A versatile Pseudomonas putida KT2440 with new ability: selective oxidation of 5-hydroxymethylfurfural to 5-hydroxymethyl-2-furancarboxylic acid

Currently, biotransformation of 5-hydroxymethylfurfural (HMF) into a series of high-value bio-based platform chemicals is massively studied. In this study, selective biooxidation of HMF to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) by Pseudomonas putida KT2440 with superior titer, yield, and productivity was reported. The biocatalytic performances of P. putida KT2440 were optimized separately. Under optimal conditions, 100% yield of HMFCA was obtained when HMF concentration was less than 150 mM, while the maximum concentration of 155 mM was achieved from 160 mM HMF in 12 h. P. putida KT2440 was highly tolerate to HMF, up to 190 mM. Besides, it was capable of selective oxidation of other furan aldehydes to the corresponding carboxylic acids with good yield of 100%. This study further demonstrates the potential of P. putida KT2440 as a biocatalyst for biomass conversion, as this strain has been proved the capacity to convert and utilize many kinds of biomass-derived sugars and ligin-derived aromatic compounds.

     

Sequential oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid by an evolved aryl-alcohol oxidase

Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-alcohol oxidase (AAO) and 5-hydroxymethylfurfural oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.     

Oxidation of 5-hydroxymethylfurfural with a novel aryl alcohol oxidase from Mycobacterium sp. MS1601

Bio-based 5-hydroxymethylfurfural (HMF) serves as an important platform for several chemicals, among which 2,5-furan dicarboxylic acid (FDCA) has attracted considerable interest as a monomer for the production of polyethylene furanoate (PEF), a potential alternative for fossil-based polyethylene terephthalate (PET). This study is based on the HMF oxidizing activity shown by Mycobacterium sp. MS 1601 cells and investigation of the enzyme catalysing the oxidation. The Mycobacterium whole cells oxidized the HMF to FDCA (60% yield) and hydroxymethyl furan carboxylic acid (HMFCA). A gene encoding a novel bacterial aryl alcohol oxidase, hereinafter MycspAAO, was identified in the genome and was cloned and expressed in Escherichia coli Bl21 (DE3). The purified MycspAAO displayed activity against several alcohols and aldehydes; 3,5 dimethoxy benzyl alcohol (veratryl alcohol) was the best substrate among those tested followed by HMF. 5-Hydroxymethylfurfural was converted to 5-formyl-2-furoic acid (FFCA) via diformyl furan (DFF) with optimal activity at pH 8 and 30–40°C. FDCA formation was observed during long reaction time with low HMF concentration. Mutagenesis of several amino acids shaping the active site and evaluation of the variants showed Y444F to have around 3-fold higher k_cat/K_m and ~1.7-fold lower K_m with HMF.     

Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688

In the search for useful and renewable chemical building blocks, 5-hydroxymethylfurfural (HMF) has emerged as a very promising candidate, as it can be prepared from sugars. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a substitute for petroleum-based terephthalate in polymer production. On the basis of a recently identified bacterial degradation pathway for HMF, candidate genes responsible for selective HMF oxidation have been identified. Heterologous expression of a protein from Methylovorus sp. strain MP688 in Escherichia coli and subsequent enzyme characterization showed that the respective gene indeed encodes an efficient HMF oxidase (HMFO). HMFO is a flavin adenine dinucleotide-containing oxidase and belongs to the glucose-methanol-choline-type flavoprotein oxidase family. Intriguingly, the activity of HMFO is not restricted to HMF, as it is active with a wide range of aromatic primary alcohols and aldehydes. The enzyme was shown to be relatively thermostable and active over a broad pH range. This makes HMFO a promising oxidative biocatalyst that can be used for the production of FDCA from HMF, a reaction involving both alcohol and aldehyde oxidations.     

Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran

Renewable lignocellulosic materials are attractive low-cost feedstocks for bioethanol production. Furfural and 5-hydroxymethylfurfural (HMF) are among the most potent inhibitory compounds generated from acid hydrolysis of lignocelluloses to simple sugars for fermentation. In Saccharomyces cerevisiae ATCC 211239 and NRRL Y-12632 and Pichia stipitis NRRL Y-7124, furfural and HMF inhibition were determined to be dose-dependent at concentrations from 10 to 120 mM. The yeast strains were more sensitive to inhibition by furfural than HMF at the same concentration, while combined treatment of furfural and HMF synergistically suppressed cell growth. A metabolite transformed from HMF by strain NRRL Y-12632 was isolated from the culture supernatant, and conclusively identified as 2,5-bis-hydroxymethylfuran, a previously postulated HMF alcohol, with a composition of C₆H₈O₃ and a molecular weight of 128. It is proposed that, in the presence of HMF, the yeast reduces the aldehyde group on the furan ring of HMF into an alcohol, in a similar manner as for furfural. The accumulation of this biotransformed metabolite may be less toxic to yeast cultures than HMF, as evidenced by the rapid yeast fermentation and growth rates associated with HMF conversion. The ability of yeasts to adapt to and transform furfural and HMF offers the potential for in situ detoxification of these inhibitors and suggests a genetic basis for further development of highly tolerant strains for biofuel production.     

Efficient microwave-assisted synthesis of 5-hydroxymethylfurfural from concentrated aqueous fructose

Studies on the HCl-catalysed microwave-assisted dehydration of highly concentrated aqueous fructose (27 wt %) to 5-hydroxymethylfurfural (HMF) revealed a significant increase in the fructose conversion rate over the conventional heated systems. Water, being the most benign solvent and therefore ideal for green and sustainable chemistry, normally is a poor solvent for the dehydration process resulting in low HMF selectivities and yields. However, reaction at 200 °C with microwave irradiation with a short reaction time of only 1 s resulted in good HMF selectivity of 63% and fructose conversion of 52%, while prolonged irradiation for 60 s (or more) resulted in nearly full fructose conversion (95%) but lower HMF yield (53%). Decreasing the fructose concentration significantly improved the HMF selectivity, but possibly made the production route less attractive from an industrial point of view due to the resultant low throughput.     

Production of 5-hydroxymethylfurfural in ionic liquids under high fructose concentration conditions

Acid-promoted, selective production of 5-hydroxymethylfurfural (HMF) under high fructose concentration conditions was achieved in ionic liquids (ILs) at 80 °C. A HMF yield up to 97% was obtained in 8 min using 1-butyl-3-methylimidazolium chloride ([C₄mim]Cl) catalyzed with 9 mol % hydrochloric acid. More significantly, an HMF yield of 51% was observed when fructose was loaded at a high concentration of 67 wt % in [C₄mim]Cl. Water content below 15.4% in the system had little effect on HMF yield, whereas a higher water content was detrimental to both reaction rate and HMF yield. In situ NMR analysis suggested that the transformation of fructose to HMF was a highly selective reaction that proceeded through the cyclic fructofuranosyl intermediate pathway. This work increased our capacity to produce HMF, and should be valuable to facilitate cost-efficient conversion of biomass into biofuels and bio-based products.     

糠醛和5-羟甲基糠醛对大肠杆菌产丁二酸的影响

利用可再生生物质特别是木质纤维素水解液来生产平台化合物丁二酸,是目前研究的热点。虽然许多研究者相继报道了木质纤维素水解液对菌株生长和丁二酸生产存在一定抑制作用,但并没有水解液中各种抑制物对菌株影响的相关动力学研究及机理研究。我们选择了两种代表性木质纤维素水解液抑制物,即糠醛和5-羟甲基糠醛,系统研究了它们对大肠杆菌的生长和丁二酸生产的影响。结果表明：糠醛和5-羟甲基糠醛的初始抑制浓度均为0.8 g/L。当糠醛浓度大于6.4 g/L,5-羟甲基糠醛浓度大于12.8 g/L时,菌株生长完全受到抑制。在最高耐受浓度下,糠醛的存在使菌株生物量比对照菌株下降77.8%,丁二酸产量下降36.1%。5-羟甲基糠醛的存在使菌株生物量比对照菌株降低13.6%,丁二酸产量降低18.3%。糠醛和5-羟甲基糠醛具有明显的协同作用。体外酶活测定表明丁二酸生产途径中关键酶磷酸烯醇式丙酮酸羧化酶、苹果酸脱氢酶、富马酸还原酶均受糠醛和5-羟甲基糠醛抑制。研究结果对丁二酸生产用纤维素水解液的预处理和脱毒工艺开发具有指导作用,有利于实现丁二酸发酵生产的工业化。     

Physiological effects of 5-hydroxymethylfurfural on Saccharomyces cerevisiae

 The physiological effects of 5-hydroxymethylfurfural (HMF) on Saccharomyces cerevisiae CBS 8066 in the presence and absence of furfural were studied. Experiments were carried out by pulse addition of HMF (2–4 g/l) as well as HMF (2 g/l) together with furfural (2 g/l) to batch cultivations of S. cerevisiae. Synthetic medium with glucose (50 g/l) as carbon and energy source was used. Addition of 4 g/l of HMF caused a decrease (approx. 32%) in the carbon dioxide evolution rate. Furthermore, the HMF was found to be taken up and converted by the yeast with a specific uptake rate of 0.14 (±0.03) g/g · h during both aerobic and anaerobic conditions, and the main conversion product was found to be 5-hydroxymethylfurfuryl alcohol. A previously unreported compound was found and characterized by mass spectrometry. It is suggested that the compound is formed from pyruvate and HMF in a reaction possibly catalysed by pyruvate decarboxylase. When HMF was added together with furfural, very little conversion of HMF took place until all of the furfural had been converted. Furthermore, the conversion rates of both furfural and HMF were lower than when added separately and growth was completely inhibited as long as both furfural and HMF were present in the medium. Received: 16 December 1998 / Received revision: 30 November 1999 / Accepted: 19 December 1999     

Biotransformation of 5‐Hydroxymethylfurfural to 2,5‐Furandicarboxylic Acid by a Syntrophic Consortium of Engineered Synechococcus elongatus and Pseudomonas putida

2,5‐furandicarboxylic acid (FDCA) is one of the top platform chemicals that can be produced from biomass feedstock. To make the cost of industrial FDCA production compatible with plastics made from fossils, the price of substrates and process complexity should be reduced. The aim of this research is to create a CO₂‐driven syntrophic consortium for the catalytic conversion of renewable biomass‐derived 5‐hydroxymethylfurfural (HMF) to FDCA. Sucrose produced from carbon fixation by the engineered Synechococcus elongatus serves as the sole carbon source for the engineered Pseudomonas putida to catalyze the reaction of HMF to FDCA. The yield of FDCA by the consortium reaches around 70% while the conversion of HMF is close to 100%. With further surface engineering to clump the two strains, the FDCA yield is elevated to almost 100% via the specific association between an Src homology 3 (SH3) domain and its ligand. The syntrophic consortium successfully demonstrates its green and cost‐effective characteristics for the conversion of CO₂ and biomass into platform chemicals.     

Catalytic hydrolysis of lignocellulosic biomass into 5-hydroxymethylfurfural in ionic liquid

Production of 5-hydroxymethylfurfural (HMF) from cellulose catalyzed by solid acids and metal chlorides was studied in the 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) under microwave irradiation. Among the applied catalysts, the use of CrCl₃/LiCl resulted in the highest yield of HMF. The effects of catalyst dosage (mole ratio of catalyst to glucose units in the feedstock) and reaction temperature on HMF yields were investigated to obtain optimal process conditions. With the 1:1 mol ratio of catalyst to glucose unit, the HMF yield reached 62.3% at 160 °C for 10 min. Untreated wheat straw was also investigated as feedstock to produce HMF for the practical use of raw biomass, in which the HMF yield was comparable to that from pure cellulose. After the extraction of HMF, [BMIM]Cl and CrCl₃/LiCl could be reused and exhibited no activity loss after three successive runs.     

Efficient and selective conversion of sucrose to 5-hydroxymethylfurfural promoted by ammonium halides under mild conditions

The highly efficient and selective production of 5-hydroxymethylfurfural (HMF) from sucrose has been achieved in the presence of metal chlorides and ammonium halides under mild conditions. Notably, an 87% yield of HMF from sucrose was obtained with a catalyst system composed of CrCl(3) and NH(4)Br at 100°C for 1.0 h in N,N-dimethylacetamide (DMAc) solvent. The effect of the reaction temperature and time was investigated in detail, and a possible mechanism for this catalytic process has been proposed. In addition, NH(4)Br is an effective promoter in the conversion of glucose and fructose to HMF.     

Conversion of hexose into 5-hydroxymethylfurfural in imidazolium ionic liquids with and without a catalyst

Conversion of fructose and glucose into 5-hydroxymethylfurfural (HMF) was investigated in various imidazolium ionic liquids, including 1-butyl-3-methylimidazolium chloride (BmimCl), 1-hexyl-3-methylimidazolium chloride (HmimCl), 1-octyl-3-methylimidazolium chloride (OmimCl), 1-benzyl-3-methylimidazolium chloride (BemimCl), 1-Butyl-2,3-dimethylimidazolium chloride (BdmimCl), and 1-butyl-3-methylimidazolium p-toluenesulfonate (BmimPS). The acidic C-2 hydrogen of imidazolium cations was shown to play a major role in the dehydration of fructose in the absence of a catalyst, such as sulfuric acid or CrCl₃. Both the alkyl groups of imidazolium cations and the type of anions affected the reactivity of the carbohydrates. Although, except BmimCl and BemimCl, other four ionic liquids could only achieve not more than 25% HMF yields without an additional catalyst, 60–80% HMF yields were achieved in HmimCl, BdmimCl, and BmimPS in the presence of sulfuric acid or CrCl₃ in sufficient quantities.     

Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural

An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerevisiae, has been shown to mitigate the toxicity of FFA and HMF by catalyzing the NADPH-dependent conversion to corresponding alcohols, furfuryl alcohol (FFOH) and 5-hydroxymethylfurfuryl alcohol (HMFOH). At pH 7.0 and 25°C, purified Ari1p catalyzes the NADPH-dependent reduction of substrates with the following values (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFA (23.3, 1.82, 12.8), HMF (4.08, 0.173, 23.6), and dl-glyceraldehyde (2.40, 0.0650, 37.0). When acting on HMF and dl-glyceraldehyde, the enzyme operates through an equilibrium ordered kinetic mechanism. In the physiological direction of the reaction, NADPH binds first and NADP(+) dissociates from the enzyme last, demonstrated by k(cat) of HMF and dl-glyceraldehyde that are independent of [NADPH] and (K(ia)(NADPH)/k(cat)) that extrapolate to zero at saturating HMF or dl-glyceraldehyde concentration. Microscopic kinetic parameters were determined for the HMF reaction (HMF+NADPH?HMFOH+NADP(+)), by applying steady-state, presteady-state, kinetic isotope effects, and dynamic modeling methods. Release of products, HMFOH and NADP(+), is 84% rate limiting to k(cat) in the forward direction. Equilibrium constants, [NADP(+)][FFOH]/[NADPH][FFA][H(+)]=5600×10(7)M(-1) and [NADP(+)][HMFOH]/[NADPH][HMF][H(+)]=4200×10(7)M(-1), favor the physiological direction mirrored by the slowness of hydride transfer in the non-physiological direction, NADP(+)-dependent oxidation of alcohols (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFOH (0.221, 0.00158, 140) and HMFOH (0.0105, 0.000104, 101).     

Production of 5-hydroxymethylfurfural from glucose catalyzed by hydroxyapatite supported chromium chloride

Production of 5-hydroxymethylfurfural (HMF) from glucose was studied in ionic liquids in the presence of hydroxyapatite supported chromium chloride (Cr-HAP) using oil-bath heating and microwave irradiation (MI). Compared with oil-bath heating, the MI way obviously increased HMF yield and reduced the reaction time from days to several minutes. A maximum HMF yield of 40% was obtained from the dehydration of glucose under MI in 2.5 min. This method is potential as an energy-efficient and cost-effective approach for the conversion of biomass into platform chemicals.     

Effect of Formic Acid on Conversion of Fructose to 5-Hydroxymethylfurfural in Aqueous/Butanol Media

Acid-catalyzed dehydration of carbohydrates into 5-hydroxymethylfurfural (HMF), a valuable biomass-derived intermediate, has received increasing attention. Efficient methods for HMF production are needed for successful commercialization of HMF in the near future. A new process for the dehydration of sugars into 5-hydroxymethylfurfural in aqueous/butanol media enhanced by using formic acid was developed. The effects of formic acid concentration, reaction temperature, and reaction time on the fructose conversion and HMF yield showed the significant influences of these process variables. The optimum conditions were found to be 2.5?mol/L formic acid concentration, 170°C and 70?min. Under such conditions, a fructose conversion of 98.3% with a HMF yield of 69.2% was achieved. The application of the butanol solvent and formic acid led to the conversion of fructose to HMF with high yield. The catalytic system in this study has prospects for commercial application due to its less corrosion and convenient downstream separation.     

Nutrient regulation of bacterial growth and production of anti-tumor metabolites in <Emphasis Type="Italic">Stigmatella</Emphasis> WXNXJ-B fermentation

The metabolites produced by Stigmatella WXNXJ-B inhibited the growth of tumor cells. The aims of this research were to evaluate the inhibition potency to different tumor cell lines and to study the effects of ammonium, phosphate and iron salts on bacterial growth and production of bioactive metabolites in Stigmatella WXNXJ-B fermentation. The results showed that the chloroform extract (CE-ME) showed the strongest growth inhibition bioactivity on mouse melanoma cell line (B16), murine colon carcinoma cell line (CT-26), human liver carcinoma cell line (HepG2) and human breast cancer cell line (MDA-MB231) in vitro and the IC₅₀ values were 9.94, 7.33, 11.34 and 11.66 μg ml⁻¹ respectively. The IC₅₀ value was above 700 μg ml⁻¹ on normal mouse spleen cells. Morphology happened changes in B16 cells treated with CE-ME. The anti-tumor metabolites were mainly produced during the stationary phase of the bacterial growth. Cell growth was stimulated at the phosphate concentration below 5 mM, but it was inhibited partly with 10 mM phosphate. The production of bioactive substances was inhibited by the phosphate. Ammonium increased the cell growth by 250% at 5 mM addition. The inhibition rate to B16 cells was increased to 89% at the concentration of 40 mM ammonium. The bacteria showed the best growth with 4 mM iron. Iron had little effect on the production at 2 mM, but bigger inhibition effect at higher iron concentration.     

Dehydration of fructose to 5-hydroxymethylfurfural by rare earth metal trifluoromethanesulfonates in organic solvents

The catalytic dehydration of fructose to 5-hydroxymethylfurfural (HMF) was investigated by using various rare earth metal trifluoromethanesulfonates, that is, Yb(OTf)₃, Sc(OTf)₃, Ho(OTf)₃, Sm(OTf)₃, Nd(OTf)₃ as catalysts in DMSO. It is found that the catalytic activity increases with decreasing ionic radius of rare earth metal cations. Among the examined catalysts, Sc(OTf)₃ exhibits the highest catalytic activity. Fructose conversion of 100% and a HMF yield of 83.3% are obtained at 120 °C after 2 h by using Sc(OTf)₃ as the catalyst. Moreover, the catalytic dehydration of fructose was also carried out in different solvents, for example, DMA, 1,4-dioxane, and a mixture of PEG-400 and water. The results show that among the solvents DMSO is the most efficient in promoting the dehydration of fructose to HMF, and no rehydration byproducts such as levulinic acid and formic acid are detected.     

Microwave-assisted conversion of lignocellulosic biomass into furans in ionic liquid

Production of 5-hydroxymethylfurfural (HMF) and furfural from lignocellulosic biomass was studied in ionic liquid in the presence of CrCl₃ under microwave irradiation. Corn stalk, rice straw and pine wood treated under typical reaction conditions produced HMF and furfural in yields of 45–52% and 23–31%, respectively, within 3 min. This method should be valuable to facilitate energy-efficient and cost-effective conversion of biomass into biofuels and platform chemicals.