Adaptive Evolution Relieves Nitrogen Catabolite Repression and

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Food and Beverage Chemistry/Biochemistry

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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Journal of Agricultural and Food Chemistry

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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

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Weiping Zhang†,‡,#, Yan Cheng†, Yudong Li§, Guocheng Du†, Guangfa Xie¶, Huijun

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Zouǁ, Jingwen Zhou*,†,‡,#, and Jian Chen*,†,#

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†

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Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China;

Key Laboratory of Industrial Biotechnology, Ministry of Education and School of

10

‡

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University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China

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§

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Zhejiang Gongshang University, Hangzhou 310018, China

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¶

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China

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#

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Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.

18

ǁ

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Zhejiang, China.

20

*

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Jian Chen, Jingwen Zhou

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Mailing address: School of Biotechnology, Jiangnan University, 1800 Lihu Road,

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Wuxi, Jiangsu 214122, China.

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Phone: +86-510-85918312, Fax: +86-510-85918309

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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

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have been made to decrease urea accumulation, few methods can be applied to the

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industrial food production due to potential safety concerns. In this study, adaptive

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laboratory evolution (ALE) followed by high-throughput screening was used to

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identify low urea-accumulating strains derived from the industrial Chinese rice wine

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yeast strain Saccharomyces cerevisiae XZ-11. Three evolved strains were obtained

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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

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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

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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.

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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

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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

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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

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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).

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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.

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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).

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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.

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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.

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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%,

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respectively, after 360 rounds of transfer. These results show that stronger selective

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pressure led to faster adaptation to culture condition.

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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

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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

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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.

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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

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(Table 2), suggesting that beneficial mutations may spread evenly across different

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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%,

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and 12.4% lower than that of the wild-type strain, respectively (Figure 2e). Moreover,

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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

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in the cultures of the evolved strains 4B, 7H, and 10G were 40.5%, 28.7%, and 18.6%

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lower than that of the wild-type strain, respectively (Figure 2g).

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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

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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).

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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

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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

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variants in the genomes of strains 4B, 7H, and 10G were approximately 76.8%, 40.9%,

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and 41.1%, respectively. Considering that non-coding sequences account for only

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25% of the S. cerevisiae genome, genomic variants appeared to be enriched in

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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

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site (Ka) to the number of synonymous substitutions per synonymous site (Ks), Ka/Ks,

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is used to estimate the evolutionary rate of genes under selection.44 We identified 15,

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718, and 831 fast-evolving genes (Ka/Ks value > 1) in strains 4B, 7H, and 10G,

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respectively. Some of these genes are involved in the amino acid metabolism pathway,

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including AAT2, which is involved in converting α-ketoglutarate to glutamate,45 and

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PRO1, which is involved in converting glutamate to ornithine and entering into the

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urea cycle. ARG2, another gene involved in the generation of ornithine from

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glutamate,46 was identified as a fast-evolving gene in strains 4B and 7H but not in

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strain 10G. The duplicated chromosomal regions in the genomes of the evolved strains

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are shown in Figure 3. In general, these were evenly distributed throughout the

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genomes of the three evolved strains. Remarkably, a large region (approximately 38.5

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kb) in chromosome XII was found to undergo massive amplification with a final copy

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number of approximately 10 (Figure 3). Twenty-two annotated genes are located in

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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

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utilization

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The transcriptional level of genes related to urea utilization, including genes

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involved in urea metabolism and regulatory pathways (Figure 4), were examined after

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culture in YPD media. In the three evolved strains, these genes were significantly

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upregulated compared to the wild-type strain (Figure 5). Genes directly involved in

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the metabolism of urea, such as DUR1,2, DUR3, and CAR1, were upregulated by

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81.2-, 8.1-, and 24.8-fold, respectively, in strain 4B compared to the wild-type strain.

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Furthermore, genes encoding the positive regulators such as GLN3 and GAT1 were

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upregulated to a greater extent than those encoding the negative regulators such as

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GZF3, DAL80, and URE2. These results may suggest that beneficial mutations in

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GLN3 and GAT1 coding regions contributed to the derepression of NCR. In addition,

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an obvious correlation between the expression levels of urea metabolism genes and

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the urea consumption ability of strains was observed (Figure 5). Generally,

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upregulation of genes was highest in strain 4B and lowest in strain 10G (Figure 5).

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DISCUSSION

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In this study, a novel strategy integrating ALE and high-throughput screening

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was established to identify Chinese rice yeast strains that accumulate low urea

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concentrations during fermentation. Three adaptive evolved strains, named 4B, 7H,

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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

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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

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18.6% than that in culture of wild-type strain. NCR regulators and genes involved in

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urea catabolism were significantly upregulated in low urea-accumulating strains.

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Genome resequencing and comparative genomic analysis revealed that many variants

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accumulated in the genomes of evolved strains during ALE. Genes involved in linking

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carbon metabolism and the urea cycle pathway, including ATT1, PRO1, and ARG2,

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evolved more quickly.

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Urea is considered to be the major precursor of EC in Chinese rice wine.32

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Previously, the reduction of urea in alcoholic beverages was achieved mainly by

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genetically modifying fermentation strains. For example, promoting urea

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degradation by enhancing the expression of DUR1,2 and blocking urea formation by

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deleting CAR1 decreased urea accumulation by 75.6% and 86.9%, respectively.17, 19

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Similarly, stabilizing the urea permease Dur3 by mutating its potential ubiquitination

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sites decreased urea accumulation by 29.7%.47 Furthermore, eliminating the

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repression of urea utilization by mutating potential phosphorylation sites and

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truncating the localization regulation signal of the NCR regulator Gln3 reduced urea

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concentrations by 63%.10 Although these methods greatly reduce urea concentrations,

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the potential safety risks associated with GMOs limit their application in industrial

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processes. In contrast, strains screened after ALE exhibit much lower urea

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accumulation than the wild-type strain, are free from genetic modification, and are

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safe for use in fermented beverage production. Moreover, adaptive evolved strains

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exhibit similar fermentation properties to the wild-type strain in simulations of

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Chinese rice wine fermentation.

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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

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significant selection pressure, which promotes the enrichment of beneficial

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mutations and allows for the cells to adapt to the selection pressure. However, a

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simple high-throughput screening method is essential to isolate adaptive evolved

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strains with desired characteristics. Based on our previous work, we implemented a

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flow cytometry-based sorting method to isolate single colonies from large evolved

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populations.34 This is a much simpler and more convenient method than the

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traditional dilution separation method, which requires single colonies to be picked up,

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which is laborious. Moreover, urea concentrations can be rapidly and conveniently

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determined by measuring the absorbance of each well at 520 nm. By combining flow

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cytometry sorting with a convenient urea detection method, nine evolved strains with

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low urea accumulation levels were easily selected from 4,721 candidate colonies in

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this study.

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The utilization of urea is controlled by the systematic regulation of nitrogen

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metabolism. Many regulatory pathways have been found to be involved in nitrogen

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metabolism, including the SPS-sensor system,48 the TOR pathway,49 the general

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amino acid pathway,50-51 and NCR.7, 10, 52 In this study, the transcription levels of urea

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cycle genes and regulators of the NCR and TOR pathways, including CAR1, were

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found to be upregulated, suggesting that, overall, transcriptional activity was activated

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after ALE. However, greater activation of DUR1,2 compared to CAR1 may lead to

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overall reductions in urea concentrations after ALE. In addition, the upregulation of

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IDP2 and the fast-evolving genes ATT2, PRO1, and ARG2 were confirmed by

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comparative genomics analysis. These genes are involved in the metabolic pathway

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by which α-ketoglutarate is converted to glutamate and finally directed to ornithine.

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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

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plays an important role in reducing urea accumulation during ALE.

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Three Chinese rice wine strains with low urea accumulation properties were

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selected using ALE, a genetic modification-free approach. However, the level of urea

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reduction was still relatively low compared to that achieved by genetic modification

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or urease addition.17-19,

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concentrations could be performed. With the help of the high-throughput screening

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method established here, large mutation libraries can be constructed using other

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efficient approaches in addition to ALE, such as random mutagenesis. Identification

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of carbon catabolism-related genes by comparative genomic analysis of the adaptive

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evolved and wild-type strains may provide new approaches for reducing urea

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accumulation and could provide new clues for coordinating the metabolic flux of

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carbon and nitrogen sources.

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Further evolution in the presence of higher urea

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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

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adaptive evolution improved strains 4B, 7H, and 10G; Table S3, functional annotation

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of the genes in the massive amplified region in the chromosome XII of evolved strains;

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and Figure S1, comparative analysis of the variants among three evolved strains.

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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

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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.


Page 17 of 36


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54. Liu, Z.; Butow, R. A., Mitochondrial retrograde signaling. Annu. Rev. Genet. 2006, 40, 159-185. 55. Yang, L. Q.; Wang, S. H.; Tian, Y. P., Purification, properties, and application of a novel acid urease from Enterobacter sp. Appl. Biochem. Biotechnol. 2010, 160 23 ACS Paragon Plus Environment


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(2), 303-313.

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



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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Page 27 of 36


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.



Page 28 of 36

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


Page 29 of 36


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.



FIGURE GRAPHICS Figure 1


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Figure 2




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Figure 3



Figure 4


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Figure 5



The TOC graphic


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Adaptive Evolution Relieves Nitrogen Catabolite Repression and

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