Combinatorial metabolic engineering of Escherichia coli for de novo production of structurally defined and homogeneous Amino oligosaccharides.

Amino oligosaccharides (AOs) possess various biological activities and are valuable in the pharmaceutical, food industries, and agriculture. However, the industrial manufacturing of AOs has not been realized yet, despite reports on physical, chemical, and biological approaches. In this study, the de novo production of chitin oligosaccharides (CHOS), a type of structurally defined AOs, was achieved in Escherichia coli through combinatorial pathway engineering. The most suitable glycosyltransferase for CHOS production was found to be NodCL from Mesorhizobium Loti. Then, by knocking out the nagB gene to block the flow of N-acetyl-d-glucosamine (NAG) to the glycolytic pathway in E. coli and adjusting the copy number of NodCL-coding gene, the CHOS yield was increased by 6.56 times. Subsequently, by introducing of UDP-N-acetylglucosamine (UDP-GlcNAc) salvage pathway for and optimizing fermentation conditions, the yield of CHOS reached 207.1 and 468.6 mg/L in shake-flask cultivation and a 5-L fed-batch bioreactor, respectively. Meanwhile, the concentration of UDP-GlcNAc was 91.0 mg/L, the highest level reported in E. coli so far. This study demonstrated, for the first time, the production of CHOS with distinct structures in plasmid-free E. coli, laying the groundwork for the biosynthesis of CHOS and providing a starting point for further engineering and commercial production.

Co-utilization of carbon sources in microorganisms for the bioproduction of chemicals.

Carbon source is crucial for the cell growth and metabolism in microorganisms, and its utilization significantly affects the synthesis efficiency of target products in microbial cell factories. Compared with a single carbon source, co-utilizing carbon sources provide an alternative approach to optimize the utilization of different carbon sources for efficient biosynthesis of many chemicals with higher titer/yield/productivity. However, the efficiency of bioproduction is significantly limited by the sequential utilization of a preferred carbon source and secondary carbon sources, attributed to carbon catabolite repression (CCR). This review aimed to introduce the mechanisms of CCR and further focus on the summary of the strategies for co-utilization of carbon sources, including alleviation of CCR, engineering of the transport and metabolism of secondary carbon sources, compulsive co-utilization in single culture, co-utilization of carbon sources via co-culture, and evolutionary approaches. The findings of representative studies with a significant improvement in the bioproduction of chemicals via the co-utilization of carbon sources were discussed in this review. It suggested that by combining rational metabolic engineering and irrational evolutionary approaches, co-utilizing carbon sources can significantly contribute to the bioproduction of chemicals.

Co-utilization of wastewater sludge and heavy metals for single-atom electrocatalytic reduction of gaseous CO2.

Synergetic management of waste activated sludge, heavy metals (HMs) and CO2 for their valorization and cyclic utilization is rarely reported. Herein, we employed sludge-derived extracellular polymeric substances (EPS) and HMs in wastewater to fabricate a gas diffusion electrode (GDE) for electrochemical CO2 reduction. This approach atomically dispersed Ni at each nanofiber of the GDE. Abundant N element in the EPS proved to play a key role in the formation of Nx-Ni (mixture of N3-Ni and N4-Ni) sites for highly efficient CO2 to CO conversion. The atomical Ni3+ shows high catalytic activity. Direct gaseous CO2 reduction in a membrane electrode assembly generated a current density up to 50 mA·cm-2 with CO:H2 ratio of ∼100 and ∼75% FECO under 2.69 cell voltage. This strategy takes advantage of all waste streams generated on site and consolidates traditionally separated treatment processes to save costs, produces value-added products and generates carbon benefits during wastewater treatment.

Bioconversion of chitin into chitin oligosaccharides using a novel chitinase with high chitin-binding capacity.

Chitin is the second largest renewable biomass resource in nature, it can be enzymatically degraded into high-value chitin oligosaccharides (CHOSs) by chitinases. In this study, a chitinase (ChiC8-1) was purified and biochemically characterized, its structure was analyzed by molecular modeling. ChiC8-1 had a molecular mass of approximately 96 kDa, exhibited its optimal activity at pH 6.0 and 50 °C. The Km and Vmax values of ChiC8-1 towards colloidal chitin were 10.17 mgmL-1 and 13.32 U/mg, respectively. Notably, ChiC8-1 showed high chitin-binding capacity, which may be related to the two chitin binding domains in the N-terminal. Based on the unique properties of ChiC8-1, a modified affinity chromatography method, which combines protein purification with chitin hydrolysis process, was developed to purify ChiC8-1 while hydrolyzing chitin. In this way, 9.36 ± 0.18 g CHOSs powder was directly obtained by hydrolyzing 10 g colloidal chitin with crude enzyme solution. The CHOSs were composed of 14.77-2.83 % GlcNAc and 85.23-97.17 % (GlcNAc)2 at different enzyme-substrate ratio. This process simplifies the tedious purification and separation steps, and may enable its potential application in the field of green production of chitin oligosaccharides.

Detecting radius of curvature (ROC) mismatch among primary mirror (PM) segments.

A segmented primary mirror (PM) is an efficient solution to the problems of a monolithic PM manufacture, testing, transportation, and launch. However, the problem of the radius of curvature (ROC) matching among PM segments will arise, which if not solved will seriously degrade the final imaging quality of the system. Accurately detecting ROC mismatch among PM segments from the wavefront map is of crucial importance for efficiently correcting this kind of manufacturing error, while currently there are few related studies. Based on the inherent relation between the PM segment's ROC error and corresponding sub-aperture defocus aberration, this paper proposes that the ROC mismatch can be accurately estimated from the sub-aperture defocus aberration. Secondary mirror (SM) lateral misalignments will influence the accuracy of estimating ROC mismatch. A strategy is also proposed to reduce the influence of SM lateral misalignments. Detailed simulations are performed to demonstrate the effectiveness of the proposed method for detecting ROC mismatch among PM segments. This paper paves a road for detecting ROC mismatch using image-based wavefront sensing methods.

De novo biosynthesis of 2'-fucosyllactose in engineered Pichia pastoris.

PURPOSE: Pichia pastoris is well known for its ability to produce short and low-immunogenic humanized glycosyl chains onto recombinant glycoproteins, it was thus speculated to be applicable to synthesize oligosaccharides. In this study, generally recognized as safe (GRAS) microorganism Pichia pastoris GS115 was tested for its potential to be used as a new synthetic chassis to produce the most abundant human milk oligosaccharide 2'-fucosyllactose (2'-FL). METHODS: To enable the de novo synthesis of 2'-FL, lactose transporter lac12, two enzymes of gmd, gmer, and fucosyltransferases futC were integrated into the genome of P. pastoris, under the control of constitutive PGAP promoter. RESULTS: The resulting recombinant yeasts yielded up to 0.276 g/L through culture optimization in a 5 L bioreactor. CONCLUSION: To our knowledge, this is the first report of 2'-FL production in engineered Pichia pastoris. This work is a good starting point to produce 2'-FL using Pichia pastoris as a viable chassis.

[Thermal Environment Evolution and Response Mechanism of Urban Sprawl Based on Multi-source Data].

Improving the urban thermal environment and improving the quality of human settlements are important prerequisites for creating ecologically livable cities. The current research on the relationship between urban expansion and the thermal environment is mostly based on remote sensing data, and the application of multi-source data is weak. Here, we selected the Xi'an metropolitan core area, measured the urban expansion and temporal and spatial evolution of the thermal environment based on Landsat remote sensing images in 2010 and 2020, and used multi-source data, such as interest points and the Baidu thermal index, to study the response mechanism of the urban thermal environment through geoscience statistical analysis methods. The results showed that:① the construction land in the study area had expanded by 200.84 km2, and the area and intensity showed that "the center and the periphery are weaker, and the difference between the two" characteristics, and the expansion mode was mainly edge type and infill type. ② Between 2010 and 2020, the overall thermal environment in the study area deteriorated, and the area of the heat island area increased by 282.65 km2. The spread of the heat island area was in the same direction as the urban expansion, and the distribution pattern evolved from "southeast-northwest" to "northeast-southwest;" however, the average temperature in the central area of the city decreased 1.09℃. ③ Urban expansion was strongly positively correlated with the deterioration of the urban thermal environment. The expansion of urban space had a contribution rate of 60.40% to the deterioration of the thermal environment, and various socio-economic factors had a weaker effect, with an overall contribution rate of 39.60%. The vegetation water body had an obvious cooling effect; under the influence of multiple factors, the surface temperature increased by 0.241 units. In the process of urban expansion, changes in surface parameters and two-dimensional urban morphology were still the main factors for thermal environment changes, whereas three-dimensional morphology had a small effect on the warming of social and economic activities, and the contribution of water bodies and vegetation to cooling was prominent.

Active alignment of space astronomical telescopes by matching arbitrary multi-field stellar image features.

Space-based optical astronomical telescopes are susceptible to mirror misalignments due to space disturbance in mechanics and temperature. Therefore, it is of great importance to actively align the telescope in orbit to continuously maintain imaging quality. Traditional active alignment methods usually need additional delicate wavefront sensors and complicated operations (such as instrument calibration and pointing adjustment). This paper proposes a novel active alignment approach by matching the geometrical features of several stellar images at arbitrary multiple field positions. Based on nodal aberration theory and Fourier optics, the relationship between stellar image intensity distribution and misalignments of the system can be modeled for an arbitrary field position. On this basis, an objective function is established by matching the geometrical features of the collected multi-field stellar images and modeled multi-field stellar images, and misalignments can then be solved through nonlinear optimization. Detailed simulations and a real experiment are performed to demonstrate the effectiveness and practicality of the proposed approach. This approach eliminates the need for delicate wavefront sensors and pointing adjustment, which greatly facilitates the maintainance of imaging quality.

Deep learning wavefront sensing for fine phasing of segmented mirrors.

Segmented primary mirror provides many crucial important advantages for the construction of extra-large space telescopes. The imaging quality of this class of telescope is susceptible to phasing error between primary mirror segments. Deep learning has been widely applied in the field of optical imaging and wavefront sensing, including phasing segmented mirrors. Compared to other image-based phasing techniques, such as phase retrieval and phase diversity, deep learning has the advantage of high efficiency and free of stagnation problem. However, at present deep learning methods are mainly applied to coarse phasing and used to estimate piston error between segments. In this paper, deep Bi-GRU neural work is introduced to fine phasing of segmented mirrors, which not only has a much simpler structure than CNN or LSTM network, but also can effectively solve the gradient vanishing problem in training due to long term dependencies. By incorporating phasing errors (piston and tip-tilt errors), some low-order aberrations as well as other practical considerations, Bi-GRU neural work can effectively be used for fine phasing of segmented mirrors. Simulations and real experiments are used to demonstrate the accuracy and effectiveness of the proposed methods.

Aberration compensation strategy for the radius of curvature error of the primary mirror in off-axis three-mirror anastigmatic telescopes.

This paper discusses compensation strategies for the aberration fields caused by the error in the radius of curvature (ROC) of the primary mirror (PM) in pupil-offset off-axis three-mirror anastigmatic (TMA) astronomical telescopes. Based on the nodal aberration theory framework, the specific astigmatic and coma aberrations of the off-axis three-mirror system in the presence of the ROC error of the PM are derived. It is demonstrated that some field-dependent aberration components can be induced by ROC error in the off-axis TMA telescopes, apart from the dominating field-constant aberration terms. To reduce the influence of the ROC error on the aberration fields, we propose two aberration compensation strategies: adjusting the position of the PM and introducing axial misalignment of the secondary mirror (SM). Through theoretical analysis and simulations, we conclude that the compensation strategy of changing the axial position of the PM can make the aberration distribution close to the nominal state; the compensation strategy of axially adjusting the SM can make the aberration distribution meet the observation requirements, which is more suitable for space applications. We also discuss compensating the effect of the ROC error using lateral misalignments.

A Novel GH Family 20 ß-N-acetylhexosaminidase With Both Chitosanase and Chitinase Activity From Aspergillus oryzae.

Aminooligosaccharides possess various biological activities and can exploit wide applications in food, pharmaceutical and cosmetic industries. Commercial aminooligosaccharides are often prepared by the hydrolysis of chitin and chitosan. In this study, a novel GH family 20 ß-N-acetylhexosaminidases gene named AoNagase was cloned from Aspergillus oryzae and expressed in Pichia pastoris. The purified AoNagase had maximal activity at pH 5.5 and 65°C. It exhibited good pH stability in the range of pH 6.0-7.5 and at temperatures below 50°C. AoNagase was capable of hydrolyzing not only colloidal chitosan (508.26 U/mg) but also chitin (29.78 U/mg). The kinetic parameters (K m and V max ) of AoNagase were 1.51 mM, 1106.02 U/mg for chitosan and 0.41 mM, 40.31 U/mg for colloidal chitin. To our knowledge, AoNagase is the first GH family 20 ß-N-acetylhexosaminidase capable of hydrolyzing both chitosan and chitin. AoNagase is an endo-type ß-N-acetylhexosaminidases and can potentially be used for the manufacturing of aminooligosaccharides.

Inhibitory effects of Coptidis Rhizoma on the intestinal absorption and metabolism of Scutellariae Radix.

ETHNOPHARMACOLOGICAL RELEVANCE: The Scutellariae Radix (SR) and Coptidis Rhizoma (CR) herb couple is widely used in traditional Chinese medicine prescriptions for the treatment of diabetes mellitus due to its interaction and synergistic effect compared to either herb alone, but the underlying mechanism of interaction between these herbs is unclear. This study aimed to investigate the effects of CR on the metabolism and absorption of SR. MATERIALS AND METHODS: After rats were treated with normal saline (NS group) or the CR extract (CR-treated group) for seven consecutive days, the intestinal flora was extracted from rat faeces for a co-incubation with the SR extract to investigate the metabolism of SR flavonoids, and a non-everted gut sac was prepared in vitro to evaluate the intestinal absorption of the SR extract. The components of the SR extract, the metabolites of the SR extract that was co-incubated with intestinal flora, and the dialysate acquired from non-everted gut sacs were identified and determined by an HPLC-MS/MS method. The absorption rate constant (Ka) and the apparent permeability (Papp) of each compound were calculated, and the effects of CR on the metabolism and absorption of flavonoids in SR were evaluated, by comparison the Ka and Papp between two groups using Student's t-test. RESULTS: Twenty-nine flavonoids were detected and identified in the SR extract, including 16 glycosides and 13 aglycones. In the co-incubation with the intestinal flora, differences in metabolite classes were not observed between the NS group and CR-treated group; however, the metabolic rates of 17 flavonoids in the CR-treated group were significantly higher than the NS group. The Papp of 11 compounds (4 glycosides and 7 aglycones) across the gut sac were greater than 2 × 10-5 cm/s in both groups, while the Papp values of 7 compounds including wogonoside (WG) and other aglycones were significantly decreased in the CR-treated group. CONCLUSION: Based on these results, CR decreased the metabolism and absorption of SR flavonoids, and exerted much greater inhibitory effects on aglycones than glycosides, which may be one of the potential mechanisms underlying the therapeutic effects of the combination of SR and CR on diabetes mellitus.

Stability of fat globules in UHT milk during proteolysis by the AprX protease from Pseudomonas fluorescens and by plasmin.

Fat separation is a limiting factor for the shelf life of UHT milk. It may be promoted by the proteolysis of fat surface-adsorbed proteins (FSAP) by proteases that remain active after UHT treatment. The aim of this research was to explore the relationship between the proteolysis of FSAP and fat destabilization. In this study, we developed a full-fat UHT milk-based model system and added either the major bacterial protease AprX from Pseudomonas fluorescens or the major native milk protease plasmin at high levels to induce fast destabilization of the milk fat globules. We monitored changes in physical properties and FSAP composition, and structural changes in fat globules, over 24 h. Our results showed that AprX-induced sedimentation as a result of the flocculation of fat globules, and plasmin induced cream to float as a result of the coalescence of fat globules. This study confirmed that AprX and plasmin can both lead to fat destabilization in full-fat UHT milk, and it provides insights in the underlying mechanisms.

Object-independent image-based wavefront sensing approach using phase diversity images and deep learning.

This paper proposes an image-based wavefront sensing approach using deep learning, which is applicable to both point source and any extended scenes at the same time, while the training process is performed without any simulated or real extended scenes. Rather than directly recovering phase information from image plane intensities, we first extract a special feature in the frequency domain that is independent of the original objects but only determined by phase aberrations (a pair of phase diversity images is needed in this process). Then the deep long short-term memory (LSTM) network (a variant of recurrent neural network) is introduced to establish the accurate non-linear mapping between the extracted feature image and phase aberrations. Simulations and an experiment are performed to demonstrate the effectiveness and accuracy of the proposed approach. Some other discussions are further presented for demonstrating the superior non-linear fitting capacity of deep LSTM compared to Resnet 18 (a variant of convolutional neural network) specifically for the problem encountered in this paper. The effect of the incoherency of light on the accuracy of the recovered wavefront phase is also quantitatively discussed. This work will contribute to the application of deep learning to image-based wavefront sensing and high-resolution image reconstruction.

Self-generated carbon nanotubes for protecting active sites on bifunctional Co/CoOx schottky junctions to promote oxygen reduction/evolution reactions via efficient valence transition.

Protecting active species from aggregation and corrosion may be feasible to obtain stable catalytic activities for oxygen reduction reaction (ORR) and oxygen evolution reaction (OER). Herein, bamboo-shaped N-doped carbon nanotubes (hollow BS-NCNTs as shells) are self-generated to in situ wrap the Co/CoOx schottky junctions (cores) to obtain the Co/CoOx@BS-NCNTs as bifunctional ORR/OER catalysts by using the Co-chelated melamine precursor. For ORR, Co/CoOx@BS-NCNTs (700 °C) exhibits more positive peak (0.822 V vs. RHE) and half-wave (0.842 V vs. RHE) potential than those of commercial Pt/C (10 wt%). Superior ORR activity is mainly attributed to the enriched coordination-unsaturated Co2+ (tetrahedral CoTd2+) in the CoOx wrapped in the tubular structure of BS-NCNTs featuring high electrical conductivity and active N species. Moreover, the π-π bonds of CNTs are activated by N substitution, which provides a stunning electron capture and transmission capability for enhancing ORR activity. For OER, Co/CoOx@BS-NCNTs (700 °C) obtains a smaller potential (1.590 V vs. RHE) than that of RuO2/C at 10 mA cm-2. The outstanding OER activity and durability of Co/CoOx@BS-NCNTs (700 °C) originates from strong interactions between C-skeleton and Co species, and efficient Co3+/Co4+ (Co4+OOH as active sites) transition protected by the externally-grown CNTs. Furthermore, abundant oxygen vacancies on CoOx surface can facilitate the adsorption of OH-/or OER-related intermediates to improve OER activity. Therefore, this study provides a promising strategy to develop NCNTs-wrapped Co species with high catalytic activity and stability for energy conversion.

Genetic characterization and recombinant history of a novel HIV-1 circulating recombinant form (CRF101_01B) identified in Yunnan, China.

The high frequency of multiple HIV-1 recombinant events among the B, C and CRF01_AE were constantly occurring in Yunnan China. Here, we characterized a novel HIV-1 circulating recombinant form (CRF) consisting of CRF01_AE and subtype B (CRF101_01B) from three epidemiologically unlinked individuals. Phylogenetic analysis based on near full length genome (NFLG) sequences revealed that CRF101_01B formed a distinct monophyletic cluster supported by a high bootstrap value of 100%, distantly related to all known HIV-1 CRFs. CRF101_01B had a CRF01_AE backbone with two B segments inserted, respectively, in the gag and pol region. Further, subregion tree analysis showed that CRF01_AE backbone and subtype B segment inserted originated from a Thailand lineage. In addition, our study found that CRF101_01B originated around the year 1996-1998. This findings described a novel HIV-1 CRF, and highlighted the importance of continual monitoring of genetic diversity and complexity of HIV-1 strains in Yunnan, China.

The Extracellular Protease AprX from Pseudomonas and its Spoilage Potential for UHT Milk: A Review.

The negative effects of proteases produced by psychrotrophic bacteria on dairy products, especially ultra-high-temperature (UHT) milk, are drawing increasing attention worldwide. These proteases are especially problematic, because it is difficult to control psychrotrophic bacteria during cold storage and to inactivate their heat-resistant proteases during dairy processing. The predominant psychrotrophic species with spoilage potential in raw milk, Pseudomonas, can produce a thermostable extracellular protease, AprX. A comprehensive understanding of AprX on the aspects of its biological properties, regulation, proteolytic potential, and its impact on UHT milk can contribute to finding effective approaches to minimize, detect, and inactivate AprX. AprX also deserves attention as a representative of all extracellular metalloproteases produced by psychrotrophic bacteria in milk. The progress of current research on AprX is summarized in this review, including a view on the gap in current understanding of this enzyme. Reducing the production and activity of AprX has considerable potential for alleviating the problems that arise from the instability of UHT milk during shelf-life.

Destabilization of UHT milk by protease AprX from Pseudomonas fluorescens and plasmin.

Destabilization of UHT milk during its shelf life is mainly promoted by the residual proteolytic activity attributed to the psychrotrophic bacterial proteases and native milk proteases. In this study, we built skim UHT milk-based model systems to which either the major bacterial protease (AprX from Pseudomonas fluorescens), or the major native milk protease (plasmin) was added, to allow a direct comparison between the destabilization of skim UHT milk by both categories of enzymes. The physical and chemical properties were studied during 6 weeks. Our results showed AprX induced compact gels when almost all the κ-casein was hydrolyzed and the degree of hydrolysis (DH) exceeded 1.3%. Plasmin induced soft gels when around 60% of both ß- and αs1-casein were hydrolyzed and the DH reached 2.1%. The knowledge gained from this study may be used for developing diagnostic tests for determining the protease responsible for UHT milk destabilisation.

High seroprevalence of hepatitis E virus in the ethnic minority populations in Yunnan, China.

Hepatitis E virus (HEV) infection is relatively high in the southern regions of China. Yunnan, located in southwestern China, has the highest number of ethnic groups. However, HEV infection in the ethnic population is largely unknown. Therefore, we aimed to investigate the seropositive rate, risk factor, and clinical impact of HEV infection in the ethnic groups of Yunnan. We recruited 1912 individuals from four minority groups in three prefectures of Yunnan province. Epidemiological records on potential risk factors for exposure to HEV and blood biochemical index were analyzed. All the serum samples were tested for anti-HEV IgM/IgG by enzyme-linked immunosorbent assay, and the IgM-positive samples were subjected to nested reverse transcription-PCR to detect HEV RNA. Overall, 1273 individuals (66.58%) were positive for anti-HEV IgG, 16 (0.84%) for anti-HEV IgM, and 64 (3.35%) for anti-HEV IgG and IgM both; none of them had detectable HEV RNA. Multivariate analysis revealed a strong statistical association between ethnic origin and HEV IgG seroprevalence. Anti-HEV IgG reactivity in the Hani ethnic (82.3%; 401/487) population was higher than that in the Naxi (71.9%, 340/473), Bulang (65.1%; 302/464), and Wa (60.2%; 294/488) ethnic populations (p < 0.0001). Older age and male sex were independently associated with the risk of past HEV infection. Moreover, anti-HEV IgG-positive individuals showed significantly higher levels of total and direct bilirubin and alanine amino transferase but significantly lower levels of globulin and low-density lipoprotein, than the respective levels in anti-HEV IgG-negative individuals. Thus, the seroprevalence of HEV infection is high in the ethnic populations of Yunnan, China. It is therefore necessary to increase the surveillance of specific risk groups and raise awareness about the possible infectious diseases to help limit the HEV transmission here.