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Adaptive Evolution Relieves Nitrogen Catabolite Repression and Decreases Urea Accumulation in Cultures of the Chinese Rice Wine Yeast Strain Saccharomyces cerevisiae XZ-11 Weiping Zhang, Yan Cheng, Yudong Li, Guocheng Du, Guangfa Xie, Huijun Zou, Jingwen Zhou, and Jian Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01313 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018
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Adaptive Evolution Relieves Nitrogen Catabolite Repression and Decreases Urea
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Accumulation in Cultures of the Chinese Rice Wine Yeast Strain Saccharomyces
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cerevisiae XZ-11
4 5
Weiping Zhang†,‡,#, Yan Cheng†, Yudong Li§, Guocheng Du†, Guangfa Xie¶, Huijun
6
Zouǁ, Jingwen Zhou*,†,‡,#, and Jian Chen*,†,#
7 8
†
9
Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China;
Key Laboratory of Industrial Biotechnology, Ministry of Education and School of
10
‡
11
University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China
12
§
13
Zhejiang Gongshang University, Hangzhou 310018, China
14
¶
15
China
16
#
17
Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
18
ǁ
19
Zhejiang, China.
20
*
21
Jian Chen, Jingwen Zhou
22
Mailing address: School of Biotechnology, Jiangnan University, 1800 Lihu Road,
23
Wuxi, Jiangsu 214122, China.
24
Phone: +86-510-85918312, Fax: +86-510-85918309
25
E-mail:
[email protected],
[email protected].
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan
Department of Bioengineering, School of Food Sciences and Biotechnology,
College of Shaoxing Rice Wine, Zhejiang Shuren University, Shaoxing 312028,
Jiangsu Provisional Research Center for Bioactive Product Processing Technology,
Zhejiang Guyuelongshan Shaoxing Wine Company, 13 Yangjiang Road, Shaoxing,
Corresponding authors.
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ABSTRACT
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Urea is the major precursor of ethyl carbamate in Chinese rice wine. Although efforts
28
have been made to decrease urea accumulation, few methods can be applied to the
29
industrial food production due to potential safety concerns. In this study, adaptive
30
laboratory evolution (ALE) followed by high-throughput screening was used to
31
identify low urea-accumulating strains derived from the industrial Chinese rice wine
32
yeast strain Saccharomyces cerevisiae XZ-11. Three evolved strains were obtained
33
that had 47.9%, 16.6%, and 12.4% lower urea concentrations than the wild-type strain.
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Comparative genomics analysis revealed that genes involved in carbon and nitrogen
35
metabolism evolved quickly. Transcription levels of genes involved in urea
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metabolism were dramatically upregulated after ALE. This work describes a novel
37
and safe strategy to improve nitrogen utilization of industrial yeast strains involved in
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food fermentation. The identified genomic variations may also help direct rational
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genetic engineering of nitrogen metabolism processes to achieve other goals.
40 41
Keywords: Adaptive laboratory evolution; Comparative genomics; High-throughput
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screening; RT-qPCR; Saccharomyces cerevisiae; Urea
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INTRODUCTION
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Chinese rice wine is an important traditional alcoholic beverage in China. It has
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been widely consumed for the past 5,000 years because of its unique flavor and high
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nutritional value.1-2 However, harmful components have been detected in Chinese rice
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wine, of which ethyl carbamate (EC) is the most common.3 EC, also known as
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urethane, is naturally formed in fermented foods and has been detected in many kinds
50
of alcoholic beverages.3 In 2007, the carcinogen classification of EC was upgraded
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from Group 2B to Group 2A by the International Agency for Research on Cancer
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(IARC).4-5 EC is formed by the spontaneous reaction between urea and ethanol and
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the accumulation of urea is one of the major causes of overaccumulation of EC in
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Chinese rice wine.3 The average concentration of EC in Chinese rice wine was
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reported to be 160 µg/kg.6 However, the maximum level of EC in sake, which is
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another kind of rice wine similar with Chinese rice wine, is limited to 100 µg/kg in
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America. Although there has yet no limitation about the maximum level of EC in
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Chinese rice wine, decreasing the concentration of EC is urgent to improve the quality
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of Chinese rice wine. The key point of reducing EC in Chinese rice wine is how to
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reduce the accumulation of urea during fermentation process.
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Urea is considered a non-preferred nitrogen source for yeast.7 When preferred
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nitrogen sources such as glutamate and glutamine are present, the utilization of
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non-preferred nitrogen sources is repressed through a process known as nitrogen
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catabolite repression (NCR).7-10 In Saccharomyces cerevisiae, NCR is regulated by
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four global transcription factors (TFs), including the two positive TFs, Gln3 and Gat1,
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and the two negative TFs, Gzf3 and Dal80.11-12 In the presence of preferred nitrogen
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sources, Gln3 and Gat1 are phosphorylated by the target of rapamycin complex 1
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(TORC1) and sequestered in the cytoplasm with the help of the repressor, Ure2.13 3 ACS Paragon Plus Environment
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However, under conditions of nitrogen starvation, Gln3 and Gat1 can be translocated
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from the cytoplasm into the nucleus, which facilities their interaction with promoter
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regions of non-preferred nitrogen utilization genes to activate their expression.10, 14-15
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In addition to NCR, nitrogen metabolism is also regulated by carbon sources.
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α-ketoglutarate, an intermediate of carbon metabolism, can be converted into
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glutamate and glutamine. Thus, it is the major amide group donor in amino acid
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biosynthesis and links carbon and nitrogen metabolisms.12, 16
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A series of efforts have been made to reduce NCR to decrease the accumulation
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of urea in Chinese rice wine,3 including remodeling the regulatory and metabolic
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pathways of nitrogen metabolism.7, 10, 17-20 However, genetic modification methods
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such as these are not suitable for the optimization of strains used in the food industry
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due to public safety concerns related to genetically modified organisms (GMOs).
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Adaptive laboratory evolution (ALE), which does not require genetic modification,
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has been developed as a powerful tool to domesticate strains and obtain desired
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characteristics.21 During ALE, cells are cultured under conditions that promote the
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accumulation of beneficial mutations that allow the cells to adapt to the selection
85
pressure. ALE has been applied to many microbes,21 including Escherichia coli22-26
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and S. cerevisiae,27-31 to gain desirable characteristics such as increased growth rates
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and resistance to environmental stresses. Moreover, with the rapid development of
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massively parallel DNA sequencing technologies, the cost of resequencing microbial
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genomes has decreased dramatically, facilitating the identification of causal mutations
90
and the investigation of genotype-phenotype relationships.24-26, 30-31
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In this study, the industrial Chinese rice wine strain S. cerevisiae XZ-11 was
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propagated for several hundred generations in parallel serial cultures in which urea
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was the sole nitrogen source to improve its ability to utilize urea. Genome 4 ACS Paragon Plus Environment
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resequencing and comparative genomics analysis revealed genetic variants to account
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for the low urea accumulation in cultures of these evolved strains. The strains
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screened in this study could be used during the fermentation process to improve the
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quality of Chinese rice wines. Additionally, the results of genomic analysis could
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serve as a guide for genetic modification of strains in other non-food industries.
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MATERIALS AND METHODS
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Strains, media, and culture conditions
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The S. cerevisiae diploid strain XZ-11 used in this study was an industrial
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Chinese rice wine strain provided by the Guyuelongshan Shaoxing Wine Company
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(Shaoxing, China).
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Media for seed culture (YPD) contained 20 g/L glucose, 20 g/L peptone, and 10
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g/L yeast extract; 20 g/L agar was added when necessary. Nitrogen-free media
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contained 20 g/L glucose and 1.74 g/L yeast nitrogen base (YNB) without amino acids
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or ammonium ion. Media for ALE contained nitrogen-free media supplemented with
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0.03, 0.3, or 1.5 g/L urea as required. Prescreening media contained nitrogen-free
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media supplemented with 5 g/L urea. First-round screening media contained
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nitrogen-free media supplemented with 5 g/L ammonium sulfate, 5 g/L glutamate, 5
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g/L glutamine, and 5 g/L urea. Second-round screening media contained YPD media
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supplemented with 0.6 g/L urea. Urea detection media contained nitrogen-free media
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supplemented with 5 g/L urea. Sporulation media contained 20 g/L potassium acetate
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supplemented with 20 g/L agar. All strains were cultured at 30°C with shaking at 220
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rpm. Urea concentration measurements were performed after culture for 54 h. For
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spore dissection, strains were firstly cultured in the YPD medium at 30°C for 24 h.
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Then cells were collected by centrifugation at 3000 g for 5 min and washed with 5 ACS Paragon Plus Environment
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sterile water twice. Next, cells were incubated on the sporulation plate at 30°C for 96
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h.
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Adaptive laboratory evolution
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Primary ALE experiments were started from wild-type S. cerevisiae XZ-11
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frozen stocks after overnight activation on YPD plates. A single colony was then
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inoculated into a 250 mL flask containing 20 mL YPD medium and cultured for 24 h.
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Three 20 µL aliquots were transferred into three 14 mL test tubes containing 2 mL
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nitrogen-free media supplemented with 0.03, 0.3, or 1.5 g/L urea for ALE. A total of
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20 µL of culture was serially passaged after culture for 2 days (1% of the total culture
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volume was transferred to the subsequent culture).
130 131
Urea detection by high-performance liquid chromatography
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Urea was detected using a high-performance liquid chromatography32 system
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(Agilent 1260, Agilent Biotechnologies, Santa Clara, CA, USA) equipped with a
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ZORBAX Eclipse XDB C18 column (250×4.6 mm, 5 µm; Agilent Biotechnologies).
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Twenty microliters samples were injected with the mobile phase at 1 mL/min at 35°C.
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The mobile phase gradient is shown in Table 1. Urea was detected using a
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fluorescence detector with excitation wavelength of 213 nm and an emission
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wavelength of 308 nm.
139 140
The high-throughput urea detection method
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In order to determine urea concentrations using high-throughput screening, a
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method from previous studies was optimized.33-34 Briefly, 80 µL of culture
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supernatant was collected and mixed with 100 µL iron phosphate solution (6 g/L), 20
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µL mixture of diacetyl monoxime (6 g/L) and thiamine (0.3 g/L) in 96-well plates.
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Then, the plates were incubated at 100°C for 10 min. The absorbance at 520 nm of
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each well was immediately measured using a microplate spectrophotometer (BioTek
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Instruments, Inc., Winooski, VT, USA).
148 149
Single live cell sorting after evolution using flow cytometry
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Cultures were diluted with saline to 1×105 cells/mL. An equal volume of
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propidium iodide (PI, 8 µmol/L) was then added to the samples, followed by
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incubation at 4°C for 20 min. Cells were gated on a forward and side scatter plot and
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then classified as either PI-stained or non-stained based on their 617 nm fluorescence
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and side scatter. Non-stained cells were individually sorted into 96-well plates
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containing 700 µL prescreening media in each well. Finally, the 96-wells plates were
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incubated at 30°C for 54 h.
157 158
DNA preparation, genome resequencing, and data analysis
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Genomic DNA was isolated from the adaptive evolved strains 4B, 7H, and 10G
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using a PureLink™ Genomic Plant DNA Purification Kit (Thermo Fisher Scientific,
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Waltham, MA, USA) following the manufacturer’s protocol. Paired-end libraries with
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300 bp inserts were constructed and sequenced using an Illumina Hiseq 2500
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sequencer (Illumina, San Diego, CA, USA) at Shanghai Biochip Co., Ltd. (Shanghai,
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China). The quality of raw data was monitored using FastQC35 and filtered using
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PRINSEQ.36 The resulting clean data sets were mapped to the genome sequence of
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the wild-type strain XZ-11 using Bowtie2.37 Genomic variants were analyzed using 7 ACS Paragon Plus Environment
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samtools38 and VCFtools.39 HMMcopy was used to analyze chromosome copy
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numbers.40 KaKs_Caculator was applied to measure the gene evolutionary rates of the
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evolved strains based on genetic variation.41
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RNA preparation, cDNA synthesis, and RT-qPCR assays
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After overnight incubation on YPD plates, single colonies were cultured to the
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early log phase in liquid YPD medium for 12 h. Total RNA was extracted from each
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sample using a RiboPure RNA Purification Kit (Thermo Fisher Scientific) following
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the manufacturer’s instructions. The cDNA was synthesized using the PrimeScript™
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RT Reagent Kit with gDNA Eraser (Takara-Bio, Dalian, China). RT-qPCR
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experiments were performed on a LightCycler 480 system (Roche Applied Science,
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Mannheim, Germany) using a SYBR® Premix Ex Taq™ Kit (Takara-Bio). Primers
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used in this assay are listed in Table S1. ACT1 was selected as the housekeeping gene
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and fold-change was calculated using the 2-∆∆Ct method. Biological triplicates for each
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sample were analyzed.
182 183
Simulation of Chinese rice wine fermentation
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Simulations of Chinese rice wine fermentation performed in this study were
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similar to those described in previous research.42 First, 0.75 kg glutinous rice was
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soaked for 48 h in 0.9 L water at 28°C. Next, the glutinous rice was washed and
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steamed for 20 min. Then, 1.5 mL yeast cultured overnight was mixed with the
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steamed rice and incubated at 28°C. Forty-eight hours later, 75 g Chinese koji
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(provided by the Guyuelongshan Shaoxing Wine Company) and 0.9 L water were
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added and incubated at 28°C for 2–3 days to complete the mashing process. The
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temperature was then lowered to 18°C for post-fermentation for 16–20 days. The
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concentration of EC in the stimulated Chinese rice wine was determined by gas
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chromatography with mass spectrometry (GS/MS) as described by Ryu et al..43
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RESULTS
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Acquisition of low urea-accumulating strains by adaptive laboratory evolution
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S. cerevisiae XZ-11 was subjected to continuous cultivation under selective
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culture conditions in which urea was the sole nitrogen source presented at one of three
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different concentrations. During the ALE process, a constant passage size (1% of the
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total culture volume) was used. Log phase cultures were transferred to tubes
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containing fresh media every 48 h. Cultures were sampled and subjected to batch
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fermentation every 60 transfers. As mutations accrued and gained dominance within
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the separate populations, the amount of residual urea decreased (Figure 1). Compared
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to the initial culture, the percentage of urea residue in cultures containing 0.03, 0.3,
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and 1.5 g/L urea as sole nitrogen source decreased by 30.1%, 34.0%, and 34.7%,
206
respectively, after 360 rounds of transfer. These results show that stronger selective
207
pressure led to faster adaptation to culture condition.
208 209
High-throughput screening and characteristics of low urea-accumulating strains
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Using flow cytometry sorting, a total of 4,721 single colonies were isolated from
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the three end-point evolved populations. Results of high-throughput screening after
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culture in first-round screening media showed that the amount of residual urea in the
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cultures of most evolved strains was significantly lower than that in cultures of the
214
wild-type strain (Figure 2a). Preliminary screening revealed nine strains with low urea
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concentrations that were then subjected to further fermentation. After 54 h of batch 9 ACS Paragon Plus Environment
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fermentation using YPD medium, the urea concentrations in cultures of four of the
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nine selected strains were reduced by 5.1%−53.9% (Figure 2b). Further fermentation
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analysis of the three strains with the lowest urea concentration, named 10G, 7H, and
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4B, in medium containing 0.6 g/L urea as the sole nitrogen source revealed that their
220
urea consumption capabilities were almost doubled and their growth rates were much
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faster than the wild-type strain XZ-11 (Figure 2c and d). Compared to the wild-type
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strain, the residual urea concentration in cultures of 10G, 7H, and 4B were 37.4%,
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39.9%, and 43.9% lower, respectively.
224
Beneficial mutations gained from ALE were likely located on only one copy of
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the chromosome and could be lost during budding dissection. Therefore, the three
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evolved strains were subjected to serial spore dissection. Urea consumption of the
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homozygotes of each budding generation was found to be stable after six generations
228
(Table 2), suggesting that beneficial mutations may spread evenly across different
229
chromosomes. In addition, in simulated Chinese rice wine fermentation, the final urea
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concentrations in cultures of the evolved strains 4B, 7H, and 10G were 47.9%, 16.6%,
231
and 12.4% lower than that of the wild-type strain, respectively (Figure 2e). Moreover,
232
the volume percent of ethanol, one of the most important indicators of the quality of
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Chinese rice wine, was not significantly changed (Figure 2f). The EC concentrations
234
in the cultures of the evolved strains 4B, 7H, and 10G were 40.5%, 28.7%, and 18.6%
235
lower than that of the wild-type strain, respectively (Figure 2g).
236 237
Adaptive laboratory evolution triggers multiple genomic variants
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Genome resequencing was performed to analyze the genome of strains as they
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adapted to the urea media. After mapping sequence reads of evolved strains to the
240
genome of wild-type strain XZ-11, many genetic variants were detected (Table 3 and 10 ACS Paragon Plus Environment
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Table S2). The number of detected genetic variants in the genome of strain 4B (388)
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was much lower than that in the genomes of strains 7H (19,169) and 10G (19,312).
243
Fifty-five unique variants corresponding to changes in the coding sequences of 19
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genes were identified in the genome of strain 4B (Figure S1, Table S2). Functional
245
annotation of these 19 genes revealed that most were involved in transportation of
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hexoses, including fructose, glucose, and mannose. The percentages of non-exonic
247
variants in the genomes of strains 4B, 7H, and 10G were approximately 76.8%, 40.9%,
248
and 41.1%, respectively. Considering that non-coding sequences account for only
249
25% of the S. cerevisiae genome, genomic variants appeared to be enriched in
250
non-coding regions, suggesting they were not randomly distributed.
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The ratio of the number of non-synonymous substitutions per non-synonymous
252
site (Ka) to the number of synonymous substitutions per synonymous site (Ks), Ka/Ks,
253
is used to estimate the evolutionary rate of genes under selection.44 We identified 15,
254
718, and 831 fast-evolving genes (Ka/Ks value > 1) in strains 4B, 7H, and 10G,
255
respectively. Some of these genes are involved in the amino acid metabolism pathway,
256
including AAT2, which is involved in converting α-ketoglutarate to glutamate,45 and
257
PRO1, which is involved in converting glutamate to ornithine and entering into the
258
urea cycle. ARG2, another gene involved in the generation of ornithine from
259
glutamate,46 was identified as a fast-evolving gene in strains 4B and 7H but not in
260
strain 10G. The duplicated chromosomal regions in the genomes of the evolved strains
261
are shown in Figure 3. In general, these were evenly distributed throughout the
262
genomes of the three evolved strains. Remarkably, a large region (approximately 38.5
263
kb) in chromosome XII was found to undergo massive amplification with a final copy
264
number of approximately 10 (Figure 3). Twenty-two annotated genes are located in
265
this region (Table S3), including IDP2, which encodes the isocitrate dehydrogenase 11 ACS Paragon Plus Environment
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responsible for converting oxaloacetate into α-ketoglutarate.
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Adaptive laboratory evolution leads to upregulation of genes related to urea
269
utilization
270
The transcriptional level of genes related to urea utilization, including genes
271
involved in urea metabolism and regulatory pathways (Figure 4), were examined after
272
culture in YPD media. In the three evolved strains, these genes were significantly
273
upregulated compared to the wild-type strain (Figure 5). Genes directly involved in
274
the metabolism of urea, such as DUR1,2, DUR3, and CAR1, were upregulated by
275
81.2-, 8.1-, and 24.8-fold, respectively, in strain 4B compared to the wild-type strain.
276
Furthermore, genes encoding the positive regulators such as GLN3 and GAT1 were
277
upregulated to a greater extent than those encoding the negative regulators such as
278
GZF3, DAL80, and URE2. These results may suggest that beneficial mutations in
279
GLN3 and GAT1 coding regions contributed to the derepression of NCR. In addition,
280
an obvious correlation between the expression levels of urea metabolism genes and
281
the urea consumption ability of strains was observed (Figure 5). Generally,
282
upregulation of genes was highest in strain 4B and lowest in strain 10G (Figure 5).
283 284
DISCUSSION
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In this study, a novel strategy integrating ALE and high-throughput screening
286
was established to identify Chinese rice yeast strains that accumulate low urea
287
concentrations during fermentation. Three adaptive evolved strains, named 4B, 7H,
288
and 10G, were selected using this strategy. Compared to the wild-type strain XZ-11,
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the residual concentrations of urea in cultures of 4B, 7H, and 10G were reduced by
290
47.9%, 16.6%, and 12.4%, respectively. Meanwhile, the fermentation performances of 12 ACS Paragon Plus Environment
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these strains remained similar to that of strain XZ-11, while the EC concentrations in
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cultures of evolved strains 4B, 7H, and 10G were reduced by 40.5%, 28.7%, and
293
18.6% than that in culture of wild-type strain. NCR regulators and genes involved in
294
urea catabolism were significantly upregulated in low urea-accumulating strains.
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Genome resequencing and comparative genomic analysis revealed that many variants
296
accumulated in the genomes of evolved strains during ALE. Genes involved in linking
297
carbon metabolism and the urea cycle pathway, including ATT1, PRO1, and ARG2,
298
evolved more quickly.
299
Urea is considered to be the major precursor of EC in Chinese rice wine.32
300
Previously, the reduction of urea in alcoholic beverages was achieved mainly by
301
genetically modifying fermentation strains. For example, promoting urea
302
degradation by enhancing the expression of DUR1,2 and blocking urea formation by
303
deleting CAR1 decreased urea accumulation by 75.6% and 86.9%, respectively.17, 19
304
Similarly, stabilizing the urea permease Dur3 by mutating its potential ubiquitination
305
sites decreased urea accumulation by 29.7%.47 Furthermore, eliminating the
306
repression of urea utilization by mutating potential phosphorylation sites and
307
truncating the localization regulation signal of the NCR regulator Gln3 reduced urea
308
concentrations by 63%.10 Although these methods greatly reduce urea concentrations,
309
the potential safety risks associated with GMOs limit their application in industrial
310
processes. In contrast, strains screened after ALE exhibit much lower urea
311
accumulation than the wild-type strain, are free from genetic modification, and are
312
safe for use in fermented beverage production. Moreover, adaptive evolved strains
313
exhibit similar fermentation properties to the wild-type strain in simulations of
314
Chinese rice wine fermentation.
315
In the past decades, ALE has been applied to many microbes to obtain desired 13 ACS Paragon Plus Environment
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characteristics without genetic modification.21 During ALE, cells are cultured under
317
significant selection pressure, which promotes the enrichment of beneficial
318
mutations and allows for the cells to adapt to the selection pressure. However, a
319
simple high-throughput screening method is essential to isolate adaptive evolved
320
strains with desired characteristics. Based on our previous work, we implemented a
321
flow cytometry-based sorting method to isolate single colonies from large evolved
322
populations.34 This is a much simpler and more convenient method than the
323
traditional dilution separation method, which requires single colonies to be picked up,
324
which is laborious. Moreover, urea concentrations can be rapidly and conveniently
325
determined by measuring the absorbance of each well at 520 nm. By combining flow
326
cytometry sorting with a convenient urea detection method, nine evolved strains with
327
low urea accumulation levels were easily selected from 4,721 candidate colonies in
328
this study.
329
The utilization of urea is controlled by the systematic regulation of nitrogen
330
metabolism. Many regulatory pathways have been found to be involved in nitrogen
331
metabolism, including the SPS-sensor system,48 the TOR pathway,49 the general
332
amino acid pathway,50-51 and NCR.7, 10, 52 In this study, the transcription levels of urea
333
cycle genes and regulators of the NCR and TOR pathways, including CAR1, were
334
found to be upregulated, suggesting that, overall, transcriptional activity was activated
335
after ALE. However, greater activation of DUR1,2 compared to CAR1 may lead to
336
overall reductions in urea concentrations after ALE. In addition, the upregulation of
337
IDP2 and the fast-evolving genes ATT2, PRO1, and ARG2 were confirmed by
338
comparative genomics analysis. These genes are involved in the metabolic pathway
339
by which α-ketoglutarate is converted to glutamate and finally directed to ornithine.
340
Importantly,
α-ketoglutarate
is essential for linking nitrogen and carbon 14
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metabolism.53-54 These results suggested that the remodeling of carbon metabolism
342
plays an important role in reducing urea accumulation during ALE.
343
Three Chinese rice wine strains with low urea accumulation properties were
344
selected using ALE, a genetic modification-free approach. However, the level of urea
345
reduction was still relatively low compared to that achieved by genetic modification
346
or urease addition.17-19,
347
concentrations could be performed. With the help of the high-throughput screening
348
method established here, large mutation libraries can be constructed using other
349
efficient approaches in addition to ALE, such as random mutagenesis. Identification
350
of carbon catabolism-related genes by comparative genomic analysis of the adaptive
351
evolved and wild-type strains may provide new approaches for reducing urea
352
accumulation and could provide new clues for coordinating the metabolic flux of
353
carbon and nitrogen sources.
55
Further evolution in the presence of higher urea
354
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ASSOCIATED CONTENT
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Supporting Information
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Table S1, primers used in this study; Table S2, genetic variants in the genome of
359
adaptive evolution improved strains 4B, 7H, and 10G; Table S3, functional annotation
360
of the genes in the massive amplified region in the chromosome XII of evolved strains;
361
and Figure S1, comparative analysis of the variants among three evolved strains.
362 363 364
AUTHOR INFORMATION
365
Corresponding Authors
366
*Phone:
367
[email protected] (Jingwen Zhou),
[email protected] (Jian Chen).
+86-510-85914317,
Fax:
+86-510-85914317.
E-mail:
368 369
Funding
370
This work was supported by The National Key Research and Development Program
371
of China (2017YFC1600403), the National Natural Science Foundation of China
372
(31670095, 31770097, 31671836), the Key Research and Development Program of
373
Jiangsu Province (BE2016689), the Fundamental Research Funds for the Central
374
Universities (JUSRP51701A), the Six Talent Peaks Project in Jiangsu Province
375
(2015-JY-005), the Distinguished Professor Project of Jiangsu Province.
376 377
Notes
378
The authors declare no conflict of competing financial interests.
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555
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556
FIGURE CAPTIONS
557
Figure 1. Urea residue percentages of evolved populations. The percentage of urea
558
in cultures of evolved populations tested every 60 transfers compared to the original
559
strain. Square, circle, and triangle symbols indicate populations that evolved with 0.03,
560
0.3, and 1.5 g/L urea as the sole nitrogen source, respectively. Error bars represent
561
standard deviations from three biological replicates.
562 563
Figure 2. Fermentation profiles of evolved strains. a, The percentage of urea
564
relative to the wild-type (WT) strain XZ-11 of 4,721 single colonies cultured for 54 h
565
after adaptive laboratory evolution in nitrogen-free media containing 5 g/L each
566
glutamine, glutamate, (NH4)2SO4, and urea. b, the concentration of urea in the media
567
of nine evolved strains and the WT strain XZ-11 after culture for 54 h in YPD
568
containing 5 g/L each glutamine, glutamate, (NH4)2SO4, and urea. c, d, The growth
569
and urea concentration curves of the three selected evolved strains and the WT strain
570
after culture in nitrogen-free media containing 5 g/L urea. e, f, g, Final concentrations
571
of urea, ethanol and EC of the three selected evolved strains and the WT strain after
572
Chinese rice wine fermentation simulations. Error bars represent standard deviations
573
from three biological replicates.
574 575
25 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
576
Figure 3. Chromosomal copy number variants in the genomes of evolved strains.
577
Three adaptive evolved strains, 4B, 7H, and 10G, were selected after adaptive
578
laboratory evolution. Chromosome copy numbers were analyzed based on sequencing
579
data using HMMcopy. Individual chromosomes, indicated by Roman numerals, are
580
separated by dashed lines.
581 582
Figure 4. The metabolic and regulatory pathways of urea. Urea is mainly
583
generated from the catabolism of arginine in the urea cycle. The product of DUR1,2
584
mediates the complete degradation of urea to CO2. DUR3 encodes the permease
585
responsible for urea import and export. The activation of these urea metabolism genes
586
is dependent on the positive regulators of nitrogen catabolite repression (NCR), Gln3
587
and Gat1. However, when nitrogen sources are replete, Gln3 and Gat1 are sequestered
588
in the cytoplasm, which represses expression of genes involved in urea metabolism.
589
The TOR pathway triggers phosphorylation of Gln3 and Gat1, blocking their
590
translocation from the cytoplasm into the nucleus.
591 592 593
Figure 5. Transcriptional analysis of genes involved in urea metabolism and
594
regulatory pathways. The three evolved strains, 4B, 7H, and 10G, were cultured in
595
YPD media supplemented with 5 g/L urea for 12 h. The expression levels of the genes
596
involved in urea metabolism and regulatory pathways were measured by RT-qPCR
597
using the wild-type strain XZ-11 as the control. Data were normalized to the
598
expression level of the ACT1 gene. Error bars represent standard deviations of three
599
biological replicates. Black, dark gray, and light gray bars indicate the 7H, 4B, and
600
10G strains, respectively. 26 ACS Paragon Plus Environment
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TABLES Table 1. Mobile phase gradients for urea detection
a
Time (min)
A%a
B%b
C%c
0.00
80
20
0
0.06
80
20
0
12.06
50
50
0
12.80
0
50
50
15.60
0
100
0
21.60
0
100
0
23.80
0
20
80
24.50
80
20
0
30.00
80
20
0
31.00
80
20
0
20 mM sodium acetate; bacetonitrile; cH2O.
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Table 2. Genetic stability of adaptive evolved strains
Strains
Residual urea (mg/L) 1st
2nd
3rd
4th
5th
6th
Generation
Generation
Generation
Generation
Generation
Generation
WT
15.48 ± 0.16
15.2 ± 0.12
15.6 ± 0.11
15.3 ± 0.15
15.5 ± 0.08
15.4 ± 0.08
4B
9.5 ± 0.15
9.6 ± 0.15
9.5 ± 0.17
9.6 ± 0.24
9.7 ± 0.16
9.6 ± 0.19
7H
10.7 ± 0.06
10.6 ± 0.10
10.6 ± 0.11
10.5 ± 0.07
10.5 ± 0.10
10.6 ± 0.07
10G
10.4 ± 0.07
10.6 ± 0.10
10.7 ± 0.15
10.7 ± 0.16
10.6 ± 0.10
10.8 ± 0.09
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Table 3. Genetic variations identified in adaptive evolved strains
4B
7H
10G
Total
Hom a
Het b
Total
Hom
Het
Total
Hom
Het
90
39
51
11324
10628
696
11373
10683
690
Deletion
3
3
0
110
98
12
115
107
8
Insertion
20
18
2
170
138
32
175
143
32
MNV c
0
0
0
198
131
67
196
138
58
Replacement
1
1
0
9
6
3
11
8
3
SNV d
66
17
49
10837
10255
582
10876
10287
589
298
195
103
7845
7809
36
7939
7912
27
Exonic
Non-Exonic a
Hom, homozygous; bHet, heterozygous; cMNV, multi-nucleotide variant; dSNV,
single-nucleotide variant.
29 ACS Paragon Plus Environment
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FIGURE GRAPHICS Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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The TOC graphic
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