Natural microbial consortia could efficiently produce 1,3-propanediol , a most promising bulk biochemical derived from glycerol that can be used as a monomer in the synthesis of polytrimethylene terephthalate (PTT). While natural microbial communities are made up of a diverse range of microbes with frequently unknown functions, the construction of synthetic microbial consortia allows for the creation of more defined systems with lower complexity.Klebsiella pneumoniae DUT2 (KP) and/or Escherichia coli DUT3 (EC) cultures with the strictly anaerobic microbe of Clostridium butyricum DUT1 (CB) cultures under micro-aerobic conditions. The function of EC and KP during the fermentation process was to deplete oxygen and create an anaerobic environment for CB. Furthermore, KP competes with CB for the consumption of crude glycerol and the production of 1,3-PDO. The interaction of commensalism and competition resulted in the construction of synthetic microbial consortia capable of efficiently converting crude glycerol to 1,3-PDO even under micro-aerobic conditions. In a batch fermentation, the synthetic CB:KP co-culture at an initial abundance ratio of 92.5:7.5, yielded a maximum 1,3-PDO concentration of 52.08 g/L, with a yield of 0.49 g/g and a productivity of 1.80 g/(L.h), which increased by 10%, 9%, and 12%, respectively, when compared to the CB mono-culture under strictly anaerobic conditions. The final 1,3-PDO concentration, yield, and productivity by the synthetic CB:KP consortia increased by 16%, 19%, and 84%, respectively, when compared to the KP mono-culture. At an initial abundance ratio of 85:7.5:7.5, the synthetic CB:KP:EC co-culture achieved the highest 1,3-PDO flux of 49.17%, while 7.43%, 5.77%, 3.15% 4.24%, and 2.13% of flux was distributed to butyric acid, acetic acid, lactic acid, ethanol, and succinic acid pathways. In a fed-batch fermentation, the synthetic CB:KP:EC co-culture demonstrated a maximum 1,3-PDO concentration of 77.68 g/L with a yield of 0.51 g/g which is 30% and 13% higher than the production by the CB mono-culture at 0.02 vvm (nitrogen volume/culture volume/min) N2 supply. The initial abundance of CB, which is guaranteed to be at least 85%, enables efficient 1,3-PDO production from crude glycerol via the development of synthetic microbial consortia.In this study, the synthetic microbial consortia were constructed by combining facultative microbes of The synthetic microbial consortia demonstrated excellent performance on 1,3-propanediol production under micro-aerobic conditions through the interaction of commensalism and competition. The experimental results demonstrated the potential benefit of using synthetic microbial consortia to produce 1,3-propanediol from crude glycerol. Microbial consortia have been widely used in traditional food and beverage fermentation for thousands of years due to their resistance to environmental fluctuations and excellent performance for complex tasks , 2. NatuKlebsiella pneumoniae achieved a relatively high 1,3-PDO titer of 81.4 g/L with a yield of 0.52 g/g [Clostridiaceae, Enterococcaceae, and Enterobacteriaceae families produced the highest 1,3-PDO production yield of 0.64 mol/mol at pH 7 and 8. Furthermore, an anaerobic microbial consortium C2-2 M enriched from anaerobic activated sludge with 94.64% abundance of Clostridium butyricum produced 60.61 and 82.66 g/L 1,3-PDO in batch and fed-batch fermentation, with productivity of 3.79 and 3.06 g/(L.h), respectively [−1 and an initial glycerol concentration of 130 g/L [C. butyricum, facultative microbes of K. pneumoniae and E. coli was able to efficiently convert crude glycerol to 1,3-PDO [1,3-Propanediol is a promising bulk biochemical that can be used as a monomer for the synthesis of polytrimethylene terephthalate (PTT) , 7–12. D0.52 g/g . The eff0.52 g/g . The pre 130 g/L . FurtherDespite the fact that these natural microbial consortia could efficiently convert crude glycerol to 1,3-PDO, there are still significant limitations in answering fundamental ecological and evolutionary questions, as well as the interaction mechanism surrounding natural microbial consortia. Even the most basic natural microbial consortia characterized to date contain tens to thousands of species, making it difficult to experimentally verify which species in such characterizations are actively part of the community or perform key functions . The creC. butyricum DUT1 and facultative K. pneumoniae DUT2 [C. butyricum and facultative microbes of K. pneumoniae and/or E. coli were investigated to improve the high oxygen tolerance of the strict anaerobes under micro-aerobic conditions. Furthermore, the distribution of metabolic flux distribution and the abundance of synthetic microbial consortia were discussed.In our previous work, we isolated 1,3-PDO producing microbes from natural microbial consortium DUT08, including anaerobic iae DUT2 . In thisClostridium butyricum (CB) and K. pneumoniae (KP), each with a significant ability of 1,3-PDO production, have attracted much attention. We chose a CB:KP co-culture in this study to take advantage of these two 1,3-PDO producing microorganisms. Klebsiella pneumoniae is a facultative strain that can grow on glycerol under aerobic, micro-aerobic, and anaerobic conditions. As a result, we hope that the growth of K. pneumoniae will promote the growth of C. butyricum by creating an anaerobic environment. The production of 1,3-PDO using crude glycerol under mono-culture and CB:KP co-culture conditions at the anaerobic serum vial level was tested, and the results are presented in Table Clostridium butyricum can grow well with 0.2 vvm (nitrogen volume/culture volume/min) nitrogen supply for 3 min, yielding 10.79 g/L 1,3-PDO with a glycerol yield of 0.54 g/g. In the absence of N2, the mono-culture of C. butyricum produced no 1,3-PDO. The CB:KP co-culture produced nearly identical amounts of 1,3-PDO at different inoculation ratios. 6.84 g/L 1,3-PDO was produced after 24 h cultivation without N2 at the optimal ratio of 3:2. The level of 1,3-PDO production in the CB:KP co-culture was comparable to the KP mono-culture but lower than the CB mono-culture. Many byproducts were produced for the KP mono-culture and the CB:KP co-culture, including acetic acid, lactic acid, succinic acid, and ethanol. However, butyric acid was not detected in the CB:KP co-culture. This was interpreted to indicate that dissolved oxygen in the medium inhibited the growth of C. butyricum in the CB:KP co-culture, even though K. pneumoniae was inoculated to create the anaerobic conditions. Klebsiella pneumoniae was primarily responsible for the ability of the CB:KP co-culture to produce 1,3-PDO without N2 supply. Under micro-aerobic conditions, K. pneumoniae performed worse that C. butyricum under anaerobic conditions in producing 1,3-PDO. As a result, the synthetic microbial consortium of C. butyricum and K. pneumoniae under micro-aerobic conditions demonstrated a weak 1,3-PDO production ability that was inferior to the CB mono-culture under anaerobic conditions.E. coli (EC), a microbe that does not produce 1,3-PDO but consumes oxygen, was inoculated to the mono-culture and co-culture cultivation. To that end, the results obtained for 1,3-PDO by different synthetic microbial consortia are represented in Fig. To improve 1,3-PDO production under micro-aerobic conditions, 2 supply, the results showed that E. coli could significantly increase 1,3-PDO production in the CB:EC co-culture at different inoculation ratios. After 24 h of fermentation in the CB mono-culture and the CB:EC co-culture with 0.2 vvm N2 supply, glycerol was almost depleted. In the CB:EC co-culture with a 1:1 inoculation ratio and no N2 supply, 16.38 g/L glycerol remained in the fermentation media, but only 1.72 g/L 1,3-PDO was produced within 24 h. Glycerol was nearly depleted in the CB:EC co-culture after 48 h of fermentation in the absence of N2. In the absence of N2, the presence of E. coli causes C. butyricum to grow and produce 1,3-PDO slowly. When compared to anaerobic conditions (0.2 vvm N2 supply), the fermentation time for the CB:EC co-culture was extended under micro-aerobic conditions (no N2 supply), and the yield of 1,3-PDO to glycerol decreased by 24% at the inoculation ratio of 1:1. Regardless of the CB:EC inoculation ratios, the CB:EC co-culture could efficiently produce 1,3-PDO once the anaerobic conditions was established by E. coli. At a CB:EC inoculation ratio of 3:2, The optimal 1,3-PDO concentration of 8.48 g/L with a yield of 0.42 g/g of glycerol was obtained at a CB:EC inoculation ratio of 3:2. Furthermore, butyric acid was the most abundant byproduct in both the CB mono-culture and the CB:EC co-culture, followed by acetic acid.When compared to the CB mono-culture without NC. butyricum in the CB:KP:EC co-culture. The CB:KP:EC co-culture demonstrated a greater ability to remove oxygen due to E. coli inoculation than the CB:KP co-culture. As a result, at a CB:KP:EC inoculation ratio of 13:1:1, the highest 1,3-PDO concentration of 7.02 g/L was obtained in the CB:KP:EC co-culture. Because C. butyricum and K. pneumoniae competed for glycerol to produce 1,3-PDO, the CB:KP:EC co-culture reduced the final 1,3-PDO concentration compared to the CB:EC co-culture. The ability of KP to produce 1,3-PDO is weaker than the ability of CB, and KP is more competitive than CB under micro-aerobic conditions, causing KP to become the dominant in the CB:KP:EC co-culture.The KP:EC co-culture was similar to the metabolic profile of the KP mono-culture. Lactic acid, succinic acid, acetic acid, and ethanol were among the byproducts, with lactic acid having the highest concentration. When EC was inoculated to the CB:KP co-culture, butyric acid and lactic acid were produced as the main byproduct, indicating that anaerobic cultivation conditions were established for good growth of K. pneumoniae and strictly anaerobic C. butyricum. The abundance of C. butyricum DUT1 decreased from 85.25  to 60.48% (long-term preservation). Meanwhile, the abundance of K. pneumonia DUT2 increased from 0.007 to 38.64%, and the abundance of E. coli DUT3 decreased from 12.54 to 0.41%. Furthermore, investigating and determining the function of other unidentified organisms in consortium DUT08, which had a low abundance, was difficult. As a result, the construction and maintenance of a stable, controlled, and robust microbial consortium is critical for efficient 1,3-PDO production. In this case, we constructed and investigated several synthetic microbial consortia composed of strictly anaerobic C. butyricum, facultative K. pneumoniae, and facultative E. coli with or without N2 supply to efficiently produce 1,3-PDO. The co-production of 1,3-PDO, butyric acid and lactic acid by the synthetic co-culture in batch fermentation under micro-aerobic and anaerobic conditions was investigated, and the results are shown in Fig. In a previous study, we discovered that the natural microbial consortium DUT08 isolated from wastewater samples could efficiently convert crude glycerol to 1,3-PDO under anaerobic and micro-aerobic conditions. The organism compositions in DUT08 changed during the long-term preservation process due to the coexistence of facultative Clostridium butyricum grew well, with no evidence of substrate inhibition. An excellent glycerol consumption rate of 3.55 g/(L.h) was obtained at an initial glycerol concentration of 106.01 g/L and 0.20 vvm N2 supply , respectively. Furthermore, C. butyricum did not grow under 0.02 vvm N2 supply for 3 h at the same initial glycerol concentration, implying that a strictly anaerobic environment did not develop. It has been reported that C. butyricum exhibits substrate inhibition at initial glycerol concentration greater than 70 g/L [C. butyricum at an initial glycerol concentration of 125.18 g/L [C. butyricum grew well with no substrate inhibition at an initial glycerol concentration of 106.01 g/L and a supply of 0.2 vvm N2. However, the cells ceased to grow when supplied with 0.02 vvm N2. Once KP and EC were induced into the mono-culture system, the inhibition of dissolved oxygen was alleviated. As a result, it is safe to keep the glycerol concentration below 110 g/L and no dissolved oxygen in medium to achieve a high 1,3-PDO yield and productivity by C. butyricum.n 70 g/L . Dietz en 70 g/L . In our C. butyricum, K. pneumoniae demonstrated weaker glycerol consumption ability and lower 1,3-PDO productivity than C. butyricum. The fermentation with the KP mono-culture lasted 46 h. The final 1,3-PDO concentration, yield, productivity, and glycerol consumption rate were 45.03 g/L, 0.41 g/g, 0.98 g/(L.h), and 2.37 g/(L.h), respectively. When compared to C. butyricum, 1,3-PDO yield, productivity, and glycerol consumption rate by K. pneumoniae decreased by about 9%, 39%, and 33%, respectively. Lactic acid was obtained as the main byproduct by K. pneumoniae at a concentration of 25.92 g/L. The critical concentration of lactic acid for inhibiting K. pneumoniae cell growth was determined to be 19 g/L under anaerobic conditions and 26 g/L under aerobic conditions, respectively [K. pneumoniae DUT2. Most K. pneumoniae produce 2,3-butanediol  as a byproduct, and the similarity of 2,3-BDO and 1,3-PDO -causes the separation difficulties [Klebsiella pneumoniae DUT2 differs from other K. pneumoniae strains in that it does not produce 2,3-BDO during the fermentation process. The dynamic dissolved oxygen and oxidation–reduction potential (ORP) variation of the KP mono-culture during the fermentation process was studied in a 5-L bioreactor. Within 0.5 h, dissolved oxygen was depleted in the KP mono-culture. ORP fell from − 250 to – 580 mv in 8 h, then remained at − 580 mv for 15 h before rising to − 500 mv at the end of fermentation. According to the findings, K. pneumoniae scavenged oxygen and could create an anaerobic environment for C. butyricum growth in order to produce 1,3-PDO.The performance of the KP mono-culture was also investigated and the results are presented in Fig. ectively . Obviousiculties . KlebsieClostridium, 12.54% Escherichia, and 0.007% Klebsiella [2 supply. Furthermore, byproducts such as 7.14 g/L butyric acid, 2.93 g/L lactic acid, and 6.39 g/L acetic acid were produced . The results showed that E. coli scavenged oxygen faster than K. pneumoniae and provided an anaerobic environment for C. butyricum growth, allowing it to produce 1,3-PDO more quickly. In the CB:EC co-culture with initial inoculation ratio of 92.5:7.5, 51.33 g/L 1,3-PDO with a yield of 0.49 g/g, a productivity of 1.47 g/(L.h) was obtained at an initial glycerol of 107.18 g/L with 0.02 vvm N2 supply  was obtained at an initial glycerol of 107.18 g/L and a NThe metabolic flux distribution of the products was investigated in different cultures, as shown in Fig. 2 supply, which lasted for at least 12 h. The inoculation of KP and EC resulted in a high cell density in the synthetic CB:KP:EC co-culture. When compared to the synthetic consortia, the natural consortium DUT08 demonstrated similar cell growth to the KP mono-culture and a weak 1,3-PDO production capability. Only 44.29 g/L 1,3-PDO was produced, with a yield of 0.36 g/g and a productivity of 0.94 g/(L.h). The results were very similar to the KP mono-culture, indicating that K. pneumoniae has replaced C. butyricum as the dominant bacterium in natural consortium DUT08. The final butyric acid concentration of 1.75 g/L and lactic acid concentration of 22.91 g/L obtained by DUT08 also confirmed this deduction. The results presented here are consistent with those presented in the preceding section, indicating that the CB inoculation ratio was an important parameter in the development of synthetic consortia capable of producing 1,3-PDO efficiently from crude glycerol. Clostridium butyricum was the dominant bacterium in our previous report, accounting for 85.25% of the proportion in DUT08 [A high initial glycerol concentration, as previously stated, inhibits cell growth and reduces glycerol conversion yield. As a result, fed-batch cultivation by various synthetic consortia was developed and compared to evaluate consortia performance. As shown in Fig. 2 supply. The synthetic CB:KP:EC consortium obtained a higher 1,3-PDO concentration of 81.39 g/L under 0.2 vvm N2 supply. However, when N2 supply increased from 0.02 to 0.2 vvm, the 1,3-PDO yield decreased from 0.51 to 0.49 g/g. Butyric acid was the main byproduct when synthetic consortia were used. A synthetic CB:KP:EC co-culture produced more than 17 g/L butyric acid. Furthermore, the synthetic CB:EC co-culture and CB:KP co-culture produced 14.57 g/L and 15.60 g/L butyric acid, respectively. The synthetic consortia are primarily composed of C. butyricum, which is producer of butyric acid. The natural consortium DUT08 and the KP mono-culture were used to produce lactic acid as the primary byproduct. Lactic acid concentrations are 22.91 g/L in the natural consortium DUT08 and 20.81 g/L in the KP mono-culture, respectively.Synthetic consortia of CB:KP, CB:EC, and CB:KP:EC co-cultures produced the strongest 1,3-PDO formation capability. Among the three constructed synthetic consortia, synthetic CB:KP:EC showed the maximum 1,3-PDO concentration of 77.68 g/L, which is 30% higher than CB mono-culture production at 0.02 vvm N2 supply. The results showed that CB growth was inhibited for the first 4 h in two synthetic consortia mentioned above. Following that, the abundance of CB increased to 91.82% and 89.71% within 8 h, respectively, and remained high at 89.27% and 90.27% at 12 h. Under 0.02 vvm N2 supply, E. coli created an anaerobic environment for the CB:EC co-culture in 10 min. As a result, the abundance of CB has always been exceeded than 85%.As shown in Fig. C. butyricum DL07 showed the highest 1,3-PDO concentration even with crude glycerol as feedstock. A final yield of 0.52 g/g with a productivity of 2.85 g/(L.h) was obtained. Compared to pure cultures, the natural microbial consortium C2-2 M and synthetic consortium CB:KP:EC in this study also showed excellent performance for converting crude glycerol to 1,3-PDO. As a result, 82.7 g/L and 81.4 g/L 1,3-PDO were obtained by consortium C2-2 M and synthetic consortium CB:KP:EC under anaerobic conditions, respectively. Among these reported studies, natural microbial consortium C2-2 M performed the highest yield and productivity. Synthetic consortium CB:KP:EC retaining key features of their natural counterparts is a promising approach to overcoming the challenges associated with the natural communities. In this study, synthetic consortium CB:KP:EC reserved the characters of high glycerol utilization efficiency by CB, the adaptability of KP and EC to oxygen. As a result, a strictly anaerobic microbe of CB was successfully achieved to cultivate under aerobic conditions and a comparable 1,3-PDO concentration of 77.7 g/L with a yield of 0.51 g/g was obtained.Up to date, many attempts have been reported to produce 1,3-PDO from glycerol. Table C. butyricum DUT1, and facultative microbes of K. pneumoniae DUT2 and E. coli DUT3 Different from the strictly anaerobic C. butyricum, the synthetic microbial consortia showed low oxygen sensitivity and were able to produce 1,3-PDO under micro-aerobic conditions. In a batch fermentation, the synthetic CB:KP co-culture at an initial abundance ratio of 92.5:7.5, yielded a maximum 1,3-PDO concentration of 52.08 g/L, with a yield of 0.49 g/g and a productivity of 1.80 g/(L.h), which increased by 10%, 9%, and 12%, respectively, when compared to the CB mono-culture under strictly anaerobic conditions. Furthermore, 7.14 g/L butyric acid, 2.93 g/L lactic acid, and 6.39 g/L acetic acid were produced as the main byproducts in the CB:KP co-culture. In a fed-batch fermentation under micro-aerobic conditions, the synthetic CB:KP:EC co-culture showed the maximum 1,3-PDO concentration of 77.68 g/L, which is 30% higher than CB mono-culture. In contrast, a higher 1,3-PDO concentration of 81.39 g/L was obtained by the synthetic CB:KP:EC co-culture under strictly anaerobic conditions. However, the 1,3-PDO yield decreased from 0.51 to 0.49 g/g when cultivation varied from micro-aerobic conditions to strictly anaerobic conditions. In the constructed microbial consortia, the microbes of EC and KP were found to deplete oxygen and provide the anaerobic environment for CB. The investigated results also indicated that CB growth was inhibited for the first 4 h and the abundance of CB exceeded 85% within 12 h. The results demonstrated that the synthetic microbial consortium was an effective and easily controlled cultivation for 1,3-PDO production under micro-aerobic conditions.In this study, the synthetic microbial consortia were successfully constructed by co-culture of three different specified microbes, strictly anaerobic microbe of Clostridium butyricum DTU1, K. pneumoniae DTU2 and E. coli DTU3 were isolated from the natural microbial consortium DUT08, which was enriched and isolated from wastewater in the sewage treatment of a petrochemical company [Clostridium butyricum DTU1 was anaerobically pre-cultured in a serum bottle containing 100 mL nitrogen-gassed sterilized seed medium at 200 rpm and 37 °C for 12 h. Klebsiella pneumoniae DTU2 and E. coli DTU3 were aerobically pre-cultured in a serum bottle containing 100 mL sterilized seed medium at 200 rpm and 37 °C for 12 h, respectively. Seed and fermentation media, as well as crude glycerol used were described as our previous report [ company . Clostris report . Crude gThe synthetic microbial consortia were inoculated to the fermentation medium to determine their ability to convert crude glycerol to 1,3-PDO. The co-culture fermentation was performed as follows. The seed culture for CB, KP, and EC was cultivated separately for 12 h. The optical density at 650 nm for CB and KP was about 2.5 and for EC about 1.5. The total inoculation volume of synthetic consortium for co-culture was 10% (v/v) for shake flask and bioreactor. The inoculation ratio of synthetic consortium refers to the inoculation volume ratio for each microorganism. The OD value for the synthetic seed culture was different with different inoculation ratios. To keep a constant initial OD for fermentations, the synthetic seed cultures were diluted to a similar OD value.Shake-flask culture was carried out in 250-mL serum bottles with medium volume of 200 mL at 37 °C and 200 rpm. The fermentations with the KP mono-culture, the CB mono-culture, the synthetic CB:EC co-culture under anaerobic conditions were lasted for 24 h, and other groups were 48 h. Three replicates were performed for shake-flask cultures.2 supply. Micro-aerobic fermentation was achieved with or without 0.02 vvm N2 supply for 1 h before and 2 h after inoculation. Samples were taken at regular intervals to analyze the concentrations of biomass, glycerol, and products. Two replicates were performed for the batch and fed-batch fermentations. The metabolic flux distributions of the products were calculated by dividing the product formation rates (qp) by the glycerol uptake rate (qs) and multiplying by 100.The batch and fed-batch fermentations were carried out in a 5-L bioreactor with the working volume of 2 L at 37 °C and 200 rpm. The pH was automatically set to 7.0 by adding 5 mol/L NaOH. When the residue glycerol concentration was less than 20 g/L in fed-batch fermentations, continuous feeding was initiated, and the feeding rate was adjusted to keep the glycerol concentration between 20 and 30 g/L. Anaerobic fermentation was performed for 1 h before and 2 h after inoculation with 0.2 vvm NC. butyricum DUT1, K. pneumoniae DUT2, and E. coli DUT3 listed in Table Quantitative real-time PCR was used to examine the bacterial compositions of synthetic microbial consortia. The manufacturer’s instructions were followed when extracting genomic DNATaKaRa MiniBest Bacterial Genome DNA Extraction Kit Ver. 3.0). Samples were taken at regular intervals from batch and fed-batch fermentation . The purity of DNA was checked by using a NanoDrop ND-2000 spectrophotometer  and electrophoresis. Primers were designed based on the differences in 16S rRNA sequences among The reaction was performed in a final volume of 20 μL containing 10 μL of SYBR Premix EX Taq (1X) , 0.8 μL of each primer (0.4 μM), 0.4 μL of ROX Reference Dye II (1X), and 2 μL of genomic DNA (1 ng/μL).E. coli DH5α. The copy number was calculated as follows:The PCR was performed using the following protocol: 95 °C for 30 s, followed by 40 cycles at 95 °C for 3 s, and 60 °C for 30 s. For each sample, the assay was performed at least three times. Three standard plasmids were used to generate standard curves for quantification. Standard plasmid was constructed by pESI-T vector ligation with PCR product of 16S rRNA, and then transforming it into 2–107 copies with R2 of 0.99.qPCR reactions were run on serial dilutions of each standard plasmid to correlate the threshold cycle number  to copy numbers of the target sequence and to generate standard curves for quantification in unknown samples. Standard curves were linear across five orders of magnitude of 10650). Glycerol and main products  were analyzed using HPLC (Waters 600E) equipped with an Aminex HPX-87H column with a column temperature of 65 °C [2SO4 with a flow rate of 0.6 mL/min. The samples for HPLC were centrifuged at 9720×g for 10 min and the supernatant was used for analysis. Before injection, sample solutions were diluted to appropriate concentrations and filtered through a 0.22-μm membrane filter. All data presented are the average of two or three independent experiments performed under the same culture conditions.Biomass concentration was determined by the measurement of optical density at 650 nm (ODof 65 °C . The mob


Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100°C), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90°C for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions , experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment  and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea. Bacillus licheniformis α-amylase has been the main enzyme used in starch liquefaction. This enzyme exhibits optimal activity at 90°C and pH 6.0, which can only partly satisfy the starch liquefaction requirement [4Pyrococcus furiosus, Thermococcus kodakarensis and Sulfolobus solfataricus, while the remainder come from thermophilic bacteria [P. furiosus α-amylase is a hyperthermophilic enzyme that exhibits optimal activity at about 100°C, where it has a half-life of more than 12 h [P. furiosus α-amylase exhibits optimal activity at a pH of about 5.6, with good stability and no less than 80% of optimal activity at pH values from 4.5 to 6.5. These characteristics make P. furiosus α-amylase potentially suitable for industrial applications.Alpha-amylase E.C. 3.2.1.1) is an enzyme that catalyzes the hydrolysis of α-1,4 linkages in starch and related carbohydrates and has been widely applied in starch processing, baking, brewing and the de-sizing of textiles [.1 is an bacteria -9. P. fuhan 12 h . In addiP. furiosus α-amylase has been known and characterized since 1990, but the relatively low level of heterologous expression has limited its industrial application [Escherichia coli, P. furiosus α-amylase is prone to form inclusion bodies. Thus, the highest reported yield has been 11.4 U/ml [P. furiosus α-amylase is also prone to form inclusion bodies when produced in Bacillus using a signal peptide with low secretion efficiency. When using a signal peptide with high secretion efficiency, P. furiosus α-amylase is prone to be degraded by the quality control proteases HtrA/HtrB, or the remaining extracellular proteases, because it folds slowly after translocation across the cytoplasmic membrane [Bacillus subtilis and Bacillus amyloliquefaciens, the highest reported yields have been 0.7 and 2,714 U/ml, respectively. The highest reported production of P. furiosus α-amylase in yeast yielded 220 U/ml [P. furiosus α-amylase in these mesophilic host strains may be caused by poor protein folding in these strains.lication . When pr1.4 U/ml . P. furimembrane -14. When220 U/ml . The relMethanocaldococcus jannaschii, a thermophilic archaean, is a homohexamer that assembles into a filamentous structure. This protein exhibits activity similar to those of the hetero-hexameric prefoldins [cis-trans isomerase (PPIase) is another important type of chaperone. These enzymes catalyze the cis-trans isomerization of prolyl imide bonds in protein, which is a rate-limiting step in protein folding [P. furiosus α-amylase in commonly used mesophilic prokaryotic and eukaryotic host strains.Thermophilic archaea and mesophilic prokaryotic and eukaryotic microbes all express molecular chaperone systems that assist protein folding, but they differ in type, structure, and number , 17. Forefoldins , 22. Pep folding . PPIases folding , 25. TheP. furiosus α-amylase inclusion bodies produced in E. coli into soluble, active enzyme. They include co-expression of heterologous chaperones, like P. furiosus prefoldins, chaperonins and small heat shock proteins, and overexpression of homologous chaperones like GroEL/GroES. Both of these strategies have been shown to reduce the level of inclusion body formation and increase soluble activity, but co-expression of P. furiosus prefoldins has given the best results [E. coli is improved using these methods, E. coli presents a food safety issue that restricts its application in the food processing industry.Many strategies have been employed to convert the  results . Other s results , 28. EveB. subtilis shows great potential for the production of P. furiosus α-amylase because it is non-pathogenic, has powerful secretory ability and can be grown to high cell density [P. furiosus α-amylase production in B. subtilis was improved from 18.3 to 135.1 U/mL through signal peptide optimization and overexpression of the extracellular chaperone PrsA. However, even in this system substantial amounts of inclusion bodies could be found in the cytoplasm. In this study, P. furiosus α-amylase production in B. subtilis was improved using three strategies . Soy meal 1 was purchased from Shandong Longkete Enzyme Preparations Co., Ltd. (China). Soy peptone and soy meal 2 were purchased from Xi Wang Co., Ltd. (China). Yeast extract, peptone, industrial peptone, fish meal peptone, beef extract fermentation, cottonseed meal, bone peptone, tryptone, beef meal, corn steep powder, yeast extract fermentation and beef peptone were purchased from Sinopharm Chemical Reagent Co., Ltd. (China).E. coli JM109 was used as the cloning host for routine plasmid construction. Construction of B. subtilis WS9, used as an expression host and stored in our library, has been described in the literature [P. furiosus α-amylase expression plasmid pHY300PLK/pfa, which contains the dual promoter PHpaII-PamyQ’, a mutant B. subtilis signal peptide AspB (B. subtilis codon usage) α-amylase gene (GenBank No. MT988065), were constructed previously and stored in our laboratory  M. jannaschii prefoldins gene  KH2PO4 2.3, K2HPO4 16.4, glycerol 5, yeast extract 24, and peptone 12, was used for B. subtilis shake-flask fermentation. The medium used for 3-L fermenter cultivation contained (g/l) soy peptone 18, yeast extract powder 9, glucose 5, (NH4)2SO4 2.7, (NH4)2-H-citrate 1, NaH2PO4•H2O 4, K2HPO4 14.6, MgSO4•7H2O 1, Na2SO3 2, AlCl3 0.4, and trace element solution 3 ml/l [2 0.5, ZnSO4•7H2O 0.18, MnSO4•H2O 0.1, Na2-EDTA 10.05, FeCl3 8.35, CuSO4•5H2O 0.16, and C°Cl2•6H2O 0.18. The feeding solution used for 3-L fermenter cultivation contained (g/l) glucose 500, soy peptone 66.7, yeast extract powder 33.3, MgSO4•7H2O 7.9, and trace element solution 40 ml/l. The media described were supplemented with 100 mg/l ampicillin or 20 mg/l tetracycline as needed. Expression plasmid and CRISPR/Cas9 gene insertion plasmid were transformed into B. subtilis using the chemical method of Anagnostopoulos and Spizizen, which is based on the mechanism whereby B. subtilis cell membrane and cell wall form defects under the effects of transformation cultivation medium and condition [LB medium containing (g/l) NaCl 10, peptone 10, and yeast extract 5, was used for routine n 3 ml/l . The traondition .Shake-flask cultivations. For routine plasmid construction and seed culture, E. coli JM109 or B. subtilis strains in 10 ml or 50 ml of LB medium were inoculated with a 0.2% (v/v) portion of inoculum, and then cultivated at 37°C and 200 rpm for 10 h. For shake-flask fermentation, 50 ml of TB medium was inoculated with a 5% (v/v) portion of the seed culture described above and cultivated at 37°C and 200 rpm for 2 h, and then 33°C and 200 rpm for 60 h.3-L fermenter cultivations. A 3-L fermenter  containing 0.9 L of fermentation medium was inoculated with a 10% (v/v) portion of seed culture. After inoculation, the dissolved oxygen (DO) decreased slowly from 100%, and when it fell below 30%, the impeller speed was automatically adjusted between 300 and 700 rpm, and pure oxygen was automatically injected into the fermenter as needed to maintain the DO at about 30%. Approximately about 7 h after inoculation, the DO exhibited a sudden increase, indicating that the glucose in the medium had been nearly exhausted and feeding solution should be added. The feeding rate was increased slowly from 2 to 9 ml/h, such that the glucose concentration remained below 0.5 g/l. The glucose concentration was measured with a SBA-40C biosensor . The temperature was kept at 33°C, and the pH was maintained at 7.0 through the addition of NH4OH and 20% (v/v) H3PO4. Tetracycline (20 mg/l) was added every 48 h and antifoam was added as needed. Culture samples were collected at certain times.prsA2 was constructed through replacing the original promoter of plasmid pBE-S194/prsA (Papr) with the xylose-inducible promoter Pxyl . Plasmid pBE-S194/prsA2 contains a MluI site upstream and a SacI downstream of the prsA gene. Genes encoding M. jannaschii prefoldins and P. furiosus PPIase were inserted into cloning vector pMD18-T, which contains an MluI site upstream and a SacI site downstream of the inserted sequence. These sites were used to excise the desired genes and replace the prsA gene in the plasmid pBE-S194/prsA2, yielding plasmids pBE-S194/pre and pBE-S194/PPI, respectively.The primers used in this study are listed in ter Pxyl . This waxyl, the prsA gene and the terminator were inserted between the upstream and downstream segments of amyE homologous repair templates through overlap PCR with primer pairs P07/P08, P09/P10 and P11/P12, respectively. The overlap nucleotide sequence was then ligated into the NcoI restriction site of plasmid pET24a, yielding plasmid pET24a/prsA. The homologous repair template in plasmid pET24a/prsA was amplified using primer pair P13/P14 and was used to replace the original homologous repair template of pHYcas9damy1, yielding plasmid pHYcas9prsA, of which the other part of pHYcas9damy1 was amplified using primer pair P15/P16. Then, the nucleotide sequences of promoter Pxyl, prefoldins or PPIase encoding gene and terminate were amplified using primer pair P07/P08 and were used to replace the corresponding part of plasmid pHYcas9prsA, yielding plasmids pHYcas9pre and pHYcas9PPI, respectively.CRISPR/Cas9 gene insertion plasmids pHYcas9pre and pHYcas9PPI were constructed by replacing the N20 sequence and homologous repair template of CRISPR/Cas9 disruption plasmid pHYcas9dapr. The N20 sequence of pHYcas9dapr was replaced through inverse PCR with primer pair P05/P06, yielding plasmid pHYcas9damy1. The nucleotide sequences of the promoter PB. subtilis WS9 and transformants were spread onto LB agar containing 20 mg/l tetracycline. The gene insertion mutants were identified using colony PCR with primer pair P17/P18. The PCR products of the desired transformants appeared about 2,000 bp longer than those of transformants containing the normal strain in a gel electrophoresis analysis. Promising PCR products were characterized using DNA sequencing. Finally, the mutant strains were cultivated at 51°C for 10 h to cure the CRISPR/Cas9 gene insertion plasmids, yielding host strains B. subtilis WS9pre and B. subtilis WS9PPI, respectively.CRISPR/Cas9 gene insertion plasmids pHYcas9pre and pHYcas9PPI were individually transformed into The dry cell weight (DCW) was determined as follows: Cells in a 5 ml sample of CB were pelleted by centrifugation at 13,800 g and 4°C for 5 min. The pellet was resuspended with 0.9% (w/v) NaCl solution, re-pelleted, and then dried to constant weight at 105°C.Bacterial cell disruption was performed using ultrasound as follows: Cells in the CB were pelleted by centrifugation at 13,800 g and 4°C for 5 min, and then resuspended in equal volume of deionized water. Lysozyme (0.9 mg/ml) was added to the suspension and the mixture was incubated at 37°C for 30 min. The mixture was then disrupted in an ice water bath using an Ultrasonic Homogenizer , at power setting of 20%, and pulse mold of working 2 s and stopping 3 s, for 10 min. The yielded solution was centrifuged at 13,800 g and 4°C for 5 min to produce an ultrasonic disruption supernatant (UDS) and disruption pellets. The disruption pellets were resuspended with equal volume of deionized water to produce an ultrasonic disruption sediment solution (UDSS).Alternatively, bacterial cells were disrupted using the Bacterial Protein Extraction Kit  as follows: Cells in the CB were pelleted by centrifugation at 13,800 g and 4°C for 5 min. The pelleted cells were resuspended in equal volume of bacterial protein extraction reagent, which contained 0.1 mg/ml of lysozyme and a nonionic detergent. This suspension was incubated at 37°C and 200 rpm for 2 h to disrupt the cells. The yielded solution was centrifuged at 13,800 g and 4°C for 5 min to produce the Bacterial Protein Extraction Kit disruption supernatant (KDS) and disruption pellets. The disruption pellets were resuspended with equal volume of deionized water to produce Bacterial Protein Extraction Kit disruption sediment solution (KDSS).A certain volume of the CB was centrifuged at 13,800 g and 4°C for 5 min to produce culture media (CM) and bacterial cell pellets . The bacThe α-amylase activity was measured by determining the concentration of reducing sugars liberated from soluble starch. The reaction was initiated by adding 0.1 ml of appropriately diluted enzyme solution to a pre-heated assay mixture containing 1 ml of 1% (w/v) soluble starch and 0.9 ml of sodium citrate buffer . The assay solution was then incubated at 100°C in a boiling water bath for 10 min. The assay was terminated by adding 3 ml of 3,5-dinitrosalicylic acid solution. The resulting mixture was boiled for 7 min, and then immersed in ice water. Finally, the mixture was diluted with 10 ml of deionized water and the absorbance at 540 nm was measured with a spectrophotometer. One unit of α-amylase activity was defined as the amount of enzyme that produced 1 μmol of reducing sugar per minute from soluble starch under the condition described above .Protein analysis was conducted by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were analyzed using a 5% stacking gel and a 12.5% separating gel. CB supernatants (20 μl) or UDSs (20 μl) were mixed with 5 μl of SDS-PAGE buffer (5×) and then placed in a boiling water bath for 5 min. Cell ultrasonic disruption sediments of 1 ml CB were mixed with 20 μl of SDS-PAGE buffer (5×), and then placed in a boiling water bath for 5 min. Then, 8 μl of supernatant mixtures and 1 μl of sediment mixtures were loaded onto the gel for analysis. After electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 dye. To determine the identity of the protein present in inclusion bodies identified using SDS-PAGE analysis, the corresponding band was excised and analyzed using matrix-assisted laser desorption/ionization-tandem time of flight mass spectrometry (MALDI-TOF/TOF) by Shanghai Haoze Biomedical Technology Co., Ltd. (China).t test, and differences resulting in values of p <0.05 were considered statistically significant.All experiments were conducted independently at least three times. The results are presented as the mean ± SD. Statistical analyses were conducted using Student’s B. subtilis WS9amy, the P. furiosus α-amylase-producing strain, which was constructed through transforming expression plasmid pHY300PLK/pfa into host strain B. subtilis WS9. An SDS-PAGE analysis of these inclusion bodies contained two bands  of xylose. In the absence of added xylose, the extracellular α-amylase activities of B. subtilis WS9amy, B. subtilis WS9preamy and B. subtilis WS9PPIamy were 135.1, 145.5, and 156.7 U/ml, respectively (B. subtilis WS9amy produced the highest α-amylase activity (161.8 U/ml) with 0.5% xylose, B. subtilis WS9preamy produced the highest α-amylase activity (149.4 U/ml) with 0.25% xylose, and B. subtilis WS9PPIamy showed the highest α-amylase activity without added xylose. It has been reported that promoter Pxyl has weak transcription activity, even in the absence of xylose [B. subtilis WS9PPIamy to assist α-amylase in an unfolded export-competent state, and increased PPIase expression had a negative effect on α-amylase production. Since the α-amylase activity produced by B. subtilis WS9PPIamy in the absence of added xylose was 1.2-fold greater than that produced by B. subtilis WS9amy under the same conditions, and the addition of xylose increases the complexity and cost of fermentation, use of B. subtilis WS9PPIamy in the absence of xylose seems the best choice for the industrial production of P. furiosus α-amylase.The levels of α-amylase produced by ectively -2D. B. sB. subtilis WS9PPIamy, the extracellular activity increased with fermentation time, reaching 1,141.1 U/ml at 310 h (P. furiosus α-amylase is produced within a hyperthermophilic environment (~100°C) and possesses a high level of molecular rigidity, and that its denaturation requires considerable energy (316 kJ/mol), the correct folding of P. furiosus α-amylase may need a high level of energy. Heat treatment can provide energy in a physical way. Meanwhile, the high thermostability of P. furiosus α-amylase protects it from thermal deactivation. It seemed reasonable to investigate whether heat treatment can increase the yield of soluble, active P. furiosus α-amylase through the transfer of kinetic energy , while is much higher than the sum of α-amylase activities in heat-treated CM, UDS and UDSS (654.2 U/ml). These results strongly suggest that whole bacteria must be present for heat treatment to significantly augment α-amylase activity. An alternative explanation, that ultrasonic disruption was too harsh and prevented the heat activation of cellular components, was tested by disrupting the cells using the Bacterial Protein Extraction Kit, which performs a more mild disruption using lysozyme and nonionic detergent. Under these conditions, the α-amylase activities of the KDS and KDSS were 145.6 and 357.8 U/ml, respectively. These results are quite similar to those obtained using ultrasonic disruption. After heat treatment, the enzyme activities of the KDS (148.3 U/ml) was similar with before, while the enzyme activities of the KDSS increased from 357.8 to 515.8 U/ml. Although the total activity of heat-treated CM, KDS and KDS (956.5 U/ml) is still much lower than the activity obtained by heat treating the intact CB, it is 46.2% higher than that of the ultrasonic disruption (654.2 U/ml). These results are partly consistent with the above speculation that ultrasonic disruption was too harsh and prevented the heat activation of cellular components.For the sake of comparison, the α-amylase activity of heat-treated BCS supernatant  was 75.7% of the α-amylase activity of heat-treated CB supernatant , while the α-amylase activity of heat-treated CM (292.4 U/ml) was 20.1% of the α-amylase activity of heat-treated CB supernatant . These results are consistent with the hypothesis that heat treatment might accelerates the unfolding and export of a part-intracellular The α-amylase in the supernatant of heat-treated CB was stored at 4°C or 25°C and samples were assayed at regular intervals, during which the untreated extracellular α-amylase was assayed as control. As shown in P. furiosus α-amylase production by B. subtilis WS9PPIamy, the culture conditions were optimized in shake-flask experiments with a fixed fermentation time of 60 h, during which the extracellular α-amylase activities were measured . Fermentation would be conducted at 33°C and pH 7.0.Based on these experiments, scale-up fermentation was designed to include a fermentation medium containing soy peptone (18 g/l) and yeast extract powder (9 g/l) as its complex nitrogen source, glucose (5 g/l) as its carbon source, and AlP. furiosus α-amylase in a 3-L fermenter was envisioned as an initial fermentation stage (ca. 7 h) followed by a second phase in which the culture was sustained using a feeding solution. Both phases were conducted at pH 7.0 and 33°C. The initial medium was that described in the previous section. The feeding contained soy peptone and yeast extract powder in a 2:1 (w/w) ratio with a total concentration of 100 g/l. The feeding solution also contained 500 g/l glucose. During the cultivation process, the glucose concentration was measured through a biosensor and controlled below 0.5 g/l. Beginning at 60 h of culture, samples were assayed for α-amylase before and after heat treatment. The results are presented in Producing P. furiosus α-amylase export from the cell in an unfolded export-competent state is a slow process that normally leads to inclusion body formation. However, heat treatment adds kinetic energy that might accelerates the process in both shake-flask and 3-L fermenter, causing a part of the α-amylase that would have been lost to inclusion body formation or proteolysis to be exported into the CM as active, properly folded enzyme.In 3-L fermenter cultivation, the appearance of peak heat-treated activity before that of untreated activity was similar to the results seen with shake-flask cultures, supporting our model that B. subtilis has its own powerful secretory ability for its single membrane structure. In order to control the quality of secreted recombinant proteins, B. subtilis uses its own quality control proteases HtrA/HtrB, which can degrade unfolded and misfolded proteins in the interface of membrane and cell wall [P. furiosus α-amylase increased from 18.3 to 135.1 U/ml, and SDS-PAGE analysis showed that substantial amounts of inclusion bodies formed in the cytoplasm, which was similar with that in E. coli [ell wall . The expell wall . Therefoell wall . In our  E. coli . The for E. coli .P. furiosus α-amylase in B. subtilis  may be relatively low and influence the delivery efficiency, which demonstrates that chaperones may be co-expressed with their synergistic chaperones to sufficiently function.In this study, three strategies of thermophilic archaeal chaperone co-expression, heat treatment and fermentation optimization were applied to improve the soluble production of subtilis . The co-one PrsA . Our preficiency , 39. TheP. furiosus α-amylase is natively produced within a hyperthermophilic environment (~100°C) and possesses a high level of molecular rigidity, and as its denaturation requires considerable energy (316 kJ/mol), the correct folding of P. furiosus α-amylase may need a high level of energy [P. furiosus α-amylase is prone to aggregate for its high hydrophobic characteristic, and a substantial portion of P. furiosus α-amylase has been synthesized in 72 h as an inclusion body that slowly unfolded to the export-competent state and secreted into the extracellular space. The heat treatment of intact CB may promote this process through providing kinetic energy. Heat treatment at 90°C for 15 min was shown to be enough for heat activation, demonstrating that the refolding of intracellular P. furiosus α-amylase at high temperature occurred much more quickly, which was consistent with the previous report [P. furiosus β-glucosidase in Saccharomyces cerevisiae was increased 4.4-fold by increasing the cultivation temperature from 30°C to 40°C, and that the increased cultivation temperature promoted the folding and secretion of intracellular β-glucosidase [Besides the above strategy to convert inclusion bodies to soluble form through improving the intracellular folding state, the strategy of heat treatment was applied subsequently. Since f energy , 40, 41.cosidase . The hypcosidase . TherefoThe ultrasonic disruption of the bacterial cell was too harsh and prevented the heat activation of cellular components, while the Bacterial Protein Extraction Kit disruption was mild and partly maintained the heat activation of cellular components. Compared with heat treatments of CM and BCS separately or CM and disruption components separately, heat treatment of intact CB shows the best activation effect. Meanwhile, the mold of heat treating intact CB was simple for application in industrial production and the yielded extracellular enzyme shows good storage stability at both 4°C and 25°C.E. coli (11.4 U/ml) [The shake-flask culture conditions were optimized and included nitrogen source, carbon source, metal ion, fermentation temperature and pH. The 3-L fermenter cultivation condition was then chosen on the base of shake-flask optimization. The highest activity obtained using the 3-L fermenter  was 3.3-fold higher than that obtained prior to heat treatment 1,155.1 U/ml) and 28.2-fold higher than that obtained using shake-flask cultivation 135.1 U/ml). This was also much higher than yields previously reported in .4 U/ml) , B. amyl5.1 U/ml.5.1 U/ml P. furiosus α-amylase in B. subtilis was increased 1.2-fold by co-expression of P. furiosus PPIase and 6.2-fold through heat treatment at 90 °C for 15 min. After optimization of the culture conditions using shake-flask cultures, scale-up in a 3-L fermenter yielded the highest level of soluble, extracellular P. furiosus α-amylase activity yet reported: 3806.7 U/ml. These results demonstrate the effectiveness of thermophilic archaeal chaperone co-expression and heat treatment on hyperthermophilic protein production by B. subtilis. These simple methods may impact the starch processing industry and improve the production of other industrially useful enzymes.In summary, the extracellular production of http://jmb.or.kr.Supplementary data for this paper are available on-line only at


In ovarian cancer (OC), platinum-based therapy remains the front-line therapy, but drug resistance is common. In the current study, we analyzed two Gene Expression Omnibus (GEO) datasets to identify the responsible genes involved in the mechanism of platinum resistance. Thirteen co-upregulated genes and one co-downregulated gene were obtained. Among them, NR2F1 revealed the highest correlation with a poor prognosis. Mechanism research revealed that NR2F1 promotes epithelial-mesenchymal transition (EMT) through TGFβ-1 signaling, participating in platinum resistance. We also found that NR2F1 was positively correlated with the infiltration of immunosuppressive cancer-associated fibroblasts (CAFs). Higher expression of NR2F1 was correlated with a poorer response to immune check blockades including anti-PD-L1. In the future, by analyzing the expression status of NR2F1 and the effect of platinum and immunotherapy in the clinical setting, it is expected that NR2F1 will be established as an effective drug selection marker, guiding treatment selection in OC patients.The mechanism underlying platinum resistance in ovarian cancer (OC) remains unclear. We used bioinformatic analyses to screen differentially expressed genes responsible for platinum resistance and explore NR2F1′s correlation with prognostic implication and OC staging. Moreover, Gene-set enrichment analysis (GSEA) and Gene Ontology (GO) analyses were used for pathway analysis. Epithelial-mesenchymal transition (EMT) properties, invasion, and migration capacities were analyzed by biochemical methods. The association between NR2F1 and cancer-associated fibroblast (CAF) infiltration and immunotherapeutic responses were also researched. A total of 13 co-upregulated genes and one co-downregulated gene were obtained. Among them, NR2F1 revealed the highest correlation with a poor prognosis and positively correlated with OC staging. GSEA and GO analysis suggested the induction of EMT via TGFβ-1 might be a possible mechanism that NR2F1 participates in resistance. In vitro experiments showed that NR2F1 knockdown did not affect cell proliferation, but suppressed cell invasion and migration with or without cisplatin treatment through the EMT pathway. We also found that NR2F1 could regulate TGF-β1 signaling, and treating with TGF-β1 could reverse these effects. Additionally, NR2F1 was predominantly associated with immunosuppressive CAF infiltration, which might cause a poor response to immune check blockades. In conclusion, NR2F1 regulates TGF-β1-mediated EMT affecting platinum sensitivity and immune response in OC patients.  Ovarian cancer (OC) currently ranks fifth in cancer-related mortalities among women and accounts for more deaths relative to any other malignancy influencing the female reproductive system . The staThe nuclear receptor subfamily 2 group F member 1 (NR2F1), a well-established orphan nuclear receptor, was first cloned in 1986 ,8. In paOvarian cancer, which is highly metastatic, displayed epithelial to mesenchymal transition (EMT) features and was characterized by the loss of epithelial marker E-cadherin and the acquirement of mesenchymal markers vimentin and N-cadherin . TGF-β1 http://www.pubmed.com/geo, accessed on 26 March 2022) [p-value < 0.05 and fold-change > 1.5). Venn analysis was performed via Omicstudio  to identify the common DEGs (co-DEGs). We also utilized heatmaps to visualize the expression data of co-DEGs between resistant and sensitive samples among the two datasets, separately.Potential datasets from the GEO database ch 2022)  were sel22  were ch 2022) . GSE5137http://gepia2.cancer-pku.cn/#analysis, accessed on 20 May 2022) portal [https://www.xiantao.love/, accessed on 30 June 2022) and Kaplan-Meier plotter  [To examine the prognostic impacts of co-DEGs in OC patients, the overall survival (OS) significance heatmap data and NR2F1’s survival plots were attained from the GEPIA2 (ne 2022)  to confihttp://cis.hku.hk/TISIDB/index.php, accessed on 30 June 2022) [https://rookieutopia.com/app_direct/BEST/#PageHomeAnalysisModuleSelection, accessed on 5 September 2022) portal. Finally, a chemotherapy-related dataset, GSE47856, was then used to investigate the impact of NR2F1 on the chemotherapy response of OC [The NR2F1 expression pattern in different tissues, including normal ovary tissues, was analyzed through Human Protein Atlas (HPA) , Genotypne 2022)  to compane 2022) , which ine 2022)  and Intene 2022) . In the se of OC . p < 0.05 and false discovery rate (FDR) < 0.25 were considered significantly associated. Only phenotypes and signal pathways of interest were exhibited.To determine the NR2F1-related phenotype and signal pathways, GSEA and GO analysis through the Xiantao tool was performed based on the DEGs between platinum-resistant and -sensitive groups in GSE51373 and GSE131978, respectively. Phenotypes showing adjusted http://timer.cistrome.org/, accessed on 2 July 2022) [Tumor-infiltrating immunocytes linked with NR2F1 expression were investigated by exploiting Tumor Immune Estimation Resource 2.0  . We firsly 2022) , stromally 2022) . Higher ly 2022)  and Wolfly 2022)  were obt2 sterile incubator. Cisplatin was purchased from Sigma. Recombinant human TGF-β1, derived from HEK293 cells, was purchased from R&D system . OC cell lines (A2780 and SKOV3), provided by the Center for Molecular Medicine at Xiangya Hospital, were placed in Dulbecco’s modified eagle medium  with 10% fetal bovine serum  and 1% penicillin-streptomycin  at 37 °C within a 5% CONegative control (NC) and si-NR2F1-1 (targeting sequence: AGCTTCAACTGGCCTTACA), si-NR2F2-2 (targeting sequence: GCAAGCACTACGGCCAATT) were purchased from Suzhou Ribo Life Science . The vectors and siRNAs were transfected, separately, into A2780 and SKOV3 cells utilizing lipofectamine 3000. Cells were stratified into (1) NC group, (2) si-NR2F1 group, and (3) si-NR2F1 group. To explore the role of NR2F1 in cisplatin sensitivity, we treated A2780 and SKOV3 cells with or without cisplatin 24 h after the transfection of siRNAs. Cisplatin was dissolved in DMSO . 3 cells/well). Then, 20 μM cisplatin and a control medium were added for an additional 24 h. For dose-dependent experiments, the cells were separately cultured with cisplatin at 0, 10, and 20 μM for 24 h. CCK-8 test solution  was used to detect cell proliferation in response to cisplatin. After one hour of incubating, cell viability was determined by recording the optical density values at a test wavelength of 450 nm utilizing a VICTOR X2 microplate reader .After 24 h of transfection, A2780 and SKOV3 cells were seeded in 96 well culture plates (2 × 10v/v). Next, the membranes were rinsed thrice in PBST for 3 min, incubated for 1 h with HRP-conjugated secondary antibodies , and finally, protein band intensities were assessed through the Image Lab software . β-actin  served as a normalized control. Protein extracts of OC cells were prepared by applying a RIPA lysis buffer. The proteins were quantified employing a BCA kit, separated electrophoretically on 10% SDS-PAGE gels, electro-transferred to PVDF membranes, and blocked utilizing 5% skimmed milk. Afterwards, membranes were incubated with anti-NR2F1 , TGF-β1 , E-cadherin , N-cadherin , and vimentin  primary antibodies with a dilution of 1:1000  with 10% FBS was pipetted into the bottom chamber. Plates were subsequently incubated within a 5% CO2-contained atmosphere at 37 °C. On the next day, the transwell chambers were fixated utilizing 4% paraformaldehyde for 30 min before undergoing 0.1% crystal violet staining. Invasive cells were determined through cell counting within 5 randomly selected fields per well exploiting the inverted microscope. Taking the group with the largest amount of invading cells as 100%, the relative invasion rate was calculated as follows: relative invasion rate (%) = the number of invading cells in the treated group/the number of invading cells in the control group ×100%.Matrigel was diluted in a serum-free medium to 0.1 mg/mL and immediately put into the apical chamber. The Matrigel was cured by incubating at 37 °C for 30 min. Prior to starting the assay, Matrigel invasion chambers were hydrated for 30 min. After a 24-h serum starvation, A2780 and SKOV3 cells were trypsinized and re-suspended in a serum-free medium at 5 × 106 cells/mL. A 20 μL micropipette tip was adopted to generate a linear wound between cells via scratching the cell monolayers. The cells were then washed and grown in DMEM without FBS. Images were captured at 0 h and after 24 h with a microscope and the data were analyzed utilizing ImageJ Software.Cells were inoculated into 6-well-culture plates at a density of 1.2 × 10−ΔΔct method was exploited to decide gene expression.Forty-eight hours after transfection, the RNA extraction was performed utilizing TRIzol reagent . Reverse transcription was conducted utilizing the PrimeScriptTM RT reagent kit . Afterward, the PCR reaction was done with the following thermal program: 95 °C (30 s), 40 cycles of 95 °C (5 s), and 60 °C (30 s). Data were normalized to β-actin. The primers for TGFβ-1 are 5′-CTAATGGTGGAAACCCACAACG-3′ and 5′-TATCGCCAGGAATTGTTGCTG-3′. The primers for β-actin are 5′-CATGTACGTTGCTATCCAGGC-3′ and 5′-CTCCTTAATGTC ACGCACGAT-3′. The 2The OC tissue array (Cat NO: HOvaC154Su01) was acquired from Shanghai Outdo Biotech. The related clinicopathological details were also offered by this company. Immunohistochemical (IHC) was conducted as previously depicted . Brieflyt-test was employed to determine statistical differences, and for analyses involving three or more groups, one-way ANOVA was employed. The log-rank test was adopted for comparing survival differences. All experiments were repeated thrice, with data reported as mean ± standard deviations (SD). Quantitative data were compared and graphically represented utilizing SPSS 26.0 and GraphPad Prism 8. Significant differences were accepted when p < 0.05.For two-group analyses, Student’s p < 0.05 and FC > 1.5 as cutoff threshold). Using the GEO2R tool, platinum-resistant groups have total of 993 upregulated genes and 832 down-regulated genes in GSE51373, and 126 upregulated genes and 117 down-regulated genes in GSE131978 relative to platinum-sensitive groups , whereas others DEGs showed no obvious significance (p > 0.05) (p = 0.003) and DSS  based on Xiantao tool (p = 0.00022), PFS , and PPS  in patients with OC (p = 9.3 × 10−5), PFS , and PPS  in OC patients receiving platinum (The prognostic significance of co-DEGs expression in OC patients was explored by applying the GEPIA2. As illustrated in  > 0.05) a,b. As wtao tool c,d. Cons with OC e–g. Addiplatinum h–j. The platinum . Taken tp = 0.0566) (p < 0.05) (p < 0.001) as well as the T stage (p = 0.003), yet showed a negative relation to N (p < 0.001) and M stages (p = 0.001). Moreover, there was no significant relationship between NR2F1 expression and age (p = 0.054), ki67 intensity (p = 0.140) and extent (p = 0.057), and EGFR intensity (p = 0.614) and extent (p = 0.450). Additionally, NR2F1 expression and specific genetic mutation related to OC were acquired from TCGA and ICGC cohorts. Based on the TCGA database, no significant difference was observed in BRCA1 (p = 0.3512), BRCA2 (p = 0.6746), and TP53 (p = 0.9223) mutant patients compared to wild-type (p = 0.0238) biased with relatively small sample sizes (p = 0.9626), BRCA2 (p = 0.5586), and ARID1A mutant (p = 0.9225) patients compared to wild-type (p < 0.05) (p < 0.05) (p < 0.05)  a. Simila < 0.05) b. The coild-type –c, whilele sizes . As for ild-type –g. Then, < 0.05) c. In par < 0.05) d,e. More < 0.05) f–h. The p = 0.041) and GSE131978 (p = 0.030). Normalized enrichment scores for invasiveness were 1.927 and 2.706, respectively, indicating that EMT positively correlated with platinum resistance in OC (p < 0.05)  c,d. The  < 0.05) . In concTo assess the effect of cisplatin on NR2F1 expression, A2780 and SKOV3 cells were treated with 0–20 μM cisplatin. As shown in As a critical step for supporting tumor invasion and migration, EMT has been validated as an essential pathway in OC platinum resistance . To explConsidering that the TGF-β pathway serves a pivotal role in EMT progression and TGFβ-1 is a key modulator of the canonical TGF-β signaling cascades , we hypop = 5.96 × 10−10), MCPCOUNTER , and TIDE  algorithms (p < 0.0001) (p =1.4 × 10−36), PDGFRB , alpha-smooth muscle actin (ACTA2) , caveolin-1 (CAV1) , and fibroblast activation protein (FAP)  (p < 0.01) (p < 0.001) (p < 0.001), PFS (p = 0.004) (p < 0.001). The expression of NR2F1 was also increased in anti-PD-L1 nonresponders in the IMvigor210 cohort (p = 0.0043) and Wolf cohort (p = 0.016)  e. Correl× 10−06) f–j. Resu× 10−06) . The CAF× 10−06) k. The er < 0.01) l. Moreov< 0.001) m. Surviv= 0.004) n,o. Take= 0.016) c,d. The = 0.016) e,f.Ovarian cancer, a life-threatening gynecological malignancy, yields a 5-year survival rate of nearly 47%, a number that has been invariable throughout the past two decades . ClinicaUp to now, BRCA1/2, TP53, and ARID1A mutations are commonly used in the diagnosis and prognosis predictions of OC patients. BRCA mutations cause homologous recombination defects (HRD) and are associated with improved sensitivity toward DNA-damaging therapies including platinum chemotherapy in OC patients . TP53 isNR2F1 is an established biomarker of tumor dormancy . StudiesTGF-β1, a multipotent cytokine in the TGF-β family, is overexpressed in OC patients’ serum . IncreasAccumulative studies have revealed that cancer-associated fibroblasts are essential components of the tumor microenvironment (TME), promoting primarily the maintenance, progression, metastasis and therapeutic resistance of OC . MoreoveAlthough this study included analyses on human patient samples and in vitro experiments, additional mice models injected with NR2F1 knockdown cell lines or using NR2F1 knockout mice are required to further investigate the intrinsic mechanisms of NR2F1 in OC.In summary, this study demonstrated that NR2F1 is an upregulated gene in OC platinum-resistant tissues. The augmented expression of NR2F1 indicated dismal prognoses in OC patients. The mechanism by which NR2F1 confers platinum sensitivity in OC may be associated with the induction of EMT through the elevation of TGF-β1. Of importance, our study also discussed that NR2F1 could promote the activation of CAFs by inducing TGFβ signaling, which is associated with a poor immunotherapeutic response. Our study indicated that NR2F1 represents a promising therapeutic target for OC patients to overcome resistance toward chemotherapy and immunotherapy.


To explore the diagnostic value of dynamic enhanced magnetic resonance imaging (MRI) combined with serum CA15-3, CYFRA21-1, and TFF1 for breast cancer.  By means of a retrospective study, 60 breast cancer patients treated in our hospital from January 2018 to December 2020 were selected as the breast cancer group, 60 patients with benign breast lesions were selected as the benign group, and 60 healthy individuals who received physical examination in our hospital in the same period were selected as the control group. All study subjects received dynamic enhanced MRI scan and serological tests, their serum CA15-3 and CYFRA21-1 levels were measured with the electrochemiluminescence instrument and original auxiliary reagent, and the TFF1 level was measured with enzyme-linked immunosorbent assay (ELISA). The MRI performance variation in breast lesion patients was analyzed, the serum CA15-3, CYFRA21-1, and TFF1 levels of study subjects were compared among the three groups, and the efficacy of single diagnosis by dynamic enhanced MRI, CA15-3, CYFRA21-1, or TFF1 as well as combined diagnosis was explored by ROC curves. P < 0.05); 57 breast cancer patients were diagnosed by a combined examination, with a sensitivity of 95.0%, specificity of 83.3%, positive predictive value of 74.0%, negative predictive value of 97.1%, accuracy rate of 87.2%, and AUC (95%CI) = 0.892 (0.840–0.943), indicating a significantly higher diagnostic value of the combined examination than the single examination by CA15-3, CYFRA21-1, TFF1, or MRI.  Dynamic enhanced MRI showed that malignant lesion had obscure boundary, irregular margin, and heterogeneity after enhancement, and the time-signal intensity curve presented fast-in fast-out; the benign lesion had a clear boundary and smooth margin, 25 cases showed homogeneity after enhancement, and the time-signal intensity curve presented slow-in slow-out; the CA15-3, CYFRA21-1, and TFF1 levels were significantly different among the breast cancer group, benign group, and control group (33.81 ± 12.46 vs 19.02 ± 6.47 vs 9.55 ± 2.64, 4.08 ± 1.41 vs 1.96 ± 1.19 vs 0.99 ± 0.21, 1.39 ± 0.54 vs 1.04 ± 0.26 vs 0.89 ± 0.12,  Combining dynamic enhanced MRI with serum CA15-3, CYFRA21-1, and TFF1 has good efficacy in diagnosing breast cancer, which can be applied in clinical diagnosis of breast cancer. Breast cancer is a malignant tumor originating from mammary epithelial cells, and its early manifestations mainly include breast masses and auxiliary lymphadenectasis. With disease progression, distant metastasis of cancer cells may occur, leading to multiple organ lesions, which seriously affect the life health of patients . AccordiA total of 60 breast cancer patients treated in our hospital from January 2018 to December 2020 were selected as the breast cancer group, 60 patients with benign breast lesions were selected as the benign group, and 60 healthy individuals who received physical examination in our hospital in the same period were selected as the control group. Patients in the benign group and control group were pathologically diagnosed to exclude malignant lesions and had normal communication skills. All breast cancer patients met the following inclusion criteria: (1) the patients were diagnosed with breast cancer after pathological examination ; (2) theP > 0.05), presenting value of research.All patients in the three groups were females. In the breast cancer group, patients' mean age was (45.37 ± 5.23) years, body mass was (62.12 ± 2.32) kg, and tumor diameter was (17.65 ± 1.23) mm; according to breast cancer TNM staging of Union for International Cancer Control (UICC), there were 4 cases in stage I, 26 cases in stage II, 25 cases in stage III, and 5 cases in stage IV; there were 15 cases with papillary carcinoma, 12 cases with intraductal carcinoma, 23 cases with invasive ductal carcinoma, and 10 cases with carcinoma simplex. In the benign group, patients' mean age was (45.65 ± 5.29) years and body mass was (62.35 ± 2.21) kg; there were 12 cases with breast cyst, 20 cases with fibromatosis of the breast, 5 cases with benign phyllodes tumor of the breast, 15 cases with hyperplasia of mammary glands, and 8 cases with mastitis. In the control group, patients' mean age was (45.38 ± 5.21) years and body mass was (62.41 ± 2.30) kg. No statistical differences in patients' general data including age and body mass among the three groups were observed , and the1WI sequence, cross-sectional STIR sequence, and sagittal T2WI with fat suppression sequence were conducted in turn. Then, dynamic enhanced scanning was performed, the parameters of sagittal plane scanning were TR/TE 4.5 ms/min, FOV 22 cm, matrix 288×160, layer thickness of 3 mm, and flip angle of 15°. 0.2 mL/kg of gadopentetate dimeglumine injection  was administered to patients at a rate of 3 mL/s, and bolus injection was followed by continuous acquisition for 60 s per phase.The 1.5 T superconducting MRI scanner  manufactured by Siemens AG, Germany, was adopted. The study subjects were in the prone position, with the bilateral mammary glands being naturally symmetric and overhanging within the examining hole of a 4-channel phase array surface coil for the breast, and the examinations of cross-sectional TThe scanned images were entered into the workstation for image processing, and image evaluation was performed by 3 highly qualified radiologists to observe the boundary, morphology, signal, etc. Of the tumor, after the enhancement, in addition to the routine observation of indicators, the enhancement form of the lesions, the time-signal curve, etc., were observed.TFF1. Four ml of fasting venous blood was drawn from the study subjects, let to stand for 20 min, and then centrifuged for 5 min under 3,000 r/min to extract the supernatant, and the TFF1 level was measured with enzyme-linked immunosorbent assay (ELISA) .CA15-3 and CYFRA21-1. Five ml of fasting venous blood was drawn from the study subjects, let to stand for 20 min, and then centrifuged for 10 min under 3,000 r/min to extract the supernatant, and the CA15-3 and CYFRA21-1 levels were measured with a Switzerland Roche electrochemiluminescence instrument  20113402843).MRI. The results were determined by 3 radiologists through discussion.Serological Indicators. CA15-3 ≥ 25 U/ml indicated positive, CYFRA21-1 ≥ 3.5 ng/mL indicated positive, and TFF1 S/N value  ≥ 2.1 indicated positive. When combining the three serological indicators and MRI in diagnosis, if any result was positive, it was determined as positive, and if four results were negative, it was determined as negative.Diagnostic Efficacy. ① Sensitivity: the number of true positive cases/ ∗ 100%; ② specificity: the number of true negative cases/ ∗ 100%; ③ positive predictive value (PPV): the number of true positive cases/; ④ negative predictive value (NPV): the number of true negative cases/.2 test and t-test, and differences were considered statistically significant at P < 0.05.In this study, the data processing software was SPSS20.0, the picture drawing software was GraphPad Prism 7 , the items included were enumeration data and measurement data, the methods used were XT1 and long T2, 5 cases showed short T1 and long T2, and 58 cases showed hyperintensity on T2WI and in 42 cases, early enhancement was seen on contrast-enhanced scans and the enhancement was not uniform. In terms of the time-signal intensity curves, there were 3 cases of the inflow type, 15 cases of the plateau type, and 42 cases of the outflow type.MRI of the breast cancer group showed an obscure boundary and irregular margin. On routine MRI, 40 cases showed long 1WI. No enhancement was seen on contrast-enhanced scans of 35 cases, and in 25 cases, the enhancement was uniform. In terms of the time-signal intensity curves, there were 45 cases of the inflow type, 13 cases of the plateau type, and 2 cases of the outflow type.MRI of the benign group showed a clear boundary and smooth margin, in a quasi-circular, circular, or leaf shape, and hyperintensity, isointensity, or hypointensity on TP < 0.05)  .P < 0.05).The CA15-3, CYFRA21-1, and TFF1 levels were significantly different among the breast cancer group, benign group, and control group (33.81 ± 12.46 vs 19.02 ± 6.47 vs 9.55 ± 2.64, 4.08 ± 1.41 vs 1.96 ± 1.19 vs 0.99 ± 0.21, 1.39 ± 0.54 vs 1.04 ± 0.26 vs 0.89 ± 0.12, For results of single diagnosis by CA15-3, CYFRA21-1, TFF1, or MRI and of combined examination, see For the diagnosis efficacy of CA15-3, CYFRA21-1, TFF1, and MRI, see Breast cancer, a malignancy resulting from uncontrolled proliferation of mammary epithelial cells, occurs more often in women and less frequently in men. The morCurrently, CA15-3 is one of the most common serum markers of breast cancer, which is a variant of the epithelial surface glycoprotein of breast cells, was first found on the breast cancer cell pellicle, and exhibits a high expression status in breast cancer tissue, so it can be used to determine the condition of breast cancer lesions. The diaIn conclusion, breast cancer is a relatively common malignancy in the clinical field and increasing the detection rate of this disease is beneficial for reducing the medical burden of breast cancer in China. The study found that combining dynamic enhanced MRI with serum CA15-3, CYFRA21-1, and TFF1 has a good efficacy in diagnosing breast cancer, which can be applied in the clinical diagnosis of breast cancer.


Mycobacterium tuberculosis that removes 4′-phosphopantetheine (Ppt) from holo-carrier proteins (CPs) and thereby opposes the action of phosphopantetheinyl transferases (PPTases). PptH is the first structurally characterized enzyme of the phosphopantetheinyl hydrolase family. However, conditions for optimal activity of PptH have not been defined, and only one substrate has been identified. Here, we provide biochemical characterization of PptH and demonstrate that the enzyme hydrolyzes Ppt in vitro from more than one M. tuberculosis holo-CP as well as holo-CPs from other organisms. PptH provided the only detectable activity in mycobacterial lysates that dephosphopantetheinylated acyl carrier protein M (AcpM), suggesting that PptH is the main Ppt hydrolase in M. tuberculosis. We could not detect a role for PptH in coenzyme A (CoA) salvage, and PptH was not required for virulence of M. tuberculosis during infection of mice. It remains to be determined why mycobacteria conserve a broadly acting phosphohydrolase that removes the Ppt prosthetic group from essential CPs. We speculate that the enzyme is critical for aspects of the life cycle of M. tuberculosis that are not routinely modeled.Phosphopantetheinyl hydrolase, PptH (Rv2795c), is a recently discovered enzyme from IMPORTANCE Tuberculosis (TB), caused by Mycobacterium tuberculosis, was the leading cause of death from an infectious disease before COVID, yet the in vivo essentiality and function of many of the protein-encoding genes expressed by M. tuberculosis are not known. We biochemically characterize M. tuberculosis’s phosphopantetheinyl hydrolase, PptH, a protein unique to mycobacteria that removes an essential posttranslational modification on proteins involved in synthesis of lipids important for the bacterium’s cell wall and virulence. We demonstrate that the enzyme has broad substrate specificity, but it does not appear to have a role in coenzyme A (CoA) salvage or virulence in a mouse model of TB. Ppt, also called P-PantSH, is the nonadenosyl moiety of CoA that is essential for the synthesis of fatty acids, polyketides, and nonribosomal peptides in microorganisms . The covMycobacterium tuberculosis are family I AcpS and family II PptT  and that purified, recombinant Rv2795c removed Ppt from the holo-CP AcpM  and NRPS (EcEntB [E. coli]) families. Lysates from M. tuberculosis lacking PptH lost the ability to dephosphopantetheinylate holo-AcpM. However, no role could be ascribed to PptH in CoA salvage, nor did the enzyme provide a survival benefit for M. tuberculosis during infection of mice. Current efforts involve investigating the role of PptH in the life cycle of M. tuberculosis that are not routinely modeled, such as transmission.Here, we have characterized the metallophosphodiesterase activity of E. coli BL21-AI and purified as reported (p-nitrophenyl phosphate (pNPP) and bis(p-nitrophenyl) phosphate (bis-pNPP), respectively. PptH failed to hydrolyze pNPP but hydrolyzed bis-pNPP to the chromogenic p-nitrophenol (pNP) product as detected by its absorbance at 405 nm. The reaction was carried out at pH 7.0 in the presence  was overexpressed in reported   by 2-fold or 3-fold, respectively, with maximum activity observed with 62.5 μM Mn2+ (8-fold increase) or Fe2+ (11-fold increase)  . Mn2+ anpNPP by PptH was tested over the pH range of 6 to 9, using MES (morpholineethanesulfonic acid) as the buffer at pH 6.0 and 6.5 and Tris over the range of 7.0 to 9.0. Phosphodiesterase activity was optimal at pH 7.0 to 7.5  of both polyP and AMP were high  , arguing against their potential to serve as physiologic modulators. We cannot rule out the possibility that some of these small molecules serve as substrates for PptH, as is the case for AcpH and Rv0805, another annotated metallophosphoesterase from M. tuberculosis , ATP, ADP, AMP, cAMP, guanosine tetraphosphate (ppGpp), coenzyme A (CoA) and polyphosphate (polyP). Only polyP and AMP reduced enzyme activity by >50% at 1 mM  . The 50% inhibitory concentrations , K. pneumoniae (KpACP), and P. aeruginosa (PaACP). Sfp catalyzed the transfer of *Ppt from *CoA to each of the CPs to form *holo-CPs  results in conditional knockdown (cKD) of panB to deplete the cell’s CoA pool   , and res0, 3 μM) . In the  formats  and C, a formats  and C. TMycobacterium leprae with its greatly reduced genome, suggesting that PptH has a critical function that might be manifest in an infected host. To test this, we infected C57BL/6 mice with M. tuberculosis strains WT, ΔpptH, ΔpptH:WTpptH, and ΔpptH:pptHH246N. All strains achieved a comparable bacterial burden in lungs through 150 days of infection  of PptH is similar to that of PaAcpH (Ppt hydrolase from P. aeruginosa) on the artificial substrate bis-pNPP  and its low maximal velocity  compared to those of Rv0805 . Although PptH phosphodiesterase activity is inhibited by polyP and AMP, this effect is only evident at concentrations above the physiologic range.Biologic and biochemical evidence indicated that PptH catalyzes the removal of Ppt from holo-CPs and converts at least one of them into apo-CPs . The crybis-pNPP . A compar center , 16 undeM. tuberculosis PptH catalyzes the removal of Ppt from the M. tuberculosis holo-PKS13-ACP domain. PKS13 is essential for mycolic acid biosynthesis and is activated by PptT  for an assay to visualize the release of Ppt from CPs. This probe is reminiscent of those developed previously , 18 but  by PptT . M. tubeificity 35.M. tuberculosis does not encode a homolog of ATPHs, and since PptH has broad specificity, we reasoned that it may be the only Ppt hydrolase in M. tuberculosis. Whole-cell lysates from wild-type M. tuberculosis hydrolyzed holo-AcpM, whereas lysates from M. tuberculosis lacking PptH or lacking functional PptH (H246N) did not. These results suggest that PptH is likely the only protein responsible for removing Ppt from holo-AcpM in M. tuberculosis and may be the primary Ppt hydrolase in M. tuberculosis. It is possible that under other conditions, including conditions of stress, another hydrolase can compensate for the loss of PptH and perform an analogous function.AcpH appears to be limited to Gram-negative bacteria, and PptH, to mycobacteria , 7, 8. MM. tuberculosis whose growth is limited by reduced synthesis of CoA. Jackowski and Rock found that the rate of turnover of Ppt from holo-ACP is increased when CoA is low  (600) of 0.8 followed by cold shock on ice for 20 to 30 min. Inducers  were added to the culture, which was kept shaking overnight at 18°C. Bacteria were pelleted by centrifugation and lysed using a French press by resuspending the cell pellet in 50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5 mM MgCl2, 10% glycerol, and 5 mM imidazole. Cell debris was separated by centrifugation, and the supernatant was incubated with Ni-nitrilotriacetic acid (NTA) beads for 1 h at 4°C. Beads were loaded onto a gravity column and washed with buffers of 50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5 mM MgCl2, and 10% glycerol containing 10 mM, 20 mM, or 50 mM imidazole. Protein was eluted using 50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5 mM MgCl2, 10% glycerol, and 250 mM imidazole. The eluted protein was dialyzed in 50 mM Tris-HCl, pH 8.0, 20 mM NaCl, 5 mM MgCl2 and 10% glycerol, applied to a HiTrap Q HP anion exchange column, and eluted with a linear gradient of 0 to −500 mM NaCl. The protein yield was ∼2.2 mg/liter of culture. The protein was flash-frozen in liquid N2 and stored at −80°C until use.An overnight culture of  to 310)  was diluE. coli , K. pneumoniae (ACP), and P. aeruginosa (ACP) were cloned in a pET 28a vector and expressed in BL21-AI (primers are listed in Table S1). M. tuberculosis PKS13 ACP domain was cloned into pMCSG7 (primers are listed in Table S1) and expressed in BL21-AI. Overnight cultures of the transformants were inoculated into fresh 2YT medium and grown to log phase. The culture was put on ice for 20 to 30 min and induced with 0.3 to 1 mM IPTG and 0.2% arabinose for 3 h at 30°C with shaking. AcpM was expressed and purified as reported  and lysed by French press. Cell debris was separated by centrifugation, and supernatant was incubated with Ni-NTA beads for 1 h at 4°C with shaking. Protein-bound beads were washed with 50 mM Tris pH 7.5, 1 M NaCl, 10% glycerol, 5 mM DTT, and 20 mM imidazole. Protein was eluted in fractions using 50 mM Tris, pH 7.5, 200 mM NaCl, 10% glycerol, 5 mM DTT, and 250 mM imidazole, dialyzed in low-salt buffer  applied to a HiTrap Q HP anion exchange column, and eluted as for PptH. Purification of EcEntB, EcACP, KpACP, and PaACP yielded ∼5 to 10 mg/liter of culture, and PKS13 yielded ∼3 mg/liter of culture. All proteins were flash-frozen in liquid NSfp phosphopantetheinyl transferase (Sfp) was prepared as described  and stored at −80°C until purification. Cells were thawed in cold lysis buffer  and lysed by sonication. The cell lysate was cleared of debris by centrifugation  before incubation with 50% TALON cobalt affinity-resin (4 ml/liter culture) for 1 to 2 h at 4°C in a batch-binding format. The protein-immobilized resin was loaded on a gravity column and washed with lysis buffer containing 10 mM imidazole at room temperature. Bound Sfp was eluted with lysis buffer supplemented with 100 mM imidazole. Fractions containing purified Sfp, as determined by SDS-PAGE with Coomassie staining, were combined and concentrated using an Amicon Ultra 0.5 ml 10K MWCO filtration device  before dialysis into 50 mM Tris and 5% glycerol, pH 7.5. Protein concentration was determined by Bradford assay.escribed . Brieflyp-nitrophenyl phosphate (pNPP) or phosphodiesterase substrate (bis (p-nitrophenyl) phosphate [bis-pNPP]) in 50 mM Tris-HCl, pH 7.0, with or without 500 μM MnCl2 and 500 μM FeCl2 at 37°C in a 384-well plate, measuring absorbance of the p-nitrophenol (pNP) product at 405 nm for 5 min. The molar extinction coefficient of pNP 18,000 M−1 cm−1 was used to calculate specific activity.PptH activity was tested using 500 nM PptH and 20 mM phosphomonoesterase substrate 2+ in all steps of the purification, and incubated with 2 mM EDTA for 3 h at 25°C, followed by dialysis against 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 10% glycerol before assay with bis-pNPP as described above, adding metal ions at a final concentration of 0 to 500 μM or at 500 μM if not otherwise indicated.PptH was purified as described, excluding Mg2, and 20 mM bis-pNPP in 50 mM buffers adjusted to the indicated pHs in a 25-μl reaction volume in 384-well assay plates. Reactions were stopped with 40 mM EDTA and 1/10th reaction volume of 2 N NaOH after 3 min. NaOH was added to alkalinize the reaction mixture and convert p-nitrophenol to p-nitrophenolate, whose absorbance was monitored at 405 nm.PptH activity was tested at 37°C using MES (pH 6 to 6.7) and Tris-HCl (pH 7.0 to 9.0) buffers with 500 nM PptH, 500 μM MnClpNPP were incubated in 50 mM Tris-HCl, pH 7.0, and 500 μM MnCl2 with NaCl (100 to 500 mM), and formation of pNP was monitored at 405 nm for 5 min.PptH (500 nM) and 20 mM bis-2), 500 nM PptH was added along with increasing concentrations of bis-pNPP in 25-μl reaction volumes at 37°C, monitoring absorbance of pNP at 405 nm for 5 min. mK and Vmax were determined by fitting data to the Michaelis-Menten equation with nonlinear regression analysis using GraphPad Prism software.Under optimized reaction conditions , ppGpp, 3′,5′-cAMP, 3′,5′-cGMP, or CoA with 500 nM PptH in 50 mM Tris-HCl, pH 7.0, 100 mM NaCl, 500 μM MnCl2. The IC50 was calculated using GraphPad Prism software.The assay with bis-18 column, eluting with a 5 to 40% ACN/10 mM NH4HCO3 gradient and a flow rate of 20 ml/min for 12-min runs to afford the desired biotin-PEG-ACM-CoA as the ammonium salt (7 mg [33% yield]). Biotin-PEG-ACM-CoA 1H NMR spectrum peaks : δ 8.60 , 8.34 , 6.15 , 4.80 , 4.51 , 4.32 , 4.18 , 3.97 , 3.78 , 3.59 to 3.53 , 3.40 to 3.28 , 3.21 , 2.91 , 2.70 , 2.63 , 2.40 , 2.19 , 1.64 to 1.49 , 1.31 , 0.86 , 0.73 ; 31P NMR δ 0.17 to 11.10, −11.20, –11.70, –11.79; electrospray MS calculated for C39H66N11O21P3S2, 1,181.31, found 1,182.72 M+H, 591.83 M/2+H.All the solvents were deoxygenated with argon prior to additions. A solution of iodoacetyl-PEG2-biotin  in DMSO (1 ml) was added to the mixture of CoA tri-lithium salt  in 8 mM phosphate buffer . The reaction mixture was stirred in the dark for 6 h at ambient temperature. Reaction progression was monitored by ultra-high-pressure liquid chromatography (UPLC), and the crude mixture was diluted with 3 ml water and was purified directly via preparative high-pressure liquid chromatography (HPLC) on an AutoPure system from Waters Corp.  using an Atlantis C2 containing 10 μM *CoA probe (biotin-PEG-ACM-CoA). The reactions were maintained at 37°C in a heat block for 1 h. Product was visualized using IRDye 800CW streptavidin (LI-COR Biosciences) following SDS-PAGE and transfer to nitrocellulose. Images were scanned using an Azure c600 gel imaging system in the near-infrared region.CPs (each 10 μM) were incubated with 1 μM Sfp in 50 mM Tris-HCl, pH 7.5, 25 mM NaCl, and 15 mM MgCl2, washed in 10K MWCO Amicon centrifugal filters, rediluted to the original volume, and used as candidate substrates for PptH. The in vitro reactions used approximately 10 μM labeled CPs, 500 nM PptH in 50 mM Tris-pH 7.5, 100 mM NaCl, 1 mM DTT, and 100 μM MnCl2 at 37°C in a heat block. At 0, 6, 30, 90, and 180 min (AcpM) and 0, 30, 120, 210, and 300 min , samples were removed and mixed with Laemmli buffer to stop the reactions. As a control, labeled CPs were incubated without PptH. Samples were run on 15% SDS-PAGE and transferred to nitrocellulose membranes. Following ponceau S staining, the blot was blocked in Pierce protein-free phosphate-buffered saline (PBS) blocking buffer for 1 h at room temperature (RT), rinsed with Tris-buffered saline with Tween 20 (TBST), and probed with IRDye 800CW streptavidin  for 45 min at RT. The antibody was removed, the blot was washed 4 times (5 min each time) with TBST, and the image was captured using an Azure c600 gel imaging system in the near-infrared region.Labeled CPs obtained from the Sfp-catalyzed reactions described above were diluted approximately 10-fold with 50 mM Tris-pH 7.5, 100 mM NaCl, 1 mM DTT, and 100 μM MnCl2 and reacted with 500 nM PptH in 50 mM Tris-pH 7.5, 1 mM DTT, and 100 μM MnCl2. At 0, 6, 30, 90, and 180 min, samples were removed and processed for mass spectrometry as follows. Each ultra-high-pressure liquid chromatography (UPLC) sample was prepared by addition of 20 μl of phosphate buffer (pH 7.2) to sample solution (50 μl). Injections were set at 5 μl each on an Acquity UPLC connected to a single quadrupole (SQ) mass detector, C18 column  from Waters Corp.  using the gradient elution system with 2 to 25% acetonitrile in water (both containing 0.01% formic acid) for an 8-min run.Sfp-catalyzed labeled AcpM (biotin-labeled holo-AcpM) was washed with 50 mM Tris-pH 7.5, 1 mM DTT, and 100 μM MnClM. tuberculosis cell lysates were prepared by growing WT, ΔpptH, ΔpptH:pptHWT, and ΔpptH:pptHH246N strains in 7H9 medium (40 ml) to an OD600 of 0.8. Cells were pelleted and washed twice with PBS-0.02% tyloxapol. Pellets were resuspended in buffer , and ∼200 μl of zirconium beads was added for bead beating 3 times for 30 s each time at 400 × g with 1-min intervals on ice. The lysates were separated from the beads by centrifugation at 800 × g for 5 min, and supernatant was passed through a spin-X centrifuge tube filter (0.22-μm cutoff). Reactions  used 18 μg protein of lysate from each strain and 1.5 μg holo-AcpM in 50 mM Tris-pH 7.5, 100 mM NaCl, 1 mM DTT, and 100 μM MnCl2. After 0, 3, and 18 h of incubation at 37°C, samples were removed and mixed with Laemmli buffer for SDS-PAGE followed by immunoblotting with anti-His antibody .M. tuberculosispanB cKD was a kind gift of V. Mizrahi -3-N-ethylcarbamimodoyl]urea) at final concentrations of 12 μM (4× MIC) and 25 μM (8× MIC). Plates were incubated for 3 to 4 weeks at 37°C, and the surviving colonies were isolated. Colonies were expanded. We extracted DNA from strains with confirmed resistance to 8918 but without resistance to isoniazid (INH), rifampicin (RIF), or moxifloxacin (MOXI) and sequenced the region of the genome encoding pptH and pptT (rv2795c and rv2794c). We selected a clone with the point mutant PptH C225R. To test the effect of transcriptional silencing of Tet-OFF panB cKD and panB cKD pptH C225R, cultures were grown in 7H9 medium with 0 to 1,250 ng/ml of anhydrotetracycline (Atc). At day 11, the OD600 was measured. From the same inoculating cultures containing Atc, 10 μl was spotted on 7H10 agar plates and incubated for 2 to 4 weeks. In a separate experiment, we also spotted strains grown in the absence of Atc onto 7H10 agar plates containing 0 to 1,250 ng/ml Atc. These experiments gave the same results. Mizrahi . To selepptH, ΔpptH:pptHWT, and ΔpptH:pptHH246N strains were grown as suspension cultures in 7H9 medium containing 0.5% glycerol and 0.05% Tween 80 and were harvested while in the logarithmic growth phase. The bacteria were pelleted by centrifugation; the pellet was resuspended in phosphate-buffered saline (PBS) supplemented with 0.05% Tween 80 and was centrifuged at 800 rpm for 10 min. The supernatant, containing a single cell suspension of M. tuberculosis, was diluted in PBS for infection via the respiratory route in an aerosol infection apparatus in a biologic safety level 3 facility. The apparatus is a self-contained inhalation exposure system (IES) from Glas-Col  that employs a Venturi-nebulizer unit to expose mice to aerosolized M. tuberculosis. The 8-week-old female C57BL/6 mice (Jackson Laboratory) inhaled 100 to ∼200 CFU during a 40-min nebulization. Groups of 5 mice (4 on day 1) were euthanized with CO2 at days 1, 21, 122, and 150. At each time point, lungs, liver, and spleen were harvested. Each organ (except the upper lobe of the left lung) was homogenized, serially diluted, and plated for determination of CFU on 7H10 agar containing 10% oleic acid-albumin-dextrose-catalase (OADC) (Difco) and 0.5% glycerol, and the colonies were counted at 3 weeks. For histopathology, at each harvest time point, the upper lobe of the left lung was fixed in 10% formalin in PBS for 24 h at room temperature, followed by embedding, sectioning, staining with hematoxylin and eosin, and photomicroscopy.The WT, ΔAll data are contained within the manuscript.