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DNA Damage in Circulating Hematopoietic Progenitor Stem Cells as Promising Biological Sensor of Frailty

Abstract Frailty is an age-related syndrome that exposes individuals to increased vulnerability. Although it is potentially reversible, in most cases it leads to negative outcomes, including mortality. The different methods proposed identify frailty after the onset of clinical manifestations. An early diagnosis might make it possible to manage the frailty progression better. The frailty pathophysiology is still unclear although mechanisms, in particular, those linked to inflammation and immunosenescence, have been investigated. A common feature of several clinical aspects involved in senescent organisms is the increase of oxidative stress, described as one of the major causes of deoxyribonucleic acid (DNA) damage accumulation in aged cells including the adult stem cell compartment. Likely, this accumulation is implicated in frailty status. The oxidative status of our frail, pre-frail, and non-frail population was characterized. In addition, the DNA damage in hematopoietic cells was evidenced by analyzing the peripheral blood mononuclear cell and their T lymphocyte, monocyte, circulating hematopoietic progenitor stem cell (cHPSC) subpopulations. The phosphorylation of C-terminal of histone H2AX at amino acid Ser 139 (γ-H2AX), which occurs at the DNA double-strand break focus, was evaluated. In our frail population, increased oxidative stress and a high level of DNA damage in cHPSC were found. This study may have potential implications because the increment of DNA damage in cHPSC could be suggestive of an organism impairment preceding the evident frailty. In addition, it may open the possibility for attenuation of frailty progression throughout specific drugs acting on preventing DNA damage or removing damaged cells

Frailty is an age-related syndrome, which results from a decline in physiological capacities across several organ systems and exposes the individual to an increased vulnerability toward internal and external stressors (1). It is a potentially reversible condition, but in most cases it progresses toward disability and/or leads to several negative outcomes, including falls and fractures, hospitalizations, iatrogenic complications, and mortality (1)(2)(3). The risk of these ad-verse events can occur independently of the presence of comorbidity (4). Several methods have been proposed to assess frailty in research and in clinical practice, without clear evidence that one method is preferable to the others. Fried et al. (4) developed the phenotype model of frailty that is one of the most widely employed in clinical research. It considers frailty as a biological entity and classifies the individuals as frail when three or more of the following five criteria are present: Unintentional weight loss, exhaustion, slow walking speed, low level of physical activity, and low muscle strength. A classification of pre-frailty is also possible when only one or two of these criteria are present. The pathophysiological mechanisms underlying the frail phenotype are still unclear although mechanisms, in particular, those linked to inflammation and immunosenescence, have been investigated (5,6). However, the complex interactions between the studied molecules and these mechanisms during the aging process are yet to be defined.
A common feature of several clinical aspects involved in senescent organisms is the increase of oxidative stress that is described as one of the major causes of deoxyribonucleic acid (DNA) damage accumulation, well documented in the different tissues of older people (7). Likely, this accumulation might be implicated in frailty status (8) because DNA damage can lead to molecular and cellular alterations (genomic instability, altered gene expression, loss of cell division potential, cell death, and organ dysfunctions) (7,9) that are a good background on which frailty can arise.
The phosphorylation of the C-terminal of histone H2A, variant H2AX, at the highly conserved amino acid Ser 139 (γ-H2AX) is an early response to DNA double-strand breaks (DSBs). γ-H2AX and DSB correlate in a 1:1 manner (10), and transient and persistent γ-H2AX foci can be present in the cells. In the persistent foci, DSB are not repairable and appear to be common in senescent cells and to increase in older subjects (11)(12)(13). γ-H2AX foci detection is one of the most sensitive ways to examine DNA damage (14). In recent years, the role of DNA damage has been investigated on whole blood (15) and lymphocytes (16), evaluating the relationship between the level of persistent γ-H2AX foci and frail status of older patients.
With our study, we wanted to deepen the characterization of DNA damage in hematopoietic cells, analyzing the peripheral blood mononuclear cells (PBMCs) and the T lymphocyte, monocyte, and circulating hematopoietic progenitor stem cell (cHPSC) subpopulations present in PBMC of frail, pre-frail, and non-frail older adults.

Study Population
A group of 85 subjects (40 frail, 13 pre-frail, 32 non-frail) aged 65 years and above were recruited from the Geriatric Unit, San Gerardo Hospital ASST Monza. All of them were outpatients. The exclusion criteria for the recruited subjects were: (a) illiteracy, (b) psychiatric disorders, (c) Parkinson's disease, (d) cognitive function impairment, (e) multiple sclerosis, (f) neurodegenerative diseases, (g) hip fracture or other severe traumas affecting mobility. The identification of frail phenotype in all subjects occurred at recruitment and was based on the presence of five criteria (4): (i) shrinking, which was determined as an unintentional weight loss of 5% or more than usual, over the last year; (ii) self-reported exhaustion, which was evaluated as present or absent by asking the individual: "In the last month, do you have less energy to do the things of daily living you want?"; (iii) slow walking speed, which was defined as present when an individual took more than 7 s to cover a distance of 4 m (17); (iv) low physical activity, which was assessed using Physical Activity Scale for the Elderly (PASE) over 1 week. The PASE score combines information on leisure, household, and occupational activity (18); (v) weakness, which was measured using the dynamometer handgrip strength for three consecutive times on the dominant hand. The criterion is positive if the best value is less than the cutoff value (<27 for males and <16 for females) (19). Participants were considered frail if they fulfilled three or more criteria, pre-frail if they fulfilled one or two criteria, and robust or non-frail if they did not meet any criteria. Forty-six young subjects, aged between 25 and 35 years, without functional disability, acute pathologies in progress or abuse of drugs, alcohol, smoke, and with a healthy lifestyle, were recruited among blood donors as the control group. The study was conducted according to the principles of the Declaration of Helsinki II and approved by the Ethics Committee Brianza (Monza, Italy) code FRA-ARSC on March 22, 2018. The participation in the study was on a voluntary basis, all subjects were fully informed about the aims of the study and about the nature of their participation, and all of them signed the informed consent.

Comprehensive Geriatric Assessment
After recruitment, all 85 older adults underwent a comprehensive geriatric assessment (CGA) including a survey on demographics (age and sex) and life habits (smoking and alcohol consumption), and several tools to evaluate nutritional, cognitive, functional, and somatic health status. Nutritional status was evaluated using the Mini Nutritional Assessment-short form (MNA-sf) (20) and cognitive status using the Mini-Mental State Examination (21). The functional status was assessed with the modified Barthel Index (22), the Lawton and Brody's Instrumental Activity of Daily Living (23), and the Short Physical Performance Battery (17). Lastly, the somatic health status was assessed with the Charlson Comorbidity Index (CCI) (24). Also the number of drugs taken at home was determined.

PBMC Collection, Cell Characterization, DNA Damage Evaluation
Whole blood from frail, pre-frail, non-frail, and young subjects was collected into BD Vacutainer CPT Cell Preparation Tubes with sodium heparin (Becton Dickinson, Franklin Lakes, New Jersey, USA). The isolation of PBMC by Ficoll-Paque (GE Healthcare Bio-Sciences AD, Uppsala, Sweden) density gradient separation was performed according to the manufacturer's instructions. The isolated PBMC were suspended in blocking solution (PBS with 5% fetal bovine serum) at room temperature (RT) for 15 min then treated following a protocol modified by Beaton et al. (25). Briefly, membrane cell staining was performed for 15 min at RT with the following antibodies: APC Mouse Anti-Human CD3 (Clone UCHT1;   For the characterization of cHPSC on fresh whole blood, 700 µL of peripheral blood were lysed using erythrocyte lysis buffer (BioLegend, San Diego, CA, USA). Then the white blood cells underwent membrane cell staining as described above using the following antibodies: APC anti-human Lineage cocktail against CD3/CD14/CD16/CD19/ CD20/CD56 (Lin1) (BioLegend; 1:5); APC/Fire 750 anti-human CD45 (Clone 2D1, BioLegend; 1:20); PE anti-human CD34 (Clone 8G12; BD Pharmingen; 1:100). Flow cytometry analysis was performed on the basis of the gating strategy described in Supplementary Figure 2. A minimum of 2 × 10 5 events were acquired in the PBMC region.

Statistical Analysis
Continuous variables were reported as mean ± standard deviation and as median with first quartile-third quartile. Categorical variables were reported as absolute and relative frequencies. The comparison between groups was performed with the one-way analysis of variance test with Tukey's test correction and with pairwise multiple comparison procedures (Holm-Sidak method), with Wilcoxon-Mann-Whitney test in case of non-Gaussian distribution. When significant, the single pairs (frail, pre-frail, non-frail, and young) were also compared. Significance was for p < .05. Receiver operating characteristic (ROC) curve analysis was performed using one-way analysis (Wilson-Brown method) and the results were reported as a fraction. The GraphPad Prism program has been used (GraphPad Software, Inc., La Jolla, CA, USA).
As expected, age was higher in frail and pre-frail adults in comparison to non-frail older adults. Furthermore, frail and pre-frail subjects had lower scores at the MNA-sf, were more impaired in cognitive and functional status, and had a higher CCI score. Lastly, non-frail subjects took a lower number of drugs when compared with both frail and pre-frail subjects. The distribution of chronic diseases (most of them used to calculate the CCI score) and the classes of drugs taken in the frail, pre-frail, and non-frail groups are described in Supplementary Table 2. Note: Values are expressed as mean ± SD, unless otherwise specified. *Comparison between pre-frail vs. frail. † Comparison between non-frail vs. frail. ‡ Comparison between non-frail vs. pre-frail.

Hematological Data of Study Population
The hematological parameters were obtained on fresh blood of the 85 older and 46 younger adults (Supplementary Table 1). There were no differences in all variables considered, with two exceptions. The hemoglobin levels were lower in frail older adults (11.0 ± 1.6 g/dL) than in pre-frail (12.9 ± 1.3 g/dL), non-frail (14.1 ± 1.0 g/dL), and young (14.7 ± 1.3 g/dL) adults. The lymphocyte counts were lower in frail subjects. In a set of randomly chosen frail (5 samples), nonfrail (4 samples), and young (5 samples) subjects, we analyzed the cHPSC (Lin1-/CD45+/CD34+) present in the gate of mononucleated cells (Supplementary Figure 2). The results showed a percentage of cHPSC in the range of 0.040-0.071 (Supplementary Table 1). In all 85 older adults and 46 young adults (Table 2), we performed the separation of PBMC by Ficoll-Paque sedimentation, with a yield of around 40%. By FACS analysis, using the gating strategy shown in Supplementary Figure 1, we evaluated the PBMC, T lymphocytes as CD3+ cells, monocytes as CD14+ cells, CD3−/CD14− cells considered B-lymphocytes for the majority and cHPSC as CD34+/CD3−/CD14− cells ( Table 2). In the Ficoll-Paque isolated PBMC, the ratio between the T and B lymphocytes was maintained in favor of T lymphocytes. There was a slight reduction of monocytes in the separated PBMC (Table 2) compared with monocytes of the fresh whole blood (Supplementary Table  1), probably due to their typical stickiness. In any case, there were no significant differences in the monocyte counts of the different subject groups but only a trend toward higher counts was found in frail patients. The percentage of the cHPSC CD34+/CD3−/ CD14− subpopulation was around 0.068-0.099 (Table 2). This percentage is close to the one seen in the fresh whole blood, in which the more restricted Lin1-/CD45+/CD34+ phenotype has been evaluated (Supplementary Table 1).
In conclusion, the separation by Ficoll-Paque maintained the reciprocal ratios among the different cell populations in the different groups.

DNA Damage in PBMC and in the Respective Cell Subpopulations
We detected the percentage of cells positive for γ-H2AX foci in the frail, pre-frail, non-frail older adults and young individuals, assessing the DNA damage in the Ficoll-Paque isolated PBMC (Figure 1). We evaluated by FACS the C-terminal phosphorylation of H2AX histone at serine 139 (γ-H2AX), the early event in response of DSB, using the gating strategy shown in Supplementary Figure 1. Our data showed an evident significant increase of cells positive for γ-H2AX in the PBMC, cHPSC, and T cells of the frail patients (Figure 1). The difference between frail and pre-frail groups was significant only in cHPSC and not in PBMC and CD3+ T cells. The percentage of γ-H2AX+ cells in CD14+ monocytes increased in the frail group without a statistical significance compared with all the other groups ( Figure 1). However, this finding on monocytes was also strengthened by the non-significant differences in the number of monocytes in the PBMC of all studied groups, after Ficoll-Paque sedimentation ( Table 2). In monocytes, the DNA damage in pre-frail and non-frail subjects had the same level as the young individuals. The differences of age and gender, already observed among our older adult groups at recruitment, could be potential confounders for the differences observed in DNA damage. For this reason, at the initial evaluation of our data, we analyzed the frail, pre-frail, non-frail people also stratifying them according to their age class and gender. We did not have evidence that these variables had a specific influence on DNA damage, but the number of cases was low in such stratified groups. Therefore, we analyzed the groups of our older adults without Notes: PBMC = peripheral blood mononuclear cell. Samples obtained from 85 older adults divided according to the frail, pre-frail, non-frail status and from a group of 46 young subjects. Values are expressed as mean ± SD and median (first-third quartile). Statistical analysis was not significant among all groups.
*PBMC were analyzed with the non-parametric Wilcoxon-Mann-Whitney test.
additional stratification, even to achieve a more solid statistical evaluation. We also evaluated the mean fluorescence intensity (MFI) assessing the amount of DNA damage foci per cell in each specific cell population of the different subject groups (Figure 2). The quantification of the MFI provided a distinct and additional parameter to the percent of cells positive for DNA damage. In PBMC, CD3+, and CD14+ cells, we observed higher MFI in frail subjects compared with other groups, although nonsignificantly different (Figure 2). Instead, the MFI significantly increased in cHPSCs γ-H2AX+ of frail subjects compared with both pre-frail and non-frail groups and to young individuals (Figure 2).
The ROC curve analysis (Supplementary Figure 3), assessing the percentage of DNA damaged cHPSC in our frail and non-frail subjects, showed that the area under the curve (AUC) was 0.835 (95% C.I. 0.740-0.931). This suggested a good discriminating power of the percentage of DNA damaged cHPSC to potentially discriminate between frail and non-frail subjects. The Youden Index that determines the optimal cutoff value was 0.611. The comparison between frail and pre-frail subjects gave an AUC of 0.780 (95% CI 0.640-0.920) with a Youden Index of 0.548.
These data showed that cHPSC of the frail group had both a higher percentage of cells with DNA damage and an increased MFI, as an indicator of the median number of γ-H2AX foci per cell, compared with the other groups.

Increment of Oxidative Stress in Frail Population
We evaluated the oxidative status in a set of plasma of our subjects to verify whether in the frail subjects an increase of oxidative stress was detectable. We observed significant increments of auto-oxidation of oxysterols 7KC and 7βOHC, markers of oxidative stress (29)(30)(31) in the frail plasma with respect to pre-frail and non-frail plasma ( Figure  3A). Even the oxysterols 5α6αEC, 3β5α6β-3OHC, and 5β6βEC significantly increased in plasma of frail subjects (Supplementary Figure  4). These findings were suggestive for increased oxidative stress in frail compared with pre-frail, non-frail, and young individuals.
To reinforce the hypothesis that the DNA damage found in the hematopoietic cells had an oxidative origin, we also evaluated in a different set of plasma the 8-OH-dG, which is produced by the oxidative damage of DNA (32). We observed a significant increase of 8-OH-dG in frail compared with pre-frail, non-frail, and young subjects ( Figure 3B) as with 7KC and 7βOHC.

Discussion
The daily intake of drugs, malnutrition, together with comorbidity, are significantly represented in our frail patients and these may contribute to the shift to a higher level of their oxidative metabolism. In fact, the oxysterols analysis evidenced increased oxidative stress in frail individuals. We found that the increased oxidative status and the increased 8-OH-dG matched with DNA damage in the hematopoietic cells of frail patients. We observed a trend of an increased percentage of PBMC, CD34+ cHPSC, and CD3+ T lymphocytes with DNA damage in non-frail older adults compared with young controls. This finding was in accordance with the literature (33,34) and can be considered as an event associated with aging. However, our work also showed a significant percentage increment of PBMC, CD34+ cHPSC, and CD3+ T cells with DNA damage in frail in comparison to non-frail individuals. In case of cHPSC, this significant increment was evident even in comparison to the pre-frail group. Instead, the CD14+ monocytes in the frail group showed only a trend to the increment of DNA damage compared with the other older adult groups. It was similarly very interesting that the number of DNA damage foci per cell, evidenced by the MFI, was significantly higher in the CD34+ cHPSC of frail patients compared with prefrail, non-frail, and young subjects. In PBMC, CD3+ T lymphocytes,  and CD14+ monocytes, the MFI value was higher in the frail group but not significantly. It has to be noted that T lymphocytes are considered long-lived and quiescent cells (35) and their high DNA damage level may represent the accumulation of DNA damage foci that occurred over the lifespan. Instead, the monocytes are typically proliferating short-lived cells (36,37) and DNA damage foci occur in these cells that die precociously. Likely, monocytes do not have time to accumulate foci during lifespan, except in the frail period in which the risk for DNA damage increases.
In aging, genomic instability has raised a great and persistent interest and it has also been recognized as a determinant of age-related stem cell decline that accelerates age-related pathologies in several tissues and organs (38,39). It is known that genomic alterations accumulate at a linear rate in human HPSC during life, similarly to adult stem cells of other tissues (33). It is also known that the long-lived CD34+ hematopoietic stem cells accumulate DNA damages, particularly during aging, when they come out of their dormant state by cell-intrinsic mechanisms (40) and by the shift of the basal metabolism to a higher level of oxidative metabolism (41,42). Both, the loss of dormant status and the increment of ROS, may have a direct consequence in the progressive increase of γ-H2AX leading toward frailty status. It has to be mentioned that, in mice, part of the γ-H2AX foci found in aged hematopoietic stem cells can be due to ineffective dephosphorylation rather than persistent DNA damage. In any case, this situation pushes the cells to transcriptional silencing and functional decline (43).
In conclusion, in our frail population, the clinical characteristics (age, malnutrition, and drug intake) and comorbidity (diabetes mellitus, cardiovascular diseases, kidney diseases, and obstructive pulmonary diseases) causing oxidative stress can justify the high level of DNA damage in cHPSC. In addition, the oxidative DNA damage probably affects several other adult stem cells and the DNA damage, as a process interconnected with other biological processes, may induce an amplification of dangerous effects. Therefore, our findings suggest that, in the tissues of older adults, the occurrence of DNA damage accumulation, the DNA damage repair deficiency, as well as the cellular functional decline can limit the adult stem cell compartment functions (44). All these factors may be implicated in the decline of tissue renewal capacity, in the appearance of aging-related pathology (45) and in a more severe and progressive frailty.
Our study has potential implications: (i) the increment of DNA damage in cHPSC might be a suggestive signal of organism impairment that precedes the evident frailty in older people. The evaluation of cHPSC CD34+/γ-H2AX+ can be improved for its eventual clinical use. However, it is promising that the ROC curve analysis showed the possibility to distinguish the frail from non-frail and pre-frail subjects by the percentage of DNA-damaged cells in the hematopoietic adult stem cell compartment. (ii) Future studies could replicate or corroborate our findings, also recruiting individuals of similar age and gender and stratifying them according to the presence of frailty. In this case, it would be possible to hypothesize that progression of frailty may be attenuated through specific drugs that act on preventing DNA damage or on removing damaged cells. Indeed, drug therapies (eg, geroprotectors, senolytic drugs, and repurposed drugs) have been shown to attenuate frailty in preclinical and/or clinical studies (46).


s2fieldsofstudy: Biology Medicine

text:
MIC17A is a novel diagnostic marker for feline toxoplasmosis

Toxoplasma gondii is a widespread parasitic pathogen that infect humans and all warm-blooded animals, causing abortion and stillbirth in pregnant women and animals, as well as life threatening toxoplasmosis in immune com-promised individuals. Felines are the only definitive hosts of Toxoplasma and oocysts shed by infected felines are the major source of infection for humans and other animals. Given the critical role of felines for T. gondii transmission, control of feline toxoplasmosis has significant impacts on reducing the overall prevalence of animal and human toxoplasmosis. However, reliable diagnosis of feline toxoplasmosis is still challenging. In this study, we found that the putative micronemal protein 17A (MIC17A) that was abundantly expressed in Toxoplasma merozoites is a good diagnostic marker for serological diagnosis of Toxoplasma infection in felines. T. gondii encodes four paralogs of MIC17A in total and the expression of three of them is drastically upregulated in merozoites than in tachyzoites. In contrast, when proteins like GRA1 and MIC3 that are more abundantly expressed in tachyzoites than in merozoites were used as diagnostic antigens to test feline toxoplasmosis, they reacted with Toxoplasma specific IgG antibodies poorly. Taken together, these results suggest that merozoite antigens are better suited for the diagnosis of feline toxoplasmosis than antigens that are highly expressed at tachyzoite or bradyzoite stages.

Introduction
T. gondii is an obligate intracellular parasite infecting a wide variety of animals, including humans, birds, and mammals (Hill et al. 2005). It is estimated that up to one-third of the world's human population may be infected with T. gondii (Montoya and Liesenfeld 2004;Dubey 2008). The vast majority of these infections are at the chronic stage where the infected individuals do not show obvious symptoms (Desmonts and Couvreur 1974). Nonetheless, the parasites are living in the central nerve system and muscles and replicating at a very slow rate . Once the immune functions are compromised, such as those with HIV infection or organ transplantation, chronic infections may be reactivated to acute infections and cause serious complications (Wang, Wang et al. 2017). In US, T. gondii is ranked second in terms of the annual burden of disease caused by all major foodborne pathogens . It causes more than 300 deaths and 3 billion dollars of illness cost each year ). In addition, vertical transmission during pregnancy is another major risk of Toxoplasma infection, which can cause abortion and still birth of pregnant women and animals (Song et al. 2005). In some regions like Ireland and Northern Ireland, toxoplasmosis is the No.1 cause of abortion in ewes, accounting for up to 24% of all abortion cases (Innes et al. 2009). As such, T. gondii is a zoonotic pathogen of great medical and veterinary significance.
One key reason for the high prevalence of T. gondii is its complex life cycle and multiple routes of transmission.

A complete life cycle involves intermediate hosts and
Open Access *Correspondence: shenbang@mail.hzau.edu.cn definitive hosts (Miller et al. 1972). While essentially all warm-blooded animals can serve as intermediate hosts of T. gondii, felines are the only definitive hosts. T. gondii reproduces asexually in the intermediate host and exists in the forms of tachyzoites and bradyzoites, which are responsible for acute and chronic infections respectively. One unique aspect of Toxoplasma is that it can be transmitted between intermediate hosts, without the need of definitive hosts to complete the life cycle. This is due to its ability to interconvert between tachyzoites and bradyzoites (Skariah et al. 2010). For instance, one major route of human infection is through the ingestion of undercooked meat (pork and lamb etc.) that contains Toxoplasma bradyzoites (Cook et al. 2000). In addition, transmission of Toxoplasma between intermediate hosts through predation is also very common among wild and domestic animals .
Felines as the only definitive hosts of Toxoplasma have a major and unique role during its transmission. Toxoplasma undergoes sexual replication in intestinal epithelial cells of felines to produce oocysts, which are highly infectious and environmentally resistant after sporulation. One infected feline can excrete millions of oocysts in a short period of time (Dubey 2005). As such, oocyst contaminated water and fresh produce are a main source of infection for humans and animals. Oocyst production is a very complicated process and very little is known about the underlying biology. The most efficient way of oocyst shedding is when felines are infected with bradyzoites (Dubey 1998). After infection, the parasites convert to merozoites and proliferate rapidly in intestinal epithelial cells through endopolygeny. Then, some merozoites are differentiated into male and female gametes, which then fuse to produce oocysts. Mature oocysts are released from epithelial cells and shed to the environments along with feline feces (Black and Boothroyd 2000). Under appropriate humidity and temperature conditions in the environment, oocysts quickly sporulate and become infectious to other animals. However, the detailed mechanisms that govern each of the developmental steps during oocyst formation are poorly understood. Transcriptomic analyses have clearly shown that gene expression in merozoites and during sexual reproduction is significantly differently than that in tachyzoites or bradyzoites (Behnke et al. 2014). On the other hand, felines may also act as intermediate hosts for T. gondii. But the relationship of this role of felines has not been well connected to their role as definitive hosts.
Healthy felines infected with Toxoplasma often do not show obvious symptoms and the diagnosis of feline toxoplasmosis is still challenging (Dubey 1995). In the literature, many different methods have been used to assess the serologic prevalence of Toxoplasma infection in felines. These include indirect fluorescent tests, indirect hemagglutination, modified agglutination tests (MAT), Enzyme-Linked Immunosorbent Assay (ELISA) and others (Liesenfeld et al. 1996). Using these tests, the prevalence of Toxoplasma infection in felines varied from 0 to 100%, depending on the geographic region and the source of the felines (stray felines and home owned pet felines etc.) (Dubey et al. 2020). One common problem of these studies is that the accuracy of the corresponding methods is often a concern. Taking ELISA for example, many different antigens have been used but how well they agreed with each other in terms of the testing results is not known. The majority of these methods use either total soluble antigens (TSA) of tachyzoites or recombinant proteins as diagnostic antigens (Choi et al. 1992). For recombinant antigens, Toxoplasma surface antigens or secretory proteins from specialized organelles like dense granules, rhoptries, micronemes are preferred choices, due to their high levels of expression and immunoreactivity. While native TSA seems to be the most reliable antigen for diagnostic purposes, to use it is not as convenient as recombinant antigens because its production involves culture of live parasites that are of safety concerns. In this study, we have tried to find a recombinant antigen that can replace TSA for the diagnosis of feline toxoplasmosis. To our surprise, the proteins like GRA1, GRA7 and MIC3 that are commonly used for the diagnosis of Toxoplasma infection in intermediate hosts (Pietkiewicz et al. 2007;Jiang et al. 2008), did not perform well for the diagnosis of feline toxoplasmosis. On the other hand, we found that the MIC17A protein highly expressed in the merozoite stage of Toxoplasma was a suitable marker for the diagnosis of toxoplasmosis in felines.

Feasibility of GRA1, MIC3, BAG1, GRA7, and GRA10 as antigens for the diagnosis of feline toxoplasmosis
To find a recombinant protein suitable as a diagnostic antigen for feline toxoplasmosis, we first selected five antigens from commonly used iELISA methods that are used to detect toxoplasmosis in intermediate hosts like sheep and swine. These include GRA1, MIC3, BAG1, GRA7 and GRA10. The five recombinant proteins were expressed in E. coli and purified to nearly homogeneity, using the methods described previously. Then, 2 μg/ mL proteins were coated into ELISA plate wells (100 μL per well). To test whether these proteins could be used for the diagnosis of cat diagnosis, six cat serum samples that were confirmed to be positive for T. gondii IgG by MAT and TSA-iELISA were examined using the above protein coated ELISA plates. The results showed that the S/N (OD 630 of samples / OD 630 of negative control) values were very low when using these five proteins as diagnostic antigens (Table 1). In contrast, the S/N numbers from TSA-iELISA were close to or above 10, much higher than those obtained from GRA1, MIC3, BAG1, GRA7, or GRA10 based ELISA. These results suggest that these antigens may not be good for serological diagnosis of feline toxoplasmosis.

Looking for novel diagnostic markers for feline toxoplasmosis
The above five antigens were frequently used in the lab as diagnostic markers to check Toxoplasma infection in intermediate hosts. They are abundantly expressed either at the tachyzoite or the bradyzoite stage. Since the above results showed that they were not suitable for the detection of feline toxoplasmosis, we sought to identify other antigens that could service this purpose. We speculated that when T. gondii infects the definitive hosts, cats, tachyzoites or bradyzoites might not be the main parasite forms that activate immune responses and stimulate antibody production. On the other hand, merozoites are the critical stage for T. gondii to enter sexual reproduction in the intestinal epithelial cells of cats. In this regard, we hypothesized that proteins that are highly expressed in merozoites might be useful for the diagnosis of cat toxoplasmosis. To this end, we used the transcriptomic data available in ToxoDB (https:// toxodb. org/ toxo/ app) and looked for proteins that are highly expressed in merozoites and predicted to be surface or secretory antigens. After comparison, we selected the protein MIC17A (TGME49_200250) to test our hypothesis.
MIC17A was predicted to be a secretory protein localized to the secretory organelle micronemes. MIC17A has a plasminogen apple nematode (PAN) domain that is commonly found in MIC proteins. Transcriptomic data from ToxoDB show that MIC17A is highly expressed in merozoites, reaching an expression percentile of 99.99% with a transcripts per million (TPM) value of more than 10,000. In addition, T. gondii encodes four paralogs of MIC17A, three of which (MIC17A, MIC17B and MIC17D) are also significantly upregulated in merozoites than in tachyzoites. Expression of MIC17C was low in both tachyzoites and merozoites (Fig. 1A). Sequence alignment of the four MIC17 paralogs showed that they are highly similar to each other (Fig. 1B). As such, the antibodies generated from one paralog are expected to react with all four proteins. MIC17A sequence was also used in a Blast search to see if there are homologous sequences in other organisms. The results show that highly similar sequences could be found in Neospora caninum, Hammondia hammondi and Besnoitia besnoiti, which are closely related to T. gondii. Although other coccidian parasites like Cystoisospora, Eimeria and Cyclospora may also encode MIC17 homologs, the sequence identity with Toxoplasma proteins is below 30% and the antibodies generated are less likely to cross-react.

MIC17A reacted strongly with T. gondii positive cat sera
To test whether MIC17A can be used for the diagnosis of Toxoplasma infection in cats, it was expressed recombinantly in E. coli and tested with cat serum samples that are confirmed to be positive for Toxoplasma specific IgG. First, the coding sequence of MIC17A was cloned from the cDNA of the type II strain ME49 and cloned into the pET-28a to express it as a 6 x His fusion protein.
The resulting pET-28a-MIC17A plasmid was introduced into BL21 (DE3) cells and expression of the His-MIC17A fusion protein was induced by IPTG. Subsequently the bacterial cells were lysed and expression of proteins were examined by SDS-PAGE. The results showed that the recombinant His-MIC17A was mainly expressed in an insoluble form in the inclusion bodies ( Fig. 2A). To purify the recombinant protein, pelleted inclusion bodies were first denatured with 0.3% sarkosyl and then renatured in PBS. Then, the solubilized proteins were loaded to a nickel column for affinity purification. Imidazole eluted fractions were analyzed by SDS-PAGE and pure His-MIC17A fractions were collected and dialyzed in PBS before use (Fig. 2B). To test whether the purified recombinant His-MIC17A could react with the IgG antibodies in cat sera, it was subjected to Western blot analyses using Toxoplasma positive and negative sera respectively. The results clearly showed that His-MIC17A could be easily detected with the Toxoplasma positive serum, but not with the negative serum (Fig. 2C), suggesting that MIC17A is able to distinguish the infection status of cats.
To further check whether MIC17A can be used as a diagnostic antigen in ELISA tests, we coated the ELISA plates with 2.25 μg/mL recombinant MIC17A and used them to test six cat serum samples (the same samples as those used in Table 1) that were confirmed to be positive for Toxoplasma specific IgG. The calculated S/N values are all above nine (Table 2), much higher than those obtained when using GRA1, MIC3, BAG1, GRA7,  or GRA10 as diagnostic antigens (Table 1). These results further suggest that MIC17A is a feasible antigen for serological diagnosis of cat toxoplasmosis.
The results of MIC17A-based ELISA tests agree well with that of MAT and TSA based ELISA From the above tests on a small number of serum samples, it seems like the merozoite antigen MIC17A based ELISA has a higher positive detection rate than those based on tachyzoite or bradyzoite antigens like GRA1 and BAG1. To check whether the high positive detection rate is real or false positive, we compared the MIC17A-iELISA to other tests that are frequently used for the diagnosis of Toxoplasma infection. First, it was compared to the TSA based iELISA, which is a widely accepted method to test Toxoplasma specific antibodies in host serum samples. A total of 36 samples were analyzed by both optimized MIC17A-iELISA and TSA-iELISA. The results showed that 19 tested positive and 11 were negative in both tests (Table 3). Only six samples displayed discrepancy, which tested positive in MIC17A-iEL-ISA but negative in TSA-iELISA. Therefore, the overall agreement rate between these two methods is 83.3% ((19 + 11)/36 = 83.3%). MAT is another commonly used approach to examine the presence of T. gondii specific antibodies. A comparison between MIC17A-iELISA and MAT involving 106 samples found that there is a good agreement between these two methods as well. Of the samples analyzed, 24 tested positive and 67 tested negative in both tests (Table 4). The overall agreement is 85.8%. Lastly, we compared MIC17A-iELISA to a commercial kit (ID Screen Toxoplasmosis Indirect Multi-species from Innovative Diagnostics), which is an indirect ELISA test using SAG1 as the diagnostic antigen (therefore we call it SAG1-iELISA). Interestingly, of the 60 samples tested, only eight samples had the same results from both tests (four were positive and the other four were negative). Fifty-two samples that tested positive by MIC17A-iELISA tested negative by the commercial SAG1-iELISA kit (Table 5), suggesting a much lower positive detection rate of SAG1-iELISA than MIC17A-iELISA. Given the decent agreement between MIC17A-iELISA and MAT as well as TSA-ELISA, these results suggest that the low positive detection rate of SAG1-iEL-ISA kit is likely due to the low sensitivity of this kit. SAG1 is a surface antigen highly expressed in tachyzoites, similar to GRA1 in Fig. 1A. These results are consistent with the data above showing that tachyzoite or bradyzoite antigens like GRA1, MIC3, BAG1, GRA7, and GRA10 were not ideal for the diagnosis of cat toxoplasmosis.

Some infected cats may not develop chronic infection
It is widely assumed that cats are both definitive and intermediate hosts for T. gondii. Once an intermediate   host is infected with Toxoplasma, the parasite will stay in the host lifelong, mostly in the form of chronic infection. As such, infected cats are assumed to contain parasites in their tissues. The big discrepancy between the MIC17A-iELISA and SAG1-iELISA, as well as ELISAs using GRA1, MIC3, BAG1, GRA7 and GRA10 as diagnostic antigens, prompted us to check the underlying reasons. One possibility is that in some infected cats, the parasites may only go through coccidian development in gut epithelial cells to produce oocysts without penetrating deeper to form tachyzoites and bradyzoites, like that in Eimeria parasites. To check this possibility, we screened a number of cats and tested the presence of Toxoplasma DNA in their heart and brain tissues, which are preferred places for chronic infection establishment. Three cats (Cat 1, 2 and 6) that tested negative in MIC17A-, TSA-and SGA1-based ELISAs were found to be free of Toxoplasma parasites, since the PCR amplification of the Toxoplasma specific 529-bp repeat failed to amplify positive products ( Supplementary Fig. S1 A&B). On the other hand, two cats that tested positive in the above three ELI-SAs did have Toxoplasma parasites in their tissues, as the 529-bp repeat was successfully amplified from their heart and brain tissues ( Supplementary Fig. S1A). Interestingly, one cat (Cat 5) that tested positive in MIC17A-and TSA-ELISA but negative in SAG1-ELISA was found to be free of Toxoplasma in tissues ( Supplementary Fig. S1B). These results indicate that this cat had a history of Toxoplasma infection but did not develop chronic infection, supporting our hypothesis that in some infected cats, Toxoplasma only undergoes coccidian development without forming tachyzoites or tissue cysts.

Toxoplasma infection is common in pet cats
While the MIC17A-iELISA was proved to be useful for the diagnosis of Toxoplasma infection in cats, it was used to estimate the prevalence of toxoplasmosis in pet cats.
A total of 143 cat serum samples were collected from a local pet hospital in Wuhan city and tested by optimized MIC17A-iELISA. The overall seroprevalence is 30%. There is a slight difference in the seroprevalence rates among different types of cats. 42.8% (3/7) of Garfield cats and 35% (7/20) British shorthair cats were seropositive, whereas only 21.4% (3/14) of American Shorthair cats and 25% (4/16) Ragdoll cats tested positive (Fig. 3A). On the other hand, gender of the cats does not seem to affect seroprevalence, as 30% female cats and 30.1% male cats were tested positive (Fig. 3B).

Discussion
Felids are the only definitive hosts of T. gondii and they play critical roles in the transmission of Toxoplasma parasites (Frenkel et al. 1970). Therefore, proper control of cat toxoplasmosis is of great significance to reduce the overall prevalence in humans and animals. Unfortunately, there is still no reliable diagnostic method for the detection of feline toxoplasmosis. Previous work from our lab has shown that the total soluble antigen (TSA) prepared from tachyzoites can be used as diagnostic antigens to examine the presence of Toxoplasma specific IgG antibodies in cat sera. This partially solved the problem of serodiagnosis of cat toxoplasmosis. However, the production of TSA requires the culture of live parasites, which may have safety concerns. In this study, we sought to find a recombinant antigen that can replace TSA for the serodiagnosis of Toxoplasma infection in cats. We found that selected tachyzoite and bradyzoite antigens that are commonly used for serodiagnosis of Toxoplasma infection in intermediate hosts were not ideal for diagnosis of cat toxoplasmosis. In contrast, the putative micronemal protein MIC17A that is highly induced in merozoites is as good as TSA for the serodiagnosis of cat toxoplasmosis (Table 3). This study not only discovered a novel diagnostic antigen for cat toxoplasmosis, but also offered In the past, the vast majority of diagnostic antigen screens focus on infections in intermediate hosts. In addition, it is generally accepted that cats are both definitive and intermediate hosts of T. gondii. As such, it is thought that the antigens used for diagnosing Toxoplasma infection in intermediate hosts may also work for the diagnosis of cat infection. For example, the commercial kit ID Screen Toxoplasmosis Indirect Multi-species ELISA is claimed to be able to detect anti-Toxoplasma antibodies in multiple species, including cats. This kit uses the tachyzoite surface antigen SAG1 as diagnostic antigen. Compared with our MIC17A-iELISA, the SAG1-iELISA had a much lower positive detection rate (Table 5). A significant number of samples that tested positive in MIC17A-iELISA were diagnosed as negative in SAG1-iELISA. On the other hand, the results of MIC17A-iELISA agreed well with that of TSA-iELISA or MAT. Together, these results suggest that SAG1, just like other tachyzoite or bradyzoite antigens tested in this study (GRA1, MIC3, BAG1, GRA7 and GRA10), is not a suitable antigen for serodiagnosis of cat toxoplasmosis.
While tachyzoite and bradyzoite proteins like SAG1 and GRA1 are promising antigens for serodiagnosis of Toxoplasma infection in intermediate hosts (Burg et al. 1988;Cesbron-Delauw et al. 1989), their incompetence in the diagnosis of cat toxoplasmosis calls into question regarding the growth and development of Toxoplasma in cats. Because as mentioned above, cats are also intermediate hosts of T. gondii. Our results have provided one possible explanation for this conundrum. We believe that a portion of infected cats do serve as both intermediate and definitive hosts, meaning that they support sexual reproduction of the parasites to produce oocysts, meanwhile they contain tachyzoites and/or bradyzoites in diverse tissues. On the other hand, certain cats may only serve as definitive hosts during the infection and they do not support tachyzoites or bradyzoites development in other tissues, similar to corresponding hosts infected with Eimeria and Cryptosporidium parasites. We have found one cat, whose serum was tested to be positive in MIC17A-iELISA and TSA-iELISA but negative in SAG1-iELISA. When PCR amplification of the 529-bp repeat was used to assess the presence of Toxoplasma parasites in its brain and heart tissues, it was found that this cat did not contain parasites in these tissues. This is one case that supports the above hypothesis. This cat might have only supported the coccidian development of Toxoplasma (therefore it was MIC17A-iELISA and TSA-iELISA positive), but not tachyzoites or bradyzoites (SAG1-iELISA negative). Together, these results also suggest that merozoite antigens are likely major drivers of cat immune responses to produce antibodies during Toxoplasma infection. As such, antigens like MIC17A that are highly expressed in merozoites are promising antigens for serodiagnosis of Toxoplasma infection in cats.

Conclusions
We found that the MIC17A protein that is abundantly expressed in Toxoplasma merozoites is a promising antigen for serodiagnosis of Toxoplasma infection in cats. MIC17A-iELISA had much higher positive detection rates than the ELISA tests using tachyzoite and bradyzoite antigens like SAG1 and GRA1. Toxoplasma parasites may undergo coccidian development in some infected cats to produce oocysts, without penetrating further to form tachyzoites and bradyzoites. This may explain why MIC17A is better suited for the serodiagnosis of cat toxoplasmosis than tachyzoite or bradyzoite antigens.

Parasite strains and source of serum samples
T. gondii strain RH and ME49 were propagated in HFF cells, which were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 0.1 mg/mL penicillin-streptomycin and 5 mM L-glutamine. Cats for experimental purposes were purchased from a cat breeding facility in Shandong Province of China. T. gondii positive and negative control sera were verified by TSA-iELISA and MAT before use. All other serum samples were collected from pet hospitals in Wuhan, China. All animal experiments were approved by the Scientific Ethic Committee of Huazhong Agricultural University (permit #: HZAUCA-2022-0007).

Construction of the pET-28a-MIC17A plasmid and purification of the recombinant MIC17A protein
The coding sequence of MIC17A was amplified from the cDNA prepared from the type II strain ME49, using the following primers: MIC17A-F: 5′-AGC AAA TGG GTC GCG GAT CCG GGC TCC GGA GAC AGC TAGTC; MIC17A-R: 5′-TCC TTT CGG GCT TTG TTT TAG CAT GTG ATA TCG CCT GCTT. Meanwhile the plasmid backbone was amplified from pET-28a using primers vet-F: 5′-AAC AAA GCC CGA AAG GAA GC and vec-R: 5′-GGA TCC GCG ACC CAT TTG C. Then the MIC17A fragment was ligated with the plasmid backbone using the Clon-Express II Kit (Vazyme, China). The resulting pET-28a-MIC17A plasmid was sequenced before use. To express the recombinant MIC17A protein, the pET-28a-MIC17A plasmid was transformed into E. coli competent cells BL21 (DE3). IPTG at the final concentration of 1.0 mM was used to induce MIC17A expression. To purify MIC17A, induced bacterial cultures were collected and lysed on a French press. The lysates were centrifugated at 14000 rpm for 10 min and the pellets were collected, denatured in 0.3% sarkosyl (Solarbio, China) and then renatured through dialysis in PBS. The renatured proteins were then loaded to a nickel column for purification and MIC17A protein was eluted with different concentrations of imidazole. Eluted MIC17A was dialyzed again in PBS, quantified by a BCA Protein Assay Kit (Beyotime, China), and then stored at − 80 °C for further use.

Western blotting
Purified MIC17A protein samples were separated on a 12% SDS-PAGE gel and then transferred to nitrocellulose membrane. Then the membrane was probed with Toxoplasma positive or negative cat sera that were validated by MAT and TSA-iELISA. HRP conjugated rabbit anti-feline IgG (Biolab, China) was used to detect primary antibodies and the blots were developed using ECL chemiluminescence reagents (Beyotime, China) and imaged with a chemiluminescence imager (Tanon, China), as previously described (Gross et al. 1992).

MAT test
Freshly egressed tachyzoites of the type I strain RH were purified and fixed with 6.7% formaldehyde solution, resuspended in alkaline buffer to a final concentration of 2 × 10 7 mL tachyzoites, to prepare the MAT antigen stock as previously described (Desmonts and Remington 1980). The MAT tests were performed in U-shaped 96-well plates using the methods described before (Dubey and Desmonts 1987). The serum samples were tested at serial 2-fold dilutions starting at 1:25. Controls using validated positive and negative cat sera were included in each plate.

Elisa
His-SUMO-GRA1, His-SUMO-GRA10, GST-MIC3, His-BAG1, GST-GRA7, His-MIC17A or TSA were coated into the microtiter ELISA plates (100 μL per well, concentration of proteins was 2 μg/mL except His-MIC17A, which required 2.25 μg/mL) and incubated at 4 °C overnight. Then the plates were blocked with 1% BSA and incubated with the serum samples to be tested (Dubey 2009). HRP conjugated rabbit anti-feline IgG (Biolab, China) was used as the secondary antibody. After washing off the unbound secondary antibody, TMB substrate solution (Solarbio, China) was added (100 μL per well) and reacted at room temperature for 10 min in the dark. Then 0.25% hydrofluoric acid (50 μL per well) was added to each well to stop the reactions. Finally, the OD 630 value of each well was measured using a cytation 5 microplate reader (BioTek, USA). Validated positive and negative sera were used as controls and the S/N value (OD 630 of a given sample / OD 630 of the negative control) was used to determine the infection status of a given sample.


Engineering of global transcription factor FruR to redirect the carbon flow in Escherichia coli for enhancing l-phenylalanine biosynthesis

Background The catabolite repressor/activator protein (FruR) is a global regulatory protein known to control the expression of several genes concerned with carbon utilization and energy metabolism. This study aimed to illustrate effects of the FruR mutant on the L-phenylalanine (L-PHE) producing strain PHE01. Results Random mutagenesis libraries of fruR generated in vitro were first integrated into the chromosome of PHE01 by CRISPR/Cas9 technique, and then the best mutant PHE07 (FruRE173K) was obtained. With this mutant, a final L-PHE concentration of 70.50 ± 1.02 g/L was achieved, which was 23.34% higher than that of PHE01. To better understand the mechanism, both transcriptomes and metabolomes of PHE07 were carried out and compared to that of PHE01. Specifically, the transcript levels of genes involved in gluconeogenesis pathway, pentose phosphate pathway, Krebs cycle, and glyoxylate shunt were up-regulated in the FruRE173K mutant, whereas genes aceEF, acnB, and icd were down-regulated. From the metabolite level, the FruRE173K mutation led to an accumulation of pentose phosphate pathway and Krebs cycle products, whereas the products of pyruvate metabolism pathway: acetyl-CoA and cis-aconic acid, were down-regulated. As a result of the altered metabolic flows, the utilization of carbon sources was improved and the supply of precursors (phosphoenolpyruvate and erythrose 4-phosphate) for L-PHE biosynthesis was increased, which together led to the enhanced production of L-PHE. Conclusion A novel strategy for L-PHE overproduction by modification of the global transcription factor FruR in E. coli was reported. Especially, these findings expand the scope of pathways affected by the fruR regulon and illustrate its importance as a global regulator in L-PHE production. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-022-01954-7.

the formation of chorismate. It begins with the condensation of the phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to form 3-deoxy-d-arabinoheptulosonate-7-phosphate (DAHP). In the L-PHE branch, L-PHE is produced from chorismate in two steps catalyzed by the enzymes encoded by pheA and tyrB [6]. Till now, considerable attention has been paid to the sustainable production of L-PHE in microbial cells using the strategies of rational metabolic engineering, including (i) alleviation of all restrictive regulations [7][8][9]; (ii) deletion of competing pathways [5]; (iii) enhancement and balancing of precursor supplements [10,11]; and (iv) removal of L-PHE degradation pathway. However, current strategies are mainly focused on the engineering of L-PHE biosynthetic pathway itself, and it is challenging to generate a high production of the desired chemical by these classical engineering approaches [5]. In recent years, transcriptional engineering approaches have been applied to strain optimization, which makes the engineering process carried out at a global and systematic level [12][13][14]. The global regulation of metabolic networks through multiple transcription factors (TFs) is one of the complex mechanisms for prokaryotes to respond to the intracellular perturbations by altering the expression of related genes [15,16]. As an important component of gene expression regulation, modification of TFs can cause changes in carbon flux in the relevant metabolic pathways; thus, engineering of TFs has been proved to be useful for redirecting the fluxes toward the desired pathway for improving the target component [17][18][19]. However, to the best of our knowledge, few work has been reported to improve the L-PHE biosynthesis by TFs engineering.
FruR (catabolite repressor/activator, also known as Cra), is known to regulate the expression of several genes concerned with carbon utilization and energy metabolism [20][21][22]. FruR is a dual transcriptional regulator and it modulates the direction of carbon flow by transcriptional activation of genes encoding enzymes concerned with the Krebs cycle and the glyoxylate shunt and by repression of those genes that are involved in the glycolytic and Entner-Doudoroff (ED) pathways [23,24]. In a previous study, the influence of fruR knockout on tryptophan biosynthesis was reported [17], and the metabolomics analysis showed that fruR knockout significantly enhanced the metabolic flow toward glycolysis, pentose phosphate pathway, and TCA cycle, increasing levels of critical precursors and substrates for L-tryptophan biosynthesis. A similar result was also reported by Zeng group [9]. However, to our surprise, it was found that inactivating the global regulator FruR in our L-PHE-producing strain PHE01 led to a significant decrease in L-PHE production. This result revealed that the regulation effects of FruR are more complex than expected and it is worth exploring the mechanism further.
In this study, the functionality of the global regulator FruR for L-PHE over production was first verified. Then, CRISPR/Cas9-facilitated engineering was combined with sensor-guided in vivo screening for engineering of protein FruR. In order to clarify the regulation mechanism of FruR, comparison of multi-omics data was performed between the best mutant and the wild-type strains.

Knockout of fruR affects the biosynthesis of L-PHE
To explore the impact of FruR on the biosynthesis of L-PHE, the gene fruR in our previously constructed L-PHE-producing strain PHE01/pCas9 was disrupted, generating the strain PHE03 (Table 4, details in Additional files 3). The capacity of L-PHE production of PHE03 was compared to that of strain PHE01 by carrying out shake-flask fermentations (Table 1). Table 1 shows that the cultivation of both strains PHE01 and PHE03 obtained almost the same amount of biomass. However, at the end of fermentation, PHE03 produced 5.75 ± 0.55 g/L of L-PHE, which is 18.10% lower than that of the strain PHE01 (7.02 ± 0.23 g/L). Meanwhile, the productivity (Vp) and specific production rate (q PHE ) were also significantly decreased. These results suggested that the disruption of regulator FruR could result in the imbalance of central carbon metabolism, and indirectly block the metabolic flow toward the L-PHE biosynthetic pathway. Also, this result demonstrated that the regulation functionality of protein FruR is critical for L-PHE overproduction, and this was not consistent with the reported case for L-tryptophan [9,17]. To find out whether modification of the regulation functionality of protein FruR could increase the production of L-PHE, a random mutagenesis strategy combined with the L-PHE biosensor was employed for the engineering and characterization of FruR mutants.

Table 1 L-PHE fermentation parameters of the strains PHE01 and PHE03
The engineered L-PHE-producing strains were cultivated in 500 mL shake flasks for 43 h The data represents the mean ± SD from three independent experiments Vp volumetric productivity, q PHE specific production rate of L-PHE

Engineering and characterization of FruR mutants
To obtain a FruR mutant with a higher production of L-PHE, the fruR variants were generated in vitro by using the error-prone PCR, and then the resulting gene variants were integrated into the chromosome of PHE01/ pCas9 using the CRISPR/Cas9 technique. Finally, the FruR mutants were screened and characterized with the aid of L-PHE biosensor and HPLC detection. Firstly, a host strain PHE01ΔfruR::Cm R (Table 4, details in Additional files 3) was constructed by inserting a chloramphenicol resistance gene Cm R into the locus of the fruR gene to offer the sgRNA target sequence for CRISPR/Cas9 application in further gene variant integration. In principle, an engineered FruR protein with a higher activity should lead to more accumulation of L-PHE, which in turn stimulate the expression of a report gene regulated by an L-PHE biosensor. The L-PHE biosensor is composed of the promoter of mtr gene (pmtr) [25] with RFP protein fused to the downstream (Fig. 1a).
In detail, the plasmid backbone, upstream of the transcriptional start site of the pmtr promoter, and the DNA fragment encoding for RFP protein were amplified from the plasmid pBR332, the genomic DNA of E. coli K-12 W3110, and the template pET28a-RFP, respectively. All fragments were then pooled to an equimolar concentration and fused together to result in the final plasmid pmtr-RFP (details in Additional files 3). To characterize the designed biosensor, the plasmid pmtr-RFP expressed with the L-PHE biosensor was introduced into the host strain E. coli W3110. As observed in Fig. 1b, the L-PHE biosensor was able to activate RFP expression by a maximum of 23.86-fold upon supplementing L-PHE (0-200 μM) to the cultivation medium. To further validate the L-PHE biosensor, we measured fluorescence output in five different genotypes cultivated in the M9 medium, e.g., W3110, PHE01, PHE03, PHE04 (PHE 01ΔaroF MT ::aroF WT , details in Additional files 3), and PHE05 (PHE01ΔpheA MT ::pheA WT , details in Additional Fluorescence normalized by OD 610 related to concentration of L-PHE supplemented into the media. c Extracellular L-PHE normalized by OD 610 related to fluorescence normalized by OD 610 (mean values with standard errors, n = 3 technical replicates). The p-value showing a significant slope is from a two-side t-test performed on means values for the five different strains, e.g., W3110, PHE01, PHE03, PHE04, and PHE05 files 3), and observed different biosensor outputs from these strains in line with the extracellular L-PHE concentration in strains with different genotypes (R 2 = 0.914 and p = 0.029, Fig. 1c). These results demonstrated that the designed L-PHE biosensor has the ability to monitor changes in endogenously produced L-PHE pools.
Having established an L-PHE biosensor (Fig. 1), we next sought to apply the L-PHE biosensor for high-throughput screening and characterization of the FruR variants. To do so, PHE06 was constructed by introducing the plasmid pmtr-RFP into PHE01ΔfruR::Cm R /pCas9. Afterwards, a mutagenesis library of the fruR variant library was generated in vitro by using a Diversify ® PCR Random Mutagenesis Kit (PT3393-2, Takara Bio) and integrated into the chromosome of host PHE06 using the CRISPR/ Cas9 technique. Finally, the mutants were screened and selected by using L-PHE biosensor-based in vivo characterization and HPLC-facilitated in vitro detection.
After the first-round screening, 279 colonies of the FruR mutants, which have relatively higher fluorescence signals, were selected and cultivated in three 96 deep-well plates. After 15 h of cultivation, cell growth and fluorescence intensity were measured and the results are presented in heat maps (Fig. 2). Among them, 50 mutants with a stronger fluorescence intensity (colored in red in Fig. 2 (Table 2). As shown in Table 2, all of the selected FruR mutants had a higher L-PHE production compared to the strain PHE01. Especially, the FruR E173K mutant exhibited the highest L-PHE production among all types of mutants. These results suggest that the variant FruR E173K has a better performance for l-PHE production than the wildtype FruR WT under the test conditions. Interestingly, the variant FruR E173K was also generated by Zhang group in an L-PHE-producing strain HDH6-D12 by using random mutagenesis [26]. To provide more direct evidence, fed-batch fermentation was performed with the FruR E173K mutant and the strain PHE01.

Fed-batch fermentation of the variant FruR E173K
To explore the performance of the best variant FruR E173K (PHE07) on enhancing the biosynthesis of L-PHE, the capacity of L-PHE production of the variant PHE07 was compared to that of the strain PHE01 by carrying out fed-batch fermentation in 50L bioreactors.
As seen in Fig. 3a, the trend of cell growth of PHE07 was significantly lower than the strain PHE01. The overall cell biomass of the strains PHE01 and PHE07 were calculated to be 41.74 ± 0.49 g/L and 37.36 ± 0.17 g/L, respectively. However, as seen in Fig. 3b, the strain PHE07 produced a significantly higher amount of L-PHE than the reference strain PHE01 after the early stationary phase (about 20 h) until the end of the fermentation. At the end of fermentation (56 h), PHE07 produced 70.50 ± 1.02 g/L of L-PHE, which is 23.34% higher than that of the control PHE01 (57.16 ± 1.16 g/L, Table 3). According to Table 3, PHE01 and PHE07 consumed almost the same amount of glucose. It was thus suggested that in PHE07 a part of carbon flux was redirected from the central metabolic pathway to the biosynthesis of L-PHE. Considering the fact that FruR, as a global transcriptional regulator, mediates the flux balances among Krebs cycle, glyoxylate shunt, glycolytic pathway, gluconeogenesis pathway, and ED pathway [21], modification of the FruR protein could result in the flux being redirected toward the glyoxylate shunt and gluconeogenesis pathway, enhancing the supplying of precursors PEP and E4P for the biosynthesis of L-PHE.  Additionally, the overall specific formation rate of L-PHE in the strain PHE07 (33.69 ± 0.13 mg/g DCW/h, Table 3) showed an obvious advantage over that of the strain PHE01 (24.45 ± 0.02 mg/g DCW/h, Table 3). Also, it was notable that the yield of L-PHE for PHE07 was increased to 0.207 g/g at 56 h (Table 3), whereas it was 0.167 g/g for the reference strain PHE01. These results clearly demonstrated that expressing the FruR E173K protein in the strain PHE07 was able to improve the specific production rate of L-PHE and the yield of L-PHE significantly (by approximately 37.79% and 23.95%, respectively). The performance of PHE07 in terms of the increased L-PHE production titer, rate, and yield makes it more attractive for industrial application. However, inconsistent with the previous reports [9,17], overproduction of L-PHE was realized by expression of the FruR E173K protein rather than knock-out of FruR in PHE07.
As seen in the schematic diagram of the interaction between FruR and its effector fructose-1,6-bisphosphate (FBP, Additional file 3: Fig. S6), the binding cavity is surrounded by residues Asn73, Tyr76, Arg197, Ser246, Phe247, Gln291, and Arg323. According to a previous study reported by Zhu et al., [14], modification of these binding sites resulted in decrease of the binding affinity between FruR and FBP [14]. In our case, E173K mutation might also result in a low protein-effector affinity, leading to enhancement of the binding affinity between FruR and its target genes. To this end, it was thus necessary to further explore the potential regulatory mechanism for FruR E173K protein in the strain PHE07 by comparative transcriptomics and metabolomics analysis.

Transcriptome and metabolome-wide effects of the regulator FruR in FruR E173K in the L-PHE producer
To assess the influence of the FruR E173K protein on gene expression and the composition of intracellular metabolites in the FruR E173K mutant, transcriptomics and metabolomics analysis were carried out for the FruR E173K mutant (MF) as well as the wildtype PHE01 (ME). For this purpose, both strains were cultivated in the modified L-PHE fermentation medium, and the samples were taken at the time point of 10 (Early-logarithmic phase), 20 (Mid-logarithmic phase), and 40 h (Stationary phase, Fig. 3a). In order to enable a direct comparison of the transcriptome and metabolome data, mRNA of the bacteria was isolated from the same culture samples as those used for intracellular metabolite extraction. According to the results of the metabolomics, 75 of the identified metabolites were significantly up-regulated and 98 of them significantly down-regulated (|fold change (fc)| ≤ 0.5 or ≥ 1.5; corrected p ≤ 0.05) between the FruR E173K mutant and the wildtype strain at the 20 h of cultivation ( Fig. 4 and Additional file 1: Dataset S1). In parallel, transcriptomic data revealed that of 3143 profiled transcripts, 421 were significantly up-regulated and 224 significantly down-regulated in the FruR E173K mutant compared to the wildtype strain, when a |log2fc| ≥ 2.0 and a corrected p ≤ 0.05 in gene expression were taken as cut-off ( Fig. 6 and Additional file 2: DataSet S2).
The most significant differences between the FruR E173K mutant and the wildtype strain occurred in the central carbon metabolism (e.g., Krebs cycle, glyoxylate shunt, and glycolytic and gluconeogenesis pathway) and amino acid biosynthesis and metabolism (Figs. 4 and 5). The carbon flux balance between glycolytic and gluconeogenesis pathway is strictly regulated at a specific node in the EMP pathway by the regulator FruR: its effector fructose-1,6-bisphosphate (FBP) [27], meanwhile the metabolites in pentose phosphate pathway: 6-phosphogluconate (fc 261.07, p 8.88 × 10 -5 ) and ribose 1-phosphate (fc 1.55, p 5.67 × 10 -4 ), were up-regulated in the FruR E173K mutant (Fig. 5a, b, Additional file 1: DataSet S1). On the transcriptome level (Fig. 6 and Additional file 2: DataSet S2), the enzymatic reactions of glycolytic and gluconeogenesis pathways were controlled correspondingly, especially the transcript levels of genes involved in the entire gluconeogenesis pathway (gpmM, pfkA, gapA, and pgk) were significantly up-regulated in the FruR E173K mutant; In parallel, the transcript levels of genes associated with pentose phosphate pathway (zwf and tkt) were up-regulated ( Fig. 6 and Additional file 2: DataSet S2). These results demonstrated that the modification of the regulator FruR probably activates the expression of genes involved in the gluconeogenesis pathway, which would convert the metabolic flux toward the pentose phosphate pathway and make the expression of genes associated with the pentose phosphate pathway were enhanced, Table 3 Comparison of the performances of the strains PHE01 and PHE07 for L-PHE production in fed-batch fermentation Fed-batch fermentations were performed in 50 L bioreactors at 37 ℃ and pH 6.8; the initial glucose concentration was 10 g/L; the initial inoculation volume ratio was 0. 25 and eventually contributing to the accumulation of E4P, a precursor to aromatic amino acid biosynthesis. Overexpression of the genes involved in the pentose phosphate pathway, such as zwf and tkt, has been widely applied for development of aromatic amino acids (AAAs)-producing strains [11,28,29], while overexpression of genes involved in the gluconeogenesis pathway (gpmM, pfkA, gapA, and pgk) for enhancing the accumulation of E4P is rare reported. Thus, these targets could be potential candidates for further improvements of L-PHE production, as well as other AAAs.
Corresponding metabolites could also be observed for the downstream Krebs cycle, where three metabolites (succinate, fumarate, and malate) were significantly up-regulated, while two metabolites (acetyl-CoA and cis-aconitic acid) were down-regulated in the FruR E173K mutant (Fig. 5 and Additional file 1: DataSet S1). In line with this observation, parts of mRNAs of the involved Fig. 4 Heatmap of metabolite changes for the control group ME20 and the treatment group MF20 at 20 h of cultivation. Six independent biological repetitions of each group were measured and are shown in columns. Hierarchical clustering analyses were performed on those 12 samples with known annotations. The well in red indicates metabolite is up-expressed, and the well in blue indicates metabolite is down-expressed. Source data is available for this figure at Additional file 1: DataSet S1 enzymes from the Krebs cycle and glyoxylate shunt (aceA, aceB, sucA, and sucD) were activated ( Fig. 6 and Additional file 2: Dataset S2), while the transcript level of genes (aceEF, acnB, and icd) was down-regulated in the FruR E173K mutant, indicating that the transcriptome data supports our metabolome data. In addition, gene pckA (encoding phosphoenolpyruvate carboxykinase, |log2fc| 2.75, p 1.72 × 10 -21 ) and gene pps (encoding phosphoenolpyruvate synthase, |log2fc| 3.40, p 3.45 × 10 -46 ) were significantly up-regulated ( Fig. 6 and Additional file 2: Dataset S2), which resulted in a strong accumulation of phosphoenolpyruvate (PEP, fc 19.15, p 0.03, Fig. 5 and Additional file 1: Dataset S1), a direct precursor to aromatic amino acid biosynthesis. These results revealed that FruR E173K mutant activates the part of the Krebs cycle and the entire glyoxylate shunt; represses the metabolism of pyruvate and oxaloacetate and both of them are converted to PEP by enzyme Pps and PckA, respectively (Fig. 6), which resulted in the accumulation of PEP and eventually conducive to the biosynthesis of L-PHE. PEP, as a key node between the central metabolic pathway and the AAAs biosynthetic pathway, has been converted to AAAs biosynthesis by overexpression of the genes involved in the AAAs biosynthetic pathway (such as aroG [9], aroF [7], or aroL [30]), and the genes related to the central metabolic pathway (e.g., pckA and pps [11,31]), or by knockout the gene pykF that encodes the pyruvate kinase I [11,32]. Moreover, as revealed in transcriptome and metabolome data in this study, overexpression of genes involved in the glyoxylate shunt (aceA and aceB) and the succinate biosynthesis (sucA and sucD), or knockout of genes aceEF, acnB, and icd, might also be novel strategies for development of AAAs-producing strains.
In addition to the central carbon metabolism, the FruR E173K mutant had a pronounced effect on the L-PHE biosynthesis pathway. As illustrated in Figs. 4 and 5, derived from the condensation of two molecules of PEP and E4P, a 94.66-fold decrease was found for shikimate (p 3.11 × 10 −5 ), an intermediate of the central pathway for    Fig. 5). Hence, despite the central carbon metabolism, FruR E173K mutant seems to also affect the AAAs pathway, especially activation of the downstream of the shikimate pathway and L-PHE pathway, which led to an accumulation of L-PHE.
As a member of the GalR-LacI family, FruR contains two functional domains: an N-terminal domain which contains a helix-turn-helix motif for DNA binding (AA 3-58 according to UniProt), and the effector binding domain located in the C terminus. FruR forms a complex with DNA in the absence of effector and acts as an activator of genes encoding gluconeogenic, Krebs cycle, and glyoxylate shunt enzymes. Upon the binding of the inducers, FruR is inactivated and the negative effect on genes encoding Entner-Doudoroff pathway and glycolytic enzymes is thus eliminated. In the previous reports on tryptophan bioproduction [17,24], knockout of fruR results in function loss of the inducers and thus the genes involved in glycolysis, the Entner-Doudoroff pathway, and the PP pathway are activated and the genes involved in gluconeogenesis and the Krebs cycle are repressed. While in the case of L-PHE biosynthesis shown in this study, it was found that the changes in transcriptional levels of fruR gene between the FruR E173K mutant (MF) and the wildtype PHE01 (ME) are not significant (Additional file 2: Dataset S2). Therefore, it is suspected that the effects of E173K mutation might be due to the decrease of the proteineffector binding affinity rather than the change of protein expression levels. As a consequence, the FruR E173K mutant activates the gluconeogenesis pathway, alternative Krebs cycle, and the entire glyoxylate shunt, and no significant positive effect on the glycolysis and Entner-Doudoroff pathway was observed, as revealed by the metabolome and transcriptome analysis (Figs. 4 and 6). The different effects of FruR in the production of tryptophan and L-PHE might be due to the fact that more precursors are required for tryptophan biosynthesis. Besides the processors of PEP and E4P, PRPP and L-serine are also needed. Therefore, the balance among the pathways regulated by FruR should be modulated in different modes in order to achieve a high production. Another important reason may come from the different genetic backgrounds of the host strains, on which the functionality of the regulatory protein is highly dependent.

Conclusion
Knockout of the fruR gene was first constructed in E. coli PHE01and the resulting strain PHE02 (PHE01ΔfruR) exhibited a reduced L-PHE production, indicating that the functionality of the global regulator FruR is necessary for L-PHE overproduction. To improve the L-PHE production of PHE01, CRISPR/Cas9-facilitated genome-integration of the fruR mutagenesis libraries was coupled with the biosensor-assisted library screening approach. The best mutant strain, PHE07 (FruR E173K ), was obtained after several rounds of screening and characterization. Metabolomics and transcriptomics analysis suggested that the FruR E173K mutant enhanced metabolic fluxes through the gluconeogenesis pathway, alternative Krebs cycle, the entire glyoxylate shunt, and the PP pathway, therefore channeling carbon fluxes to the L-PHE biosynthetic pathway. These altered metabolic flows not only improved the utilization of carbon sources, but also enhanced the supply of precursors (PEP and E4P) for L-PHE biosynthesis.

Strains and plasmids
The strains and plasmids used in this study are listed in Table 4. The primers used are summarized in Table S1. E. coli DH5α was used for plasmid construction. An L-tyrosine auxotrophic E. coli PHE01 was used as the host strain for expression and L-PHE production. To use the CRISPR/Cas9 technique for genome-editing or mutagenesis library genome-integration, the plasmid pCas9 [33] was introduced into PHE01, resulting in the strain PHE01/pCas9 ( Table 4).

Knocking-out of furR gene by using CRISPR/Cas9 technique
To realize the disruption of fruR gene, the strain PHE01/ pCas9 (PHE02) was first constructed and used as the host strain. To achieve high efficiency for genome editing, the plasmid pGRB-ΔfruR (Additional file 3: Fig.S1) was constructed. This plasmid is able to offer the sgRNA targeting to the fruR gene and the homologous arms for recombination. More detailed information for the construction of pGRB-ΔfruR is presented in the Additional file 3. We then followed the protocol reported by Chen and coworkers [34] to prepare the electroporation-competent cells and do the transformation. Specifically, an overnight culture (grown at 30 ℃) of the strain PHE02 was inoculated into 10 mL fresh SOC medium containing 50 μg/mL spectinomycin. After OD 600 value reaching to 0.4-0.5, the cells were put on ice immediately for 10 min. Then, the cells were harvested by centrifugation at 4 ℃ and washed three times with precooled 10% glycerol. Competent cells were re-suspended in 400 μL precooled 10% glycerol and divided into 200 μL for each reaction. The corresponding sgRNA plasmid was mixed with the competent cells for transformation. The electroporation was done in the 0.2 cm cuvette at 2.5 kV, and the cells were suspended in 1 mL SOB medium and recovered for 2 h at 30 ℃ before plating. Plates were incubated more than 24 h at 30 ℃.

Construction of host strain for gene variant engineering and screening
To realize CRISPR/Cas9-facilitated library integration and in vivo screening, we first constructed a PHE02/ pmtr-RFP (PHE03) strain in which an L-PHE biosensor was expressed in the plasmid pmtr-RFP (Additional file 3: Fig.S3). Afterward, the native FruR enzyme of the strain PHE02 was inactivated by replacing the fruR gene with chloramphenicol resistance gene (Cm R , as a selection maker), generating the strain PHE03ΔfruR::Cm R ( Table 4). The insertion of gene Cm R expresses sgRNA sequence targets for further library integration using CRISPR/Cas9 technique.

Construction of gene variant library in vitro
To achieve genome-integration of mutagenesis library, it is necessary to construct a plasmid pGRB-fruR MT (Additional file 3: Fig. S4), which contains the parts expressing the sgRNA targeting the Cm R gene and the DNA fragment fruR MT flanked by the corresponding homologous arms for recombination. Specifically, error-prone PCR of fruR gene was performed using a Diversify ® PCR Random Mutagenesis Kit (PT3393-2, Takara Bio) according to the manufacturer's protocols. The purified PCR products were then ligated into the plasmid pGRB-fruR MT using a seamless colony kit (D7010M, Beyotime). More detailed information is presented in the Additional file: 1, 2, 3.

Genome-integration and screening of fruR variant library
The furR variant library pGRB-fruR MT was transferred into the competent cells of PHE03ΔfruR::Cm R (containing pCas9). After incubation at 30 ℃ for more than 24 h, transformants with a stronger fluorescent signal were picked out and re-checked by streaking them on the same medium. Afterward, the candidate strains were tested by cultivation in 500 μL PHE-fermentation medium in 96-deep well plates at 30 ℃ for 24 h. Finally, the mutants giving higher medium fluorescent (MFU) and L-PHE concentration were selected for fermentation in shake flasks. Moreover, those mutants were selected for sequencing.

Method for measurement of fluorescent intensities
The mutants containing L-PHE biosensor with reporter RFP protein cultured in the LB medium were harvested by centrifugation and individually washed three times with the M9 medium to remove LB medium. Afterward, each mutant was inoculated with the same amount of cells into 10 mL fresh M9 medium in 50 mL conical tubes, and after cultivation of 10 h cells were subjected to fluorescence analysis using a multifunctional microplate reader. To this end, each culture was first washed three times using PBS buffer and diluted 100-fold and then RFP fluorescence was monitored using a microplate reader (Tecan M200 PRO) at an excitation wavelength of 540 nm. For fluorescent intensities, medium fluorescence unit (MFU) was calculated for each culture.

Fermentation
The fermentation medium with 1.5 g/L tyrosine was prepared as a previous report [28]. The condition for batch fermentation in shake flask was carried out in 500 mL conical flasks containing 50 mL fermentation medium inoculated with 5% (v/v) seed culture. All the batch fermentations were carried out at 37 ℃ and 250 rpm for 45 h. For fed-batch fermentation in the bioreactor, it was performed in a 50-L jar fermenter (BLBIO-10SJ-10SJ-50SJ-50SJ) with an initial broth volume of 20 L. The initial glucose concentration is 10 g/L, and it was maintained at 1-5 g/L by supplementing 700 g/L glucose in the fermentation process. Ammonia was used to maintain the pH at 6.8-7.0. The dissolved oxygen (DO) level was maintained at 25-30% saturation.

Sampling and LC-MS/MS analysis
Referring to the growth curve of PHE01 and PHE03 during the whole fermentation process (Fig. 3a), the samples in the time point of 10, 20, and 40 h were sampled and prepared for metabolites extraction (sextuplicate for each time point). Metabolite and transcript samples were taken simultaneously. The whole process took ≤ 5 s (metabolites) or 10 s (transcripts) per sample from sampling to flash freezing in liquid nitrogen [35]. Metabolite extraction for liquid chromatography coupled to mass spectrometry (LC-MS) analysis was performed by resuspending the cell pellet with 500 µL ice-cold acetonitrile:methanol. The cell suspension was shockfrozen again in liquid nitrogen, and 500 µL of deionized water was added. Further metabolite extraction with repeating freeze-thaw-sonification cycles followed, as described previously [36].
LC-MS/MS analyses were performed using a UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to QExactive HFX mass spectrometer (Orbitrap MS, Thermo). The mobile phase consisted of 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide in water (pH = 9.75) (A) and acetonitrile (B). The QE HFX mass spectrometer was used for its ability to acquire MS/MS spectra on information-dependent acquisition mode in the control of the acquisition software (Xcalibur, Thermo). The ESI source conditions were set as following: sheath gas flow rate as 30 Arb, Aux gas flow rate as 25 Arb, capillary temperature 350 ℃, full MS resolution as 60,000, MS/MS resolution as 7500, collision energy as 10/30/60 in NCE mode, spray Voltage as 3.6 kV (positive) or −3.2 kV (negative), respectively [37].

Data preprocessing and annotation
The raw data were converted to the mzXML format using ProteoWizard and processed with an in-house program, which was developed using R and based on XCMS, for peak detection, extraction, alignment, and integration [38,39]. Then an in-house MS2 database (BiotreeDB) was applied in metabolite annotation. The cutoff for annotation was set at 0.3.

Transcript analysis RNA isolation, stand-specific library preparation, and Illumina sequencing
As mentioned previously, the samples in the time point of 10, 20, and 40 h were selected and prepared for RNA isolation (triplicate for each time point). Extraction of total RNA was carried out by the Invitrogen ™ TRIzol ™ Reagent Kit (15596018). RNA degradation and contamination were monitored on 1% agarose gels. Total amounts and integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Total RNA was used as input material for the RNA sample preparations. For our samples, mRNA was purified from total RNA by using probes to remove rRNA. Strand-specific RNA-seq cDNA library preparation of the total RNA of the different samples was based on RNA adapter ligation as described previously [40]. Afterward, the quality of the library was subsequently quantified by Qubit2.0 Fluorometer, Agilent 2100 bioanalyzer, and qRT-PCR. After the library is qualified, the different libraries are pooled according to the effective concentration and the target amount of data off the machine, then being sequenced by the Illumina NovaSeq 6000. The basic principle of sequencing is to synthesize and sequence at the same time (Sequencing by Synthesis). The fluorescent images measured by the high-throughput sequencer are converted into sequence data (reads) by CASAVA base recognition. Raw data (raw