs.acs.org on September 4, 2019

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Journal of Agricultural and Food Chemistry

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Endophytic bacteria as contributors to theanine production in Camellia sinensis

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Jun Sun#1,2,Manman Chang#1, Haijing Li1, Zhaoliang Zhang1, Qi Chen1, Ying Chen1,

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Yu Yao2, An Pan1, Chengying Shi1, Chunling Wang1, Jian Zhao1, Xiaochun Wan*1

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1State

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University, 130 West Changjiang Road, Hefei City 230036, Anhui Province, P. R.

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China

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2College

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Hefei City 230036, Anhui Province, P. R. China

Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural

of Horticulture, Anhui Agricultural University, 130 West Changjiang Road,

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# These authors contributed equally to this work.

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* Corresponding author:

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Xiaochun Wan

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Anhui Agricultural University,

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130 West Changjiang Road, Hefei City 230036, Anhui Province, P. R. China

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Tel: 86-0551-85786468

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E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract：Theanine is the most abundant non-protein amino acid in Camellia sinensis,

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but it is not known how tea plant accumulates such high levels of theanine. The

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endophyte isolated from in vitro grown plantlets of C. sinensis cultivars were identified

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as Luteibacter spp., showing strong biocatalytic activity for converting both glutamine

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and ethylamine to theanine. The theanine was secreted outside of the bacterial.

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Endophyte isolated from in vitro plantlets of Camellia oleifera cultivar were identified

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as Bacillus safensis, not converting glutamine and ethylamine to theanine. Enzymatic

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assays in vitro indicated that γ-glutamyl transpeptidases rCsEGGTs from the endophyte

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Luteibacter strains converted glutamine and ethylamine into theanine at higher rates

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than rCsGGTs from C. sinensis. This is the first report on theanine biosynthesis by an

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endophyte from C. sinensis, which provide a new pathway to explore the mechanism

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of theanine biosynthesis in C. sinensis and the interactions between endophyte and tea

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plants.

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Keywords: Camellia sinensis; Endophytic bacteria; Luteibacter sp.; Theanine; γ-

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glutamyltranspeptidase; Glutamine synthetase; Theanine synthetase.

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1

Introduction

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Tea is the second most popular beverage worldwide, savored for its unique flavor and

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aroma. The health benefits of tea are due to several special metabolites produced by the

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tea plant Camellia sinensis (L.) O. Kuntze. Theanine (γ-glutamyl ethylamine) is one of

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these special metabolites. Theanine accumulates in tea plants but is rarely produced in

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other plants, and if it does occur, it is at low levels. Theanine is an important indicator

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in the qualitative assessment of green tea because it confers the particular umami taste

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to brewed tea.1 Consumers of tea benefit from the biological functions of theanine,

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which include improvement of memory and learning by activating relative central

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neurotransmitters, protection against vascular diseases, and promotion of relaxation and

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concentration by inhibiting the effects of caffeine.2 Theanine also has neuroprotective

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and antiobesityfunctions and enhances antitumor activities.3 Moreover, theanine

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performs important physiological functions in plants. Theanine is a unique non-protein

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amino acid and accounts for more than 70% of the total free amino acids in the shoots

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of C. sinensis.4 Similar to glutamine (Gln), theanine plays important roles in the

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nitrogen cycle, especially in ammonia detoxification via the conversion of toxic

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ammonia into a nontoxic amino acid form.5 As theanine has important functions in both

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humans and plants, there is great interest in determining its distribution, accumulation,

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and biosynthesis in tea. Theanine is present in a variety of Camellia species, and it is

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highly abundant in C. sinensis, the species used to produce tea.5 Theanine was also

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detected in several plants in the Order Ericales, a plant in the Order Aquifoliales, and

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in the edible mushroom Xerocomusbadius.6 In C. sinensis, theanine is biosynthesized 3



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by theanine synthetase (TS), which is very similar to glutamine synthetase (GS) in DNA

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sequence and enzymatic activity.5, 7 TS is the only enzyme known to produce theanine

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in plants and, thus far, has only been found in C. sinensis.8 In free-living bacteria,

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theanine is biosynthesized by GGT from Bacillus subtilis9 and GS from the strain

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Pseudomonas taetrolens Y-30.10 The transpeptidation reaction of GGT is one of the

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most effective methods to synthesize theanine, since the enzyme source is readily

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available and can be cultured and since the reaction is fast and does not require ATP.9-

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10

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until unknown why highly abundant theanine only occurs in C. sinensis plants.

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In our previous study, we found that the in vitro-grown tea plantlets could secret yellow

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endophytic bacteria into medium, and the presence of the endophyte does not cause any

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apparent symptoms of tea plantlets in vitro.11 Many endophytes do produce a number

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of important, bioactive secondary metabolites. Endophytes can provide these

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metabolites to their hosts, thereby contributing to the performance, growth and stress

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tolerance of plants.12 Some metabolites might be produced by both the endophyte and

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the host.13 The right endophyte/host pair may be able to produce higher levels of a

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natural secondary metabolite when working together. It has been reported that many

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endophytic bacteria colonize C. sinensis14 and free-living bacteria are able to produce

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theanine.15 However, there are no relevant studies on the production of theanine by

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endophytes isolated from plants, including tea trees.

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In the present study, we successfully isolated endophytic bacteria that are able to

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produce theanine from the tea cultivars ‘Shuchazao’, ‘Wuniuzao’, ‘Longjing No.43’,

While the GGT and GS enzymes were found in both bacterial and plant species, it is

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and ‘Yunkang No.10’. The endophytic bacteria were identified as members of the genus

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Luteibacter (Order Xanthomonadales, Family Rhodanobacteraceae) based on 16S

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rDNA gene sequences.16 Analysis of the enzymatic activities of CsGGTs and CsTS1,

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isolated from C. sinensis, and CsEGGTs and CsEGSs, isolated from the endophytes,

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showed that the CsEGGTs were much more active than the CsGGTs. Our results

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showed that endophytic bacteria could convert both Gln and ethylamine to theanine,

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and could also convert Gln and other amino acids to theanine without ethylamine. The

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results of this study help to explain why high theanine levels only occur in C. sinensis

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plants. The results also provide a novel insight into the mechanism of theanine

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biosynthesis and may be useful for breeding and quality improvement of C. sinensis.

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2

Materials and Methods

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2.1 Plant materials

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Leaves of tea (C. sinensis) cultivar ‘Shuchazao’ were harvested from the Dechang tea

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plantation (Shucheng, Anhui, China). Plantlets of the tea cultivars ‘Shuchazao’ from

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Anhui Province, ‘Wuniuzao’ and ‘Longjing No.43’ from Zhejiang Province, ‘Yunkang

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No.10’ from Yunnan Province and C. oleifera ‘Changlin No.4’ from Anhui Province

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were grown in vitro.17

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2.2 Isolation and identification of endophytic bacteria from C. sinensis and C.

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oleifera

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Shoots of C. sinensis ‘Shuchazao’, ‘Wuniuzao’, ‘Longjing No.43’, ‘Yunkang No.10’

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and C. oleifera ‘Changlin No.4’ were used to establish plantlets in vitro.17 From these 5



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sterile-grown plantlets, leaves of ‘Shuchazao’, ‘Wuniuzao’, ‘Longjing No.43’,

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‘Yunkang No.10’ and ‘Changlin No.4’ were removed, cut into 5 mm × 10 mm segments,

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placed on Luria–Bertani (LB) medium, and cultured at 28°C. After the emergence of

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bacteria colonies (2-10 days), the mixed bacteria were transferred onto fresh LB

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medium,18 and labeled ‘CsE-SCZm’, ‘CsE-WNZm’, ‘CsE-LJm’, ‘CsE-YKm’ and

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‘CoE-CLm’ for the cultivar from which they were recovered.

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Identification of the endophytic bacteria from C. sinensis and C. oleifera was done on

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the basis of cell morphology, gram stain19 and subsequent 16S rDNA gene sequence

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similarity. Total DNA was extracted from pure bacterial colonies using Bacteria

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Genomic DNA Kit (CWBIO, Beijing, China) and was used as the template for PCR. A

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portion of the 16S rDNA gene was amplified by PCR using the primers 16S-PA and

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16S-PB (Table S1).20 The PCR products of the expected size (about 1,500 bp) were

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purified using a DNA Gel Extraction Kit, cloned into the pEASY-Blunt Zero vector,

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and sequenced (BGI, Shanghai, China). The phylogenetic tree was generated by the

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neighbor-joining method within the MEGA 7.0 program using 1,000 bootstrap

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replications.21

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2.3 Detection methods of theanine products by Ultra Performance Liquid

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Chromatography (UPLC)-QQQ-MS/MS

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The Ultra Performance Liquid Chromatography (UPLC)-MS/MS system (Palo Alto,

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CA, USA) was used to detect theanine products in this study. A Thermo Fisher

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Accucore RP-MS column (particle size of 2.6 µm, 100 mm in length, and internal

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diameter of 2.1 mm) was used at a flow rate of 0.3 mL min-1. The method has been 6


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improved based on previous reports.22

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The column oven temperature was set at 30℃. The mobile phase consisted of 0.1%

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acetic acid in water and 100% methyl alcohol, the equivalent elution was carried out

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with 90% methanol and 10% acetic acid in water and was held for at 10 minutes. Mass

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spectra were acquired simultaneously using electrospray ionization in the positive

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ionization mode over the range of m/z 50 to 500.7, 23

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2.4 Theanine production of endophytic bacteria isolated from in vitro-grown tea

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plantlets

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The endophytic bacteria ‘CsE-SCZm’, ‘CsE-WNZm’, ‘CsE-LJm’, and ‘CsE-YKm’

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from C. sinensis and ‘CoE-CLm’ from C. oleifera were cultured to the logarithmic

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growth phase in LB medium for inoculation onto fresh LB medium at a ratio of 1:1000.

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These cultures were grown at 28℃ and 220 rpm. Once cultures reached an OD600 of

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1.0, substrates were added to a final concentration of 20 mM Gln and 20 mM

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ethylamine hydrochloride. Controls were bacterial cultures without substrates (Gln and

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ethylamine hydrochloride) and LB medium without bacteria but with 20 mM Gln and

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20 mM ethylamine hydrochloride. Cultures were incubated for 7 d. The bacterial

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solutions were subjected to thermal cracking in a 100°C water bath for 30 min,

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centrifugated at 10000 ×g for 15 min, the supernatants were filtered through a 0.22 µm

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membrane, then to identify the product of theanine by UPLC-MS/MS.

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2.5 Theanine synthesis and secretion in monoclones of endophytic bacteria

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Ten monoclones of endophytic bacteria isolated from ‘Shuchazao’ were randomly

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picked, numbered CsE1 to CsE10, and subcultured to the logarithmic growth phase. 7



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The bacterial cultures were inoculated to fresh LB medium at a ratio of 1:1000 and

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cultured again at 28℃ and 220 rpm. Once cultures reached an OD600 of 1.0, substrates

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were added to the final concentration of 20 mM Gln and 20 mM ethylamine

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hydrochloride. Cultures in LB without the substrates and uninoculated LB medium with

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20 mM Gln and 20 mM ethylamine hydrochloride were used as controls. Samples were

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removed from the cultures on days 1, 3, 5, 7, 10, and 15, prepared as above and

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subjected to UPLC-MS/MS for detection of theanine. The supernatant of bacterial

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cultures with 20 mM Gln and 20 mM ethylamine hydrochloride at 5 d and 7 d were

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used to detect the secretion of theanine from the endophytic bacteria.

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2.6 Plant RNA extraction and cDNA synthesis

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Total RNA was prepared from tea leaves harvested from ‘Shuchazao’ using RNAprep

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Pure Plant Kit (TIANGEN, Beijing, China) according to the manufacturer’s

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instructions. cDNA, synthesized by reverse transcription from the total RNA using

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PrimeScript RT Master Mix (Takara, Dalian, China), was used as the template for RT-

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PCR cloning of CsGGT2, CsGGT4 and CsTS1 using primers listed in Table S1.

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2.7 Cloning of CsGGTs, CsTS1, CsEGGTs and CsEGSs genes

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Primers CsGGT2-pMAL-SalⅠ-F/R and CsGGT4-pMAL-SalⅠ-F/R were used to

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amplify CsGGT genes from cDNA of the tea cultivar ‘Shuchazao’. CsTS1 was cloned

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using primers CsTS1-pMAL-BamHⅠ-F/R. Primers CsEGGT-pMAL-SalⅠ-F/R and

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CsEGS-pMAL-BamHⅠ-F/R were used to amplify CsEGGT and CsEGS from strain

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CsE7 and CsE8 isolates from ‘Shuchazao’, respectively (Table S1).

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2.8 Heterologous expression in Escherichia coli and recombinant protein 8


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purification

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The full-length ORFs of the CsGGTs and CsTS1 from C. sinensis and the CsEGGTs

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and CsEGSs from endophytic bacteria were subcloned into the expression vector

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pMAL-c5x, which contained an maltose binding protein (MBP) tag of size 42.5 kDa.

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The recombinant plasmids were transferred into E. coli BL21 (DE3) (Transgen, Beijing,

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China). After the sequence of target genes were confirmed, the E. coli BL21 strain

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transformed with the target genes were cultured at 37°C in the LB medium containing

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100 µg/mL ampicillin and grown to an OD600 of 0.6-0.8. Isopropyl-β-D-

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thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM was added to the cell

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culture, which was cultured at 16°C for another 24 h to induce the expression of

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recombinant proteins.7,

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chromatography using amylase resin (New England Biolabs, MA, USA) and detected

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by SDS-PAGE gel analysis.21b,

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activity assays in vitro.

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2.9 Enzymatic assays

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The products of the enzymatic reactions were detected by UPLC-MS/MS to verify the

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function of the recombinant proteins rCsGGTs, rCsEGGTs, rCsTS1 and rCsEGSs. The

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activity of GGT was determined by the method of Orlowski and Meister with slight

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modification.26 The basic reaction buffer contained 20 mM Tris-HCl (pH 7.4), 20 mM

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NaCl, 1 mM EDTA, and 1 mM DTT. Reaction mixtures of 1mL contained 100 µL of

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recombinant protein (50 µg purified recombinant protein rCsGGTs or rCsEGGTs) and

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900 µL basic reaction buffer with 20 mM Gln and 50 mM ethylamine hydrochlorideas

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The recombinant proteins were purified by affinity

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The purified recombinant proteins were used in

9



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substrate. The reaction mixtures were incubated at 37°C for 10 h and terminated by

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heating at 96°C for 2 min.27 The reactions were repeated in triplicate. For the reaction

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of recombinant proteins rCsTS1 and rCsEGS, in addition to the 100 µL of recombinant

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protein (50 µg purified recombinant protein rCsTS1 or rCsEGSs), 900 µL basic reaction

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buffer with 20 mM Glu and 50 mM ethylamine hydrochloride, 10 µM ATP was added

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as an energy source for the GS enzyme. The reaction mixtures were incubated at 30°C

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for 10 h and terminated by heating at 96°C for 2 min.28 The reactions were repeated in

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triplicate. Reactions with protein extracts from cultures carrying the pMAL-c5x vector

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were used as controls.

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2.10 Precursors of theanine synthesis in endophytic bacteria

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Colonies of strain CsE7, isolated from ‘Shuchazao’, were randomly selected and

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cultured to an OD600 of 1.0. This culture was diluted into fresh LB and cultured with 20

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mM Gln and 20 mM of anyone of 18 different amino acids, namely Gln (double supply

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of Gln), Glycine (Gly), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile),

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Phenylalanine (Phe), Asparagine (Asn), Glutamic acid (Glu), Lysine (Lys),

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Methionine (Met), Serine (Ser), Threonine (Thr), Proline (Pro), Histidine (His),

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Arginine (Arg), γ-aminobutyric acid (GABA), or Citrulline (Cit). Negative controls

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were (1) cultures in LB without Gln or an additional amino acid and (2) a set of flasks

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containing LB medium with 20 mM Gln and 20 mM of one of the 18 amino acids but

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no bacteria. The positive control was bacteria cultured with 20 mM Gln and 20 mM

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ethylamine hydrochloride. The cultures were grown for 7 d before isolation and

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detection of theanine by UPLC-MS/MS. 10


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2.11 Statistical analysis

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Data collected in this study was subjected to analysis of variance expressed as means ±

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standard deviations and statistically analysed using Duncan’s tests. All experiments

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were measured in thrice (3 replicates). One-way analysis of variance (ANOVA) and

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multiple comparisons with Latin square design (LSD) were conducted to test the

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differences between groups using SPSS 16.0 software. A p-value ≤0.01 was considered

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to be statistically significant.

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3

Results

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3.1 Isolation and identification of endophytic bacteria from C. sinensis and C.

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oleifera

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In order to understand the influence of producing areas on endophytic bacteria in C.

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sinensis and the difference between C. sinensis and C. oleifera, shoots of C. sinensis

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‘Shuchazao’ from Anhui Province, ‘Wuniuzao’ and ‘Longjing No.43’ from Zhejiang

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Province, ‘Yunkang No.10’ from Yunnan Province and C. oleifera ‘Changlin No.4’

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from Anhui Province were used to establish plantlets in vitro (Figure 1a, b, c, d, e).

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Leaves from the in vitro-grown plantlets were cut into segments and inserted into LB

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medium. After 2 to 3 days, yellow bacteria began to appear on the medium in contact

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with the four C. sinensis cultivars (Figure 1f, g, h, i). The endophytic bacteria were

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subcultured onto fresh LB medium. After 2 to 3 days on LB agar the colonies were 1.0

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to 2.0 mm in diameter, yellow-pigmented, smooth, circular, and convex (Figure 1k).

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The endophytic bacteria secreted by the C. sinensis cultivars were Gram-negative 11



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bacteria (Figure 1l).29 The endophytic bacteria from C. oleifera were secreted into the

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medium after 5 to 7 days (Figure 1j). The colonies were milky yellow, opaque, round

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or nearly round, dull, and with uneven edges and a protrusion in the center (Figure 1m).

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The endophytic bacteria secreted by C. oleifera ‘Changlin No.4’ were Gram-positive

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bacteria (Figure 1n).30

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Part of the 16S rDNA sequences of ten monoclones of the endophytic bacteria isolated

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from the four C. sinensis cultivars and the one C. oleifera cultivar were determined and

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compared with available 16S rDNA sequences. The partial 16S rDNA sequences of the

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endophytic bacteria strains from the C. sinensis cultivars showed high homology (99%

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identity) with Luteibacter sp. (GenBank accession no. KM187159). The 16S rDNA

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sequence from the endophyte isolated from C. oleifera shared 100% identity with

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Bacillus safensis (GenBank accession no. CP032830) (Figure 1o). The colony

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morphology, Gram staining and phylogenetic analyses based on 16S rDNA gene

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sequences indicated that the endophytes from C. sinensis ‘Shuchazao’, ‘Wuniuzao’,

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‘Longjing No.43’, ‘Yunkang No.10’ are belong to Luteibacter species and the

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endophytes from C. oleifera ‘Changlin No.4’ are B. safensis.

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3.2 Endophytic bacteria from C. sinensis produce theanine

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In order to understand the role of endophytic bacteria on the theanine production of C.

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sinensis and C. oleifera, seven-day-old cultures of the mixed endophytes CsE-SCZm,

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CsE-WNZm, CsE-LJm, CsE-YKm and CoE-CLm subcultured in LB medium

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containing Gln and ethylamine hydrochloride were extracted for detection of theanine

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by UPLC-MS/MS, respectively. The results showed that the endophytic bacteria 12


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isolated from the four C. sinensis cultivars could produce theanine when supplied with

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the substrates. Among them, CsE-WNZm produced a relatively high content of

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theanine, of 6.45 mg/L, followed by CsE-SCZm (4.87 mg/L), CsE-LJm (4.18 mg/L),

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and CsE-YKm (3.73 mg/L). Cultures lacking the substrates produced little theanine.

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No theanine was detected after seven days of culture of the endophyte CoE-CLm from

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C. oleifera in the presence or absence of Gln and ethylamine hydrochloride (Figure 2).

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3.3 Production and secretion of theanine by the endophytic Luteibacter

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To further study the theanine-producing abilities of the isolated endophytes, ten

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monoclones of the bacteria isolated from C. sinensis ‘Shuchazao’ were cultured in LB

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for 7 days containing Gln and ethylamine hydrochloride. The results showed that all

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ten monoclones from C. sinensis ‘Shuchazao’ had the ability to synthesize theanine,

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and there are no significant difference among them. The yield of theanine was 3.06

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mg/L - 4.23 mg/L (Figure 3a).

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In order to learn theanine production and secretion by the endophytic Luteibacter sp.,

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endophyte strain CsE7 from C. sinensis ‘Shuchazao’ was randomly selected and used

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to study a time course of theanine production (Figure3b). Liquid cultures of strain CsE7

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were incubated in LB medium containing Gln and ethylamine hydrochloride for 1, 3,

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5, 7, 10, and 15 days and were used to detect theanine. At 5 and 7 days, the theanine

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produced by strain CsE7 and secreted to the medium was detected and compared with

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the total yield of theanine in the disrupted bacteria solution. With the proper

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supplements, strain CsE7 rapidly produced theanine, yielding a maximum of 3.83 mg/L

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at 7 d of cultivation, before levels reduced to about 1.65 mg/L at 15 d of cultivation. 13



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About 90% of the produced theanine was excreted into the culture broth (Figure 3c).

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No theanine was detected in bacteria-free LB medium containing the substrates during

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cultivation. However, theanine was detected in the endophyte culture without the

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substrates, but the theanine content was highest, at 0.6 mg/L, on the first day and then

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decreased over the cultivation period (Figure 3b).

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3.4 Polymorphism analysis of CsGGTs, CsTS1, CsEGGT and CsEGS

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In the present study, two CsGGT genes from C. sinensis were obtained and identified

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as homologous to AtGGT2 and AtGGT4 of Arabidopsis, and numbered CsGGT2 (1722

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bp) and CsGGT4 (1887 bp), respectively. CsTS1 was obtained with a length of 2532

293

bp.8 CsE7GGT, CsE7GS, CsE8GGT, and CsE8GS were obtained from the endophyte

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strains CsE7 and CsE8 with the lengths of 1743 bp, 1410 bp, 1746 bp and 1044 bp,

295

respectively. Two phylogenetic trees were constructed along with previously identified

296

GGTs and GSs with known functions, respectively. Phylogenetic analysis resulted in

297

the formation of four GGT clusters, clusters I to Ⅳ (Figure 4a). Cluster I consisted of

298

GGTs from prokaryotic organisms; cluster II consisted of GGTs from animals; and

299

clusters III and Ⅳ consisted of GGTs from eukaryotic plants. CsE7GGT and CsE8GGT,

300

from the endophyte strains CsE7 and CsE8, were placed into cluster I with GGTs from

301

other prokaryotes, including EcGGT ( P18956) from E. coli K12 and BsGGT (P54422)

302

from B. subtilis 168, which had the ability to convert Gln and ethylamine to theanine.

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CsGGT2 and CsGGT4 were placed into cluster III and Ⅳ.

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For the GSs, the phylogenetic analysis based on the amino acid sequences resulted in

305

five clusters (Figure 4b). Cluster I consisted of GSs from prokaryotic organisms28a and 14


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some GSs from eukaryotic plants. Cluster II consisted of GSs from animals. The plant

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GSs were divided into three distinct clusters, III, Ⅳ and Ⅴ. Previous studies viewed

308

CsGS1 as the best TS gene candidate, and it has been renamed CsTS1, through its high

309

homology with PtGS from the bacterium P. taetrolens.8, 10 Our results showed that

310

CsTS1, from C. sinensis, was placed into cluster I with CsE7GS and CsE8GS, from the

311

endophyte from C. sinensis, and GSs from other prokaryotes.

312

Multiple sequence alignments of the CsGGT and CsEGGT homologs revealed that the

313

functional motifs were conserved, including the catalytic threonine (Thr-391 in

314

EcGGT), as the nucleophile responsible for both the autoprocessing and the enzymatic

315

activity, and the two Gly residues (Gly483–Gly484 in EcGT) proposed to have a role

316

in the stabilization of the tetrahedral transition state of the enzyme (Figure S1).31 The

317

multiple sequence alignment of CsTS1, CsE7GS and CsE8GS homologs was further

318

optimized to provide the maximum conservation of 15 key active-site residues

319

identified from the crystal structure of the S. typhimurium (SALTY) GSI (Figure S2).28b,

320

32

321

3.5 CsEGGTs from entophytic Luteibacter converted Gln and ethylamine into

322

theanine at higher rates than CsGGTs from C. sinensis

323

To compare the enzymatic synthesis of theanine with CsGGTs and CsTS1 from C.

324

sinensis and CsEGGTs and CsEGSs from entophyte Luteibacter, the recombinant

325

proteins rCsGGT2, rCsGGT4, rCsTS1, rCsE7GGT, rCsE8GGT, rCsE7GS and

326

rCsE8GS were purified by affinity chromatography using amylose resin. The molecular

327

weights of the purified rCsGGT2, rCsGGT4, rCsTS1, rCsE7GGT, rCsE8GGT, 15



328

rCsE7GS and rCsE8GS proteins were approximately 104.3 kDa, 110.5 kDa, 136.0 kDa,

329

104.3 kDa, 104.4 kDa, 94.5 kDa and 90.0 kDa, respectively, which were basically

330

consistent with their predicted molecular weights in addition to that of MBP (42.5 kDa)

331

(Figure S3).

332

A series of enzymatic activity assays were conducted on the GGTs and GSs in vitro,

333

with the products identified using UPLC-MS/MS. The relative catalytic activities of

334

rCsGGT2, rCsGGT4, rCsE7GGT and rCsE8GGT proteins were determined by

335

comparing the yield of theanine at the same unit of protein under the same in vitro

336

reaction conditions. The specific activity of the purified recombinant proteins rCsGGT2,

337

rCsGGT4, rCsE7GGT and rCsE8GGT were 1.36 µkat/μg, 0.94 µkat/μg, 431.86

338

µkat/μg, and 299.17 µkat/μg, respectively (Figure 4c). In this enzymatic assay, the

339

theanine-producing ability of rCsE7GGT, from the endophyte CsE7, was 318 times

340

higher than that of rCsGGT2 and 459 times higher than that of rCsGGT4, both from C.

341

sinensis. Theanine production of rCsE8GGT, from the endophyte CsE8, was 220 and

342

318 times higher than that of rCsGGT2 and rCsGGT4, from C. sinensis, respectively.

343

The results showed that both recombinant proteins from the endophytes, rCsE7GGT

344

and rCsE8GGT, have significant abilities to convert Gln and ethylamine to theanine in

345

vitro.

346

The relative catalytic activities of recombinant proteins rCsTS1, rCsE7GS and

347

rCsE8GS were determined in vitro. The specific activity of the purified recombinant

348

proteins rCsTS1, rCsE7GS and rCsE8GS were 0.80 µkat/μg, 0.33 µkat/μg, and 0.49

349

µkat/μg, respectively (Figure 4d). The results showed that the recombinant proteins, 16


Page 16 of 35

Page 17 of 35


350

rCsTS1 from C. sinensis and rCsE7GS and rCsE8GS from the endophytes, have the

351

ability to convert glutamic acid (Glu) and ethylamine to theanine in vitro.

352

3.6 Lys could be closer than other amino acids to theanine in biosynthetic pathway

353

in endophyte Luteibacter

354

The above results showed that the endophytic bacteria strain CsE7, isolated from C.

355

sinensis ‘Shuchazao’, had the ability to synthesize theanine in LB medium without the

356

substrates Gln or ethylamine. In order to explore the metabolic pathway of theanine in

357

the endophytic bacteria, Gln and anyone of 18 amino acids, namely Gln, Gly, Ala, Va,

358

Leu, Ile, Phe, Asn, Glu, Lys, Met, Ser, Thr, Pro, His, Arg, GABA or Cit, were provided

359

to the strain CsE7 as substrates. Cultures grown for 7 days were used to detect theanine

360

by UPLC-MS/MS. The yield of theanine by CsE7 ranged from 6.18 to 31.87 µg/L when

361

these different amino acids were used as substrates (Table S2). The highest yield of

362

theanine occurred when Lys was used as a substrate (31.87 µg/L) compared to 9.67

363

µg/L when Ala was used. The yield of theanine by CsE7 was 3.74µg/L in LB medium

364

when no other substance was added. No theanine was detected in bacteria-free LB

365

medium containing Gln and anyone of the 18 amino acids. The results showed that the

366

strain CsE7 could convert Gln and anyone of 18 different amino acids into theanine and

367

that the conversion efficiency of Lys to theanine was higher than the conversion

368

efficiencies of other amino acids, which suggests that Lys could be closer than other

369

amino acids to theanine in biosynthetic pathway in endophyte Luteibacter.

370 371

4

Discussion 17



372

Theanine is an important secondary metabolite in C. sinensis, as it affects tea leaf

373

quality while also serving as a reservoir for nitrogen, as a building block for skeletal

374

carbon compounds, and as a molecule in developmental physiology.1 Scientists have

375

sought the mechanisms of theanine biosynthesis. In the present study, theanine was

376

found to be biosynthesized by endophytic bacteria isolated from tea plants from the

377

substrates Gln and ethylamine and from Gln and other amino acids. The theanine

378

biosynthesized by endophytic bacteria was secreted out of the bacterial cells (Figure 5).

379

It has been reported that theanine is synthesized from Glu and ethylamine by theanine

380

synthetase (CsTS) in the roots of the tea plants, from where it is then translocated to the

381

apical bud and subtending three leaves.1 In the present study, endophytic bacteria in the

382

genus Luteibacter were identified by 16S rDNA sequences as isolates from in vitro-

383

grown tea plantlets derived from axillary buds of four different C. sinensis cultivars

384

from different Provinces, namely ‘Shuchazao’ from Anhui Province, ‘Wuniuzao’ and

385

‘Longjing No.43’ from Zhejiang Province, and ‘Yunkang No.10’ from Yunnan

386

Province. Further, these endophytes were shown to produce and secrete theanine.

387

Theanine-production of moderate levels (3.73 mg/L to 6.45 mg/L) could be achieved

388

by these endophytes (CsE-YKm, CsE-LJm, CsE-SCZm, and CsE-WNZm) after 7 d of

389

incubation without optimization of the bioconversion conditions. However, an

390

endophytic bacteria isolated from in vitro-grown plantlets of C. oleifera cultivar

391

‘Changlin No.4’ from Anhui Province, B. safensis, could not produce theanine. This is

392

the first documentation, to our knowledge, of a contribution of an endophytic

393

Luteibacter sp. to theanine biosynthesis in C. sinensis plants. 18


Page 18 of 35

Page 19 of 35


394

In free-living bacteria, theanine is produced by GS,10 γ-Glutamylmethylamide

395

synthetase,28b and GGT.33 GGT seems to be better at producing theanine since it can

396

use a wide range of substrates as the γ-glutamyl acceptor and since it does not require

397

ATP for its transferase activity.34 However, the above enzymes producing theanine

398

were from free-living bacteria. Our results showed that CsE7GGT and CsE8GGT,

399

isolated from endophytic Luteibacter strains CsE7 and CsE8 isolated from C. sinensis,

400

produce theanine. The enzyme activities of rCsE7GGT and rCsE8GGT from the

401

endophytic Luteibacter strains were much greater than those of rCsGGT2 and

402

rCsGGT4 from tea. This study is the first report of GGTs and GSs producing theanine

403

from an endophytic bacteria, a Luteibacter sp. isolated from C. sinensis and GGTs

404

producing theanine from C. sinensis (Figure 5). Further research is needed to reveal

405

whether there are other genes, from Luteibacter or other endophytes, that can produce

406

theanine while associated with C. sinensis.

407

Ethylamine availability is regarded as the limiting factor for accumulation of theanine

408

in C. sinesis.7 Our results showed that ethylamine could be converted to theanine by the

409

endophytes from C. sinensis. It has been proposed that Ala could be the closest

410

precursor of ethylamine, through enzymatic decarboxylation of Ala in the biosynthetic

411

pathway.25 Recently, the gene CsAlaDC encoding Ala decarboxylase (CsAlaDC) was

412

identified in tea plants and CsAlaDC could catalyze the decarboxylation of Ala.25 These

413

results provide important information about the theanine biosynthesis pathway,

414

however, the results of this study indicate that the conversion efficiency of Gln and Ala

415

to theanine by the endophyte CsE7, isolated from C. sinensis, were not the highest, 19



416

however, the conversion efficiency of Gln and Lys to theanine by the endophyte CsE7

417

was about three times the rate of Gln and Ala, which suggests that there are other

418

metabolite pathways for driving amino acids into theanine and that Ala may not be the

419

closest precursor to theanine in the biosynthetic pathway. It needs to be clarified

420

whether there is another biosynthesis pathway that produces theanine but that does not

421

rely on ethylamine.

422

As the most important abundant non-protein amino acid in tea plants, theanine is

423

involved in ammonia detoxification, via the conversion of toxic ammonia into the

424

nontoxic amino acid form, and serves as an energy source and precursor for the

425

biosynthesis of various bioactive molecules.5 The data here shows that endophytic

426

bacteria such as Luteibacter could play important roles in nitrogen assimilation in C.

427

sinensis. The results reported here merit further research to determine the mechanisms

428

responsible for moderating the endophytic relationship between C. sinensis and

429

Luteibacter and how these two organisms work together to produce theanine in tea.

430

In this study, we isolated the endophytic bacteria from in vitro grown plantlets of C.

431

sinensis cultivars were identified as Luteibacter spp.. The endophyte Luteibacter

432

showed strong biocatalytic activity for converting both glutamine and ethylamine to

433

theanine, which was secreted outside of the bacterial. But the endophytic bacteria

434

isolated from in vitro plantlets of C. oleifera cultivar were identified as Bacillus safensis,

435

not converting glutamine and ethylamine to theanine. The γ-glutamyl transpeptidases

436

rCsEGGTs from the endophytic Luteibacter strains converted glutamine and

437

ethylamine into theanine at higher rates than rCsGGTs from C. sinensis. 20


Page 20 of 35

Page 21 of 35


438 439

Acknowledgements

440

This work was supported by the Ministry of Agriculture of China through the

441

Earmarked Fund for China Agricultural Research System (No. CARS 19), and the

442

National Natural Science Foundation of China (No. 31170283).

443 444

Declaration of Interest Statement

445

The authors declare no conflict of interest.

446 447

Author contributions

448

XC Wan and J Sun conceived and designed the research; MM Chang, HJ Li, Q Chen,

449

Y Chen, Y Yao, A Pan, and CL Wang performed the research and wrote the paper; ZL

450

Zhang, CY Shi, and J Zhao revised the manuscript.

451 452

Supporting informations

453

Figure S1 Multiple sequence alignments of CsGGTs, CsE7GGT and CsE8GGT

454

(marked by light green shadows) homologs revealed the conservation in the functional

455

motifs, including the catalytic threonine (Thr-391 in EcGGT), as the nucleophile

456

responsible for both the auto processing and the enzymatic activity, and the two Gly

457

residues (Gly483–Gly484 in EcGT) proposed to have a role in the stabilization of the

458

tetrahedral transition state of the enzyme were enclosed in red boxes and marked with

459

red asterisks below the alignment. The other residues responsible for substrate binding 21



460

and catalytic activity of the γ-GGTs are highlighted in yellow (T409, N411, D433, S462,

461

and S463 in EcGGT).

462

Figure S2 Multiple sequence alignments of CsTS1, CsE7GS and CsE8GS (marked by

463

light green shadows) homologs; the percentage identity between the sequences at each

464

residue is indicated by the shading: blue-black (100%), dark gray (75%), and light gray

465

(50%). The red asterisks below the alignment indicate the amino acids in the S.

466

typhimurium GS involved in the active site.

467

Figure S3 SDS-PAGE analysis of purified recombinant proteins rCsGGT2, rCsGGT4,

468

rCsE7GGT, rCsE8GGT, rCsTS1, rCsE7GS, rCsE8GS and pMAL-c5x vector protein

469

expressed in E. coli BL21(DE3) culture supernatants. Molecular masses in kDa of

470

protein standards are designated in red letters. rCsGGT4 has two subunits (red ellipse)

471

of about 38 to 45 kDa; rCsEGGT7 and rCsEGGT8 have a large subunit (blue ellipse)

472

of about 90 kDa and a small subunit (green ellipse) of about 20 kDa.

473

Table S1 Primers sequences for PCR used in this study

474

Table S2 Role of amino acids in theanine biosynthesis by the endophytic bacteria

475

strain CsE7

476 477

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478

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614 615

Figures:

616 617

Figure 1 Isolation and identification of endophytic bacteria from C. sinensis and

618

C. oleifera. Tea cultivars ‘Shuchazao’, ‘Wuniuzao’, ‘Longjing No.43’, ‘Yunkang 29



619

No.10’ and oil tea plantlets ‘Changlin No.4’ were grown in vitro (a, b, c, d, e). Leaves

620

of the four C. sinensis cultivars and the one C. oleifera cultivar were cut into 5 mm ×

621

10 mm segments and inserted into LB medium in vitro. After 2 to 3 days, yellow

622

bacteria from C. sinensis (f, g, h, i) and, after 5 to 7 days, milky yellow bacteria from

623

C. oleifera were secreted onto medium (j). Colony morphology of the endophytic

624

bacteria isolated from C. sinensis were 1.0 to 2.0 mm in diameter, yellow-pigmented

625

smooth, circular, and convex (k). Observation of endophytic bacteria from C. oleifera

626

showed that the colony was milky yellow, opaque, round or nearly round, dull, and with

627

uneven edges, and a protrusion in the center (m); Endophytic bacteria from C. sinensis

628

were identified as Gram-negative bacteria (l) and from C. oleifera as Gram-positive

629

bacteria (n) by Gram Staining; Phylogenetic analysis of partial 16S rDNA sequence of

630

isolates from C. sinensis and C. oleifera (o). Numbers in parentheses represent the

631

sequence accession numbers in GenBank. The endophytic bacteria isolated from C.

632

sinensis and C. oleifera were classified as ‘CsE’ and ‘CoE’ (displayed in red and green

633

shadows), and marked with red and blue dots, respectively. Tree building was

634

performed using MEGA 7.0 software with the neighbor-joining method. Bar, 0.020

635

substitutions per nucleotide position. Scale bar, 2 mm in (a-j, and k, m), 20 µm in (l, n).

636

30


Page 30 of 35

Page 31 of 35


637 638

Figure 2 Identification of endophytic Luteibacter sp. from C. sinensis produce

639

theanine. The mixed endophytes CsE-SCZm, CsE-WNZm, CsE-LJm, CsE-YKm and

640

CoE-CLm, were subcultured in LB medium containing 20 mM Gln and 20 mM

641

ethylamine

642

SCZm, CsE-WNZm, CsE-LJm, CsE-YKm and CoE-CLm subcultured in LB medium

643

without substrates (CsE-SCZm/WNZm/LJm/YKm and CoE-CLm), or in LB medium

644

containing

645

SCZm/WNZm/LJm/YKm+ and CoE-CLm+) were collected for detection of theanine

646

by UPLC-MS/MS. LB with substrates was used as control (LB+). Values are means ±

647

SD, n = 3. Asterisks indicate the statistical significance between exogenous and non-

648

exogenous substrates determined by Duncan’s tests (*P < 0.05; **P < 0.01). (b) HPLC

649

chromatogram and MS/MS analyses of the theanine standard (black peak), the

650

endophytic Luteibacter sp. from C. sinensis with substrates (CsE+, red peak), without

651

substrates (CsE, blue peak) are shown, and the mass spectrometric result of the

652

chromatographic peak Indicated by black circle is shown.

hydrochloride, respectively. (a) Seven-day-old cultures of the strains CsE-

20

mM

Gln

and

20

mM

ethylamine

653

31


hydrochloride

(CsE-


Page 32 of 35

654 655

Figure 3 Production and secretion of theanine by monoclones of endophytic

656

Luteibacter sp.. (a) Ten monoclones (CsE1 to CsE10) of endophytic bacteria isolated

657

from C. sinensis ‘Shuchazao’ were cultured in LB containing 20 mM Gln and 20 mM

658

ethylamine hydrochloride (LB+) or in LB without substrates (LB). Cultures after seven

659

days were used to detect theanine by UPLC-MS/MS. Values are means ± SD, n = 3. (b)

660

Time course of theanine production by the endophytic bacteria Luteibacter CsE7

661

isolated from C. sinensis ‘Shuchazao’. Strain CsE7 was incubated in 50 mL liquid LB

662

medium containing 20 mM Gln and 20 mM ethylamine hydrochloride

663

columns) or in LB without substrates (CsE7, gray column) for 1, 3, 5, 7, 10 or 15d. The

664

theanine contents are marked with red (with substrates) or yellow (without substrates)

665

trend lines. Values are means ± SD, n = 3. Asterisks indicate the statistical significance

666

among different incubation time determined by Duncan’s tests(*P < 0.05; **P < 0.01).

667

(c) Secretion of theanine production by endophytic bacteria CsE7 isolated from C.

668

sinensis ‘Shuchazao’. Values are means ± SD, n = 3.

32


(CsE7+, black

Page 33 of 35


669 670

Figure 4 Phylogenetic relationships and the enzymatic activities of GGTs and

671

GSs/TS from C. sinensis and the endophytic Luteibacter. Two unrooted phylogenetic

672

trees were constructed using MEGA 7.0 software with the neighbor-joining method.

673

The phylogenetic tree of the GGTs was classified into four clusters (I,II, III and IV,

674

distinguish by shadows and titles with different colors) (a), while that of the GSs/TS

675

were classified into five clusters (I,II, III, IV and V) (b), based on the GGT and GS

676

amino acid sequences together with available gene sequences from prokaryotes,

677

animals, and other plants, respectively. Red asterisks represent genes from endophytes

678

and blue asterisks represent genes from tea plants. (c) Enzymatic activity of purified

679

recombinant proteins rCsGGT2, rCsGGT4, rCsE7GGT, rCsE8GGT and pMAL-c5x

680

vector protein analyzed by UPLC-MS/MS in vitro. (d) Enzymatic activity of purified

681

recombinant proteins rCsTS1, rCsE7GS, rCsE8GS and pMAL-c5x vector protein

682

analyzed by UPLC-MS/MS in vitro. Values are means ± SD, n = 3. Asterisks indicate 33



683

the statistical significance among different recombinant proteins determined by

684

Duncan’s tests (*P < 0.05; **P < 0.01).

685

686 687

Figure 5 Schematic overview showing that both the tea plant and the endophyte

688

from C. sinensis synthesize theanine. Glu: Glutamate; Gln: Glutamine; CsEGGT: γ-

689

glutamyltranspeptidase of endophyte; CsEGS: glutamine synthetase of endophyte;

690

CsGGT: γ-glutamyltranspeptidase of tea plant; CsTS: glutamine synthetase of tea plant

691

Wei et al. (2018); CsAlaDC, alanine decarboxylase of tea plant Bai et al. (2019).The

692

lines with arrows show the biosynthetic pathways of theanine in tea plants and

693

endophytic Luteibacter sp. from C. sinensis, respectively. Different color lines

694

represent different metabolic pathways. and the thickness of the lines represents the

695

strength of the metabolic function. The figure is based on the information provided in

696

Sharma et al., 2018, with permission.

697 34


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For Table of Contents Only

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Endophytic bacteria as contributors to theanine production in Camellia

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