Bacillus idriensis CT-WN-B3, Bacillus australimaris CT-WL5-10, and Pseudomonas oleovorans CT-WL5-6, were screened out under alkaline conditions from the alkali-tolerant strains distributed in carbonate saline-alkali soil and water environments in Northeast China. Under different pH (8.0–9.0), salinities (10–30 g/L NaCl), alkalinities (10–30 mmol/L NaHCO3), and AN concentrations (1–3 mg/L), corresponding to the actual conditions of saline-alkali aquaculture waters, the AN removal rates and relative characteristics of these strains were analyzed. The results showed that all of the three strains were efficient on AN removal under various conditions, and the highest removal rate reached up to 3 × 10–13 mg/cfu/h. Both CT-WL5-10 and CT-WL5-6 were most efficient under pH 9.0 with 3 mg/L initial AN, while pH 8.5 with 2 mg/L AN was the best fit for CT-WN-B3. In 96-h pure incubation of these strains in alkali media, approximately 90% AN was removed, and pH values were decreased by 2.0 units within 12 h accompanied by the growth of the strains. In addition, salinity and alkalinity slightly disturbed the removal rates of CT-WL5-10 and CT-WL5-6, but there were at least 65% AN removed by them within 24 h. These results indicated that all three strains have good application prospect in saline-alkali aquaculture waters.The toxicity of ammonia nitrogen (AN) has always caused severe harm to aquatic animals in intensive aquaculture conditions, especially in saline-alkali aquaculture waters. The application of AN removal bacteria is a safe and effective method for controlling the AN concentration in aquaculture water through direct conversion to bacterial protein. However, there is still a lack of AN removal bacteria that are appropriate for saline-alkali aquaculture conditions. In this study, three AN removal strains, namely,  Ammoniaia (NH3) . Then, sia (NH3) .3) direction in NH3⋅H2O⇌NH4+ + OH– and could enhance the proportion of ammonia in the same concentration of AN  containing 10.0 g of tryptone, 5.0 g of yeast, and 10.0 g of NaCl in 1,000 ml of filtered water was prepared, and the pH was adjusted to 7.0 with NaOH.4)2SO4 (corresponding to 50 mg/L of AN), 1.0 g of NaCl, 0.5 g of K2HPO4, 0.25 g of MgSO4⋅7H2O, and 15.0 g of agar powder in 1,000 ml of filtered water was prepared, and the pH was adjusted to 7.0 with NaOH.A primary screening medium containing 5.0 g of glucose, 0.183 g of (NH4)2SO4 (corresponding to 10 mg/L of AN), 1.0 g of NaCl, 0.5 g of K2HPO4, and 0.25 g of MgSO4⋅7H2O in 1,000 ml of filtered water was prepared, and the pH was adjusted to 7.0 with NaOH.A rescreening medium containing 5.0 g of glucose, 0.037 g of , and a certain amount of glucose (guaranteed to achieve C/N 20:1) in 1,000 ml of filtered water was prepared, and the pH levels were adjusted to the required values  with HCl or NaOH. To explore the effect of salinity and alkalinity on the removal rate, a certain amount of NaCl  or NaHCO3  was added to the media as required, and NaOH was used to guarantee pH 9.0.An AN removal detection medium containing 1.0 g of NaCl; 0.5 g of K–4, 10–5, and 10–6 with aseptic water. A volume of 100 μl of each of the diluted solutions of the 24 strains was spread on the primary screening media, respectively, and incubated at 30°C for 5 days. The strains that grew well were picked out.Primary screening: In total, 24 alkali-tolerant strains were purely cultured in activation media overnight at 30°C and 150 rpm, and centrifuged at 12,000 rpm and 4°C for 10 min. The supernatants were discarded, washed two times with aseptic water, and resuspended in the same volumes of aseptic water. Then, the suspensions were gradient diluted to 10Rescreening: The screened strains were reactivated, and the cell suspensions were prepared according to the same method as used in the “primary screening” step. Then, the bacterial suspension was inoculated into the rescreening medium at a 1% inoculation amount and incubated at 30°C and 150 rpm for 24 h. The cultures were centrifuged at 12,000 rpm and 4°C for 10 min, and the supernatants were taken to determine the AN concentrations. The AN removal strains were picked out on the basis of AN removal rates (R).600 nm values of cultures were directly measured at 0, 4, 8, 12, 24, 48, 72, and 96 h by pH meter  and spectrophotometer from Shanghai Yoke Instrument Co., Ltd. (752N), respectively. The experiment included three replicates.The cultures incubated in different media were centrifuged at 12,000 rpm and 4°C for 10 min. The AN concentrations of the supernatants were determined using Nessler reagent colorimetry according to the description by –2, 10–3, 10–4, and 10–5. A volume of 100 μL of each of the diluted solutions was spread on the activation media and incubated at 30°C for 24 h. Then, the colony counter was used to calculate the viable count of the cultures.The biomass of the culture was detected using the plate colony counting method; 10 ml culture was centrifuged at 12,000 rpm and 4°C for 10 min; the sediments were resuspended in the same volume of aseptic water; and the gradient was diluted to 10AN removal rates (R) were calculated using the following formulas:where A is the initial AN concentration and B is the residual AN concentration.P < 0.05.The experimental data are expressed as the mean ± standard error (mean ± S.E.). All statistical analyses were performed using SPSS 19.0 for Windows. Data obtained from the experiment were analyzed by one-way ANOVA after the homogeneity of variance test. When significant differences were found, Duncan’s multiple range tests were used to identify differences among the experimental groups. Differences were considered significant at 1 and the accession numbers are listed in To analyze the phylogenetic relationships of the AN removal strains reported previously  and screBacillus idriensis CT-WN-B3, Bacillus australimaris CT-WL5-10, and Pseudomonas oleovorans CT-WL5-6 were significantly higher than those of the other strains (P < 0.05). Among them, P. oleovorans CT-WL5-6 removed more than 80% of AN within 24 h    . However < 0.05) , indicat < 0.05) . IncreasN groups . These rN groups , and thiP < 0.05) (P < 0.05), which reached the stable stage in approximately OD600 nm 0.1 within 24 h. While in 1 or 2 mg/L group, the biomass did not reach the maximum until 72 h (P > 0.05). As shown in To analyze the dynamic situation of AN removal process, this study monitored the growth of the strains within 96 h, as well as the accompanying changes of AN concentrations and pH values in pH 9.0 media. As shown in  < 0.05) . The bestil 72 h . Accompatil 72 h , no signP > 0.05), an approximate 0.3 mg/L of final AN was guaranteed (P < 0.05) (P < 0.05)  . Additio < 0.05) . The hig < 0.05) . These rP > 0.05)  . Among d > 0.05) . This in > 0.05) .3) in alkali media (pH 9.0). When NaCl was supplemented into media, invariant percentages of AN were removed after a 24-h incubation of CT-WN-B3 or CT-WL5-6 (P > 0.05) (P > 0.05)  or alkalinities (10–30 mmol/L NaHCO > 0.05) . That sh > 0.05)  compared > 0.05) , and thelemented , even inlemented . Howeverlemented , and thelemented . These r4)2SO4 as the only nitrogen source was used to isolate AN removal strains from the alkali-tolerant bacteria that are distributed in carbonate saline-alkali soils and water environments in Northeast China , salinities (10–30 g/L NaCl), alkalinities (10–30 mmol/L NaHCO3), and AN concentrations (1–3 mg/L).In this study, an oligotrophic medium with . CT-WL5-6 was clustered in a stable manner with the reported Pseudomonas AN removal bacteria (the threshold was 99%)  . These r600 nm 0.04 and 0.10. These results are consistent with the levels reported for other strains, for example, Delftia lacustria SF9 and Acinetobacter sp. Sxf14  can be found in the article/RZ: writing-original draft, investigation, data analysis, writing-review, and project. LL: resources, investigation, and software. SW: visualization and project. KG: methodology and data analysis. WX: writing-review. ZZ: writing-review, editing, and project. All authors contributed to the article and approved the submitted version.


Thermus thermophilus MAT72 phage vB_Tt72. The enzyme shows low sequence identity (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia colipolA− mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3′-5′ exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysis proved that highly-conserved residues D15, E17, D78, D180, and D184 in 3′-5′ exonuclease and D384 and D615 in the nucleotidyltransferase domain are critical for the enzyme’s activity. Despite the source of origin, the Tt72 DNA polymerase has not proven to be highly thermoresistant, with a temperature optimum at 55 °C. Above 60 °C, the rapid loss of function follows with no activity > 75 °C. However, during heat treatment (10 min at 75 °C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at Tm = 74.6 °C  and circular dichroism spectra > 60 °C indicate the enzyme’s moderate thermal stability.We present a structural and functional analysis of the DNA polymerase of thermophilic  DNA replication, an essential feature of all life forms, relies on a set of specialized proteins that, complexed in the replisome network, can coordinate and execute the entire multi-step process in a highly concerted and accurate manner ,2,3,4. ApolA gene  and transformants were grown at 30 °C and 42 °C. The test revealed that the Tt72 pol could not complement the E. coli polA gene [pRARE] harboring pET15b_polTt72  contains an N-terminal portion of Tt72 pol , characteristic of enzymes with 3′-5′ exonuclease activity. The activity was assayed with the use of the 3′end-labeled DNA restriction fragments. In the case of Tt72 pol, we observed strong exonucleolytic activity, which was indicated by the release of about 95% of the -dTTP into activated calf thymus DNA. The effect of temperature was tested in the range of 25–80 °C. It was found that Tt72 pol was active over a wide temperature range, with the highest activity at 55 °C , and betaine at a concentration of 0.5 M protected the enzyme against thermal inactivation. We observed that in the case of the first two extremolytes, the enzyme retained >80% of its original activity 2SO4, which corresponds to 25 mM and 10 mM, respectively  Tt72 pol activity b. We alsactivity d. An anaactivity e. The fomM MgCl2 f. In theectively g,h.i index for Tt72 pol was calculated to be 6.01, which corresponds with proteins from hyperthermophiles (r > 4.5). This parameter indicates a strong preference of thermophilic organisms for Glu, Lys, Tyr, and Ile over Gln, His, Ala, and Cys, whose high content is noted in proteins from mesophilic sources -methionine incorporation experiment, bacteria were cultivated in an M-9 minimal medium  cells were transformed with plasmid pHSG576 , pHSG_polTt72 (Tt72 pol), pHSG_polTth (Tth pol), or pHSG_polTaq (Taq pol). The transformed cells were cultivated at 30 °C in LB medium supplemented with Ap, Cm, and Tc to an A600 of 0.5. To determine complementation efficiency of Tt72 pol or bacterial enzymes, Eco pol I, Tth pol I, and Taq pol I, and their respective cultures, were serially diluted 10-fold with LB medium, and 5 µL of each dilution was spotted on LA plates supplemented with Ap, Cm, Tc, and isopropyl-β-D-thiogalactopyranoside . Duplicate plates were incubated for 48 h at 30 °C or 42 °C.ication; ) and itsE. coli Bl21(DE3)[pRARE][pET15b_polTt72], pulse-labeling with [35S]-methionine was performed. The cells were grown at 30 °C in an M9 medium supplemented with Ap and Cm to A600 of 0.5. At this point, the overproduction of Tt72 pol was induced by adding IPTG to the final concentration of 1 mM. The E. coli RNA polymerase inhibitor rifampicin was added 30 min after induction to a concentration of 200 µg/mL. A hundred and fifty µL samples of culture were labeled with 2.5 µCi [35S]-methionine  for 5 min at 30 °C in 30 min intervals. Cells were centrifuged and resuspended in SDS/PAGE loading dye (125 mM Tris-HCl (pH 6.8), 5% [v/v] SDS, 10% [v/v] 2-mercaptoethanol, 10% [v/v] glycerol and 0.1% [w/v] bromophenol blue). Then, samples were analyzed on 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane . Protein bands were detected by autoradiography.To estimate the rates of protein synthesis in E. coli Bl21(DE3)[pRARE] cells harboring pET15_polTt72 were cultivated at 30 °C in an LB medium supplemented with Ap and Cm to an A600 of 0.5. The temperature was reduced to 18 °C and the Tt72 pol overproduction was induced by adding IPTG to 1 mM. The culture was grown for an additional 16 h at 18 °C, and the cells were harvested by centrifugation and stored at −80 °C. The His-tagged recombinant enzyme was purified using affinity chromatography and the ÄKTA Pure chromatography system . All purification procedures were carried out at 4 °C. For the standard purification procedure, 4 g of cell paste was resuspended in 6 mL buffer A (50 mM NaH2PO4 (pH 8.0), 500 mM NaCl, 0.1% [v/v] Triton X-100, 10% [v/v] glycerol) containing 10 mM imidazole. The cells were disrupted by sonication , and the resulting lysate was clarified by centrifugation . In the next step, the lysate was incubated for 20 min at 60 °C to remove host proteins. The precipitated proteins were discarded by centrifugation . The clarified lysate was loaded onto a HiTrapTM TALON column  equilibrated with buffer A. The column was washed with buffer A and the bound protein was eluted in the same buffer supplemented with 200 mM imidazole. Peak fractions containing substantial quantities of the target protein were pooled and dialyzed against buffer B (20 mM Tris-HCl (pH 8.0), 50 mM NaCl and 5% [v/v] glycerol). Then, proteins were loaded onto the HiTrapTM Heparin HP column  equilibrated with buffer B. The column was washed with the same buffer. Bound proteins were eluted with a 50 to 1000 mM NaCl gradient in buffer B. Fractions containing the highest activity of Tt72 pol were pooled and dialyzed against 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA, and 50% [v/v] glycerol, and then stored at −20 °C. The protein concentration was determined by the Bradford assay -thymidine 5′-triphosphate , and 0.2 mg/mL of activated calf thymus DNA. The reactions started after adding aliquots of the Tt72 pol on ice; samples were immediately transferred to a preheated thermal cycler and incubated for 10 min at 55 °C, then stopped by adding 200 µL of 10% [w/v] trichloroacetic acid (TCA) and kept on ice for 10 min. After termination, an aliquot was spotted on a GF/C filter disc . The filters were washed four times with 5 mL of 5% [w/v] TCA, then washed twice with 5 mL of 70% [v/v] ethanol. The filters were air-dried and counted in a liquid scintillation counter (PerkinElmer). One unit of the enzyme was defined as the amount of enzyme that incorporated 10 nmol of dNTPs into acid-insoluble material in 30 min at 55 °C. The incorporation of radiolabeled nucleotides was used to assay the polymerase activity of the Tt72 pol. The reaction mixture (50 µL) consisted of Tt72 buffer (10 mM Tris-HCl (pH 8.5), 25 mM KCl, 0.5 mM MgCl2, 1 mM DTT, 100 µM dATP, 100 µM dGTP, 100 µM dCTP, 1 µCi [methyl-3H]-thymidine 5′-triphosphate , 4 µg λ/HindIII, and 1 U of Klenow Fragment (Thermo Scientific). After labeling , DNA fragments were purified by a Clean-up AX kit . The 3′-5′ exonuclease activity of the Tt72 pol was assayed in a 50 µL reaction mixture containing 10 mM Tris-HCl (pH 8.5), 25 mM NaCl, 0.5 mM MgCl2, 10 mM (NH4)2SO4, and 0.5 µg of the labeled substrate at 55 °C for 10 min in the presence or absence of dNTPs. The reaction was stopped by cooling samples on ice and adding 200 µL of 10% [w/v] TCA. After termination, an aliquot was spotted at a GF/C filter disc (Whatman). The filters were washed four times with 5 mL of 5% [w/v] TCA, then washed twice with 5 mL of 70% [v/v] ethanol. The filters were air-dried and counted in a liquid scintillation counter (Perkin Elmer). This allowed us to calculate the percentage of radioactivity released from labelled DNA fragments.To prepare the 3′ end-labeled DNA substrate, sticky ends of DNA restriction fragments (λ/HindIII) were filled in a reaction mixture containing 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, (pH 8.0)) and scanned using a Typhoon 9200 phosphorimager .Oligonucleotides Tt72-Cy3 (24-mer) and Tt72-blunt (24-mer) were annealed at a 1:1 molar ratio  to give 2HPO4, 1.8 mM KH2PO4, 0.05% [v/v] Tween 20, (pH 7.4)) with 3% skimmed milk for 1 h at room temperature. Next, the membrane was incubated overnight at 4 °C with a specific primary antibody  in PBS buffer ) with 3% skimmed milk. After three washing cycles with PBS-T with 3% skimmed milk, the membrane was incubated with the secondary antibody  for 1 h at room temperature. Finally, the membrane was washed three times in PBS-T buffer for 10 min at room temperature for each cycle. X-ray film and PierceTM ECL Plus Western Blotting Substrate  have been used to detect a Western blot chemiluminescent signal.Purified protein samples were separated by electrophoresis (10% SDS-PAGE) and electroblotted onto a PVDF membrane using a dry transfer system . Subsequently, the membrane was blocked in the PBS-T buffer . After loading into the centrifuge and reaching a temperature of 4 °C, the cells were equilibrated for 1 h. Scans were collected at 6 min intervals between measurements in absorbance mode at 280 nm. Buffer density (d = 1.019 g/mL) and viscosity (viscosity = 1.895 mPas) were measured at 4 °C using an Anton Paar  DMA 5000 densitometer and Lovis 2000 M rolling-ball viscometer. The partial specific volume, v-bar = 7382 g/mL, and extinction coefficient for protein were calculated using SEDNTERP software version 1.09 . Data were analyzed using the “Continuous c(s) distribution” model of the SEDFIT (version 16.1c) program. The confidence level (F-ratio) was specified as 0.6.Sedimentation velocity experiments were performed using a ProteomeLab XL-I analytical ultracentrifuge  equipped with an An-60, 4-hole analytical rotor, and double-sector charcoal-Epon cells (12-mm path length). The experiments were carried out at 4 °C at 50,000 rpm, using the continuous scan mode and a radial spacing of 0.003 cm. Cells were loaded with 400 μL of sample and 410 μL of buffer (20 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 5% [Calorimetric measurements were made with a VP-DSC microcalorimeter  at a scanning rate of 90 °C /1 h. Scans were obtained at a protein concentration of 9.1 µM. The cell volume was 0.5 mL. All scans were run in 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl in a temperature range from 25 °C to 90 °C. The reversibility of the transition was checked by cooling and reheating the same sample. These measurements were recorded three times. Differential Scanning Calorimetry (DSC) measurements were analyzed using Origin 7.0 software from MicroCal . The qua2 × dmol−1]. CD measurements were made at a protein concentration of 3.6 µM in 20 mM MES (pH 5.0) and 50 mM NaCl. The volume of samples was 0.3 mL. The secondary structure content was estimated from all the CD spectra using the CONTIN/LL program, a variant of the CONTIN method provided within the CDPro software package [Circular dichroism (CD) spectra were recorded in the water on a Jasco-715 automatic recording spectropolarimeter  for Tt72 pol in 20 mM MES (pH 5.0) and 50 mM NaCl. The experiments were performed in the range of 20–80 °C every 10 degrees. Measurements were made in 1 mm quartz cuvettes. The spectra were recorded in the 200–260 nm wavelength range, using a sensitivity of five millidegrees and a scan speed of 50 nm/min. The results were plotted as the mean residue ellipticity Θ [degree × cm package .Pico instrument . The substrate oligo was assembled by annealing polTt72-Cy5  and polTt72-long  2SO4, and 0.05% Tween 20 at a volume of 20 µL. Reactions were carried out with a constant amount of substrate oligo (1 nM), which was titrated against a decreasing concentration of Tt72 pol or its substitution variants. After incubation for 10 min at 55 °C, the standard capillaries NT.115 (NanoTemper Technologies) were filled with the reaction mixture. The infrared laser power was 40%, and 20% LED power was used. Data from a minimum of three replicate binding assays were analyzed, and the KD was determined by non-linear fitting of the thermophoresis responses using the NTAnalysis software (Nano Temper Technologies).Microscale thermophoresis (MST) was used to analyze the binding affinity between purified Tt72 pol, and its substitutive variants, and partially double-stranded primer/template oligo with a fluorescent label. Assays were performed with the Monolith NT.115 strand)  at a 1:1The nucleotide sequence of Tt72 pol was deposited to GenBank under the accession number ON714139.


Hydroxyl acid is an important platform chemical that covers many industrial applications due to its dual functional modules. At present, the traditional technology for hydroxyl acid production mainly adopts the petroleum route with benzene, cyclohexane, butadiene and other non-renewable resources as raw materials which violates the development law of green chemistry. Conversely, it is well-known that biotechnology and bioengineering techniques possess several advantages over chemical methods, such as moderate reaction conditions, high chemical selectivity, and environmental-friendly. However, compared with chemical engineering, there are still some major obstacles in the industrial application of biotechnology. The critical issue of the competitiveness between bioengineering and chemical engineering is products titer and volume productivity. Therefore, based on the importance of hydroxyl acids in many fields, exploring a clean, practical and environmental-friendly preparation process of the hydroxyl acids is the core purpose of this study.Gluconobacter oxydans was constructed. In the study, we found a critical point of chain length determine the end-products. Gluconobacter oxydans catalyzed diols with chain length ≤ 4, forming hydroxyl acids, and converting 1,5-pentylene glycol and 1,6-hexylene glycol to diacids. Based on this principle, we successfully synthesized 75.3 g/L glycolic acid, 83.2 g/L 3-hydroxypropionic acid, and 94.3 g/L 4-hydroxybutyric acid within 48 h. Furthermore, we directionally controlled the products of C5/C6 diols by adjusting pH, resulting in 102.3 g/L 5‑hydroxyvaleric acid and 48.8 g/L 6-hydroxycaproic acid instead of diacids. Combining pH regulation and cell-recycling technology in sealed-oxygen supply bioreactor, we prepared 271.4 g 5‑hydroxyvaleric acid and 129.4 g 6-hydroxycaproic acid in 6 rounds.To obtain high-purity hydroxyl acid, a microbiological regulation for its bioproduction by G. oxydans as biocatalyst for superior-quality hydroxyl acids (C2–C6) production is raised up. The proposed strategy commendably demonstrated a novel technology with simple pH regulation for high-value production of hydroxyl acids via green bioprocess developments.In this study, a green scheme of employing The online version contains supplementary material available at 10.1186/s13068-022-02171-5. Consumption of traditional petrochemical resources leads to environmental pollution, causing toxicity, carcinogenicity, and biological aggregation of harmful chemical substances. Moreover, excessive use of coal, oil and natural gas makes the non-renewable resources on earth increasingly scarce , 2. Henc2–(CH2)n–COOH) is an important bioresource intermediates containing both hydroxyl and carboxyl groups at terminal positions [Escherichia coli [Klebsiella pneumoniae [, and Corynebacterium glutamicum [Hydroxyl acid  is a Gram-negative bacterium known for its incomplete oxidation capability, attributed to the membrane-bound dehydrogenases [G. oxydans to be employed as a common industrial biocatalyst for the industrial production of vitamin C [G. oxydans, have facilitated the large-scale production of hydroxyl acids. In addition, depending on G. oxydans, Sang-Hyun et al. have realized the bio-preparation of 6-hydroxycaproic acid (6-HCA) recently. During 100 h continuous whole-cell catalysis, about 350 mg 6-HCA was selectively produced in 10 mL broth with a fed-batch mode (35 g/L) [Biocatalysis, a promising approach for sustainable development of industry in the future, has applied in various fields, including food, chemical, medicine, environmental, and energy , 28. Henogenases , 30 suchogenases , aldehydogenases , glyceroogenases , and sorogenases . These mogenases . These pitamin C , xylonicitamin C , and gluitamin C . In addiitamin C , 40. The(35 g/L) . HoweverG. oxydans whole-cell catalysis of primary diols for hydroxyl acids production. As results, combined SOS technology, we successfully achieved high-titer of GA, 3-hydroxypropionic acid (3-HPA), and 4-hydroxybutyric acid (4-HBA). Furthermore, a green route for whole-cell catalysis can precisely control the production of 5‑hydroxyvaleric acid (5-HVA) and 6-HCA by pH regulation.Considering the bottlenecks in the industrial-scale preparation of hydroxyl acids, it is important to develop an efficient and green method by investigating the reaction mechanism of 4, KH2PO4, K2HPO4, (NH4)2SO4, and CaCO3 were obtained from Sinopharm Chemical Reagent Co., Ltd. (China). All other chemicals were of analytical grade and were commercially available.Ethylene glycol (EG), 1,3-propylene glycol , 1,4-butylene glycol , 1,5-pentylene glycol , 1,6-hexylene glycol , GA, 3-HPA, 4-HBA, 5-HVA, 6-HCA, glutaric acid (GTA) and adipic acid (AA) were obtained from Aladdin Chemical Reagent Corporation (China). Yeast Extract was purchased from Sigma-Aldrich. Sorbitol, MgSOGluconobacter oxydans NL71 was isolated from G. oxydans ATCC 621 using the crude lignocellulosic hydrolysate domestication process. The strain was preserved in sorbitol–agar medium containing 50 g/L sorbitol, 5 g/L yeast extract, and 15 g/L agar, at 4 ℃. The inoculum was cultivated in an Erlenmeyer flask at 30 ℃ for 24–36 h, with continuous agitation at 220 rpm using a mechanical shaker (New Brunswick Scientific). The nutrient medium was composed of 100 g/L sorbitol and 10 g/L yeast extract. Cultured cells were centrifuged at 6000 rpm at 4 ℃, for 5–10 min in a freezing centrifuge . Centrifuged cells were washed 3 times with sterile saline and sterile water, respectively [ectively .G. oxydans with OD600 = 10, 5 g/L yeast extract as the nitrogen source, and 0.5 g/L MgSO4, 1 g/L KH2PO4, 2 g/L K2HPO4, and 5 g/L (NH4)2SO4 as nutrients. Biocatalysis temperature was maintained at 30 °C and a stirring speed of 500 rpm. In addition, as G. oxydans is an obligate microorganism, oxygenation (purity ≥ 99.9) was maintained throughout the bioprocess. However, excessive oxygenation not only increases the cost but also leads to the wastage of resources due to low oxygen utilization. Considering that the whole-cell catalysis by G. oxydans is resting-cell catalysis without generating waste gas such as CO2. Hence, we employed a sealed oxygen-supplied bioreactor (SOS-BR), as shown in Fig. G. oxydans was harvested via centrifugation at 6000–8000 rpm for 5 min under 4 °C and cell pellets were washed with deionized water. Finally, the bacteria were re-inserted into the fresh medium for a new round of whole-cell catalysis.Bioprocess for whole-cell catalysis of 1,6-HG was carried out in a 3-L bioreactor with 1 L broth containing 2SO4 was used as mobile phase at a flow rate of 0.6 mL/min. Moreover, the yield of hydroxyl acid was calculated according to Formula The titer of EG, 1,3-PG, 1,4-BG, 1,5-PG, 1,6-HG, hydroxyl acids and diacids was detected by high-performance liquid chromatography  equipped with a differential detector. The column used for separation was the Aminex Bio-Rad HPX-87H and 5 mM HDue to the difference in experiment data, three parallel assays were performed for each experiment to ensure the reliability of results.G. oxydans as a core catalyst to design a green and environmental-friendly hydroxyl acids synthesis method is an effective approach to solve the current industrial bottlenecks. To explore the reaction mechanism of G. oxydans whole-cell catalysis, five linear diols were selected as substrates for kinetic study with OD600 = 2. Figure G. oxydans. The final catalytic products were GA, 3-HPA, and 4-HBA, respectively, without the formation of diacids. The average consumption rates of EG, 1,3-PG, and 1,4-BG were 0.68, 1.55, 2.23 g/L/h, respectively. Compared with the chemical method, the purity of products was satisfactory, meeting the core requirements of green chemistry, and the HPLC chromatograms in Additional file Dual-functional modules of hydroxyl acids alleviate their application prospects in many high-end fields . HoweverG. oxydans, respectively. Surprisingly, when the carbon chain length was ≥ 5, the end products showed qualitative differences. 1,5-PG and 1,6-HG catalysis followed a step-by-step process, further catalyzing to GTA and AA when primary diols were oxidized to hydroxyl acids. Moreover, the catalytic rates for 1,5-PG and 1,6-HG were very similar. The substrate consumption time was less than 3 h and 4 h, while the average substrate consumption rates reached 3.25 and 5.38 g/L/h, respectively. Although the reaction efficiency of C5/C6 was promising, the product quality was not satisfactory due to the formation of by-products (diacids). At present, hydroxyl acids have attracted much attention due to their dual-functional modules, which exhibit excelling properties. Hence, we need further explored directional regulation for hydroxyl acids production by G. oxydans to lay a theoretical foundation for the industrial production.Figure G. oxydans could bio-transform EG, 1,3-PG, and 1,4-BG into hydroxyl acids. Although the purity of the products met the green chemistry and industrial production requirements, the catalytic efficiency was low to strengthen the production economy. G. oxydans takes oxygen as the final electron receptor, so the whole-cell catalysis requires a lot of oxygen carriers to enhance the catalytic rate [Previous studies indicated that tic rate , 46. Howtic rate .G. oxydans OD600 = 2. Results indicated that GA, in the 48 h bioprocess, 3-HPA and 4-HBA accumulated 75.3 g/L, 83.2 g/L, and 94.3 g/L respectively. In terms of GA production, the highest quantity of GA obtained was 74.5 g/L with a productivity of 1.49 g/L/h by Wei et al. [Escherichia coli that produce 38.7 g 3-HPA with an average yield of 35% in 72 h fermentation [Escherichia coli to produce 103.4 g/L under the microaerobic conditions with a productivity of 0.844 g/L/h. Although the concentration of 4-HBA was slightly higher than that of whole-cell catalysis, its productivity was only 43% of SOS technology. Furthermore, a lot of inhibitors such as acetic acid were generated in the fermentation broth of recombinant Escherichia coli, which seriously affected the product quality. Apparently, the whole-cell catalysis with G. oxydans, combined with SOS technology has realized the continuous and efficient preparation of high-purity hydroxyl acids (C2–C4), which provided solid technical support for their industrial production.As shown in Fig. i et al. , which wG. oxydans, its production level was rather unsatisfactory, and GTA was accumulated in the system as a by-product. Moreover, the traditional chemical methods cannot prepare high-quality 5-HVA due to high cost, which limits their application in advanced fields such as medicine and material science. Therefore, targeted regulation for selective catalysis of 1,5-PG is a promising approach for the industrial production. As shown in Fig. G. oxydans lost catalytic activity after 2 h. Therefore, the proposed scheme of pH-regulated whole-cell catalysis provided a green and high-quality approach for the industrial production of 5-HVA without any by-products. To directionally obtain an ultra-high titer of 5-HVA, we selected pH = 5.5 for the bioreactor experiment.A previous study showed that 5-HVA could be produced by Corynebacterium glutamicum, and 55 g/L 5-HVA and 10 g/L GTA were produced after 28 h fermentation [G. oxydans. At the same time, due to the strict sealing environment of the entire system, the cost of oxygen utilization was greatly saved and the economy of the entire bioprocess was improved. Moreover, we successfully performed cell-recycling technology with 6 rounds in SOS-BR  and high pressure (10 ~ 300 bar), but the products were accompanied by abundant 1,6-HG and esters . TherefoAccording to the results of pH regulation experiment, the whole-cell catalysis for the preparation of 6-HCA was performed at pH = 7  and whole-cell catalysis for GA production. Figure S2. HPLC chromatograms of standard sample (4 g/L) and whole-cell catalysis for 3-HPA production. Figure S3. HPLC chromatograms of standard sample (4 g/L) and whole-cell catalysis for 4-HBA production. Figure S4. HPLC chromatograms of standard sample (4 g/L) and whole-cell catalysis for 5-HPA production with pH regulation. Figure S5. HPLC chromatograms of standard sample (4 g/L) and whole-cell catalysis for 6-HCA production with pH regulation.


Combined with the reduced carrier concentration via the suppressed Ge vacancies by high-entropy effect and Sb donor doping, a large Seebeck coefficient is attained. Meanwhile, the severe lattice distortions and micron-sized Zn0.6Cd0.4Te precipitations restrain the lattice thermal conductivity approaching to the theoretical minimum value. Finally, the maximum zT of Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10 reaches 1.24 at 723 K via the trade-off between the degraded carrier mobility and the improved Seebeck coefficient, as well as the depressed lattice thermal conductivity. These results provide a reference for the implementation of entropy engineering in GeTe and other thermoelectric materials.In thermoelectrics, entropy engineering as an emerging paradigm-shifting strategy can simultaneously enhance the crystal symmetry, increase the solubility limit of specific elements, and reduce the lattice thermal conductivity. However, the severe lattice distortion in high-entropy materials blocks the carrier transport and hence results in an extremely low carrier mobility. Herein, the design principle for selecting alloying species is introduced as an effective strategy to compensate for the deterioration of carrier mobility in GeTe-based alloys. It demonstrates that high configurational entropy via progressive MnZnCdTe For TE materials undergoing phase transition, implementing phase engineering can also expand the phase favorable to thermoelectric and restrain the negative phase [On account of the rising energy costs and increasing global warming, the development of eco-friendly and sustainable energy technologies is a global challenge. Among them, thermoelectrics (TE) have been considered as one of the most compelling technologies due to the capability of direct conversion between heat and electricity, vibration-free operation, high reliability, and low environmental impact ,2. The eectively . Aiming ineering ,5,6 and ineering ,9,10,11 ve phase ,13.S characterizes the disorder of the system, and it is also an important parameter for developing high-performance TE materials [S < 1 R, high-entropy alloys  consist of five or more major elements with a percentage of each atom between 5% and 35% [2Te3 [The configurational entropy Δaterials . Differe and 35% . High en and 35% ,19,20,21 and 35% ,23,24, I and 35% , GeTe [1 and 35% ,26, and  and 35% ,28,29), 5% [2Te3 , half-He5% [2Te3 , etc.S has a profound effect on the TE parameters. First, the materials tend to form the high-symmetric cubic phase structure as long as the ΔS is high enough; meanwhile, high ΔS can reduce the structural phase transition temperature Tc [NV and hence the large density-of-states’ effective mass m* [S is conducive to increasing the solubility limit of specific elements, which in turn extends the phase space for performance optimization [α can be expected with higher ΔS value. Furthermore, the large differences of both atomic radius and mass among various components at the same sublattice site in multicomponent materials can result in a severe lattice distortion, which reduces the phonon velocities and enhances the phonon scattering simultaneously [κL can be reduced. However, it is noteworthy that although the multi-principal-element alloying can lower the κL towards the theoretical minimum as well as improve the α [μH and σ that are bad for the zT advance. Consequently, the successful implementation of the entropy engineering presupposes that the fall in μH can be offset by the enhancement of α and the reduction of κL [In multicomponent materials, Δature Tc ,20,26. T mass m* . Moreoveaneously ,34,35. Hve the α ,16,19, tμH reduction in multicomponent TE materials. For TE materials with carrier mean free path approaching to the Mott-Ioffe-Regel limit, multi-principal-element alloying does not further impair μH, while only significantly reduces the κL and enhances α [μH. Second, when designing high-entropy TE materials, the selected alloying element should have a small covalent radius difference in comparison with the host atoms in order to minimize the alloy scattering potential Eal and avoid the detrimental effect of high ΔS on μH. Under this circumstance, only a few alloying elements are available for many TE systems, which also leads to a significant limitation in the implementation of entropy engineering. Last but not least, if the chosen alloying elements can enhance the m*, optimize the carrier concentration nH and decline the κL as much as possible to compensate for the deterioration of μH, the figure of merit can be surely enhanced [There are a number of strategies to solve the dilemma of hances α ,20,30,31enhanced ,20. AppanH (~1021 cm−3) at room temperature, giving rise to an extremely low α (~34 μV/K) and high κe (~5.65 W/mK) [nH [GeTe is a promising group IV-VI TE material for medium-temperature power generations . Owing t65 W/mK) . In addi/mK) [nH ,38,39,40/mK) [nH . Up to n/mK) [nH ,45,46,47S of GeTe-based materials is regulated and the zT value is enhanced by progressive MnZnCdTe3 and Sb co-alloying, attesting to the efficacy of the design principle of rational selecting alloying elements in high-entropy thermoelectrics. Previous studies have demonstrated that sole Mn [m*. In the context of electron counting, replacing divalent Ge by isovalent Mn/Zn/Cd contributes no net nH and therefore retains the nH to the first order. To reduce the otherwise excessive nH of GeTe, the introduction of trivalent Sb doping is a natural option [3 and Sb co-alloying can increase the ΔS and lower Tc towards room temperature, which is beneficial to obtain large NV and thus increase m* [μH reduction and the α enhancement in the view of increased m* and optimized nH. Combined with the substantially diminished κL stemming from the severe lattice distortions and the micron-sized Zn0.6Cd0.4Te precipitations, the maximum zT ~1.24 at 723 K is attained in Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10. These results deepen the understanding of the rational selection of alloying elements in high-entropy TE materials.In this work, the Δ sole Mn ,47, Zn [ sole Mn , or Cd [ sole Mn ,46 dopinl option . In addirease m* . This ofx1−(MnZnCdTe3)x , as well as Ge0.90−ySbyTe0.90(MnZnCdTe3)0.10 , sealed into quartz ampoules at 10−3 Pa, melted at 1373 K for 10 h and then quenched in cold water. The solidified ingots were ball milled  in a vacuum at 1200 rpm for 20 min to fine powders. Subsequently, the obtained fine powders were spark plasma sintered (SPS) into high-density cylinders with a diameter of 20 mm, with a thickness of 3 mm at 773 K for 5 min under the uniaxial pressure of 50 MPa.Appropriate amounts of high-purity (99.999%) element chunks of Ge, Te, Mn, Zn, Cd and Sb were weighed according to the nominal composition of (GeTe)®, Tokyo, Japan). The data analysis was performed via JADE 6.0 and the lattice parameters were calculated based on the Rietveld refinement method using the Topas 3.1 software. The phase transition temperature Tc from rhombohedral to cubic was inspected with a differential scanning calorimeter . The microstructures were investigated by scanning electron microscopy , and the chemical composition was examined by an energy dispersive spectrometer (EDS).The phase structures of all the GeTe-based samples were analyzed via the X-ray diffraction  under a protective helium atmosphere. The measurement of thermal diffusivity D was performed on a Netzsch LFA 467 HT laser flash apparatus. The specific heat CP was estimated by the Dulong-Petit law and the sample density ρ was determined via the Archimedes method. The thermal conductivity κ was then calculated as: κ = ρCPD. The carrier thermal conductivity κe was evaluated by the Wiedemann-Franz law, κe = LσT, where L is the Lorenz number and can be roughly expressed by L is in 10−8 WΩK−2 and α in μV/K) [κL was calculated by subtracting κe from the total κ. The room temperature Hall coefficient RH was measured on a PPMS system (Quantum Design®) with a magnetic field scanned between ±5 T. Subsequently, the carrier concentration nH and Hall mobility μH were calculated according to the equation nH = 1/eRH and μH = σRH, respectively.The measurements of the Seebeck coefficient in μV/K) . The latS is sufficiently high [S can still lead to the decrease in the structural transition temperature to some extent for GeTe-based alloys [n is the number of alloying components in the solid solution, xi is the mole fractions of the ith component, and R is the gas constant [S increases from 0 R for binary GeTe to 0.84 R for (GeTe)0.90(MnZnCdTe3)0.10 and then to 1.06 R with Sb co-doping in Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10, reaching the medium-entropy region 0.90(MnZnCdTe3)0.10 sample. Further increasing ΔS to 1.06 R, a rises to 4.205 Å and c drops to 10.486 Å for the Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10 sample, demonstrating the promotion of the rhombohedral-cubic phase transition with the increasing ΔS.In fact, all the multicomponent GeTe-based alloys are inclined to form the high-symmetric cubic structures as long as the Δtly high . Even ifd alloys . Accordiconstant ,15. Obviy region a. As obsy region b, the doine GeTe ,50. As Δo C-GeTe ,26. The Tc upon progressive MnZnCdTe3 and Sb co-alloying. As depicted in Tc of pristine GeTe is 655 K, and gradually falls to 552 K for (GeTe)0.90(MnZnCdTe3)0.10 with the increasing ΔS. When further boosting ΔS, the Tc of Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10 sample declines to 320 K, which is close to the room temperature. This indicates that the increase in ΔS can observably depress the Tc and is beneficial to obtain large NV and thus high m* [Differential scanning calorimetry (DSC) analysis is carried out to further detect the variation of  high m* ,26, whicS also inclines to extend the solubility limits of specific elements [S. For the multicomponent system, the Gibbs free energy of mixing ΔG is determined by H is the enthalpy of mixing. Obviously, if the ΔH keeps constant, a higher ΔS will give rise to a lower ΔG [S may promote the Ge precipitations to be dissolved into the matrix, which in turn reduces the excessive Ge vacancies and thus mildly decreases the nH. Furthermore, Zn0.6Cd0.4Te precipitations are observed when the MnZnCdTe3 alloying content x reaches 5%, indicating the low solubility limit of MnZnCdTe3 in GeTe. The SEM-EDS images further confirm that the dark grey regions of 0.1–2.0 μm is Zn0.6Cd0.4Te, which are embedded in the host matrix of GeTe-based multicomponent alloys x samples are measured. As presented in nH falls mildly with increasing MnZnCdTe3 alloying content. For example, the room temperature nH decreases from 8.4 × 1020 cm−3 for GeTe to 6.0 × 1020 cm−3 for (GeTe)0.90(MnZnCdTe3)0.10. Theoretically, single element doping with the isovalent Zn, Cd or Mn at Ge site will not have a significant effect on nH [nH [nH after Mn-Zn-Cd co-alloying may be ascribed to the re-dissolution of Ge precipitations into the GeTe matrix via a high entropy effect, which is in line with the XRD results. Subsequently, the donor doping of trivalent Sb further reduces the nH substantially to 1.7 × 1020 cm−3 for Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10, approaching to the optimal carrier concentration of nopt (~2 × 1020 cm−3) for rhombohedral GeTe-based alloys [In order to analyze the mechanism underlining the variation of electrical transport properties, the room temperature ct on nH ,45,46,47n nH [nH ,47. In td alloys ,52,53.μH [3 alloying dramatically deteriorates the μH of GeTe. Typically, the room temperature μH drastically reduces from 57.3 cm2V−1s−1 for pristine GeTe to 20.8 cm2V−1s−1 for (GeTe)0.95(MnZnCdTe3)0.05 and further to 15.8 cm2V−1s−1 for (GeTe)0.90(MnZnCdTe3)0.10. Moreover, Mn alloying will flatten the valence band edge and largely increase the band effective mass mb* of GeTe, leading to a remarkable deterioration in μH. For instance, Zheng et al. [m* increased quickly from 1.44 m0 for binary GeTe to 6.15 m0 for Ge0.85Mn0.15Te, corresponding to a catastrophic reduction of room temperature μH from 54.17 cm2V−1s−1 to 4.41 cm2V−1s−1. Similarly, Liu et al. found that the room temperature m* for pristine GeTe was only 1.6 m0, which was then markedly enhanced to 36.7 m0 after 30 at% Mn alloying. Meanwhile, the room temperature μH diminished rapidly from 95.3 cm2V−1s−1 to 0.5 cm2V−1s−1 [μH approaching to the Mott-Ioffe-Regel limit upon MnZnCdTe3 alloying, further Sb alloying does not impair the μH, but Sb alloying can concurrently suppress the rhombohedral-cubic phase transition, optimize the nH and reduce the κL [Severe lattice distortion induced by multi-principal-element alloying can impede the carrier transport and hence degrade the μH ,20. As ig et al.  reportedm2V−1s−1 . In viewe the κL ,31,48.S is adverse to high σ [σ for all the GeTe-based samples. The σ of pristine GeTe is extremely high because of the high nH and μH. After MnZnCdTe3 alloying, the σ decreases dramatically due to the simultaneous reduction of nH and μH, which originates from the dissolution of Ge precipitations, band flattening and severe lattice distortion. Typically, the room temperature σ decreases from 774 × 103 Sm−1 for binary GeTe to 152 × 103 Sm−1 for (GeTe)0.90(MnZnCdTe3)0.10. In light of the μH approaching to the Mott-Ioffe-Regel limit, further Sb alloying reduces the nH but has a weak impact on carrier scattering, thereby bringing out a further decreased σ. Therefore, the room temperature σ of the Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10 sample decreases to 30 × 103 Sm−1. In addition, the σ of pristine GeTe exhibits a temperature dependence of T−1.48, indicating that the acoustic phonon scattering (σ ~T−1.5) is the dominant scattering mechanism in this system. The power law exponent of the σ gradually decreases with increasing ΔS because of the enhanced point defect scattering. Thus, the temperature dependence of T−0.4 is obtained in Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10, which is consistent with the alloy scattering mechanism (σ ~−T0.5) [In general, the high Δo high σ ,20. Figu ~T−0.5) .3 and Sb increases ΔS and thus suppresses Tc near room temperature, which in turn enhances the m* and hence α [α boosts mildly near room temperature upon MnZnCdTe3 alloying while increases substantially in the whole temperature range after Sb doping. For example, the room temperature α sharply increases from 32.5 μVK−1 for binary GeTe to 156.4 μVK−1 for Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10.The progressive alloying of MnZnCdTe hence α ,26. As eα, the Pisarenko plots are calculated according to the single parabolic band (SPB) model [m* rises from 1.4 m0 for the GeTe sample to 1.8 m0 for the MnZnCdTe3 alloyed sample and finally increases to 2.4 m0 for the Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10 sample. On the one hand, the increase in ΔS lowers the Tc of GeTe, resulting in large NV and thus m*. On the other hand, the co-alloying of Mn, Zn and Cd is conductive to promoting the multivalence band convergence and valence band flattening, which further enhances m* [3 in GeTe is detrimental to band structure modulation. Besides the increase in m*, both the suppression of Ge vacancies via high entropy effect and the trivalent Sb donor doping contribute to a large reduction of nH, giving rise to a huge enhancement of α. The increased m* and optimized nH partially compensate for the deterioration of μH, thus attaining a decent power factor PF in the whole temperature region (−3 Wm−1K−2 for Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10.To gain insight into the beneficial effect of band structure variation on B) model . As demoances m* ,44,45,47e region d as wellκ (8 Wm−1K−1 at 300 K) limits the TE performance of p-type binary GeTe [κ in GeTe-based alloys. The severe lattice distortion induced by multi-principal-element alloying affords a natural option [κ for all the GeTe-based alloys is plotted in κ of multi-principal-elements alloys are much lower than the pristine GeTe, because of the concurrent reduction of both κe (due to the declined σ) and κL. The room temperature κ substantially drops from 6.76 Wm−1K−1 for binary GeTe to 1.43 Wm−1K−1 for (GeTe)0.90(MnZnCdTe3)0.10 and then to 0.98 Wm−1K−1 for Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10.The high ary GeTe . Therefol option ,19,20. TκL of all the samples is presented in κL of GeTe [0.6Cd0.4Te secondary phases were caused by the low solubility limit of MnZnCdTe3 in GeTe 0.90(MnZnCdTe3)0.10 and further falls to 0.66 Wm−1K−1 for Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10. In particular, the minimum κL of 0.45 Wm−1K−1 at 723 K is achieved in Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10, approaching the theoretical minimum κL (~0.3 Wm−1K−1) of GeTe [κL decreases from −1.25 for binary GeTe to −0.45 for Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10, indicating that the dominant phonon scattering alters from acoustic phonon scattering (κL ~T−1.0) to point defect scattering (κL ~T−0.5) with increasing alloying species and content [The  of GeTe ,20. More in GeTe  that is  of GeTe . Moreoveα, the depressed κL and the deteriorated μH determines the variation of zT values with increasing ΔS (zT of 1.24 at 723 K is attained in the Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10 sample through entropy engineering, about 22% increment over the pristine GeTe. The peak zT in this study is much higher than those of the high-entropy Ge1/3Sn1/3Pb1/3Te1/3Se1/3S1/3 (~0.51) [0.25Sn0.25Pb0.25Mn0.25Te alloys (~0.92) [zTave value between 298 K and 723 K is 0.77, which is 113% higher than that of binary GeTe  and Ge0.25Sn0.25Pb0.25Mn0.25Te alloys (~0.63). These results demonstrate the validity of the screening principle for alloying species in high-entropy TE materials.The trade-off between the enhanced asing ΔS c. Notabl (~0.51)  and Ge0. (~0.92)  without ary GeTe d. In par3 and Sb co-alloying based on the nominal composition of (Gex−y1−SbyTex1−)(MnZnCdTe3)x. Multi-principal-element alloying can regulate the configurational entropy and thus improve the thermoelectric properties of the GeTe-based materials. The phase structures, microstructures and thermoelectric properties of the obtained alloys are systematically studied. The high configurational entropy reduces the rhombohedral-cubic phase transition temperature to near room temperature and Mn-Zn-Cd co-alloying promotes the multivalent bands’ convergence, which are both conducive to obtaining a high density-of-state effective mass. Meanwhile, the synergy of the decreased Ge vacancies content via the dissolution of Ge precipitations and trivalent Sb donor doping reduces the excessive carrier concentration of GeTe-based alloys. The optimized carrier concentration and the increase in the density-of-state effective mass improve the Seebeck coefficient substantially, which counteracts the decrease in electrical conductivity. Moreover, the severe lattice distortions and micron-sized Zn0.6Cd0.4Te second phases result in a significant reduction of lattice thermal conductivity. As a result, the maximum zT value of 1.24 at 723 K is attained in Ge0.82Sb0.08Te0.90(MnZnCdTe3)0.10. These results not only prove the validity of rational screening principle for alloying species in high-entropy thermoelectric materials, but also trigger profound thoughts on the application of emerging entropy engineering in the field of thermoelectric materials.In summary, we demonstrate the power of entropy engineering on balancing the power factor and lattice thermal conductivity of GeTe in line with the rationally screening alloying species. To establish the structure-property correlation in GeTe-based multicomponent materials, entropy engineering is adopted to design the GeTe-based alloys with progressive MnZnCdTe


Saccharomyces cerevisiae.CYP725A4 catalyses the conversion of the first Taxol® precursor, taxadiene, to taxadiene-5α-ol (T5α-ol) and a range of other mono- and di-hydroxylated side products (oxygenated taxanes). Initially known to undergo a radical rebound mechanism, the recent studies have revealed that an intermediate epoxide mediates the formation of the main characterised products of the enzyme, being T5α-ol, 5(12)-oxa-3(11)-cyclotaxane (OCT) and its isomer, 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) as well as taxadienediols. Besides the high side product: main product ratio and the low main product titre, CYP725A4 is also known for its slow enzymatic activity, massively hindering further progress in heterologous production of Taxol® precursors. Therefore, this study aimed to systematically explore the key parameters for improving the regioselectivity and activity of eukaryotic CYP725A4 enzyme in a whole-cell eukaryotic biocatalyst, Investigating the impact of CYP725A4 and reductase gene dosages along with construction of self-sufficient proteins with strong prokaryotic reductases showed that a potential uncoupling event accelerates the formation of oxygenated taxane products of this enzyme, particularly the side products OCT and iso-OCT. Due to the harmful effect of uncoupling products and the reactive metabolites on the enzyme, the impact of flavins and irons, existing as prosthetic groups in CYP725A4 and reductase, were examined in both their precursor and ready forms, and to investigate the changes in product distribution. We observed that the flavin adenine dinucleotide improved the diterpenoids titres and biomass accumulation. Hemin was found to decrease the titre of iso-OCT and T5α-ol, without impacting the side product OCT, suggesting the latter being the major product of CYP725A4. The interaction between this iron and the iron precursor, δ-Aminolevulinic acid, seemed to improve the production of these diterpenoids, further denoting that iso-OCT and T5α-ol were the later products. While no direct correlation between cellular-level oxidative stress and oxygenated taxanes was observed, investigating the impact of salt and antioxidant on CYP725A4 further showed the significant drop in OCT titre, highlighting the possibility of enzymatic-level uncoupling event and reactivity as the major mechanism behind the enzyme activity. To characterise the product spectrum and production capacity of CYP725A4 in the absence of cell growth, resting cell assays with optimal neutral pH revealed an array of novel diterpenoids along with higher quantities of characterised diterpenoids and independence of the oxygenated product spectra from the acidity effect. Besides reporting on the full product ranges of CYP725A4 in yeast for the first time, the highest total taxanes of around 361.4 ± 52.4 mg/L including 38.1 ± 8.4 mg/L of T5α-ol was produced herein at a small, 10-mL scale by resting cell assay, where the formation of some novel diterpenoids relied on the prior existence of other diterpenes/diterpenoids as shown by statistical analyses.This study shows how rational strain engineering combined with an efficient design of experiment approach systematically uncovered the promoting effect of uncoupling for optimising the formation of the early oxygenated taxane precursors of Taxol®. The provided strategies can effectively accelerate the design of more efficient Taxol®-producing yeast strains.The online version contains supplementary material available at 10.1186/s12934-022-01922-1. Taxus brevifolia [Escherichia coli [Saccharomyces cerevisiae [Nicotiana benthamiana [Nicotiana sylvestris [Solanum lycopersicum [Yarrowia lipolytica [Physcomitrella patens [Bacillus subtilis [Arabidopsis thaliana [E. coli [S. cerevisiae [E. coli. Therefore, recent studies skipped to investigate the ideal conditions for T5α-ol formation and reported on the total oxygenated taxanes instead [Taxol® (Paclitaxel), a tricyclic drug originally extracted from evifolia , is one evifolia . The curhia coli –9, Sacchrevisiae –17, Nicothamiana , 19, Niclvestris , Solanumpersicum , 22, Yarpolytica , Physcoma patens , Bacillusubtilis , Arabidothaliana  or co-cuthaliana , have bethaliana , 27. Thethaliana , 15, 20 thaliana , 14. In [E. coli  and 129 revisiae  have rep instead , 14, 26.As a result of such discrepancies, different proposals have been discussed on the CYP725A4 mechanism of activity, which have questioned the existence of radical rebound mechanism  for CYP7S. cerevisiae at both enzymatic and whole-cell levels, which would accelerate the integration of the rest of the P450-dominated Taxol® biosynthetic pathway genes. While exploring the conditions for increasing the main product titre, several lines of evidence clearly showed that the uncoupling reaction by the Taxus CYP725A4 and POR interaction potentially mediates the formation of oxygenated taxanes in S. cerevisiae and the T5α-ol titre can be likely improved through optimal fusion protein construction.Building on previous studies, our study aimed to systematically find the optimal conditions for favourable activity of CYP725A4 in CYP725A4 in microbial chassis have so far focused on modifications to the P450 and cognate reductase sequences for improved solubility and expression (6), tandem P450-reductase expression (14) and constructing P450-reductase chimeras using strong eukaryotic reductases from human, rat liver or Stevia rebaudiana and cytochrome b5 [S. cerevisiae cell factories and therefore, the first question we sought to answer was to determine the rate-limiting gene through a gene dosage study.Major studies in expression of CYP725A4 or Taxus POR gene copies was evaluated in a three-day run where BN1 strain, expressing an extra copy of CYP725A4, improved the diterpenoid I concentration significantly (p = 2.5e−02), proposed to be a potential isomer to T5α-ol in our earlier study [POR, showed a significant impact on OCT (p = 8.2e−03), iso-OCT (p = 0.02) and diterpenoid I (p = 6.6e−07) formation. BN3, expressing one copy of CYP725A4 with a low-glucose inducible promoter, pHXT7, also showed significant improvement in OCT (p = 0.0001), iso-OCT (p = 0.002), and diterpenoid I formation (p = 1.3e−03). However, factoring in the impact of interaction between the production time and the strain revealed more detailed results. In more detail, the interaction between the strain type and growth time was found to significantly affect the taxadiene (p = 2.6e−08), OCT (p = 0.0015), taxadienediol , diterpenoid I (p = 3.9e−08) and iso-taxadiene (p = 7.4e−09) titres. However, this interaction was found insignificant towards the T5α-ol and iso-OCT titres .The effect of increased er study . Also, BCYP725A4 gene copy numbers in BN1 did not improve the final T5α-ol in comparison to its parent strain, LRS6. However, OCT concentration was slightly improved by 1.2-fold . Interestingly, diterpenoid I remained the major product in all the new strains with 2.6- and 1.9-fold improvement in its titre in BN1 and BN2, respectively, despite this improvement being insignificant (p = 7.8e−02 and 8.7e−01).As depicted in Fig. CYP725A4 enzyme neither favoured the taxadiene conversion nor it caused any remarkable increase in T5α-ol production, while it improved the production of side oxygenated taxane products. Furthermore, BN2, with one more copy of Taxus POR, showed a decreased final biomass accumulation by 52% (OD600: 16 ± 1.7) relative to LRS6. Similar improvement in the side oxygenated taxanes concentrations was initially observed for BN2, however, the final taxadiene concentration in this strain showed 1.5-fold improvement to that of BN1 strain, being 4.6 ± 0.2 mg/L. Despite this, due to the poor performance of BN2 in comparison to BN1, particularly its instability in later experiments and no growth, the next strains were created with focusing on P450 overexpression. In line with our results, the increased POR expression also reduced the CYP725A4 oxygenated taxanes concentration in Biggs et al. [E. coli, the phenomenon which authors attributed to resource competition, elevated burden and inefficient use of NADPH. They also noted that increasing CYP725A4 expression to POR resulted in higher accumulation of oxygenated taxanes.On the other hand, none of the strains showed any enhancement in taxadiene consumption and its titre was increased by 1.5- and 2.4-fold, in BN1 and BN2. Based on the data from Fig. TAT) expression. Despite this, to account for any product changes due to TAT expression, BN3 was created with the 1st copy of CYP725A4 under the control of a different strong constitutive promoter (pHXT7) using taxadiene-producing LRS5. HXT7 gene is known to be repressed during high glucose availability [TAT expression cassette. Hence, it is possible that the side oxygenated products also play intermediary roles in Taxol® biosynthesis pathway that need to be further investigated using the same promoter and terminator sets for the overexpression. Also, the final biomass accumulation of BN3 was reduced to 17.3 ± 1.5, compared to BN1 and LRS6 with final biomass accumulations of 28.14 ± 2.5 and 22.2 ± 0.7, respectively. Hence, it was hypothesised that potentially the taxanes and/or their producing enzymes were inducing the oxidative stress in the cell, lowering the biomass accumulation.Similar to our previous study , we did lability , and sinTRX2 gene promoter, which has been found to be responsive to hydroperoxide stresses [CYP725A4. This strain displayed no growth and therefore, no productivity. Biggs et al. has also separately reported that the higher production of CYP725A4 diterpenoids in E. coli resulted in loss of productivity [POR and CYP725A4 was deemed inefficient. The chromatograms of these strains are available in Additional file Using the stresses , BN4 structivity . In theiuctivity , they atThe production of the oxygenated taxanes, other than T5α-ol, could also be viewed to be influenced by the electron coupling and P450-reductase interactions efficiency, growth-based and/or no-growth-involved endogenous reactions, and P450/reductase enzymatic components. Therefore, in the rest of our study, we examined the accuracy of these hypotheses through a range of strain engineering and biochemical assays.Our first hypothesis was rooted in the possibility of inefficient electron coupling and P450-reductase interactions that result in the side products as the electron transfer occurs in two steps . Indeed,CYP725A4 only (BN5) was also constructed. Interestingly, the strain only expressing the P450 enzyme still produced T5α-ol, OCT and iso-OCT, although at minor quantities of 0.11 ± 0.02, 0.51 ± 0.2 mg/L, and below the limit of quantification detection for iso-OCT, which represented a reduction of 77.4- and 15.4-fold for T5α-ol and OCT, respectively, in comparison to the strain BNF-1, expressing Taxus POR , there was no significant difference between final taxadiene concentration of CYP725A4-expressing strain (BN5) and the self-sufficient strains. Hence, it is more likely that suboptimal expression of prokaryotic reductases with strong coupling capacity [Of note, the final biomass accumulations of these self-sufficient strains were not found to be significantly different despite their overall poor performance. However, the taxadiene production was found to be higher by at least 3.9-fold as in BN5 to 6.2-fold in BNF-4 compared to taxadiene-producing LRS5 strain, without significant difference in final biomass accumulation. On the contrary, around 1.2-fold reduction in taxadiene formation was noticed in BNF-1 compared to LRS5. Therefore, BNF-1 expressing  studies –43, it ccapacity , 45 causTaxus cuspidata reductase (POR). As LRS6 strain was constructed using GAL promoters for both CYP725A4 and POR genes, to minimise any delay in electron transfer, POR was expressed under a strong consecutive promoter (pTDH3), while CYP725A4 was expressed using the strong galactose promoter (pGAL1) for BN6 strain. However, to keep CYP725A4 expression stronger than that of POR, stronger terminator (RPL41Bt) and weaker terminator (ICY2t) [CYP725A4 and POR, respectively. In YPG medium, BN6 generated the highest amounts of OCT, iso-OCT and T5α-ol at the final concentrations of 14.6 ± 1.4, 12 ± 1.4 and 10.6 ± 1.8 mg/L, respectively, which represented 1.8-, 1.7-, and 1.2-fold improvement compared to BNF-1, expressing Taxus POR fused to CYP725A4. However, taxadiene was almost doubled to be 20.3 ± 0.9 mg/L and the final biomass slightly reduced to 26.5 ± 3.2 by the end of 72 h of cultivation  [600) compared to BN6 (0.90 mg/L/OD600), a phenomenon also reported for other P450-reductase fusions [Taxus CYP725A4 and POR interaction. This is also supported by the epoxidation theory [CYP725A4. However, as of now, it is unclear how the interaction between CYP725A4 and POR can be optimised to promote the uncoupling for higher oxygenated taxanes production whilst maximising the taxadiene consumption.In the previous section, we showed that the superior coupling capacity of prokaryotic reductase did not favour the CYP75A4 activity in yeast. Therefore, another strain, BN6, was constructed using  (ICY2t)  were useeptor(s) . On thiseptor(s)  by uncoueptor(s) . This pr fusions . Thus, in theory , 22, wheInterestingly, the T5α-ol: OCT: iso-OCT ratio remained very similar, 1:0.89:0.81 and 1:1.4:1.1 for BNF-1 and BN6, respectively, by the end of 72-h cultivation in YPG medium. Since uncoupling is an auxiliary mechanism of activity, its effect on product distribution cannot be inferred through in vivo studies. Instead, as results suggest, and due to unknown reasons, T5α-ol is the later product formed when taxadiene consumption is high and possibly the least affected by the decreased uncoupling rate, given the electron transfer to CYP725A4 is established. Consequently, more optimal fusion protein design can be exploited to improve the P450-POR interaction for higher taxadiene conversion and oxygenated taxanes production. Despite this, due to the superior capability of BN6 for higher oxygenated taxanes production, this strain was used for all the other experiments performed later to answer our other hypotheses.S. cerevisiae yet, we postulated that selecting a variety of microbiology media might be influential in a change in CYP725A4 product spectrum in S. cerevisiae.Our second formulated hypothesis was the cumulative effects of endogenous, underground metabolism  on the p600, highlighting that the promotion of biomass is not tightly correlated to increased oxygenated taxanes production. Despite this, these ratios changed to 1, 2, 0.7, 0.8 and 0.8 mg/L/OD600 for taxadiene, showing that the taxadiene production was coupled to biomass production [The results clearly indicated that using more enriched medium like TBMG, while promoting the biomass accumulation of BN6 Fig. H to 52.3oduction . As SDG S. cerevisiae was shown capable of producing early oxygenated taxanes, varying the electron transfer partner or reductase gene dosage did not necessarily help with higher CYP725A4 activity as shown in Figs. Taxus CYP725A4 and reductase proteins, being heme and flavins, respectively, were selected to be supplemented exogenously in the BN6 growth medium.While Despite our incomplete understanding of the underlying mechanism for heme prosthetic group incorporation into the apocytochrome form of CYP725A4, it was critical to investigate if the flavins were the controlling elements for CYP725A4 optimal activity as a result of improved POR activity. In parallel, to screen for the potential effect of each parameter on the product distribution, a three-level definitive screening design  was perfFor all the oxygenated taxanes, ALA was a significant negative predictor . However, hemin, which is the heme ferric chloride species (or ferrous protoporphyrin IX) , was fouS. cerevisiae. We noted that riboflavin exerted a negative impact on oxygenated taxanes production while it had a positive impact on increased ROS level, despite being known for its protecting effect for glutathione redox cycle improvement [Among flavins, FAD and riboflavin were found as significant factors  . Hence, a variation partitioning study was performed to examine the contribution of each predictor to the overall variance in each response. As a result, each variable was found significant at p = 0.001 by the ANOVA test, regardless of the effect of other significant predictors. Hence, the importance of each predictor reported in each model was again validated Fig. A–D. SimiTaxus CYP725A4-POR enzymes both induce oxidative stress through uncoupling, whilst protecting against it [Based on the initial conclusions drawn from the previous sections, our fourth hypothesis was that the ainst it  through ainst it  from theainst it , 68. Simainst it  and to painst it , similarTaxus CYP725A4 and POR interaction in yeast as in Kells et al. [A one-way ANOVA test revealed that except T5α-ol and taxadienediol, all other factors including OCT (p = 2.6e−05), iso-OCT (p = 0.003), taxadiene (p = 1.04e−05), diterpenoid I (p = 0.02) and biomass (p = 0.01) were significantly affected by the addition of ascorbic acid to the medium Fig. A. The pas et al.  study, ws et al. .Fig. 7Th600 unit was found to increase by around 1.4-fold upon supplementing the exogenous NaCl concentration up to 5 mM, while a sudden drop to 0.81 ± 0.07 mg/L/OD600 was observed at 10 mM NaCl. The previous studies have reported on improved P450-POR interaction and intra-reductase electron transfer due to enhanced charge pairing and/or protein conformations, as a result of which the P450s performances were improved at increased ionic strengths [E. coli cell-surface immobilised human CYP1A2 and POR [As illustrated in Fig. trengths , 72, 73.trengths , 74, 75. and POR . DespiteTaxus CYP725A4 and POR, the lipophilic nature of taxadiene and its derivatives might increase their intracellular residence, leading to cytotoxicity due to their metabolism, by which the endogenous mechanisms promote their functionalisation and conjugations to render them more polar [S. cerevisiae.Besides the potential uncoupling event by re polar . Therefore polar , 76. In Taxus POR has not been reported yet.Therefore, to illustrate all the metabolites of CYP725A4, BN6 resting cell assays in 50 mM phosphate buffer at approximate pH of 6–8 were performed to keep the effect of endogenous pathways on the enzyme activity to the minimum. Our initial results revealed that pH 6 was the least optimal for total oxygenated taxanes production (p = 0.04). Also, the yeast biomass increased after 115 h in pH = 8, while showing an almost double overall side oxygenated taxane: T5α-ol (22.5 ± 4) at time point one relative to pH = 7. However, the overall difference between pH = 7 and pH = 8 was insignificant (adjusted p = 0.69)   ) to induce the taxanes production during the resting stage. As the biomass accumulation did not change over time at pH 7, it can be inferred that the higher NADPH availability together with optimal pH favoured higher and more diverse oxygenated taxanes production, where a relatively higher total oxygenated taxanes titre at the last end point 115 h) of pre-rest galactose induction , and oxygenated taxadiene and potentially taxadiene that might have co-eluted with it, was found as the Granger-causal of diterpenoid IX (p = 0.02) and diterpenoid VII (p = 0.04). Interestingly, diterpenoid IX was also found to be associated with diterpenoid III (p = 0.045), and taxadienediol, which is a double oxygenated taxane (304 (m/z)), was Granger-caused by diterpenoid VII (p = 0.03). While the complete identity of this taxadienediol is not unknown, Edgar et al.  have proS. cerevisiae culture at 1-L scale with optimised cultivation medium [Interestingly, it was also found that diterpenoid III unidirectionally Granger-caused iso-taxadiene (p = 0.03) and similarly, diterpenoid VII and IX Granger-caused the oxygenated taxadiene . Therefore, it is also possible that these diterpenoids stimulate the higher production of taxadiene isomers. Overall, the concentration of total taxanes during post-rest stage (361.4 ± 52.4 mg/L) in a 10-mL scale was remarkable and comparable to the previous study, reporting the highest total taxanes concentration by whole n medium .Taxus cuspidata CYP725A4 and reductase (POR) copy numbers in S. cerevisiae. While using low-glucose induced promoter, pHXT7, for CYP725A4 expression almost doubled the total oxygenated products to 17.8 ± 1.3 mg/L in a taxadiene-producing yeast strain, a similar result was achieved (18.6 ± 5.5 mg/L) by expressing an extra copy of CYP725A4 under the pGAL1 promoter in a yeast also expressing the next Taxol® pathway gene, Taxa-4(20),11(12)-dien-5α-olO-Acetyl Transferase (TAT). This however did not affect the product distribution and the T5α-ol titre compared to the control strain. Interestingly, we found that TAT expression likely promoted the consumption of all major CYP725A4 diterpenoids, where further investigation would however be required. In contrast, an extra copy of POR resulted in an unstable yeast strain, highlighting the inefficiency of gene dosage variance in improving the enzyme productivity, and higher main product titre. By assuming that the epoxidation route was directly correlated to the cytochrome P450 uncoupling event, we compared the performance of the strains harbouring the tandem and fusion gene cassettes of CYP725A4 and T. cuspidata POR to those expressing the fusion gene cassettes of CYP725A4 and self-sufficient prokaryotic reductases with known strong electron coupling capacity. Interestingly, the separate expression of Taxus CYP725A4 and POR outperformed the fusion constructs in terms of whole oxygenated taxanes concentrations, being 6.1 ± 1.4 mg/L compared to 56.8 ± 17.6 mg/L, favouring the hypothesis of uncoupling being influential for the formation of whole diterpenoids. Despite this, OCT was the main product in all oxygenated taxanes-producing strains, while a shift towards higher T5α-ol formation and taxadiene consumption was noticed using T. cuspidata CYP725A4 and POR fusion. Using a variety of other growth media, despite affecting the final biomass, did not result in consensus changes in the ratio of T5α-ol: side oxygenated taxanes, and OCT remained the major product. Thus, the previous attempts to increase the biomass as a proxy for increased diterpenoid titres are questioned. To screen for the rate-limiting step in the strain expressing the Taxus CYP725A4 and POR in tandem, five cofactors involved in the structural components of their enzymes were tested. These included hemin (ready heme), iron precursor (δ-Aminolevulinic acid), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and their precursor, riboflavin, all screened by an augmented definitive screening design. Forward stepwise regression modelling revealed the positive impact of FAD in oxygenated taxanes and biomass improvement, while riboflavin and ALA were found to have negative impacts on the same responses. Also, hemin was found to further decrease the concentrations of iso-OCT and T5α-ol. However, due to positive effect of ALA and hemin, we anticipate that the two former oxygenated products were the secondary metabolites of CYP725A4. Studying the effect of antioxidant and ionic strength on P450-POR activity further strengthened the uncoupling playing the main factor in oxygenated taxanes production, releasing electron and oxygen sources for use by the enzyme. Finally, the resting cell assays were employed to study the reaction kinetics with minimised effect from the yeast cell internal metabolic processes in a strain expressing the CYP725A4 and POR in tandem. This led to the highest taxanes concentrations in S. cerevisiae (361.4 ± 52.4 mg/L) at 10-mL scale using galactose inducer before the resting stage. As a consequence of reduced activities of the growth-associated processes and other cellular mechanisms, around 4.4-fold difference in total taxanes concentration was observed in comparison to the growing cells in YPG medium. In addition, a diverse variety of products which were not detected by whole yeast culture, was revealed, rendering the resting cell assay a valuable tool for studying the activity of Taxol® downstream enzymes in yeast. Moreover, as the iron, flavins, antioxidants and salt were found to impact both CYP725A4 and the whole cell, future studies should invest on finding ways to assure sufficient reducing equivalents and cofactors are provided through more cost-effective approaches like genetic engineering for more efficient electron transfer and hydroxylation. In conclusion, based on our results and in particular due to the strong harmful effect of CYP725A4 and POR overexpression on the yeast, future studies need to construct more efficient fusion constructs to tightly regulate the oxygenated taxanes production with respect to uncoupling effect, whilst maximising the taxadiene consumption.CYP725A4 is the first committed cytochrome P450 enzyme in Taxol® (paclitaxel) biosynthetic pathway. It catalyses the conversion of taxadiene into taxadiene-5α-ol (T5α-ol), as the main product, and several other diterpenoids, mainly 5(12)-oxa-3(11)-cyclotaxane (OCT), 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) and taxadienediols. There are several reasons for which this enzyme has been known as a bottleneck to the whole Taxol® pathway integration in non-native systems, mainly due to its slow activity and the high side-product: main-product titre ratio. While this enzyme was long known to undergo radical-rebound mechanism, the recent studies have proposed the existence of an alternative catalysis route, namely the epoxidation. We therefore started by examining the effect of S. cerevisiae strain used for genomic integration of CYP725A4 and reductases was LRS5 ; his3Δ1::hphMX4-pGAL1-ERG12/pGAL10-IDI1;trp1-289::TRP1_pGAL1-CrtE(Xanthophyllomyces dendrorhous)/pGAL10-ERG20; YPRCdelta15::NatMX-pGAL1-CrtE(codon-optimised)/pGAL10-CrtE:: ARS1014a::pGal1-TASY-GFP:: ARS1622:: pGal1-MBP-TASY-ERG20*::ARS1114a::pGal1-MBP-TASY-ERG20*) previously constructed in [, originated from CEN.PK2-1C (EUROSCARF collection). To study the gene dosage effect, LRS6  was used [The parent 2HPO4, 2 g/L D( +)-glucose, 10 g/L peptone and 5 g/L sodium chloride. TBMG contained 12 g/L tryptone (pancreatic digest of casein), 24 g/L yeast extract, 9.4 g/L K2HPO4, 10 g/L glycerol, and 2.2 g/L KH2PO4. LBG contained 5 g/L NaCl, 10 g/L tryptone and 5 g/L yeast extract. Each medium was supplemented with 20 g/L galactose. A 20% (v/v) n-dodecane (ACROS Organics™) was used in all cultivations to extract taxanes and prevent their air-stripping. Gene dosage study was initially performed in 24-well deep well polypropylene plates. However, due to incompatibility of polypropylene and dodecane and massive co-elution of taxane products and contaminant peaks  powder (Formedium), 5 g/L ammonium sulphate , 1.7 g/L yeast nitrogen base without amino acids (Alfa Aesar™) and was supplemented with 2% (w/v) glucose  or galactose . The dropout medium for yeast transformation had the same formula for SDD, except that it contained 0.77 g/L CSM-URA  and 20 g/L agar (ACROS Organics™). The routinely used growth medium containing yeast extract (1%(w/v)) and casein peptone (2%(w/v)) (Merck), supplemented with 2% (w/v) glucose (YPD) or galactose (YPG) was used for inoculum preparation and production screening, respectively. Two other media, Brain Heart Infusion Broth-galactose  and Terrific Broth, Modified-galactose  were included to study the medium effect. BHIG contained 5 g/L beef heart (infusion from 250 g), 12.5 g/L calf brains (infusion from 200 g), 2.5 g/L NaS. cerevisiae codon-optimised Taxus cuspidata CYP725A4 and POR were amplified from LRS6 strain [Bacillus megaterium P450BM3 Reductase (BMR), Rhodococcus sp. P450RhF Reductase (RhFRED) and Albidovulum xiamenense CYP116B64 Reductase (AxRED), were codon optimised for S. cerevisiae using IDT Codon Optimisation Tool and all were then synthesised with natural linkers by TWIST Bioscience. Escherichia coli Flavodoxin (fldA) and Flavodoxin Reductase (Fpr) were directly amplified from DH5α cells. The truncated POR sequence was constructed by removing the transmembrane domain as predicted by TMHMM-2.0 [E. coli (NEB) according to manufacturer’s instructions. Upon initial screening of transformants on LB Lennox (Merck) medium supplemented with ampicillin (Fisher BioReagents™) for GFP reporter exclusion, the plasmids of a few selected transformants were isolated with GeneJet Miniprep kit (Thermo Scientific™) and sequenced. Together with pWS158 Cas9 vector, they were then either amplified by a 3-step PCR reaction using Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific™) or 1 µg of each were digested with EcoRV (Promega) and BsmBI (NEB) for 3–4 h, respectively, to yield homology arms for repairing into single plasmid. The correct sizes of digested plasmids were recovered from 0.7% (w/v) low-melting point TopVision agarose (Thermo Scientific™) using Zymoclean Gel DNA Recovery Kit (Zymo Research). All PCR products and donor DNAs were purified with GeneJET PCR Purification Kit (Thermo Scientific™).The native promoter and terminator sequences of yeast genome were used in this study. All primers and single stranded linker sequences were synthesised by IDT . 6 strain . BacilluMHMM-2.0 . To amplMHMM-2.0  were perMHMM-2.0  were ampMHMM-2.0  and tranSelf-sufficient CYP725A4 were constructed by phosphorylating and annealing the single-stranded linker sequences, using the same procedure described above. Following PCR amplification, this short fragment was purified with ethanol according to Sambrook and Russell . The fulURA3 selection marker and for which single stranded sgRNAs were synthesized [http://n2t.net/addgene:90517; RRID:Addgene_90517 and Addgene plasmid #90516; http://n2t.net/addgene:90516; RRID:Addgene_90516). To increase the transformation efficiency, a modified protocol by Muller et al. [The strains with extra P450 and/or reductase copy numbers were constructed using Cas9-sgRNA 2-micron plasmids harbouring a thesized . In thisthesized . Yeast fr et al.  was implr et al.  for each600 = 1, they were then inoculated into different media, namely SDG, YPG, LBG, BHIG and TBMG (as described above), all containing dodecane at final 20% (v/v) concentration. The prepared tubes were covered with cotton wool and foil, and were incubated at 30 °C, 250 rpm, for up to 72 h prior to GC–MS analysis.The BN6 cells were grown overnight in 5 mL of YPD medium, and with a starting ODA three-level definitive screening design  was creaFirst Run:A) Iron: (i) Hemin: 10, 55, 100 µM; (ii) δ-Aminolevulinic acid (ALA): 0.1, 1.55, 3 mM.B) Flavin: (i) Riboflavin: 0.1, 1.05, 2 mM; (ii) Flavin adenine dinucleotide (FAD): 5, 7.5, 10 mM; (iii) Flavin mononucleotide (FMN): 5, 7.5, 10 mM.Second Run:A) Iron: (i) Hemin: 5, 27.5, 50 µM; (ii) ALA: 0.1, 1.55, 3 mM.B) Flavin: (i) Riboflavin: 1, 4.5, 8 mM; (ii) FAD: 1, 4.5, 8 mM; (iii) FMN: 1, 4.5, 8 mM.600 = 1 and incubated at 30 °C and 1400 rpm for 72 h. A 140 µL of dodecane was also added at 20% (v/v) to reach 700 µL total volume. Each condition was tested in triplicate in a 96-well microplate with glass inserts (Hirschmann™), sealed with two layers of gas permeable adhesive plate seals (Thermo Scientific™), to minimise the dodecane evaporation. The results were analysed with GC–MS and the second run data were normalised relative to the control sets.All chemicals were sourced from Thermo Fisher Scientific (Thermo Scientific™) at the highest available purity. The starting cultures were prepared from overnight cultivation of BN6 strain in YPD. To ensure sufficient aeration and proper mixing, the total culture volumes were set to 560 µL, inoculated at ODAt the end of the cultivation, the total reactive oxygen species (ROS) was measured using Deep Red fluorometric intracellular ROS kit (Merck) according to manufacturer’s instructions in BMG Clariostar microplate reader. The relative fluorescence units were normalised by the final biomass.lmStepAIC function in caret package in R [VIF function in car R package [check_model and performance_accuracy functions in performance R package [rda function in vegan R package [cor.test function in R to calculate the Kendall’s tau correlation coefficient. The correlation plot and significance test were then visualised using corrplot R package [The entire data was subjected to forward stepwise multiple regression modelling  using lmage in R . Full quage in R  and sele package ; 4) Lowe package  to check package . The cor package .The BN6 cells were grown overnight in 5 mL of YPD medium, and inoculated into YPG media, containing different concentration of either sodium chloride or ascorbic acid at regular intervals of 0.1, 1, 2.55, 3.8, 5 and 10 mM at a total volume of 700 uL including 20% dodecane. Triplicates of each run were prepared in a 96-well microplate with glass inserts (Hirschmann™), sealed with two layers of gas permeable adhesive plate seals (Thermo Scientific™) and incubated for 72 h at 30 °C and 1400 rpm.600 units were then resuspended in 10 mL of 50 mM filter sterilised potassium phosphate buffer (pH = 7), containing 20% (v/v) dodecane, in glass tubes covered with cotton wool and aluminium foil. The cells were incubated at 30 °C, 250 rpm, and 50 µL of dodecane samples were withdrawn after 21, 28, 50, 98 and 115 h during this experiment for all three replicates. A parallel run with LRS5 was done in SDD to confirm and relate the observed peaks to CYP725A4 and POR expressions. Prior to this experiment, preliminary runs of BN6 experiment were done to determine a suitable pH for CYP725A4 activity, following the same procedure except that it was done with pH = 6, 7, and 8 with galactose induction during the resting stage and with lower oxygen availability. The possible relationship among the taxanes over time were investigated using Granger causality testing [The BN6 cells were grown overnight in 500 mL of SDD or SDG and harvested by sequential centrifugation of every 50 mL at 5000 rpm for 2 min. The cell pellets were washed twice with sterile ultrapure water. The cell pellets at 40 OD testing  by lmtes testing . For thiAt the end of each cultivation, 50 µL of dodecane overlay was extracted from the culture media and analysed by GC–MS using Trace 1300 GC (ThermoFisher Scientific), equipped with TG-SQC column (15 m × 0.25 mm × 0.25 μm) (gene dosage study) or TraceGOLD TG-5MS (30 m × 0.25 mm × 0.25 μm) . The mass spectra in the range of 40–650 m/z was recorded on a Thermo Scientific ISQ Series single quadrupole mass spectrometer using EI ionization mode. The GC temperature programme began at 120 °C (3 min) and was then raised to 250 °C at a rate of 20 °C/minute with 3-min hold time. Xcalibur™ software (ThermoFisher Scientific) was employed for data processing and AMDIS software was used for extracted ion (288 m/z) chromatograms retrieval. Geranylgeraniol  was used to quantify GGOH concentration. Pure taxadiene  and the diterpene totarol  were used as standards for quantifying taxanes using shorter and longer GC columns, respectively.The control cells used in antioxidant-ionic strength study were pelleted, vortexed and mixed with 1 mL of 99% acetone (Thermo Scientific™)-dodecane (1:1) for 16 h at 800 rpm. The extracts were centrifuged at 215 × g for 5 min, and the dodecane layer was extracted for GC–MS analysis.All statistical analyses of this study were performed in R version 4.1.3. Apart from the gene dosage study which was evaluated with two-way analysis of variance (ANOVA) to study the effect of both strain type and growth time, a one-way ANOVA was conducted to evaluate the other results with R stats package. Using Levene’s test in car R package , 96, theAdditional file 1: Table S1. Schematic representation of strains in this study. Table S2. List of primers used in the study. Table S3. Sequence of genes in this study. Table S4. List of guide RNA sequences used in this study. Table S5. List of linkers used in this study. Table S6. Augmented definitive screening designs for optimising P450-reductase expressions, including controls. Table S7. Linear model formulas for response factors in definitive screening designs. Figure S1. The messy chromatogram from 2 mL of LRS6 strain culture in polypropylene-made deepwell microplates. Figure S2. Representative chromatograms of gene dosage study. Figure S3. Representative mass spectrum for LRS6 strain cultivated in shake flask. Figure S4. Representative chromatograms of the strains expressing self-sufficient CYP725A4, CYP725A4 only and parent taxadiene-producing LRS5, all cultivated in 5 mL of YPG in 10 mL glass tubes. Figure S5. Representative chromatograms for BN6, expressing Taxus CYP725A4 and POR in tandem, in different culture media. Figure S6. Marginal model plots for design of experiment (DoE) study for all predictors (Flavin Adenine Dinucleotide (FAD), Flavin Mononucleotide (FMN), Riboflavin, Hemin and δ-Aminolevulinic acid (ALA)) and their interactions on: Figure S7. DoE study results according to run number. Figure S8. Preliminary resting cell assay to test the effect of acid–base on side-product (OCT+ iso-OCT) to main product (T5α-ol) ratio. Figure S9. Representative chromatograms for the resting cell assays. Figure S10. Representative chromatograms for the resting cell assays with extracted ion of 288 m/z for confirming the identified diterpenoids. Figure S11. Representative mass spectra from resting cell assay experiment. Figure S12. Representative mass spectra from resting cell assay experiment.