The challenge of enzyme cost in the production of lignocellulosic biofuels

With the aim of understanding the contribution of enzymes to the cost of lignocellulosic biofuels, we constructed a techno-economic model for the production of fungal cellulases. We found that the cost of producing enzymes was much higher than that commonly assumed in the literature. For example, the cost contribution of enzymes to ethanol produced by the conversion of corn stover was found to be $0.68/gal if the sugars in the biomass could be converted at maximum theoretical yields, and $1.47/gal if the yields were based on saccharification and fermentation yields that have been previously reported in the scientific literature. We performed a sensitivity analysis to study the effect of feedstock prices and fermentation times on the cost contribution of enzymes to ethanol price. We conclude that a significant effort is still required to lower the contribution of enzymes to biofuel production costs.     

Strategies for the production of cell wall‐deconstructing enzymes in lignocellulosic biomass and their utilization for biofuel production

Microbial cell wall‐deconstructing enzymes are widely used in the food, wine, pulp and paper, textile, and detergent industries and will be heavily utilized by cellulosic biorefineries in the production of fuels and chemicals. Due to their ability to use freely available solar energy, genetically engineered bioenergy crops provide an attractive alternative to microbial bioreactors for the production of cell wall‐deconstructing enzymes. This review article summarizes the efforts made within the last decade on the production of cell wall‐deconstructing enzymes in planta for use in the deconstruction of lignocellulosic biomass. A number of strategies have been employed to increase enzyme yields and limit negative impacts on plant growth and development including targeting heterologous enzymes into specific subcellular compartments using signal peptides, using tissue‐specific or inducible promoters to limit the expression of enzymes to certain portions of the plant or certain times, and fusion of amplification sequences upstream of the coding region to enhance expression. We also summarize methods that have been used to access and maintain activity of plant‐generated enzymes when used in conjunction with thermochemical pretreatments for the production of lignocellulosic biofuels.     

Selectivity in the binding of NAD(P)+ analogues to NAD- and NADP-dependent pig heart isocitrate dehydrogenases. A nuclear magnetic resonance study.

The coenzyme selectivity of pig heart NAD-dependent and NADP-dependent isocitrate dehydrogenase has been investigated by nuclear magnetic resonance through the use of coenzyme analogues. For both isocitrate dehydrogenases, more than 10-fold lower maximal activity is observed with thionicotinamide adenine dinucleotide [sNAD(P)+] than with NAD(P)+ or acetylpyridine adenine dinucleotide [acNAD-(P)+] as coenzyme. Nuclear Overhauser effect measurements failed to reveal any differences in the adenine-ribose conformations among the enzyme-bound analogues. The 2'-phosphate resonance of the enzyme-bound NADP+ analogues showed the same change in chemical shift observed for the natural coenzyme and revealed the same lack of pH dependence in the range from pH 5.4 to 8.2. NADP-dependent isocitrate dehydrogenase exhibits only small differences in Michaelis constants for the coenzymes with various nicotinamide substituents, reflecting a predominant role for the adenosine moiety in binding. The conformation of the bound nicotinamide-ribose of the natural coenzymes was appreciably different from that of the coenzyme, sNAD(P)+, which shows low catalytic activity. For both isocitrate dehydrogenases, sNAD(P)+ bound to the enzymes exhibits a mixture of syn and anti conformations while only the anti conformation can be detected for NAD(P)+. Chemical shifts of NAD(P)+ enriched with 13C in the carboxamide indicate that interaction of this group with the enzymes may play a role in positioning the nicotinamide ring to participate in catalysis. Our results suggest that, although interaction of the nicotinamide moiety with the enzymes contributes relatively little to the energy of interaction in the binary complex, the enzymes must correctly position this group for the catalytic event.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maßgeschneiderte Mikroorganismen

Tailor‐made microorganisms Microbial diversity provides unlimited resources for the development of novel industrial processes and products. Since the beginning of the 20th century microorganisms have been successfully applied for the large scale production of bio‐based products. In recent years, modern methods of strain development and Synthetic Biology have enabled biotech engineers to design even more sophisticated and tailor‐made microorganisms. These microbes serve industrial processes for the production of bulk chemicals, enzymes, polymers, biofuels as well as plant‐derived ingredients such as Artemisinin in an ecologically and economically sustainable and attractive fashion. In the future, production of advanced biofuels, microbial fuel cells, CO₂ as feedstock and microbial cellulose are research topics as well as challenges of global importance. Continuous efforts in microbiology and biotechnology research will be pivotal for white biotechnology to gain more momentum in transforming the chemical industry towards a knowledge based bio‐economy.     

Agave for tequila and biofuels: an economic assessment and potential opportunities

This paper explores the economic viability of producing biofuels from Agave in Mexico and the potential for it to complement the production of tequila or mescal. We focus on Agave varieties currently being used by the tequila industry to produce two beverages, tequila and mescal, and explore the potential for biofuel production from these plants. Without competing directly with beverage production, we discuss the economic costs and benefits of converting Agave by‐products to liquid fuel as an additional value‐added product and expanding cultivation of Agave on available land. We find that the feedstock cost for biofuel from the Agave piña alone could be more than US$3 L^?1 on average. This is considerably higher than the feedstock costs of corn ethanol and sugarcane ethanol. However, there may be potential to reduce these costs with higher conversion efficiencies or by using sugar present in other parts of the plant. The costs of cellulosic biofuels using the biomass from the entire plant could be lower depending on the conversion efficiency of biomass to fuel and the additional costs of harvesting, collecting and transporting that biomass.     

萜烯生物合成中关键酶的研究进展*

萜烯类化合物是一类高度多样化的天然产物,具有抗肿瘤、抗氧化及免疫调节等生理活性,因此被广泛应用于医药健康、食品、化妆品领域。然而,直接从自然资源中获取萜烯类化合物效率低、成本高,且往往对生态环境产生不利影响,不能实现绿色可持续生产。微生物合成萜烯类化合物近年来备受关注,研究人员从合成途径的构建与调控、关键酶的理性及半理性改造、发酵工艺优化等多个方面进行了探究,取得了丰硕的成果。其中,合成途径中关键酶的催化效率是影响微生物生产萜烯类化合物的重要因素。针对关键酶的研究对于提高微生物合成萜烯类化合物的能力,推动该类天然产物微生物生产的大规模应用具有重要意义。对萜烯类化合物合成途径中的3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)、1-脱氧-D-木酮糖-5-磷酸合酶(DXS)、异戊二烯基二磷酸合成酶(IDS)和萜烯合酶(TPS)4种关键酶的研究进行了综述,并总结讨论了如何通过代谢工程和蛋白质工程手段以及合成生物学技术调节关键酶的催化活性,提高微生物合成萜烯类化合物的效率,对未来利用微生物合成萜烯类化合物的发展进行了展望。     

Ligand binding and stabilization of malate- and lactate dehydrogenase

Binding of coenzymes, coenzyme fragments and phenolate ligands to malate- and lactate dehydrogenase was studied. From linear competition in titration experiments, the coenzyme binding site was concluded to bind all the ligands employed. The analogy between the phenolate ligands and tetraiodofluorescein which is known to bind at the adenosine binding site suggests binding of phenolates at this site. Coenzymes and coenzyme fragments retard the irreversible thermal inactivation of the enzymes. The retardation effect decreases in the order NADH greater than NAD greater than ADPR greater than or equal to AMP for both enzymes. Phenolate ligands binding to the adenosine pocket do not stabilize the enzymes. The stabilization is concluded to originate from the interaction of coenzyme phosphate and nicotinamide with the enzymes. The interactions with the adenosine moiety and with the second ribose seem to be ineffective in retardation of thermal denaturation.     

Validation of leaf enzymes in the detergent and textile industries: launching of a new platform technology

Chemical catalysts are being replaced by biocatalysts in almost all industrial applications due to environmental concerns, thereby increasing their demand. Enzymes used in current industries are produced in microbial systems or plant seeds. We report here five newly launched leaf‐enzyme products and their validation with 15 commercial microbial‐enzyme products, for detergent or textile industries. Enzymes expressed in chloroplasts are functional at broad pH/temperature ranges as crude‐leaf extracts, while most purified commercial enzymes showed significant loss at alkaline pH or higher temperature, required for broad range commercial applications. In contrast to commercial liquid enzymes requiring cold storage/transportation, chloroplast enzymes as a leaf powder can be stored up to 16 months at ambient temperature without loss of enzyme activity. Chloroplast‐derived enzymes are stable in crude‐leaf extracts without addition of protease inhibitors. Leaf lipase/mannanase crude extracts removed chocolate or mustard oil stains effectively at both low and high temperatures. Moreover, leaf lipase or mannanase crude‐extracts removed stain more efficiently at 70 °C than commercial microbial enzymes (<10% activity). Endoglucanase and exoglucanase in crude leaf extracts removed dye efficiently from denim surface and depilled knitted fabric by removal of horizontal fibre strands. Due to an increased demand for enzymes in the food industry, marker‐free lettuce plants expressing lipase or cellobiohydrolase were created for the first time and site‐specific transgene integration/homoplasmy was confirmed by Southern blots. Thus, leaf‐production platform offers a novel low‐cost approach by the elimination of fermentation, purification, concentration, formulation and cold‐chain storage/transportation. This is the first report of commercially launched protein products made in leaves and validated with current commercial products.     

Recombinant Bacillus subtilis That Grows on Untreated Plant Biomass

Lignocellulosic biomass is a promising feedstock to produce biofuels and other valuable biocommodities. A major obstacle to its commercialization is the high cost of degrading biomass into fermentable sugars, which is typically achieved using cellulolytic enzymes from Trichoderma reesei. Here, we explore the use of microbes to break down biomass. Bacillus subtilis was engineered to display a multicellulase-containing minicellulosome. The complex contains a miniscaffoldin protein that is covalently attached to the cell wall and three noncovalently associated cellulase enzymes derived from Clostridium cellulolyticum (Cel48F, Cel9E, and Cel5A). The minicellulosome spontaneously assembles, thus increasing the practicality of the cells. The recombinant bacteria are highly cellulolytic and grew in minimal medium containing industrially relevant forms of biomass as the primary nutrient source (corn stover, hatched straw, and switch grass). Notably, growth did not require dilute acid pretreatment of the biomass and the cells achieved densities approaching those of cells cultured with glucose. An analysis of the sugars released from acid-pretreated corn stover indicates that the cells have stable cellulolytic activity that enables them to break down 62.3% ± 2.6% of the biomass. When supplemented with beta-glucosidase, the cells liberated 21% and 33% of the total available glucose and xylose in the biomass, respectively. As the cells display only three types of enzymes, increasing the number of displayed enzymes should lead to even more potent cellulolytic microbes. This work has important implications for the efficient conversion of lignocellulose to value-added biocommodities.     

Physiology,Genomics, and Pathway Engineering of an Ethanol-Tolerant Strain of Clostridium phytofermentans

Novel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bacterium Clostridium phytofermentans was adapted to increased ethanol concentrations. The resulting ethanol-tolerant (ET) strain has nearly doubled ethanol tolerance relative to the wild-type level but also reduced ethanol yield and growth at low ethanol concentrations. The genome of the ET strain has coding changes in proteins involved in membrane biosynthesis, the Rnf complex, cation homeostasis, gene regulation, and ethanol production. In particular, purification of the mutant bifunctional acetaldehyde coenzyme A (CoA)/alcohol dehydrogenase showed that a G609D variant abolished its activities, including ethanol formation. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase in the ET strain increased cellulose consumption and restored ethanol production, demonstrating how metabolic engineering can be used to overcome disadvantageous mutations incurred during adaptation to ethanol. We discuss how genetic changes in the ET strain reveal novel potential strategies for improving microbial solvent tolerance.     

Purification and Characterization of Glucose 6‐Phosphate Dehydrogenase, 6‐Phosphogluconate Dehydrogenase,and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide,Digoxin, and Dopamine on the Enzymes Activities

The present study was aimed to investigate characterization and purification of glucose–6‐phosphate dehydrogenase, 6‐phosphogluconate dehydrogenase, and glutathione reductase from rat heart and the inhibitory effect of three drugs. The purification of the enzymes was performed using 2',5'‐ADP sepharose 4B affinity material. The subunit and the natural molecular weights were analyzed by SDS‐PAGE and gel filtration. Biochemical characteristics such as the optimum temperature, pH, stable pH, and salt concentration were examined for each enzyme. Types of product inhibition and K_i values with K_m and V_max values of the substrates and coenzymes were determined. According to the obtained K_i and IC₅₀ values, furosemide, digoxin, and dopamine showed inhibitory effect on the enzyme activities at low millimolar concentrations in vitro conditions. Dopamine inhibited the activity of these enzymes as competitive, whereas furosemide and digoxin inhibited the activity of the enzyme as noncompetitive.     

Engineering microbial factories for synthesis of value-added products

Microorganisms have become an increasingly important platform for the production of drugs, chemicals, and biofuels from renewable resources. Advances in protein engineering, metabolic engineering, and synthetic biology enable redesigning microbial cellular networks and fine-tuning physiological capabilities, thus generating industrially viable strains for the production of natural and unnatural value-added compounds. In this review, we describe the recent progress on engineering microbial factories for synthesis of valued-added products including alkaloids, terpenoids, flavonoids, polyketides, non-ribosomal peptides, biofuels, and chemicals. Related topics on lignocellulose degradation, sugar utilization, and microbial tolerance improvement will also be discussed.     

Interaction of folylpolyglutamates with enzymes in one-carbon metabolism

Of all the coenzymes, tetrahydrofolate exhibits the most structural diversity. The relationship of these structural forms to physiological function is under intense study by numerous research groups. In textbooks, tetrahydrofolate (tetrahydropteroylmonoglutamate) is shown as the coenzyme of one-carbon metabolism, but it has been known for several decades that the physiologically active forms of the coenzyme contain from 4 to 7 glutamyl residues linked by amide bonds through the gamma-carboxyl group. These glutamyl residues do not serve a direct function in transferring the one-carbon group. The tetrahydrofolylpolyglutamates were originally thought to be simply storage forms of the coenzyme, but studies now show that the polyglutamate chain of the coenzyme affects the transport properties of the coenzyme, alters the kinetic properties of many enzymes in one-carbon metabolism, and results in channeling of the coenzyme between several enzymes. In general, the dissociation constants of this group of enzymes for the tetrahydrofolylpolyglutamates are very low, in the 0.1 to 1 microM range. The concentration of the coenzyme in the cell appears to be similar to the concentration of folate-utilizing enzymes, suggesting that the concentration of unbound coenzyme in the cell may be very low. Several of the enzymes in one-carbon metabolism are either multifunctional proteins or multienzyme complexes. An active area of research is to determine if there is a functional relationship between these multifunctional enzymes and the polyglutamate portion of the coenzyme.     

芳香类天然产物的合成生物学研究进展

植物芳香类天然产物具有重要的药用价值,可制成具有抗菌、抗炎、镇痛、抗氧化、杀虫驱虫、祛痰止咳、安神镇静和抗肿瘤等药效的医药保健用品.然而,由于植物中芳香类天然产物含量较低并且难以提取和纯化,严重限制了其工业化生产及应用.合成生物学和代谢工程技术的发展为天然产物的生产提供了新的思路,可以利用人工微生物细胞工厂来实现多种芳...     

Molecularly Ordered Bioelectrocatalytic Composite Inside a Film of Aligned Carbon Nanotubes

Molecularly ordered composites of polyvinylimidazole‐[Os(bipyridine)₂Cl] (PVI‐[Os(bpy)₂Cl]) and glucose oxidase (GOD) are assembled inside a film of aligned carbon nanotubes. The structure of the prepared GOD/PVI‐[Os(bpy)₂Cl]/CNT composite film is entirely uniform and stable; more than 90% bioelectrocatalytic activity could be maintained even after storage for 6 d. Owing to the ideal positional relationship achieved between enzyme, mediator, and electrode, the prepared film shows a high bioelectrocatalytic activity for glucose oxidation (ca. 15 mA cm^?2 at 25 °C) with an extremely high electron‐transfer turnover rate (ca. 650 s^?1) comparable to the value for GOD solutions, indicating almost every enzyme molecule entrapped within the ensemble (ca. 3 × 10¹² enzymes in a 1 mm × 1 mm film) can work to the fullest extent. This free‐standing, flexible composite film can be used by winding on a needle device; as an example, a self‐powered sugar monitor is demonstrated.     

An ultraviolet resonance Raman study of dehydrogenase enzymes and their interactions with coenzymes and substrates

Ultraviolet resonance Raman (UVRR) spectra, with 260-nm excitation, are reported for oxidized and reduced nicotinamide adenine dinucleotides (NAD+ and NADH, respectively). Corresponding spectra are reported for these coenzymes when bound to the enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and liver and yeast alcohol dehydrogenases (LADH and YADH). The observed differences between the coenzyme spectra are interpreted in terms of conformation, hydrogen bonding, and general environment polarity differences between bound and free coenzymes and between coenzymes bound to different enzymes. The possibility of adenine protonation is discussed. UVRR spectra with 220-nm excitation also are reported for holo- and apo-GAPDH (GAPDH-NAD+ and GAPDH alone, respectively). In contrast with the 260-nm spectra, these show only bands due to vibrations of aromatic amino acid residues of the protein. The binding of coenzyme to GAPDH has no significant effect on the aromatic amino acid bands observed. This result is discussed in the light of the known structural change of GAPDH on binding coenzyme. Finally, UVRR spectra with 240-nm excitation are reported for GAPDH and an enzyme-substrate intermediate of GAPDH. Perturbations are reported for tyrosine and tryptophan bands on forming the acyl enzyme.     

Potential synergies and challenges in refining cellulosic biomass to fuels,chemicals, and power

Lignocellulosic biomass such as agricultural and forestry residues and dedicated crops provides a low-cost and uniquely sustainable resource for production of many organic fuels and chemicals that can reduce greenhouse gas emissions, enhance energy security, improve the economy, dispose of problematic solid wastes, and improve air quality. A technoeconomic analysis of biologically processing lignocellulosics to ethanol is adapted to project the cost of making sugar intermediates for producing a range of such products, and sugar costs are predicted to drop with plant size as a result of economies of scale that outweigh increased biomass transport costs for facilities processing less than about 10,000 dry tons per day. Criteria are then reviewed for identifying promising chemicals in addition to fuel ethanol to make from these low cost cellulosic sugars. It is found that the large market for ethanol makes it possible to achieve economies of scale that reduce sugar costs, and coproducing chemicals promises greater profit margins or lower production costs for a given return on investment. Additionally, power can be sold at low prices without a significant impact on the selling price of sugars. However, manufacture of multiple products introduces additional technical, marketing, risk, scale-up, and other challenges that must be considered in refining of lignocellulosics.     

Polysaccharide hydrolysis with engineered Escherichia coli for the production of biocommodities

Escherichia coli can ferment a broad range of sugars, including pentoses, hexoses, uronic acids, and polyols. These features make E. coli a suitable microorganism for the development of biocatalysts to be used in the production of biocommodities and biofuels by metabolic engineering. E. coli cannot directly ferment polysaccharides because it does not produce and secrete the necessary saccharolytic enzymes; however, there are many genetic tools that can be used to confer this ability on this prokaryote. The construction of saccharolytic E. coli strains will reduce costs and simplify the production process because the saccharification and fermentation can be conducted in a single reactor with a reduced concentration or absence of additional external saccharolytic enzymes. Recent advances in metabolic engineering, surface display, and excretion of hydrolytic enzymes provide a framework for developing E. coli strains for the so-called consolidated bioprocessing. This review presents the different strategies toward the development of E. coli strains that have the ability to display and secrete saccharolytic enzymes to hydrolyze different sugar-polymeric substrates and reduce the loading of saccharolytic enzymes.     

A systematic analysis of TCA Escherichia coli mutants reveals suitable genetic backgrounds for enhanced hydrogen and ethanol production using glycerol as main carbon source

Biodiesel has emerged as an environmentally friendly alternative to fossil fuels; however, the low price of glycerol feed‐stocks generated from the biodiesel industry has become a burden to this industry. A feasible alternative is the microbial biotransformation of waste glycerol to hydrogen and ethanol. Escherichia coli, a microorganism commonly used for metabolic engineering, is able to biotransform glycerol into these products. Nevertheless, the wild type strain yields can be improved by rewiring the carbon flux to the desired products by genetic engineering. Due to the importance of the central carbon metabolism in hydrogen and ethanol synthesis, E. coli single null mutant strains for enzymes of the TCA cycle and other related reactions were studied in this work. These strains were grown anaerobically in a glycerol‐based medium and the concentrations of ethanol, glycerol, succinate and hydrogen were analysed by HPLC and GC. It was found that the reductive branch is the more relevant pathway for the aim of this work, with malate playing a central role. It was also found that the putative C4‐transporter dcuD mutant improved the target product yields. These results will contribute to reveal novel metabolic engineering strategies for improving hydrogen and ethanol production by E. coli.     

The dilemma for lipid productivity in green microalgae: importance of substrate provision in improving oil yield without sacrificing growth

Rising oil prices and concerns over climate change have resulted in more emphasis on research into renewable biofuels from microalgae. Unlike plants, green microalgae have higher biomass productivity, will not compete with food and agriculture, and do not require fertile land for cultivation. However, microalgae biofuels currently suffer from high capital and operating costs due to low yields and costly extraction methods. Microalgae grown under optimal conditions produce large amounts of biomass but with low neutral lipid content, while microalgae grown in nutrient starvation accumulate high levels of neutral lipids but are slow growing. Producing lipids while maintaining high growth rates is vital for biofuel production because high biomass productivity increases yield per harvest volume while high lipid content decreases the cost of extraction per unit product. Therefore, there is a need for metabolic engineering of microalgae to constitutively produce high amounts of lipids without sacrificing growth. Substrate availability is a rate-limiting step in balancing growth and fatty acid (FA) production because both biomass and FA synthesis pathways compete for the same substrates, namely acetyl-CoA and NADPH. In this review, we discuss the efforts made for improving biofuel production in plants and microorganisms, the challenges faced in achieving lipid productivity, and the important role of precursor supply for FA synthesis. The main focus is placed on the enzymes which catalyzed the reactions supplying acetyl-CoA and NADPH.