Stiff gel-protected fiber-shaped supercapacitors based on CNFA/silk composite fiber with superhigh interference-resistant ability as self-powered temperature sensor.

The development of self-powered sensors with interference-resistant detection is a priority area of research for the next generation of wearable electronic devices. Nevertheless, the presence of multiple stimuli in the actual environment will result in crosstalk with the sensor, thereby hindering the ability to obtain an accurate response to a singular stimulus. Here, we present a self-powered sensor composed of silk-based conductive composite fibers (CNFA@ESF), which is capable of energy storage and sensing. The fabricated CNFA@ESF exhibits excellent mechanical performance, as well as flexibility that can withstand various deformations. The CNFA@ESF provides a good areal capacitance (44.44 mF cm-2), high-rate capability, and excellent cycle stability (91 % for 5000 cycles). In addition, CNFA@ESF also shows good sensing performance for multiple signals including strain, temperature, and humidity. It was observed that the assembly of the symmetrical device with a stiff hydrogel surface layer for protection enabled the real-time, interference-free monitoring of temperature signals. Also, the CNFA@ESF can be woven into fabrics and integrated with a solar cell to form a self-powered sensor system, which has been proven to convert and store solar energy to power electronic watches, indicating its huge potential for future wearable electronics.

Fine and combinatorial regulation of key metabolic pathway for enhanced ß-alanine biosynthesis with non-inducible Escherichia coli.

ß-Alanine is the only ß-amino acid in nature and one of the most important three-carbon chemicals. This work was aimed to construct a non-inducible ß-alanine producer with enhanced metabolic flux towards ß-alanine biosynthesis in Escherichia coli. First of all, the assembled E. coli endogenous promoters and 5'-untranslated regions (PUTR) were screened to finely regulate the combinatorial expression of genes panDBS and aspBCG for an optimal flux match between two key pathways. Subsequently, additional copies of key genes (panDBS K104S and ppc) were chromosomally introduced into the host A1. On these bases, dynamical regulation of the gene thrA was performed to reduce the carbon flux directed in the competitive pathway. Finally, the ß-alanine titer reached 10.25 g/L by strain A14-R15, 361.7% higher than that of the original strain. Under fed-batch fermentation in a 5-L fermentor, a titer of 57.13 g/L ß-alanine was achieved at 80 h. This is the highest titer of ß-alanine production ever reported using non-inducible engineered E. coli. This metabolic modification strategy for optimal carbon flux distribution developed in this work could also be used for the production of various metabolic products.

Identification of a novel growth-associated promoter for biphasic expression of heterogenous proteins in Pichia pastoris.

Pichia pastoris (P. pastoris) is one of the most popular cell factories for expressing exogenous proteins and producing useful chemicals. The alcohol oxidase 1 promoter (PAOX1) is the most commonly used strong promoter in P. pastoris and has the characteristic of biphasic expression. However, the inducer for PAOX1, methanol, has toxicity and poses risks in industrial settings. In the present study, analyzing transcriptomic data of cells collected at different stages of growth found that the formate dehydrogenase (FDH) gene ranked 4960th in relative expression among 5032 genes during the early logarithmic growth phase but rose to the 10th and 1st during the middle and late logarithmic growth phases, respectively, displaying a strict biphasic expression characteristic. The unique transcriptional regulatory profile of the FDH gene prompted us to investigate the properties of its promoter (PFDH800). Under single-copy conditions, when a green fluorescent protein variant was used as the expression target, the PFDH800 achieved 119% and 69% of the activity of the glyceraldehyde-3-phosphate dehydrogenase promoter and PAOX1, respectively. After increasing the copy number of the expression cassette in the strain to approximately four copies, the expression level of GFPuv driven by PFDH800 increased to approximately 2.5 times that of the strain containing GFPuv driven by a single copy of PAOX1. Our PFDH800-based expression system exhibited precise biphasic expression, ease of construction, minimal impact on normal cellular metabolism, and high strength. Therefore, it has the potential to serve as a new expression system to replace the PAOX1 promoter.IMPORTANCEThe alcohol oxidase 1 promoter (PAOX1) expression system has the characteristics of biphasic expression and high expression levels, making it the most widely used promoter in the yeast Pichia pastoris. However, PAOX1 requires methanol induction, which can be toxic and poses a fire hazard in large quantities. Our research has found that the activity of PFDH800 is closely related to the growth state of cells and can achieve biphasic expression without the need for an inducer. Compared to other reported non-methanol-induced biphasic expression systems, the system based on the PFDH800 offers several advantages, including high expression levels, simple construction, minimal impact on cellular metabolism, no need for an inducer, and the ability to fine-tune expression.

Breeding of Saccharomyces cerevisiae with a High-Throughput Screening Strategy for Improvement of S-Adenosyl-L-Methionine Production.

S-adenosyl-l-methionine (SAM), a vital physiologically active substance in living organisms, is produced by fermentation over Saccharomyces cerevisiae. The main limitation in SAM production was the low biosynthesis ability of SAM in S. cerevisiae. The aim of this work is to breed an SAM-overproducing mutant through UV mutagenesis coupled with high-throughput selection. Firstly, a high-throughput screening method by rapid identification of positive colonies was conducted. White colonies on YND medium were selected as positive strains. Then, nystatin/sinefungin was chosen as a resistant agent in directed mutagenesis. After several cycles of mutagenesis, a stable mutant 616-19-5 was successfully obtained and exhibited higher SAM production (0.41 g/L vs 1.39 g/L). Furthermore, the transcript levels of the genes SAM2, ADO1, and CHO2 involved in SAM biosynthesis increased, while ergosterol biosynthesis genes in mutant 616-19-5 significantly decreased. Finally, building on the above work, S. cerevisiae 616-19-5 could produce 10.92 ± 0.2 g/L SAM in a 5-L fermenter after 96 h of fermentation, showing a 2.02-fold increase in the product yield compared with the parent strain. Paving the way of breeding SAM-overproducing strain has improved the good basis for SAM industrial production.

[Microbial production of S-adenosyl-l-methionine: a review].

S-adenosyl-l-methionine (SAM) is ubiquitous in living organisms and plays important roles in transmethylation, transsulfuration and transamination in organisms. Due to its important physiological functions, production of SAM has attracted increasing attentions. Currently, researches on SAM production mainly focus on microbial fermentation, which is more cost-effective than that of the chemical synthesis and the enzyme catalysis, thus easier to achieve commercial production. With the rapid growth in SAM demand, interests in improving SAM production by developing SAM hyper-producing microorganisms aroused. The main strategies for improving SAM productivity of microorganisms include conventional breeding and metabolic engineering. This review summarizes the recent research progress in improving microbial SAM productivity to facilitate further improving SAM productivity. The bottlenecks in SAM biosynthesis and the solutions were also addressed.

Determination of ß2-Agonist Residues in Fermented Ham Using UHPLC-MS/MS after Enzymatic Digestion and Sulfonic Resin Solid Phase Purification.

ß2-agonists are a class of synthetic sympathomimetic drugs with acute poisoning effects if consumed as residues in foods. To improve the efficiency of sample preparation and to overcome matrix-dependent signal suppression in the quantitative analysis of four ß2-agonists (clenbuterol, ractopamine, salbutamol, and terbutaline) residues in fermented ham, an enzyme digestion coupled cation exchange purification method for sample preparation was established using ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS). Enzymatic digests were subject to cleanup treatment on three different solid phase extraction (SPE) columns and a polymer-based strong cation resin (SCR) cartridge containing sulfonic resin was found to be optimal compared with silica-based sulfonic acid and polymer sulfonic acid resins based SPEs. The analytes were investigated over the linear range of 0.5 to 10.0 µg/kg with recovery rates of 76.0-102.0%, and a relative standard deviation of 1.8-13.3% (n = 6). The limit of detection (LOD) and the limit of quantification (LOQ) were 0.1 µg/kg and 0.3 µg/kg, respectively. This newly developed method was applied to the detection of ß2-agonist residues in 50 commercial ham products and only one sample was found to contain ß2-agonist residues (clenbuterol at 15.2 µg/kg).

Improving ATP availability by sod1 deletion with a strategy of precursor feeding enhanced S-adenosyl-L-methionine accumulation in Saccharomyces cerevisiae.

S-adenosyl-L-methionine (SAM), used in diverse pharmaceutical applications, was biosynthesized from L-methionine (L-met) and adenosine triphosphate (ATP). This study aims to increase the accumulation of SAM in Saccharomyces cerevisiae by promoting ATP availability. Strain ΔSOD1 was obtained from the parent strain WT15-33 (CCTCC M 2021915) by deleting gene sod1, which improved the supply of ATP. The SAM content in strain ΔSOD1 exhibited a 22.3% improvement compared to the parent strain, which reached 93.6 mg g-1. The transformation of NADH (reduced nicotinamide adenine dinucleotide) and the relative expression of ATPase essential genes were investigated, respectively. The results showed that the lack of gene sod1 benefited the generation of ATP, which positively regulated the synthesis of SAM. Besides that, the production of SAM was further enhanced by improving substrate assimilation. With the infusion of 1.44 g L-1L-met and 0.60 g L-1 adenosine at 24 h (h) and 0 h following fermentation, the optimum medium could produce 1.54 g L-1 SAM. Based on the regulations mentioned above, the SAM concentration of strain ΔSOD1 enhanced from 7.3 g L-1 to 10.1 g L-1 in a 5-L fermenter in 118 h. This work introduces a novel idea for the biosynthesis of ATP and SAM, and the strain ΔSOD1 has the potential for industrial production.

Research progress of L-aspartate-α-decarboxylase and its isoenzyme in the ß-alanine synthesis.

Driven by the massive demand in recent years, the production of ß-alanine has significantly progressed in chemical and biological ways. Although the chemical method is relatively mature compared to biological synthesis, its high cost of waste disposal and environmental pollution does not meet the environmental protection standard. Hence, the biological method has become more prevalent as a potential alternative to the chemical synthesis of ß-alanine in recent years. As a result, the aspartate pathway from L-aspartate to ß-alanine (the most significant rate-limiting step in the ß-alanine synthesis) catalyzed by L-aspartate-α-decarboxylase (ADC) has become a research hotspot in recent years. Therefore, it is vital to comprehensively understand the different enzymes that possess a similar catalytic ability to ADC. This review will investigate the exploratory process of unique synthesis features and catalytic properties of ADC/ADC-like enzymes in particular creatures with similar catalytic capacity or high sequence homology. At the same time, we will discuss the different ß-alanine production methods which can apply to future industrialization.

Improvement of S-adenosyl-L-methionine production in Saccharomyces cerevisiae by atmospheric and room temperature plasma-ultraviolet compound mutagenesis and droplet microfluidic adaptive evolution.

To improve S-Adenosyl-L-methionine (a compound with important physiological functions, SAM) production, atmospheric and room temperature plasma and ultraviolet-LiCl mutagenesis were carried out with Saccharomyces cerevisiae strain ZY 1-5. The mutants were screened with ethionine, L-methionine, nystatin and cordycepin as screening agents. Adaptive evolution of a positive mutant UV6-69 was further performed by droplet microfluidics cultivation with ethionine as screening pressure. After adaptation, mutant T11-1 was obtained. Its SAM titer in shake flask fermentation reached 1.31 g/L, which was 191% higher than that of strain ZY 1-5. Under optimal conditions, the SAM titer and biomass of mutant T11-1 in 5 L bioreactor reached 10.72 g/L and 105.9 g dcw/L (142.86% and 34.22% higher than those of strain ZY 1-5), respectively. Comparative transcriptome analysis between strain ZY 1-5 and mutant T11-1 revealed the enhancements in TCA cycle and gluconeogenesis/glycolysis pathways as well as the inhibitions in serine and ergosterol synthesis of mutant T11-1. The elevated SAM synthesis of mutant T11-1 may attribute to the above changes. Taken together, this study is helpful for industrial production of SAM.

Cometabolic biodegradation system employed subculturing photosynthetic bacteria: A new degradation pathway of 4-chlorophenol in hypersaline wastewater.

4-chlorophenol (4-CP) as a toxic persistent pollutant is quite difficult treatment by using traditional biological processes. Herein, photosynthetic bacteria (PSB) driven cometabolic biodegradation system associated with exogeneous carbon sources (e.g., sodium acetate) has been demonstrated as an effective microbial technique. The biodegradation rate (ri) can be at 0.041 d-1 with degradation efficiency of 93% in 3094 lx. Through the study of subculturing PSB in absence of NaCl, it was found that 50% inoculation time can be saved but keeping a similar 4-CP biodegradation efficiency in scale-up salinity system. A new plausible biodegradation pathway for 4-CP in 4th G PSB cometabolic system is proposed based on the detected cyclohexanone generation followed by ring opening. It is probably ascribed to the increasement of Firmicutes and Bacteroidetes at phyla level classified based on microbial community. This study contributes to a new insight into cometabolic technology for chlorophenol treatment in industrial hypersaline wastewater.

Improvement of pyrroloquinoline quinone-dependent d-sorbitol dehydrogenase activity from Gluconobacter oxydans via expression of Vitreoscilla hemoglobin and regulation of dissolved oxygen tension for the biosynthesis of 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose.

The miglitol intermediate, 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose (6NSL), is catalyzed from N-2-hydroxyethyl glucamine (NHEG) by resting cells of Gluconobacter oxydans. One of the key factors limiting 6NSL production was the availability of oxygen during both cell cultivation and biotransformation of NHEG to 6NSL. Based on G. oxydans/pBBR1-sldAB-pqqABCDE-tldD (G. oxydans/AB-PQQ), the Vitreoscilla hemoglobin (VHb) was heterologously expressed in G. oxydans to enhance oxygen transfer efficiency and improve 6NSL production. The recombinant G. oxydans/AB-PQQ-VHb displayed higher biomass and NHEG oxidation activity than the control stain. The transcription levels of respiratory chain-related enzyme genes in G. oxydans/AB-PQQ-VHb exhibited up-regulation, indicating that the presence of VHb promoted the respiration. The dissolved oxygen (DO) concentration for cell cultivation was optimized in a 5-L stirred bioreactor. At a DO concentration of 20%, the maximum volumetric oxidation activity of NHEG of G. oxydans/AB-PQQ-VHb in the stirred bioreactor reached 168.3 ± 3.2 U/L. Furthermore, the biotransformation of NHEG to 6NSL using G. oxydans/AB-PQQ-VHb was carried out under different oxygen tensions to investigate the effect of oxygen on 6NSL production. Finally, up to 87.5 ± 5.9 g/L 6NSL was accumulated in the reaction mixture within 16 h when the DO was controlled at 30%.

Co-metabolic biodegradation of 4-chlorophenol by photosynthetic bacteria.

ABSTRACTEnvironmental contamination by 4-chlorophenol (4-CP) is a major concern. Photosynthetic bacteria have the ability to biodegrade 4-CP under dark aerobic conditions. In this study, we found that using different carbon sources (i.e. glucose, sodium acetate, sodium propionate sucrose, and malic acid) as co-metabolic substrates accelerated the biodegradation of 4-CP, and this acceleration was especially pronounced in the glucose treatment. A maximum degradation rate of 96.99% was reached under a concentration of 3.0 g·L-1 after 6 days of culture. The optimum conditions were pH 7.5, a temperature of 30°C, and a rotation speed of 135 rpm. The biodegradation of 4-CP was achieved at a range of salinities (0-3.0% NaCl, w/v). The biodegradation kinetics agreed with the Haldane model, and the kinetic constants were rmax = 0.14 d-1, Km = 33.9 mg·L-1, and Ki = 159.6 mg·L-1. Additionally, the coexistence of phenol or 2,4-dichlorophenol (2, 4-DCP) had a certain impact on the degradation of 4-CP under dark aerobic conditions. When the coexisting phenol concentration reached 100 mg·L-1, the maximum degradation rate of 4-CP reached 90.20%. The degradation rate of 4-CP decreased as the concentration of coexisting 2, 4-DCP increased.

Combinational expression of D-sorbitol dehydrogenase and pyrroloquinoline quinone increases 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose production by Gluconobacter oxydans through cofactor manipulation.

6-(N-hydroxyethyl)-amino-6-deoxy-l-sorbofuranose (6NSL), a key precursor in the synthesis of miglitol, is produced from N-2-hydroxyethyl-glucamine (NHEG) by the regioselective oxidation of Gluconobacter oxydans. The limitation of PQQ biosynthesis became a bottleneck for improvement of PQQ-dependent D-sorbitol dehydrogenase (mSLDH) activity. Five expression plasmids were constructed for the co-expression of the pqqABCDE gene cluster and the tldD gene on the basis of pBBR1-gHp0169-sldAB in G. oxydans to increase the biosynthesis of PQQ. The G. oxydans/pGA004, in which pqqABCDE and tldD were expressed as a cluster under the control of gHp0169 promoter, showed the optimal performance. The intracellular PQQ concentration and specific activity of mSLDH in cells increased by 79.3 % and 53.7 %, respectively, compared to that in G. oxydans/pBBR-sldAB. Then, the repeated batch biotransformation of NHEG to 6NSL by G. oxydans/pGA004 was carried out. Up to 75.0 ±â€¯3.0 g/L of 6NSL production with 94.5 ±â€¯3.6 % of average conversion rate of NHEG to 6NSL was achieved after four cycles of run. These results indicated that G. oxydans/pGA004 with high productivity had great potential for 6NSL production in industrial bioprocess.

Effects of methyl oleate and microparticle-enhanced cultivation on echinocandin B fermentation titer.

Echinocandin B (ECB) is a key precursor of antifungal agent Anidulafungin, which has demonstrated clinical efficacy in patients with invasive candidiasis. In this study, the effects of microparticle-enhanced cultivation and methyl oleate on echinocandin B fermentation titer were investigated. The results showed that the titer was significantly influenced by the morphological type of mycelium, and mycelium pellet was beneficial to improve the titer of this secondary metabolism. First, different carbon sources were chosen for the fermentation, and methyl oleate achieved the highest echinocandin B titer of 2133 ± 50 mg/L, which was two times higher than that of the mannitol. The study further investigated the metabolic process of the fermentation, and the results showed that L-threonine concentration inside the cell could reach 275 mg/L at 168 h with methyl oleate, about 2.5 times higher than that of the mannitol. Therefore, L-threonine may be a key precursor of echinocandin B. In the end, a new method of adding microparticles for improving the mycelial morphology was used, and the addition of talcum powder (20 g/L, diameter of 45 µm) could make the maximum titer of echinocandin B reach 3148 ± 100 mg/L.

Repeated production of 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose by immobilized Gluconobacter oxydans cells with a strategy of in situ exhaustive cell regeneration.

The major troubles in 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose (6NSL) production from N-2-hydroxyethyl glucamine (NHEG) by Gluconobacter oxydans were low cell yield during cell preparation and loss of cells' biocatalytic ability during biotransformation, resulting in high production cost and low 6NSL production. The target of this work was to enhance 6NSL production by reusing cells and improving the cells biocatalytic ability. First, inhibitory effects of substrate and product on 6NSL production, and optimization of cell regeneration condition were investigated, respectively. Then repeated production of 6NSL by immobilized cell using a strategy of in situ exhaustive cell regeneration in a bubble column bioreactor was developed. As a result, the bioprocess underwent nine cycles, the average 6NSL production and conversion rate of NHEG to 6NSL reached 42.6 g L-1 and 83.1% in each batch was achieved, respectively.

Mutagenesis of echinocandin B overproducing Aspergillus nidulans capable of using starch as main carbon source.

Echinocandin B, a kind of antimycotic with cyclic lipo-hexapeptides, was produced by fermentation with Aspergillus nidulans using fructose as main carbon source. The objective of this study was to screen a high-yield mutant capable of using cheap starch as main carbon source by atmospheric and room temperature plasma (ARTP) treatment in order to decrease the production cost of echinocandin B. A stable mutant A. nidulans ZJB19033, which can use starch as optimal carbon source instead of expensive fructose, was selected from two thousands isolates after several cycles of ARTP mutagenesis. To further increase the production of echinocandin B, the optimization of fermentation medium was performed by response surface methodology (RSM), employing Plackett-Burman design (PBD) followed by Box-Behnken design (BBD). The optimized fermentation medium provided the optimal yield of echinocandin B, 2425.9 ± 43.8 mg/L, 1.3-fold compared to unoptimized medium. The results indicated that the mutant could achieve high echinocandin B production using cheap starch as main carbon source, and the cost of carbon sources in fermentation medium reduced dramatically by about 45%.

Synergistic improvement of PQQ-dependent D-sorbitol dehydrogenase activity from Gluconobacter oxydans for the biosynthesis of miglitol precursor 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose.

6-(N-hydroxyethyl) amino-6-deoxy-l-sorbofuranose (6NSL) is the direct precursor of miglitol for diabetes therapy. The regio- and stereo-selective dehydrogenation offered by the membrane-bound d-sorbitol dehydrogenase (mSLDH) from Gluconobacter oxydans provides an elegant enzymatic method for 6NSL production. In this study, two subunits sldA and sldB of mSLDH were introduced into G. oxydans ZJB-605, and the specific enzyme activity of mSLDH towards NHEG was enhanced by 2.15-fold. However, the endogenous PQQ level was dramatically reduced in the recombinant strain and became a bottleneck to support the holo-enzyme activity. A combined supplementation of four amino acids (Glu, Ile, Ser, Arg) involved in biosynthesis of PQQ in conventional media effectively increased extracellular accumulation of PQQ by 1.49-fold, which further enhanced mSLDH activity by 1.33-fold. The synergic improvement of mSLDH activity provided in this study supports the superior high dehydrogenate activity towards substrate N-2-hydroxyethyl-glucamine, 184.28 g·L-1 of 6NSL was produced after a repeated bioconversion process catalyzed by the resting cells of G. oxydans/pBB-sldAB, all of which presenting a great potential of their industrial application in 6NSL biosynthesis.

Integrated strategy of temperature shift and mannitol feeding for enhanced production of echinocandin B by Aspergillus nidulans CCTCC M2012300.

The production of echinocandin B (ECB) by Aspergillus nidulans CCTCC M2012300 was improved by integrating the temperature-shift and fed-batch control strategies. The kinetic characteristics of batch cultures were analyzed at different culture temperatures, and then a two-stage temperature control strategy was established. In the first 6 days, the temperature was maintained at 30 °C to obtain the maximal cell growth rate; subsequently, 25 °C was used to gain a high ECB formation rate. On the basis of temperature control, the ECB productivity was increased to 143.3 mg/(L day), which was a 1.3-fold improvement compared with the optimal constant-temperature cultivations. The influences of fed-batch cultures were further investigated. A maximal ECB productivity of 170.8 mg/(L day) was obtained through a three-stage mannitol pulse-feeding strategy, which was another 1.2-fold improvement than that of the batch fermentation. This is the first report of the use of a two-stage temperature control fed-batch strategy in ECB fermentation. This strategy was simple and economical to operate and may provide new guidance for the industrial-scale production of ECB.

Enhanced Production of 6-(N-Hydroxyethyl)-Amino-6-Deoxy-α-L-Sorbofuranose by Immobilized Gluconobacter oxydanson Corn Stover with a pH Control Strategy in a Bubble Column Bioreactor.

6-(N-Hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose (6NSL) is a key intermediate in the synthesis of miglitol. Biotransformation of N-2-hydroxyethyl glucamine (NHEG) to 6NSL was performed by immobilized Gluconobacter oxydans, which was prepared by cultivating the cells in a home-made bubble column bioreactor where corn stover particles were loaded. The optimal carrier addition and aeration rate for 6NSL production by immobilized cells in the bioreactor were determined to be 25 g/L and 2.5 vvm respectively. The supplementation of NH4Cl was conducive to the biotransformation of NHEG and was performed by adding aqueous ammonia and HCl, which was taken as the pH controlling agents as well. An optimal pH control strategy using the mixture of aqueous ammonia and NaOH was applied, resulting in a 9.9% increased production of 6NSL, while repeated batches of biotransformation increased from three times to four times. Finally, the 6NSL concentration and the conversion rate of NHEG to 6NSLreached 44.2 ± 1.5 g/L and 88.4 ± 2.0%, respectively, in average after four cycles of biotransformation under the optimized condition.

Biosynthesis of miglitol intermediate 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose by an improved d-sorbitol dehydrogenase from Gluconobacter oxydans.

Adaptable exploitation of the catalytic potential of membrane-bound d-sorbitol dehydrogenase (mSLDH) from Gluconobacter oxydans is desperately needed in the industrial-scale production of miglitol. In the present study, a carbonyl group-dependent colorimetric quantification method was developed for the assay of miglitol key intermediate 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose (6NSL), and a high-throughput screening process of positive mutants was processed. Combined with several rounds of ultraviolet irradiation mutagenesis and screening procedure, a positive mutant strain G. oxydans ZJB16009 was obtained with significant increase in mSLDH catalytic activity by 1.5-fold, which exhibited an extremely accelerated uptake rate of d-sorbitol, and the fermentation time was significantly shortened from 22 to 11 h. In a 5-L biotransformation system, 60 g/L substrate N-2-hydroxyethyl glucamine (NHEG) was catalyzed by the resting cells of the mutant strain within 36 h and accumulated 53.6 g/L 6NSL, showing a 33.6% increase in the product yield. Therefore, it was indicated that the established high-throughput screening method could provide a highly efficient platform for the breading of G. oxydans strain for the industrial biosynthesis of miglitol intermediate 6NSL.