Recent progress in adaptive laboratory evolution of industrial microorganisms.

Adaptive laboratory evolution (ALE) is a technique for the selection of strains with better phenotypes by long-term culture under a specific selection pressure or growth environment. Because ALE does not require detailed knowledge of a variety of complex and interactive metabolic networks, and only needs to simulate natural environmental conditions in the laboratory to design a selection pressure, it has the advantages of broad adaptability, strong practicability, and more convenient transformation of strains. In addition, ALE provides a powerful method for studying the evolutionary forces that change the phenotype, performance, and stability of strains, resulting in more productive industrial strains with beneficial mutations. In recent years, ALE has been widely used in the activation of specific microbial metabolic pathways and phenotypic optimization, the efficient utilization of specific substrates, the optimization of tolerance to toxic substance, and the biosynthesis of target products, which is more conducive to the production of industrial strains with excellent phenotypic characteristics. In this paper, typical examples of ALE applications in the development of industrial strains and the research progress of this technology are reviewed, followed by a discussion of its development prospects.

De novo Synthesis of 2-phenylethanol from Glucose by Metabolically Engineered Escherichia coli.

2-Phenylethanol (2- PE) is an aromatic alcohol with wide applications, but there is still no efficient microbial cell factory for 2-PE based on Escherichia coli. In this study, we constructed a metabolically engineered E. coli capable of de novo synthesis of 2-PE from glucose. Firstly, the heterologous styrene-derived and Ehrlich pathways were individually constructed in an L-Phe producer. The results showed that the Ehrlich pathway was better suited to the host than the styrene-derived pathway, resulting in a higher 2-PE titer of ∼0.76 ± 0.02 g/L after 72 h of shake flask fermentation. Furthermore, the phenylacetic acid synthase encoded by feaB was deleted to decrease the consumption of 2-phenylacetaldehyde, and the 2-PE titer increased to 1.75 ± 0.08 g/L. As phosphoenolpyruvate (PEP) is an important precursor for L-Phe synthesis, both the crr and pykF genes were knocked out, leading to ∼35% increase of the 2-PE titer, which reached 2.36 ± 0.06 g/L. Finally, a plasmid-free engineered strain was constructed based on the Ehrlich pathway by integrating multiple ARO10 cassettes (encoding phenylpyruvate decarboxylases) and overexpressing the yjgB gene. The engineered strain produced 2.28 ± 0.20 g/L of 2-PE with a yield of 0.076 g/g glucose and productivity of 0.048 g/L/h. To our best knowledge, this is the highest titer and productivity ever reported for the de novo synthesis of 2-PE in E. coli. In a 5-L fermenter, the 2-PE titer reached 2.15 g/L after 32 h of fermentation, suggesting that the strain has the potential to efficiently produce higher 2-PE titers following further fermentation optimization.

Improvement of the catalytic performance of glycerol kinase from Bacillus subtilis by chromosomal site-directed mutagenesis.

Glycerol kinase is the key enzyme in glycerol metabolism, and its catalytic efficiency has an important effect on glycerol utilization. Based on an analysis of the glycerol utilization pathway and regulation mechanism in B. subtilis, we conducted site-directed mutagenesis of the key glycerol kinase gene (glpK) on the chromosome to improve the glycerol utilization efficiency of Bacillus subtilis. Recombinant wild-type Bacillus subtilis glycerol kinase (BsuGlpKWT) and two mutants (BsuGlpKM270I and BsuGlpKS71V) were successfully overexpressed in Escherichia coli BL21(DE3) and purified by Ni-IDA metal chelate chromatography. The specific activity of the BsuGlpKM270I mutant (62.6 U/mg) was significantly higher (296.2%) than that of wild-type BsuGlpKWT (15.8 U/mg). By contrast, the mutant BsuGlpKS71V (4.89 U/mg) exhibited lower (69.1%) activity than BsuGlpKWT, which suggested that variant S71V exhibited reduced catalytic efficiency for the substrate. Furthermore, the mutant strain B. subtilis M270I was constructed using a markerless delivery system, and exhibited a higher specific growth rate (improved by 11.3%, from 0.453 ± 0.012 to 0.511 ± 0.017 h-1) and higher maximal biomass (cell dry weight increased by 16%, from 0.577 ± 0.033 to 0.721 ± 0.015 g/L) than the parental strain with a shortened lag phase (2 ~ 4 h shorter) in M9 minimal medium with glycerol. These results indicate that the mutated glpK resulted in improved glycerol utilization, which has broad application prospects.

Bioconversion of recombinantly produced precursor peptide pqqA into pyrroloquinoline quinone (PQQ) using a cell-free in vitro system.

Pyrroloquinoline quinone (PQQ) has been recognized as the third class of redox cofactors in addition to the well-known nicotinamides (NAD(P)+) and flavins (FAD, FMN). It plays important physiological roles in various organisms and has strong antioxidant properties. The biosynthetic pathway of PQQ involves a gene cluster composed of 4-7 genes, named pqqA-G, among which pqqA is a key gene for PQQ synthesis, encoding the precursor peptide PqqA. To produce recombinant PqqA in E. coli, fusion tags were used to increase the stability and solubility of the peptide, as well simplify the scale-up of the fermentation process. In this paper, pqqA from Gluconobacter oxydans 621H was expressed in E. coli BL21 (DE3) as a fusion protein with SUMO and purified using a hexahistidine (His6) tag. The SUMO fusion protein and His6 tag were specifically recognized and cleaved by the SUMO specific ULP protease, and immobilized-metal affinity chromatography was used to obtain high-purity precursor peptide PqqA. Expression and purification of target proteins was confirmed by Tricine-SDS-PAGE. Finally, the synthesis of PQQ in a cell-free enzymatic reaction in vitro was confirmed by LC-MS.

Geographic and position-based variations in phyllospheric bacterial communities present on flue-cured tobacco.

Although tobacco leaves (TLs) contain abundant bacteria, how the geography and leaf position of TLs affect these bacteria is unclear. Here, TLs at different positions from Henan (HN, strong flavor style) and Yunnan (YN, fresh flavor style) provinces were collected, and the bacteria were characterized by Illumina sequencing at harvest and 1 year of storage. Bacterial communities were very different between TLs originating from different geographical areas and positions, and beta diversity analysis showed that leaf position was the most important factor for phyllospheric bacterial communities, followed by geographical area and storage time. At the genus level, Subdoligranulum, Thermus, and Acinetobacter were obviously more abundant in HN than in YN, while Blautia and Ruminococcus were significantly more abundant in YN. These differences in bacterial communities decreased after 1 year of storage, indicating that the microbiota tends to become similar during tobacco processing. Storage time also affected the phyllospheric bacteria of TLs, as the bacterial communities shifted significantly on both HN and YN TLs after 1 year of storage. Significant differences in the predicted genes were also observed between the different geographic locations and leaf positions. Potential human pathogens, including Acinetobacter, Methylobacterium, and Escherichia-Shigella, were greatly different between TLs originating from different areas and positions. These data suggested that geographic variations and positions were associated with phyllospheric bacterial communities on TLs, which may be related to not only the flavor style and quality of TLs but also the potential health risks to humans. KEY POINTS: • Tobacco leaf position and tobacco growth location affected bacterial communities. • Microbial communities of TLs shifted significantly after one year of storage. • Potential human pathogens differed at different leaf positions and growth locations.

The effects of threshing and redrying on bacterial communities that inhabit the surface of tobacco leaves.

Before being subjected to the aging process, raw tobacco leaves (TLs) must be threshed and redried. We propose that threshing and redrying affect the bacterial communities that inhabit the TL surface, thereby influencing the aging process. However, these effects remain unclear. In this study, Illumina sequencing was applied to analyze the bacterial communities on both raw and redried TLs. Shannon's diversity value decreased from 3.38 to 2.52 after the threshing and redrying processes, indicating a large reduction in TL bacterial diversity. The bacterial communities also largely differed between raw TLs and redried TLs. On unaged raw TLs, Proteobacteria was the most dominant phylum (56.15%), followed by Firmicutes (38.99%). In contrast, on unaged redried TLs, Firmicutes (76.49%) was the most dominant phylum, followed by Proteobacteria (21.30%). Thus, the dominant genus Proteobacteria, which includes Sphingomonas, Stenotrophomonas, and Pantoea, decreased after the threshing and redrying processes, while the dominant genus Firmicutes, which includes Bacillus and Lactococcus, increased. Changes in the bacterial communities between raw and redried TLs were also noted after 1 year of aging. The relative abundance of dominant Proteobacteria taxa on raw TLs decreased from 56.15 to 16.92%, while the relative abundance of Firmicutes taxa increased from 38.99 to 79.10%. However, small changes were observed on redried TLs after 1 year of aging, with a slight decrease in Proteobacteria (21.30 to 17.64%) and a small increase in Firmicutes (76.49 to 79.10%). Based on these results, Firmicutes taxa may have a higher tolerance for extreme environments (such as high temperature or low moisture) than Proteobacteria bacteria. This study is the first report to examine the effects of threshing and redrying on bacterial communities that inhabit TLs.

A new family-3 glycoside hydrolase from Penicillium oxalicum BL 3005 catalyzing tyrosol glucosylation to form salidroside.

A glycoside hydrolase from Penicillium oxalicum BL 3005 was purified to apparent homogeneity. Its molecular mass was estimated to be 90 kDa by SDS-PAGE. The enzyme was identified to be a new member of family-3 by peptide sequence. High transglycosylation activity was found in the hydrolytic reaction of cellobiose. In the reaction, salidroside (4-hydroxyphenethyl O-ß-d-glucopyranoside) was formed by adding tyrosol as the glycosyl acceptor. The optimum reaction pH and temperature were pH 6.5 and 55 °C, respectively. The maximum yield of salidroside was almost 20 g/L. These results indicated that the ß-glucosidase of P. oxalicum can be considered as a very promising catalyst for the synthesis of salidroside.

Degradation of phytosterols in tobacco waste extract by a novel Paenibacillus sp.

Phytosterols have been demonstrated to be precursors of polycyclic aromatic hydrocarbons (PAHs) formed during biomass pyrolysis. Here, a novel Paenibacillus sp. was evaluated for its ability to degrade phytosterols in tobacco waste extract (TWE). The optimal conditions for cell growth and stigmasterol (a representative of phytosterols) degradation were 37 °C, pH 7.0, 1.0 g/L yeast extract, and 6.0 g/L glucose. Paenibacillus sp. could degrade stigmasterol under high concentrations of glucose (up to 130 g/L) and tolerate wide pH (5.0-9.0) and temperature (25-42 °C) ranges. The new strain could degrade stigmasterol completely into CO2 and H2 O, and no intermediate steroids were detected during the degradation process. Phytosterol degradation in TWE was demonstrated by high-performance liquid chromatography-tandem mass spectrometry. Under optimal conditions (37 °C, pH 7.0, with the exponential-phase cells), the total degradation ratio of phytosterols reached 38.5% in TWE, including 45.2% of stigmasterol, 37.4% of ß-sitosterol, 27.3% of campesterol, and 28.7% of cholesterol. These results showed that Paenibacillus sp. is a candidate for phytosterol degradation in TWE and other biomass and is potentially useful in reducing the PAHs generated from biomass pyrolysis.

Bioconversion of lutein by Enterobacter hormaechei to form a new compound, 8-methyl-α-ionone.

OBJECTIVES: To investigate the final product of the bioconversion of lutein by a novel lutein-degrading bacterium, Enterobacter hormaechei A20, and the kinetics of the process. RESULTS: A new product, 8-methyl-α-ionone, was resolved by GC-MS. The compound was further identified by NMR. A conversion yield of 90% was achieved by E. hormaechei in 36 h with 10 g lutein l-1. CONCLUSIONS: This is the first report of the bioconversion of lutein to form 8-methyl-α-ionone. A degradation pathway is proposed.

Zoonotic Enterocytozoon bieneusi in raw wastewater in Zhengzhou, China.

Contamination of Enterocytozoon bieneusi Desportes, Charpentier, Galian, Bernard, Cochand-Priollet, Laverne, Ravisse, et Modigliani, 1985 in water sources may cause outbreaks of microsporidiosis. To examine the occurrence of E. bieneusi, 108 raw wastewater samples were collected from three wastewater treated plants in Zhengzhou, China. In total, 46 samples were PCR positive for E. bieneusi. A total of 15 ITS genotypes was identified, including ten known genotypes (D, BEB6, I, J, PigEbIX, PigEBITS5, EbpA, Peru6, Peru8, Type IV) and five novel genotypes (HNWW1, HNWW2, HNWW3, HNWW4, HNWW5). Nine genotypes belonged to a known zoonotic group (group 1) and the other genotypes belonged to potential zoonotic group (group 2). Most of the genotypes had been identified in wildlife or domestic animals in former reports in Zhengzhou. The occurrence of E. bieneusi in wastewater was probably related to the rainfall day before sampling. Of 36 sampling days, 20 days had rainfall on the previous day and 16 days had none. As many as 43 of 60 samples were found to be E. bieneusi-positive in the 20 days which had rainfall on the previous day. Only three of 48 samples were found to be E. bieneusi-positive in the 16 days without rainfall the day before. The significant difference of the occurrence of E. bieneusi was observed between wet days and dry days by t-test (43/60 vs 3/48, p < 0.01). This indicates that the occurrence of E. bieneusi in wastewater in Zhengzhou mainly originated from animals and was probably related to rainfall the day before sample collection. Given the zoonotic genotypes detected in wastewater, animal faeces should be treated appropriately before being drained into the water source.

A new α-amylase inhibitory peptide from Gynura medica extract.

In this study, we discovered a novel peptide, Gymepeptide A, with α-amylase inhibitory activity in the water extract of Gynura medica. The structure of Gymepeptide A was determined as CGDREETR using HR-MS, 1H NMR, 13C NMR, and 2D-NMR techniques. Notably, Gymepeptide A possesses a rare double arginine residue structure and exhibits strong α-amylase inhibitory activity. Enzyme dynamic assays, molecular docking experiments, and isothermal titration calorimetry indicated that the double arginine residue structure of Gymepeptide A interacts with amino acid residues in the nearby active site region of α-amylase through hydrogen bonds and van der Waals forces. This interaction effectively inhibits the hydrolysis activity of α-amylase. Furthermore, in vitro starch digestion tests revealed that Gymepeptide A significantly reduced the digestion rate of starch and the concentration of glucose produced after starch digestion. These findings highlight the great potential of Gymepeptide A in decreasing postprandial blood glucose levels.

A Concise Synthesis of (-)-Ambrox.

(-)-Ambrox, a highly prized and commercially significant component of ambergris, finds widespread application in perfumery, cigarettes, cosmetics, and the food industry. Despite considerable attention to this research area over the years, an environmentally friendly and practical method for synthesizing (-)-ambrox has remained elusive. This study presents a succinct and efficient approach to (-)-ambrox synthesis, involving two consecutive alkylations at C-6, followed by an acid-catalyzed cyclization to give bicyclic ketones starting from (R)-carvone. Subsequent reduction, Barton Vinyl Iodide synthesis, alkylation, and an acid-catalyzed cyclization collectively achieved the synthesis of (-)-ambrox with a satisfactory yield of 26.2 %.

Selective extraction and quantitative analysis of pyrroloquinoline quinone from food.

Pyrroloquinoline quinone (PQQ) is a bioactive compound that has attracted significant attention due to its potential health benefits. In this study, we developed a new magnetic molecularly imprinted nanoparticle (MMIN) for the selective extraction and determination of PQQ from food samples. The MMIN was synthesized using a surface molecular imprinting technique with PQQ as the template molecule, Fe3O4 nanoparticles as the magnetic core, and methacrylic acid as the functional monomer. The MMIN exhibited high selectivity and affinity towards PQQ, allowing for efficient extraction and preconcentration of PQQ from complex food matrices. The extracted PQQ was then quantified using HPLC-DAD. The developed method showed good linearity (R2 = 0.9985) and low limits of detection (0.03 µg L-1). The accuracy and precision of the method were evaluated by analyzing spiked food samples, with average recoveries close to 89.8%. The MMIN also demonstrated good reusability, with negligible decrease in extraction efficiency after five cycles of use. Overall, the developed MMIN-based method provides a reliable and efficient approach for the analysis of PQQ in food samples.

Regulation of cell proliferation and transdifferentiation compensates for ventilator-induced lung injury mediated by NLRP3 inflammasome activation.

BACKGROUND: Mechanical ventilation is an important means of respiratory support and treatment for various diseases. However, its use can lead to serious complications, especially ventilator-induced lung injury (VILI). The mechanisms underlying this disease are complex, but activation of inflammatory signalling pathways results in activation of cytokines and inflammatory mediators, which play key roles in VILI. Recent studies have demonstrated that nod-like receptor protein 3 (NLRP3) inflammasome activation mediates VILI and also accompanied by cell proliferation and transdifferentiation to compensate for alveolar membrane damage. Type I alveolar epithelial cells (AECs I), which are involved in the formation of the blood-air barrier, are vulnerable to damage but cannot proliferate by themselves; thus, replacing AECs I relies on type II alveolar epithelial cells (AECs II). OBJECTIVE: The review aims to introduce the mechanisms of NLRP3 inflammasome activation and its inhibitors, as well as the mechanisms that regulate cell proliferation and transdifferentiation. METHODS: A large number of relevant literature was searched, then the key content was summarized and figures were also made. RESULTS: The mechanism of NLRP3 inflammasome activation has been further explored, including but not limited to pathogenic and aseptic inflammatory signals, such as, pathogenic molecular patterns and host-derived danger-associated molecular patterns activate toll-like receptor 4/nuclear factor-kappaB pathway or reactive oxygen species, cyclic stretch, adenosine triphosphate induce K+ efflux through P2X7, Ca2+ inflow, mitochondrial damage, etc, eventually induce NIMA-related kinase 7/NLRP3 binding and NLRP3 inflammasome activation. Not only that, the review also described in detail the inhibitors of NLRP3 inflammasome. And the mechanisms regulating cell proliferation and transdifferentiation are complex and unclear, including the Wnt/ß-catenin, Yap/Taz, BMP/Smad and Notch signalling pathways. CONCLUSIONS: NLRP3 inflammasome activation mediated VILI, and VILI is alleviated after interfering with its activation, and inflammation and repair exist simultaneously in VILI. Clarifying these mechanisms is expected to provide theoretical guidance for alleviating VILI by inhibiting the inflammatory response and accelerating alveolar epithelial cell regeneration in the early stage.

Intraocular lens power calculation for silicone oil-dependent eyes.

Background: Silicone oil tamponade is widely used in vitreoretinal surgery. In some cases, silicone oil may not be extracted for a long time or even permanently and is referred to as silicone oil-dependent eyes. In this study, we aimed to deduce a theoretical formula for calculating intraocular lens power for silicone oil-dependent eyes and compare it with clinical findings. Methods: A theoretical formula was deduced using strict geometric optical principles and the Gullstrand simplified eye model. The preoperative and postoperative refractive statuses of patients with silicone oil-dependent eyes who underwent intraocular lens implantation were studied (Group A, n = 13). To further test our derived theoretical formula, patients with silicone oil tamponade and first-stage intraocular lens implantation were included (Group B, n = 19). In total, 32 patients (32 eyes) were included in the study. Results: In group A, the calculated intraocular lens power based on our formula was 24.96 ± 3.29 diopters (D), and the actual refraction of the patients was 24.02 ± 4.14D. In group B, the theoretical intraocular lens power was 23.10 ± 3.08D, and the clinical intraocular lens power was 22.84 ± 3.42D. There was no significant difference between the theoretical and clinical refractive powers, and the intraclass correlation coefficient was 0.771 for group A and 0.811 for group B (both p ≤ 0.001). The mean absolute error for silicone oil-dependent eyes of the formula was 1.66 ± 2.09D. After excluding data for two patients with a flat cornea (corneal refractive power < 42D), the mean absolute error decreased to 0.83 ± 0.62D. Conclusion: A strong correlation between the theoretical and clinical intraocular lens powers was observed, and the formula we deduced can be used to calculate the intraocular lens power for silicone oil-dependent eyes. This formula will help clinicians select a more appropriate intraocular lens for patients with silicone oil-dependent eyes, especially when the corneal refractive power is ≥42D.

Analysis of retinal and choroidal characteristics in patients with early diabetic retinopathy using WSS-OCTA.

Introduction: Diabetic retinopathy (DR) is one of the most common and destructive microvascular complications of DM, and has become a major cause of irreversible visual impairment. The purpose of this study was to evaluate the changes in fundus microcirculation in non-diabetic retinopathy (NDR) and mild non-proliferative diabetic retinopathy (NPDR) in patients with type 2 diabetic mellitus (T2DM) using widefield swept-source optical coherence tomography angiography (WSSOCTA), and to investigate the correlation with laboratory indices of T2DM. Methods: Eighty nine, 58 and 28 eyes were included in the NDR, NPDR and Control groups, respectively, were enrolled in this study. The 12mm×12mm fundus images obtained by WSS-OCTA were divided into 9 regions (supratemporal, ST; temporal, T; inferotemporal, IT; superior, S; central macular area, C; inferior, I; supranasal, SN; nasal, N; inferonasal, IN) to evaluate changes in vessel density (VD) of the superficial capillary plexus (SCP), deep capillary plexus (DCP), choriocapillaris, and mid-large choroidal vessel (MLCV), as well as changes in inner retinal thickness (IRT), outer retinal thickness (ORT), and choroidal thickness (CT). Results: Compared with control group, MLCV VD (I, N, IN) was significantly decreased in NDR group, SCP VD (IT, C, I) and DCP VD (T, IT, I) were significantly decreased in NPDR group. In NPDR group, DCP VD (IT) was significantly decreased compared with that in NDR group. Compared with control group, CT (ST, T, IT, S, SN, IN) was significantly declined in NDR group, and IRT (ST, IT) and ORT (ST, N) were significantly increased in NPDR group. In NPDR group, IRT (ST) and ORT (T, S) were significantly increased compared with NDR group. Correlation analysis showed that age, body mass index, fasting blood glucose, fasting insulin, fasting C-peptide, and estimated glomerular filtration rate in T2DM patients were statistically correlated with retinal and choroidal thickness/VD. Discussion: Structural and blood flow changes in the choroid occur before the onset of DR and precede changes in the retinal microcirculation, and MLCV thickness/VD is a more sensitive imaging biomarker for the clinical detection of DR. WSS-OCTA enables large-scale non-invasive visual screening and follow-up of the retinal and choroidal vasculature in DR patients, providing a new strategy for the prevention and monitoring of DR in patients with T2DM.

Investigating mechanism of sweetness intensity differences through dynamic analysis of sweetener-T1R2-membrane systems.

Knowing the mechanism of action of sweet taste receptors is important for the design of new, healthy sweeteners. However, little is known about the structures and recognition mechanisms of these receptors. 28 sweeteners were assessed by molecular docking, and 8 typical sweeteners were chosen to construct sweetener-T1R2-membrane systems to analyze interactions between receptor and sweeteners. Natural sweeteners with low-intensity sweetness, such as fructose and xylitol, were released from the Venus flytrap domain at ∼30 ns, with displacements greater than 50 Å. In contrast, artificial neotame and advantame bound stably to the receptor, within 5 Å. Van der Waals interactions were significant in high-intensity sweetener systems, imparting an energy difference of over 15 kcal/mol between neotame (artificial sweetener) and fructose (natural). These results provide a deeper understanding of the mechanisms of sweetener function and offer a new direction for the design of sweeteners.

Separation and purification of pyrroloquinoline quinone from Gluconobacter oxydans fermentation broth using supramolecular solvent complex extraction.

In this paper, new supramolecular extractants, which contained surfactant, alkane and alkanol, were designed and used to separate PQQ. After a series of tests, the optimal extractant composition was determined as benzalkalonium (C8-C16) chloride (BC): n-hexane:n-pentanol, and the highest extraction rate could reach 98%. The extraction equilibrium could be reached in five minutes. The mechanism of the extraction selectivity was inferred as an ion-pair and π-π complexation interaction between PQQ and BC, which was indicated by UV and fluorescence quenching experiments. To recycle the organic extractant, the extract was back-extracted with sodium chloride solution. After extraction, back extraction and crystallization, an isolated product with a purity of 97.5% was obtained from G. oxydans fermentation broth. The product was identified as PQQ by HPLC analysis and MS. Above all, the present research developed a simple and efficient method for the separation of PQQ from fermentation broth.

Pyrroloquinoline quinone biosynthesis in Escherichia coli through expression of the Gluconobacter oxydans pqqABCDE gene cluster.

We have expressed the pqqABCDE gene cluster from Gluconobacter oxydans, which is involved in pyrroloquinoline quinone (PQQ) biosynthesis, in Escherichia coli, resulting in PQQ accumulation in the medium. Since the gene cluster does not include the tldD gene needed for PQQ production, this result suggests that the E. coli tldD gene, which shows high homology to the G. oxydans tldD gene, carries out that function. The synthesis of PQQ activated d-glucose dehydrogenase in E. coli and the growth of the recombinant was improved. In an attempt to increase the production of PQQ, which acts as a vitamin or growth factor, we transformed E. coli with various recombinant plasmids, resulting in the overproduction of the PQQ synthesis enzymes and, consequently, PQQ accumulation--up to 6 mM--in the medium. This yield is 21.5-fold higher than that obtained in previous studies.

High cell density fermentation of Gluconobacter oxydans DSM 2003 for glycolic acid production.

Gluconobacter oxydans has a lower biomass yield. Uniform design (UD) was applied to determine the optimum composition of the critical media and their mutual interactions for increased biomass yield of Gluconobacter oxydans DSM 2003 in shake flasks. Fed-batch fermentation process for biomass was optimized in a 3.7-l fermentor. By undertaking a preliminary and improved fed-batch fermentation-process strategy, a cell density of 6.0 g/l (DCW) was achieved in 22 h and 14.1 g/l (DCW) in 35 h, which is the highest cell density of G. oxydans produced thus far in a 3.7-l bioreactor. The biomass production was increased by 135% compared with that using the original cultivation strategy. Bioconversion of ethylene glycol to glycolic acid was catalyzed by the resting cells of G. oxydans DSM 2003, and conversion rate reached 86.7% in 48 h. In summary, the approach including high-density fermentation of G. oxydans DSM 2003 and bioconversion process was established and proved to be an effective method for glycolic acid production.