Formation of 4-vinyl guaiacol as an intermediate in bioconversion of ferulic acid by Schizophyllum commune

In order to utilize phenolic compounds in unused biomass resources, the metabolic pathway of ferulic acid by way of a white-rot fungus, Schizophyllum commune, was investigated. Ferulic acid was immediately degraded, and the formation of 4-vinyl guaiacol was confirmed by GC-MS analysis. The metabolic test of ferulic acid and its degradation products indicated that S. commune converted ferulic acid into 4-vinyl guaiacol by decarboxylation. This was then oxidized to vanillin and vanillic acid. This result indicates that S. commune distinguished ferulic acid from lignins and metabolized it specifically.     

Transformation of ferulic acid to 4-vinyl guaiacol as a major metabolite: a microbial approach

The majority of the flavours and fragrances used worldwide are produced by chemical synthesis at low price. However, consumers prefer natural compounds because of increasing health and nutrition awareness in routine life. Hence, biotransformation is an alternative process to produce natural aroma compounds. Microorganisms have been gradually used more to produce natural aroma compounds with various applications in food, agriculture and pharmaceutical industries. This paper reviews the role of microorganisms in the transformation of ferulic acid to 4-vinyl guaiacol. The microbial processes based on biocatalytic method are discussed in terms of their advantages over chemical synthesis, plant cell cultures and enzyme catalyzed reactions. Thus, the transformation of ferulic acid by microorganisms could have possible use in food, pharmaceutical industry and become an increasingly important platform for the production of natural aroma compounds.     

Process design and evaluation of value-added chemicals production from biomass

Three different biodiesel production processes were simulated using the SuperPro Designer program. The process for producing biodiesel from soybean oil and methanol was designed using commercial chemical catalysts. This chemical process was compared with the biological process catalyzed by immobilized enzymes. In addition, a hybrid process consisting of catalytic biodiesel production and enzymatic glycerol carbonate production was designed and simulated for the conversion of waste glycerol to value-added chemical. Finally, the economics and productivity of these processes were evaluated to determine economic feasibility.     

Strain engineering for microbial production of value-added chemicals and fuels from glycerol

While the widespread reliance on fossil fuels is driven by their low cost and relative abundance, this fossil-based economy has been deemed unsustainable and, therefore, the adoption of sustainable and environmentally compatible energy sources is on the horizon. Biorefinery is an emerging approach that integrates metabolic engineering, synthetic biology, and systems biology principles for the development of whole-cell catalytic platforms for biomanufacturing. Due to the high degree of reduction and low cost, glycerol, either refined or crude, has been recognized as an ideal feedstock for the production of value-added biologicals, though microbial dissimilation of glycerol sometimes can be difficult particularly under anaerobic conditions. While strain development for glycerol biorefinery is widely reported in the literature, few, if any, commercialized bioprocesses have been developed as a result, such that engineering of glycerol metabolism in microbial hosts remains an untapped opportunity in biomanufacturing. Here we review the recent progress made in engineering microbial hosts for the production of biofuels, diols, organic acids, biopolymers, and specialty chemicals from glycerol. We begin with a broad outline of the major pathways for fermentative and respiratory glycerol dissimilation and key end metabolites, and then focus our analysis on four key genera of bacteria known to naturally dissimilate glycerol, i.e. Klebsiella, Citrobacter, Clostridium, and Lactobacillus, in addition to Escherichia coli, and systematically review the progress made toward engineering these microorganisms for glycerol biorefinery. We also identify the major biotechnological and bioprocessing advantages and disadvantages of each genus, and bottlenecks limiting the production of target metabolites from glycerol in engineered strains. Our analysis culminates in the development of potential strategies to overcome the current technical limitations identified for commonly employed strains, with an outlook on the suitability of different hosts for the production of key metabolites and avenues for their future development into biomanufacturing platforms.     

The potential of cloud point system as a novel two-phase partitioning system for biotransformation

Although the extractive biotransformation in two-phase partitioning systems have been studied extensively, such as the water–organic solvent two-phase system, the aqueous two-phase system, the reverse micelle system, and the room temperature ionic liquid, etc., this has not yet resulted in a widespread industrial application. Based on the discussion of the main obstacles, an exploitation of a cloud point system, which has already been applied in a separation field known as a cloud point extraction, as a novel two-phase partitioning system for biotransformation, is reviewed by analysis of some topical examples. At the end of the review, the process control and downstream processing in the application of the novel two-phase partitioning system for biotransformation are also briefly discussed.     

Recent advances in engineering propionyl-CoA metabolism for microbial production of value-added chemicals and biofuels

Diminishing fossil fuel reserves and mounting environmental concerns associated with petrochemical manufacturing practices have generated significant interests in developing whole-cell biocatalytic systems for the production of value-added chemicals and biofuels. Although acetyl-CoA is a common natural biogenic precursor for the biosynthesis of numerous metabolites, propionyl-CoA is unpopular and non-native to most organisms. Nevertheless, with its C3-acyl moiety as a discrete building block, propionyl-CoA can serve as another key biogenic precursor to several biological products of industrial importance. As a result, engineering propionyl-CoA metabolism, particularly in genetically tractable hosts with the use of inexpensive feedstocks, has paved an avenue for novel biomanufacturing. Herein, we present a systematic review on manipulation of propionyl-CoA metabolism as well as relevant genetic and metabolic engineering strategies for microbial production of value-added chemicals and biofuels, including odd-chain alcohols and organic acids, bio(co)polymers and polyketides.

     

Recent advances in metabolic engineering of Corynebacterium glutamicum for bioproduction of value-added aromatic chemicals and natural products

Recent progress in synthetic and systems metabolic engineering technologies has explored the potential of microbial cell factories for the production of industrially relevant bulk and fine chemicals from renewable biomass resources in an eco-friendly manner. Corynebacterium glutamicum, a workhorse for industrial amino acid production, has currently evolved into a promising microbial platform for bioproduction of various natural and non-natural chemicals from renewable feedstocks. Notably, it has been recently demonstrated that metabolically engineered C. glutamicum can overproduce several commercially valuable aromatic and related chemicals such as shikimate, 4-hydroxybenzoate, and 4-aminobenzoate from sugars at remarkably high titer suitable to commercial application. On the other hand, overexpression and/or extension of its endogenous metabolic pathways by integrating heterologous metabolic pathways enabled production of structurally intricate and valuable natural chemicals like plant polyphenols, carotenoids, and fatty acids. In this review, we summarize recent advances in metabolic engineering of C. glutamicum for production of those value-added aromatics and other natural products, which highlights high potential and the versatility of this microbe for bioproduction of diverse chemicals.

     

Efficient purification of recombinant proteins using hydrophobins as tags in surfactant-based two-phase systems

In this work we describe the new concept of using fungal hydrophobins as efficient tags for purification of recombinant fusion proteins by aqueous two-phase separation. Hydrophobins are a group of small surface-active proteins produced by filamentous fungi. Some characteristics of hydrophobins are that they are relatively small (approximately 100 amino acids), they contain eight disulfide-forming Cys residues in a conserved pattern, and they self-assemble on interfaces. The aqueous two-phase systems studied were based on nonionic surfactants that phase-separate at certain temperatures. We show that the use of hydrophobins as tags has many advantages such as high selectivity and good yield and is technically very simple to perform. Fusion proteins with target proteins of different molecular size were compared to the corresponding free proteins using a set of different surfactants. This gave an understanding on which factors influence the separation and what rationale should be used for optimization. This unusually strong and specific interaction between polymeric surfactants and a soluble protein shows promise for new developments in interfacing proteins and nonbiological materials for other applications as well.     

Partitioning of caseinomacropeptide in aqueous two-phase systems

This study evaluates the influence of type of salt and temperature on the partition coefficient of caseinomacropetide (CMP) to determine the best conditions for the recovery of CMP in aqueous two-phase systems (ATPS) composed by poly(ethylene glycol) (PEG) 1500 and an inorganic salt (potassium phosphate, sodium citrate, lithium sulfate or sodium sulfate). In all systems, CMP presented affinity for the PEG-rich phase. The PEG1500+lithium sulfate showed the highest values of partitioning coefficient. In addition, thermodynamic parameters (DeltaH degrees , DeltaS degrees , DeltaG degrees) as a function of temperature, were calculated for the system PEG1500-sodium citrate at different PEG concentrations and the results imply thermodynamic differences between partitioning of CMP in this system.     

Applications of aqueous two-phase systems in biotechnology

Aqueous two-phase systems can be employed in several areas of biotechnology including the purification of biomolecules, cells and organelles and increasing the speed of product-inhibited fermentations such as the production of acetone, butanol and ethanol. Furthermore, separations in aqueous two-phase systems have been successfully applied in binding assays.     

Activity of phospholipase C in two-phase systems

Although phospholipase C (PLC) is known to be activated by water-insoluble organic solvents, most activity assays have been designed to work in an aqueous milieu. Here a sensitive method is described for the determination of PLC activity in two-phase systems. The assay is based on the hydrolysis of phosphatidylcholine (PC) in chloroform/buffer. The initial rates of the reaction are determined by densitometric quantification of the product 1,2-diacylglycerol after its separation by high-performance TLC and staining with a CuSO4/H3PO4 or p-methoxybenzaldehyde/H2SO4 reagent. The method is examined for the determination of Vmax and Km values of PCs with varying length acyl chains (C10-C18). The comparison of the kinetic parameters with the Vmax and Km values of the same substrates in the conventional titrimetric assay, using sodium deoxycholate for micellization of PC, demonstrates the high efficiency of PLC in the two-phase emulsion system.     

Photosynthetic production of fuels and chemicals in immobilized systems

Whole cells of algae, cyanobacteria and photosynthetic bacteria entrapped in alginate gels or polyurethane foams can retain their photo-synthetic activities for months and in some cases for years. Such immobilized cells can be used in bioreactors for the production of H₂, NH₃, NADPH₂, carbohydrates, hydrocarbons, etc., with sunlight as the energy source.     

Intensification of mass transfer in aqueous two-phase systems

A novel technique which intensifies conventional aqueous two-phase extraction by conversion of dispersed phase into colloidal gas aphrons (CGAs) has been developed for extraction of an enzyme. In the present work, amyloglucosidase (1,4-alpha-D-glucan glucohydrolase) was extracted using a polyethylene glycol-sodium sulfate-water system. The lighter phase, i.e., polyethylene glycol (PEG) rich phase, was converted into CGAs which were then dispersed into a salt rich phase. The effect of type of surfactant and its concentration, dispersed phase velocity, phase composition, and type of sparger on the dispersed phase mass transfer coefficient was investigated. The results suggests 9-16 times higher values of mass transfer coefficient compared to spray column. The multiorifice sparger at concentrations of 0.33 g/L of cetyl trimethyl ammonium chloride yielded best results. (c) 1993 John Wiley & Sons, Inc.     

Production of flavour ketones in aqueous-organic two-phase systems by using free and microencapsulated fungal spores as biocatalysts

The formation of 2-alkanones by free and microencapsulated P. roquefortii spores in an aqueous-organic two-phase system was investigated by using substrates supplied as a solution in decane. It was shown that the spores remained catalytically active after entrapment within permeable polyamide microcapsules and readily catalyzed the formation of 2-pentanone, 2-heptanone, and 2-undecanone from short-chain alkyl esters of hexanoic, octanoic, and lauric acid, respectively, with the rate of reaction being markedly dependent on the type and concentration of the ester substrate used. In general, the optimal concentration of the esters in decane was found to be much higher than that of the respective fatty acid substrates and, in the case of alkyl dodecanoates, the biotransformation could be carried out efficiently even in the absence of added solvent. Further analysis revealed a significant difference in the reaction rates observed with free and microencapsulated spores at 0.5 but not at 3.0 M methyl dodecanoate, suggesting that at high substrate concentrations the biotransformation was no longer limited by mass transfer.     

Phase transfer and biocatalyst behaviour during biotransformation of beta-ionone in a two-phase liquid system by immobilised Aspergillus niger

The biotransformation of beta-ionone by Aspergillus niger IFO 8541 entrapped in Ca-alginate beads was investigated in a two-phase liquid system, due to the low aqueous solubility of the precursor. Modelling of phase transfer processes of the substrate demonstrated that the solute was transferred from the organic droplets to the gas, giving a loss by stripping, and then from the gas to the aqueous solution where a chemical degradation occurred. The biological reaction took place after direct precursor transfer from the organic layer to the biocatalyst by surface adsorption. Studies on the biological process demonstrated the critical effect of the biomass content in the medium at the time at which beta-ionone was added. Optimum conditions involved fed-batch feedings of both precursor and carbon source (sucrose) after the biomass concentration reached a value close to 6.8g/l. The biotransformation process then took place at a constant rate of 0.046mmol/lh with a reaction yield, defined with respect to beta-ionone metabolised by the fungus, close to unity. Best results achieved in this study allowed to obtain 3.5g/l biological compounds after 400h reaction.     

Cultivation of plant cells in aqueous two-phase polymer systems

Summary Suspension cultures ofNicotiana tabacum have been successfully grown in aqueous, two-phase systems comprised of polyethylene glycol (PEG) and dextran in a modified LS medium. Aqueous two-phase systems may be advantageous for plant tissue cultivation since cells can be immobilized in one phase while secondary products are collected and withdrawn in the other phase, thus enhancing productivity. Culture growth rate was compared in a variety of two-phase systems, covering a range of both polymer molecular weight and concentration. Systems exhibiting relatively higher phase miscibility yielded increased growth rates as compared to less miscible phase formulations. The highest observed growth rate occurred in 3% PEG 20000/5% crude dextran and approached growth rates and cell densities of cultures grown in standard LS medium.     

Immobilized metal ion affinity partitioning of cells in aqueous two-phase systems: erythrocytes as a model

Immobilized metal ion affinity partitioning of erythrocytes from different species is described. We have explored the affinity between transition metal chelates and metal-binding sites situated on the cell surface by partitioning in aqueous two-phase system composed of poly(ethylene glycol) and dextran. Soluble metal-chelate-poly(ethylene glycol) was prepared by fixing metal ions to poly(ethylene glycol) via the covalently bonded chelator, iminodiacetic acid. The partitioning behaviour of erythrocytes in systems at different concentrations of the ligand was tested. The copper-chelate-poly(ethylene glycol) was quite effective in the affinity extraction of human and rabbit erythrocytes, while the zinc-chelate-poly(ethylene glycol) displayed significant affinity only to the rabbit cells. Furthermore, the influence of various effectors such as imidazole, sialic acid on immobilized metal ion affinity partitioning of erythrocytes was examined.     

PEG-柠檬酸盐双水相体系纯化α-淀粉酶及模型构建

探索了采用PEG-柠檬酸盐双水相体系纯化α-淀粉酶的可行性和工艺条件,实验考察了PEG分子量。结线长度、pH、上清加入量和NaCl浓度对α-淀粉酶的分配的不同影响。双水相分离体系复杂,影响因素多,为进行分析、预测和优化,采用了响应面方法,以PEG3350浓度、柠檬酸盐浓度和NaCl浓度为自变量进行了α-淀粉酶分配系数、总蛋白分配系数、纯化因子和α-淀粉酶收率的优化,确定了特定的系统组成区域,通过实验验证了该区域内纯化因子大于2,收率约90％,并且具有较低的系统粘度。