Preparation and Characterization of a Novel Morphosis of Dextran and Its Derivatization with Polyethyleneimine.

Dextran, a variant of α-glucan with a significant proportion of α-(1,6) bonds, exhibits remarkable solubility in water. Nonetheless, the precipitation of dextran has been observed in injection vials during storage. The present study aimed to establish a technique for generating insoluble dextran and analyze its structural properties. Additionally, the potential for positively ionizing IS-dextran with polyethyleneimine was explored, with the ultimate objective of utilizing IS-dextran-PEI as a promising support for enzyme immobilization. As a result, IS-dextran was obtained by the process of slow evaporation with an average molecular weight of 6555 Da and a yield exceeding 60%. The calculated crystallinity of IS-dextran, which reaches 93.62%, is indicative of its irregular and dense structure, thereby accounting for its water insolubility. Furthermore, positive charge modification of IS-dextran, coupled with the incorporation of epichlorohydrin, resulted in all zeta potentials of IS-dextran-PEIs exceeding 30 mV, making it a promising supporting factor for enzyme immobilization.

Metabolic engineering of Pichia pastoris for malic acid production from methanol.

The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP-CM301 could produce 8.55 g/L malic acid from glucose, which showed a 3.45-fold increase compared to the parent strain. To improve the efficiency of site-directed gene knockout, NHEJ-related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to by-product generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose-6-phosphate isomerase was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for the metabolic engineering of P. pastoris to produce value-added chemicals from methanol.

Metabolic engineering of Escherichia coli for L-malate production anaerobically.

BACKGROUND: L-malate is one of the most important platform chemicals widely used in food, metal cleaning, textile finishing, pharmaceuticals, and synthesis of various fine chemicals. Recently, the development of biotechnological routes to produce L-malate from renewable resources has attracted significant attention. RESULTS: A potential L-malate producing strain E. coli BA040 was obtained by inactivating the genes of fumB, frdABCD, ldhA and pflB. After co-overexpression of mdh and pck, BA063 achieved 18 g/L glucose consumption, leading to an increase in L-malate titer and yield of 13.14 g/L and 0.73 g/g, respectively. Meantime, NADH/NAD+ ratio decreased to 0.72 with the total NAD(H) of 38.85 µmol/g DCW, and ATP concentration reached 715.79 nmol/g DCW. During fermentation in 5L fermentor with BA063, 41.50 g/L glucose was consumed within 67 h with the final L-malate concentration and yield of 28.50 g/L, 0.69 g/g when heterologous CO2 source was supplied. CONCLUSIONS: The availability of NAD(H) was correlated positively with the glucose utilization rate and cellular metabolism capacities, and lower NADH/NAD+ ratio was beneficial for the accumulation of L-malate under anaerobic conditions. Enhanced ATP level could significantly enlarge the intracellular NAD(H) pool under anaerobic condition. Moreover, there might be an inflection point, that is, the increase of NAD(H) pool before the inflection point is followed by the improvement of metabolic performance, while the increase of NAD(H) pool after the inflection point has no significant impacts and NADH/NAD+ ratio would dominate the metabolic flux. This study is a typical case of anaerobic organic acid fermentation, and demonstrated that ATP level, NAD(H) pool and NADH/NAD+ ratio are three important regulatory parameters during the anaerobic production of L-malate.

Techniques for enhancing the tolerance of industrial microbes to abiotic stresses: A review.

The diversity of stress responses and survival strategies evolved by microorganism enables them to survive and reproduce in a multitude of harsh environments, whereas the discovery of the underlying resistance genes or mechanisms laid the foundation for the directional enhancement of microbial tolerance to abiotic stresses encountered in industrial applications. Many biological techniques have been developed for improving the stress resistance of industrial microorganisms, which greatly benefited the bacteria on which industrial production is based. This review introduces the main techniques for enhancing the resistance of microorganisms to abiotic stresses, including evolutionary engineering, metabolic engineering, and process engineering, developed in recent years. In addition, we also discuss problems that are still present in this area and offer directions for future research.

Current status and perspectives of 2-phenylethanol production through biological processes.

2-Phenylethanol (2-PE), an important flavor and fragrance compound with a rose-like smell has been widely used in the cosmetics, perfume, and food industries. Traditional production of 2-PE was mainly through the extraction from plant materials or by chemical synthesis. However, the increasing demand of consumers for natural flavors cannot be met by these methods. Biological production of 2-PE has emerged to be an appealing solution due to an environmental friendly process and the definition of a "natural" product. In this review, we have comprehensively summarized the current status and perspectives for biological 2-PE production in terms of its advantages over classical chemical synthesis and extraction from natural plants. A comprehensive description of 2-PE synthetic pathways and global regulation mechanisms, strategies to increase 2-PE production, and the utilization of agro-industrial wastes as feedstocks has been systematically discussed. Furthermore, the application of in situ product removal techniques have also been highlighted.

Chemoenzymatic Synthesis of Branched Glycopolymer Brushes as the Artificial Glycocalyx for Lectin Specific Binding.

The artificial glycocalyx fabricated by carbohydrates is of interest because it provides a platform to simulate the cell membranes that widely exist in the nature, and thus enable extensive applications to be implantable in bioengineering. Here, we present a green strategy combining two polymerization techniques, surface-initiated atom transfer radical polymerization (SI-ATRP) and enzyme-catalyzed elongation of polysaccharide, for fabricating densely packed branched glycopolymer brushes on the gold surface as the artificial glycocalyx. In this strategy, SI-ATRP is first performed to graft a linear polymer chain for anchoring maltose, which can be used as an enzyme acceptor for dextransucrase (DSase). Under DSase, a branched polysaccharide is efficiently formed through elongation of a sucrose substrate. Undoubtedly, enzymatic transglycosylation has unique advantages, such as being green, regio-, and stereo-selective, etc. The process of DSase-catalyzed polysaccharide is well monitored by a quartz crystal microbalance, and the grafting density of the glycopolymer brushes is estimated to be 0.7 chain nm-2 with 23.0 nm dry thickness. The polysaccharide brushes display a branched structure consisting of α-d-glucose residues with 5% of α-1,3-linked shorter chain branches, and the branched structure is well characterized by X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectrometry, Fourier transform infrared/mirror reflection, water contact angle analysis, and atomic force microscopy. Compared with the linear maltose-anchored brushes, the branched glycopolymer analog prepared here shows high specific binding capacity of concanavalin A recognition, which should be of use in biomedical application.

The broad-specificity chitinases: their origin, characterization, and potential application.

Chitinases are hydrolases that catalyze the cleavage of the ß-1,4-O-glycosidic linkages in chitin, a polysaccharide abundantly found in nature. Although numerous chitinolytic enzymes have been studied in detail, relatively little is known about chitinases capable of broad specificity. Broad-specificity chitinases are a sort of novel chitinases possessing two or three different catalytic activities among exochitinase, endochitinase, and N-acetylglucosaminidase. In the light of the difference of module composition and catalytic mechanism, the broad-specificity chitinases included two broad categories, broad-specificity chitinases with a single catalytic domain or multi-catalytic domains. This broad-specificity chitinases have great potential in chitin conversion. In this review, we summarize all reported cases of broad-specificity chitinases and provide an overview of the recent findings on their origin, characterization, catalytic mechanism, and potential application. Moreover, in-depth study into these chitinases could contribute to our understanding of other broad-specificity enzymes which may have some benefits on progress of biotechnology.

The Draft Genome Sequence of Methylophilus sp. D22, Capable of Growing Under High Concentration of Methanol.

In this study, a wild type Methylophilus sp. strain D22 belonging to the family Methylophilus was isolated and characterized, which shows high tolerance towards methanol, as it can grow under 50 g/L of methanol. Methylophilus sp. strain D22 was isolated from the lake sludge in Nanjing Tech University, China. The assembled draft genome contains one circular chromosome with 3,004,398 bp, 49.7% of GC content, and 2107 predicted encoding proteins. Sequence-based genomic analysis demonstrates that the assimilation pathway of ribulose monophosphate (RuMP) pathway and dissimilation pathway of tetrahydromethanopterin (H4MPT) pathway are co-existing and contribute to the high methanol utilization efficiency.

Heterologous expression of cyclodextrin glycosyltransferase from Paenibacillus macerans in Escherichia coli and its application in 2-O-α-D-glucopyranosyl-L-ascorbic acid production.

BACKGROUND: Cyclodextrin glucanotransferase (CGTase) can transform L-ascorbic acid (L-AA, vitamin C) to 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), which shows diverse applications in food, cosmetic and pharmaceutical industries. RESULTS: In this study, the cgt gene encoding α-CGTase from Paenibacillus macerans was codon-optimized (opt-cgt) and cloned into pET-28a (+) for intracellular expression in E. coli BL21 (DE3). The Opt-CGT was purified by Ni2+-NTA resin with a 55% recovery, and specific activity was increased significantly from 1.17 to 190.75 U·mg- 1. In addition, the enzyme was adopted to transform L-AA into 9.1 g/L of AA-2G. Finally, more economic substrates, including ß-cyclodextrin, soluble starch, corn starch and cassava starch could also be used as glycosyl donors, and 4.9, 3.5, 1.3 and 1.5 g/L of AA-2G were obtained, respectively. CONCLUSIONS: N-terminal amino acid is critical to the activity of CGTase suggested by its truncation study. Furthermore, when the Opt-CGT was flanked by His6-tags on the C- and N-terminal, the recovery of purification by Ni2+-NTA resin is appreciably enhanced. α-cyclodextrin was the ideal glycosyl donor for AA-2G production. In addition, the selection of low cost glycosyl donors would make the process of AA-2G production more economically competitive.

Recent advances on conversion and co-production of acetone-butanol-ethanol into high value-added bioproducts.

Butanol is an important bulk chemical and has been regarded as an advanced biofuel. Large-scale production of butanol has been applied for more than 100 years, but its production through acetone-butanol-ethanol (ABE) fermentation process by solventogenic Clostridium species is still not economically viable due to the low butanol titer and yield caused by the toxicity of butanol and a by-product, such as acetone. Renewed interest in biobutanol as a biofuel has spurred technological advances to strain modification and fermentation process design. Especially, with the development of interdisciplinary processes, the sole product or even the mixture of ABE produced through ABE fermentation process can be further used as platform chemicals for high value added product production through enzymatic or chemical catalysis. This review aims to comprehensively summarize the most recent advances on the conversion of acetone, butanol and ABE mixture into various products, such as isopropanol, butyl-butyrate and higher-molecular mass alkanes. Additionally, co-production of other value added products with ABE was also discussed.

Opportunities, challenges, and future perspectives of succinic acid production by Actinobacillus succinogenes.

Due to environmental issues and the depletion of fossil-based resources, ecofriendly sustainable biomass-based chemical production has been given more attention recently. Succinic acid (SA) is one of the top value added bio-based chemicals. It can be synthesized through microbial fermentation using various waste steam bioresources. Production of chemicals from waste streams has dual function as it alleviates environmental concerns; they could have caused because of their improper disposal and transform them into valuable products. To date, Actinobacillus succinogenes is termed as the best natural SA producer. However, few reviews regarding SA production by A. succinogenes were reported. Herewith, pathways and metabolic engineering strategies, biomass pretreatment and utilization, and process optimization related with SA fermentation by A. succinogenes were discussed in detail. In general, this review covered vital information including merits, achievements, progresses, challenges, and future perspectives in SA production using A. succinogenes. Therefore, it is believed that this review will provide platform to understand the potential of the strain and tackle existing hurdles so as to develop superior strain for industrial applications. It will also be used as a baseline for identification, isolation, and improvement of other SA-producing microbes.

Application of eukaryotic and prokaryotic laccases in biosensor and biofuel cells: recent advances and electrochemical aspects.

Laccases exhibit a wide range of applications, especially in the electrochemical field, where they are regarded as a potential biotic component. Laccase-based biosensors have immense practical applications in the food, environmental, and medical fields. The application of laccases as biocathodes in enzymatic biofuel cells has promising potential in the preparation of implantable equipment. Extensive studies have been directed towards the potential role of fungal laccases as biotic components of electrochemical equipment. In contrast, the potential of prokaryotic laccases in electrochemistry has been not fully understood. However, there has been recent and rapid progress in the discovery and characterization of new types of prokaryotic laccases. In this review, we have comprehensively discussed the application of different sources of laccases as a biocatalytic component in various fields of application. Further, we described the potential of different types of laccases in bioelectrochemical applications.

The Draft Genome Sequence of Thermophilic Thermoanaerobacterium thermosaccharolyticum M5 Capable of Directly Producing Butanol from Hemicellulose.

A novel thermophilic and butanogenic Thermoanaerobacterium thermosaccharolyticum M5 was successfully isolated and characterized, which could produce butanol from hemicellulose via a unique ethanol-butanol (EB) pathway through consolidated bioprocessing (CBP). This represents the first wild-type bacterium which could produce butanol from hemicellulose via CBP under thermophilic conditions. The assembled draft genome of strain M5 is 2.64 Mp, which contains 2638 genes and 2465 protein-coding sequences with 33.90% G + C content. Among these annotated proteins, xylanases, xylosidases, and bifunctional alcohol/aldehyde dehydrogenase (AdhE) play key roles in the achievement of EB production from hemicellulose through CBP.

The Draft Genome Sequence of Clostridium beijerinckii NJP7, a Unique Bacterium Capable of Producing Isopropanol-Butanol from Hemicellulose Through Consolidated Bioprocessing.

A wild type solventogenic Clostridium beijerinckii NJP7 capable of converting polysaccharides, such as hemicellulose, into butanol and isopropanol via a unique acetone-isopropanol-butanol (AIB) pathway was isolated and characterized. This represents the first wild type isopropanol-butanol generating bacterium which could achieve butanol production directly from lignocellulose through consolidated bioprocessing (CBP). Strain NJP7 was isolated from decomposite soil from Laoshan Nature Park, China, and its genome shows 98.6% identical to 89.5% of the Clostridium diolis submitted genome sequence. The assembled draft genome contains 5.76 Mb and 5101 predicted encoding proteins with a GC content of 29.73%. Among these annotated proteins, hemicellulase and the secondary alcohol dehydrogenase play key roles in achievement of AIB production from hemicellulose through CBP.

The Draft Genome Sequence of Clostridium sp. Strain NJ4, a Bacterium Capable of Producing Butanol from Inulin Through Consolidated Bioprocessing.

A novel butanogenic Clostridium sp. NJ4 was successfully isolated and characterized, which could directly produce relatively high titer of butanol from inulin through consolidated bioprocessing (CBP). The assembled draft genome of strain NJ4 is 4.09 Mp, containing 3891 encoded protein sequences with G+C content of 30.73%. Among these annotated genes, a levanase, a hypothetical inulinase, and two bifunctional alcohol/aldehyde dehydrogenases (AdhE) were found to play key roles in the achievement of ABE production from inulin through CBP.

Characteristics and metabolic pathway of acetamiprid biodegradation by Fusarium sp. strain CS-3 isolated from soil.

An acetamiprid-degrading fungus was isolated from contaminated soil and identified as Fusarium sp. strain CS-3 based on physiological, biochemical, and molecular analyses. Strain CS-3 exploited 50 mg/L as the sole carbon source in liquid culture, removing 98% in 96 h. Strain CS-3 retained its acetamiprid degradation abilities over a wide range of pH (5.0-8.0) and temperature (20-42 °C). HPLC-MS analysis showed that N'-[(6-chloropyridin-3-yl)methyl]-N-methylacetamide, 2-chloro-5-hydroxymethylpyridine, and 6-chloronicotinic acid were identified as the most predominant metabolites, forming the basis for a newly described acetamiprid degradation pathway. Strain CS-3 efficiently degraded 99.6% of 50 mg/kg acetamiprid in soil, indicating potential for soil remediation.

Bio-succinic acid production from coffee husk treated with thermochemical and fungal hydrolysis.

Coffee husk (CH), a waste obtained from processing of coffee cherries via dry method, causes serious environmental problems. In this study, strategies were designed to utilize CH for succinic acid (SA) production. Three different CH hydrolysis methods: thermal, thermochemical and crude enzymes obtained by solid state fermentation of Aspergillus niger and Trichoderma reesei, were evaluated to generate fermentable feedstock for SA production using Actinobacillus succinogenes. The feasibility of these pretreatment methods was investigated. Accordingly, thermochemical hydrolysis using H2SO4 at 121 °C for 30 min, appeared the most effective method for CH hydrolysis, producing 24.4 g/L of reducing sugars (RS). Finally, 19.3 g/L of SA with yield and productivity of 0.95 g SA/g RS and 0.54 g/L/h, respectively, were obtained using CH hydrolysate. The current study revealed an alternative way of utilization coffee waste for value addition while mitigating environmental problems caused by its disposal.

Recent advances in microbial production of mannitol: utilization of low-cost substrates, strain development and regulation strategies.

Mannitol has been widely used in fine chemicals, pharmaceutical industries, as well as functional foods due to its excellent characteristics, such as antioxidant protecting, regulation of osmotic pressure and non-metabolizable feature. Mannitol can be naturally produced by microorganisms. Compared with chemical manufacturing, microbial production of mannitol provides high yield and convenience in products separation; however the fermentative process has not been widely adopted yet. A major obstacle to microbial production of mannitol under industrial-scale lies in the low economical efficiency, owing to the high cost of fermentation medium, leakage of fructose, low mannitol productivity. In this review, recent advances in improving the economical efficiency of microbial production of mannitol were reviewed, including utilization of low-cost substrates, strain development for high mannitol yield and process regulation strategies for high productivity.

Recent insights into the microbial catabolism of aryloxyphenoxy-propionate herbicides: microbial resources, metabolic pathways and catabolic enzymes.

Aryloxyphenoxy-propionate herbicides (AOPPs) are widely used to control annual and perennial grasses in broadleaf crop fields and are frequently detected as contaminants in the environment. Due to the serious environmental toxicity of AOPPs, there is considerable concern regarding their biodegradation and environmental behaviors. Microbial catabolism is considered as the most effective method for the degradation of AOPPs in the environment. This review presents an overview of the recent findings on the microbial catabolism of various AOPPs, including fluazifop-P-butyl, cyhalofop-butyl, diclofop-methyl, fenoxaprop-P-ethyl, metamifop, haloxyfop-P-methyl and quizalofop-P-ethyl. It highlights the microbial resources that are able to catabolize these AOPPs and the metabolic pathways and catabolic enzymes involved in their degradation and mineralization. Furthermore, the application of AOPPs-degrading strains to eliminate AOPPs-contaminated environments and future research hotspots in biodegradation of AOPPs by microorganisms are also discussed.

High-yield production of mannitol by Leuconostoc pseudomesenteroides CTCC G123 from chicory-derived inulin hydrolysate.

Chicory is an agricultural plant with considerable potential as a carbohydrate substrate for low-cost production of biochemicals. In this work, the production of mannitol by Leuconostoc pseudomesenteroides CTCC G123 from chicory-derived inulin hydrolysate was investigated. The bioconversion process initially suffered from the leakage of fructose to the phosphoketolase pathway, resulting in a low mannitol yield. When inulin hydrolysate was supplemented with glucose as a substrate for mannitol production in combination with aeration induction and nicotinic acid induced redox modulation strategies, the mannitol yield greatly improved. Under these conditions, significant improvement in the glucose consumption rate, intracellular NADH levels and mannitol dehydrogenase specific activity were observed, with mannitol production increasing from 64.6 to 88.1 g/L and overall yield increase from 0.69 to 0.94 g/g. This work demonstrated an efficient method for the production of mannitol from inulin hydrolysate with a high overall yield.