Metabolic engineering of bacteria for ethanol production

Technologies are available which will allow the conversion of lignocellulose into fuel ethanol using genetically engineered bacteria. Assembling these into a cost-effective process remains a challenge. Our work has focused primarily on the genetic engineering of enteric bacteria using a portable ethanol production pathway. Genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase have been integrated into the chromosome of Escherichia coli B to produce strain KO11 for the fermentation of hemicellulose-derived syrups. This organism can efficiently ferment all hexose and pentose sugars present in the polymers of hemicellulose. Klebsiella oxytoca M5A1 has been genetically engineered in a similar manner to produce strain P2 for ethanol production from cellulose. This organism has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. The optimal pH for cellulose fermentation with this organism (pH 5.0-5.5) is near that of fungal cellulases. The general approach for the genetic engineering of new biocatalysts has been most successful with enteric bacteria thus far. However, this approach may also prove useful with Gram-positive bacteria which have other important traits for lignocellulose conversion. Many opportunities remain for further improvements in the biomass to ethanol processes. These include the development of enzyme-based systems which eliminate the need for dilute acid hydrolysis or other pretreatments, improvements in existing pretreatments for enzymatic hydrolysis, process improvements to increase the effective use of cellulase and hemicellulase enzymes, improvements in rates of ethanol production, decreased nutrient costs, increases in ethanol concentrations achieved in biomass beers, increased resistance of the biocatalysts to lignocellulosic-derived toxins, etc. To be useful, each of these improvements must result in a decrease in the cost for ethanol production. Copyright 1998 John Wiley & Sons, Inc.     

Enteric bacterial catalysts for fuel ethanol production.

The technology is available to produce fuel ethanol from renewable lignocellulosic biomass. The current challenge is to assemble the various process options into a commercial venture and begin the task of incremental improvement. Current process designs for lignocellulose are far more complex than grain to ethanol processes. This complexity results in part from the complexity of the substrate and the biological limitations of the catalyst. Our work at the University of Florida has focused primarily on the genetic engineering of Enteric bacteria using genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase. These two genes have been assembled into a portable ethanol production cassette, the PET operon, and integrated into the chromosome of Escherichia coli B for use with hemicellulose-derived syrups. The resulting strain, KO11, produces ethanol efficiently from all hexose and pentose sugars present in the polymers of hemicellulose. By using the same approach, we integrated the PET operon into the chromosome of Klebsiella oxytoca to produce strain P2 for use in the simultaneous saccharification and fermentation (SSF) process for cellulose. Strain P2 has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. Recently, the ability to produce and secrete high levels of endoglucanase has also been added to strain P2, further reducing the requirement for fungal cellulase. The general approach for the genetic engineering of new biocatalysts using the PET operon has been most successful with Enteric bacteria but was also extended to Gram positive bacteria, which have other useful traits for lignocellulose conversion. Many opportunities remain for further improvements in these biocatalysts as we proceed toward the development of single organisms that can be used for the efficient fermentation of both hemicellulosic and cellulosic substrates.     

Performance of a newly developed integrant of Zymomonas mobilis for ethanol production on corn stover hydrolysate

Efficient conversion of lignocellulosic biomass requires biocatalysts able to tolerate inhibitors produced by many pretreatment processes. Recombinant Zymomonas mobilis 8b, a recently developed integrant of Zymomonas mobilis 31821(pZB5), tolerated acetic acid up to 16 g l(-1) and achieved 82%-87% (w/w) ethanol yields from pure glucose/xylose solutions at pH 6 and temperatures of 30 degrees C and 37 degrees C. An ethanol yield of 85% (w/w) was achieved on glucose/xylose from hydrolysate produced by dilute sulfuric acid pretreatment of corn stover after an overliming' process was used to improve hydrolysate fermentability.     

Extractive Synthesis of Ethyl‐Oleate Using Alginate Gel Co‐Entrapped Yeast Cells and Lipase Enzyme

To synthesize ethyl‐oleate ester, a complex Ca‐alginate gel co‐entrapped system was prepared. The gel beads contained two kinds of biocatalysts (living yeast cells and a lipase enzyme) and various amounts of glucose (100–400 g/L). These alginate beads dispersed directly in pure oleic acid. To follow the bioconversion of the cell growth, the glucose uptake of yeast cells, the concentration of ethanol inside the gel beads and the ethyl‐oleate concentration in oleic acid phase was monitored. The glucose was quantitatively taken up by yeast cells during 24–72 h, depending on the concentration of glucose. After this 24–72‐hour period, the glucose uptake was stopped. In accordance with changes in glucose concentration, the concentration of ethanol and ethyl‐oleate increased rapidly during the first day of fermentation and thereafter slowed down. It is supposed that the inhibitory effect of produced ethanol would be resolved by co‐immobilization of lipase in the same gel particles. Using lipase, one is able to transform ethanol to ethyl‐oleate, which is soluble in oleic acid. According to the data obtained a minimum of 4 U/mL lipase is required to increase ethyl‐oleate production significantly. Summing up it can be concluded that by means of this system a maximum yield of ethanol and ethyl‐oleate was achieved when gel beads containing 100 g/L glucose and 4 U/mL lipase enzyme were used.     

生物催化剂发现与改造的研究进展

生物催化是指将酶或生物有机体用于有用的化学转化的过程,在人们对传统化学催化的环境影响抱有忧虑的情况下,生物催化提供了一种有吸引力的选择。在过去的几十年里,对生物催化剂的研究每出现一次大的进步,生物催化的发展就会出现一次高潮。因此,生物催化剂的发现与改造已成为当今研究的热点。宏基因组文库技术的出现克服了许多微生物不可培养的障碍,人们能够从自然资源中获得丰富的潜在的生物催化剂。而基于理性设计的分子改造技术的发展,可以使得人们对潜在的生物催化剂进行快速而有效的改造以满足工业化生产的需求。随着生物催化剂发现与改造的手段不断进步,更多的优良生物催化剂得到了广泛的应用,生物催化在工业生产中也得到了更深入的应用。结合作者的研究工作,总结了生物催化剂发现与改良的一些研究进展,以为获得更多优良的、能够实现工业应用的生物催化剂奠定理论基础。     

Engineering ethanologenic Escherichia coli for levoglucosan utilization

Levoglucosan is a major product of biomass pyrolysis. While this pyrolyzed biomass, also known as bio-oil, contains sugars that are an attractive fermentation substrate, commonly-used biocatalysts, such as Escherichia coli, lack the ability to metabolize this anhydrosugar. It has previously been shown that recombinant expression of the levoglucosan kinase enzyme enables use of levoglucosan as carbon and energy source. Here, ethanologenic E. coli KO11 was engineered for levoglucosan utilization by recombinant expression of levoglucosan kinase from Lipomyces starkeyi. Our engineering strategy uses a codon-optimized gene that has been chromosomally integrated within the pyruvate to ethanol (PET) operon and does not require additional antibiotics or inducers. Not only does this engineered strain use levoglucosan as sole carbon source, but it also ferments levoglucosan to ethanol. This work demonstrates that existing biocatalysts can be easily modified for levoglucosan utilization.     

Investigation on macrokinetics of heterogeneous process of monosaccharide isomerization using non-growing cells of a glucoisomerase producer <Emphasis Type="Italic">Arthrobacter nicotianae</Emphasis> immobilized inside SiO<Subscript>2</Subscript>-xerogel

Macrokinetic peculiarites of heterogeneous process of monosaccharide (glucose/fructose) isomerization using biocatalysts prepared by incorporation of non-growing cells of a glucose isomerase-producing strain Arthobacter nicotianae inside SiO₂-xerogel have been investigated. It was shown that the process proceeds in kinetic regime without diffusion limitation and biocatalyst activities at 60 and 75°C were 19 and 32 U/g, respectively. Time of equilibrium in the reaction of monosaccharide isomerization was a function of starting (“triggering”) glucose isomerase activity in a unit of reaction volume. When the activity exceeds 10 U/ml, equilibrium equimolar mixture of glucose and fructose was produced within a few hours. It was established that a continuous process carried out in a plug-flow packed-bed reactor is more efficient than a batch process accompanied with recycling, first of all, to significant improvement of operation stability of the designed biocatalysts. Under model conditions of industrial heterogeneous process of producing glucose-fructose syrups, the half-life time of inactivation of the biocatalysts was more than 500 h at (65 ± 5)°C.     

Heme-iron oxygenases: powerful industrial biocatalysts?

Are cytochrome P450 enzymes powerful industrial biocatalysts? Next to market demands, well-defined enzyme functionalities and process parameters allow generalizations on the basis of process windows. These can provide useful guidelines for the design of improved biocatalysts. Oxygenase-catalyzed reactions are of special interest for selective C-H bond oxidation. The versatile class of cytochrome P450 mono-oxygenases attracts particular attention, and impressive advances have been achieved with respect to mechanistic insight, enzyme activity, stability, and specificity. Recent major achievements include significant increases in productivities, yields, and rates of catalytic turnover as well as modification of substrate specificity and efficient multistep reactions in whole-cell biocatalysts. For some biocatalysts, these parameters are already of an industrially useful magnitude.     

Extractive bioconversion in aqueous two-phase systems. A model study on the conversion of cellulose to ethanol

Summary Aqueous two-phase systems composed of dextran and poly (ethylene glycol) have been successfully used for glucose fermentation, cellulose hydrolysis and bioconversion of cellulose to ethanol. The biocatalysts are confined in the bottom phase whereas the products are extracted by the top phase.     

Effects of organic solvents on activity and stability of lipases produced by thermotolerant fungi in solid-state fermentation

Dried solid-state fermented solids (biocatalysts) produced by seven thermotolerant fungal strains were tested for lipase activity and stability in organic solvents. Two strains of Rhizopus sp. (19 and 43a) produced biocatalysts (L-19 and L-43a) that showed high lipase activities (74 and 72 U/g of dry matter, respectively) comparable to Lipozyme® RM IM (118 U/g DM). The use of the dipole moment of the organic solvents along with their classification based on the functional groups (non-polar, protic polar, aprotic polar) allowed the establishment of four different relative activity profiles for the seven biocatalysts evaluated. Compared to a biocatalyst not exposed to the organic solvent (100% relative activity), all biocatalysts showed a high relative activity (greater than 90%) in aprotic polar solvents (acetonitrile, acetone and ethyl acetate), whereas in protic polar solvents (ethanol and i-propanol) activity was reduced (lower than 40%). In addition, the incubation of biocatalysts L-19 and L-43a in i-amyl alcohol increased lipase activity in the synthesis of ethyl oleate 3.36 and 1.46 times, respectively. L-19 activity also increased after incubation in toluene (2.0 times), i-propanol (1.5 times) and acetonitrile (1.3 times) at temperatures from 30 to 50 °C. The results suggest that these biocatalysts can be used for a broad range of lipase reactions.     

Fermentations with new recombinant organisms.

United States fuel ethanol production in 1998 exceeded the record production of 1.4 billion gallons set in 1995. Most of this ethanol was produced from over 550 million bushels of corn. Expanding fuel ethanol production will require developing lower-cost feedstocks, and only lignocellulosic feedstocks are available in sufficient quantities to substitute for corn starch. Major technical hurdles to converting lignocellulose to ethanol include the lack of low-cost efficient enzymes for saccharification of biomass to fermentable sugars and the development of microorganisms for the fermentation of these mixed sugars. To date, the most successful research approaches to develop novel biocatalysts that will efficiently ferment mixed sugar syrups include isolation of novel yeasts that ferment xylose, genetic engineering of Escherichia coli and other gram negative bacteria for ethanol production, and genetic engineering of Saccharoymces cerevisiae and Zymomonas mobilis for pentose utilization. We have evaluated the fermentation of corn fiber hydrolyzates by the various strains developed. E. coli K011, E. coli SL40, E. coli FBR3, Zymomonas CP4 (pZB5), and Saccharomyces 1400 (pLNH32) fermented corn fiber hydrolyzates to ethanol in the range of 21-34 g/L with yields ranging from 0.41 to 0.50 g of ethanol per gram of sugar consumed. Progress with new recombinant microorganisms has been rapid and will continue with the eventual development of organisms suitable for commercial ethanol production. Each research approach holds considerable promise, with the possibility existing that different "industrially hardened" strains may find separate applications in the fermentation of specific feedstocks.     

Controlled pilot development unit-scale fed-batch cultivation of yeast on spruce hydrolysates

Yeast production on hydrolysate is a likely process solution in large-scale ethanol production from lignocellulose. The hydrolysate will be available on site, and the yeast has furthermore been shown to acquire an increased inhibitor tolerance when cultivated on hydrolysate. However, due to over-flow metabolism and inhibition, efficient yeast production on hydrolysate can only be achieved by well-controlled substrate addition. In the present work, a method was developed for controlled addition of hydrolysate to PDU (process development unit)-scale aerobic fed-batch cultivations of Saccharomyces cerevisiae TMB 3000. A feed rate control strategy, which maintains the ethanol concentration at a low constant level, was adapted to process-like conditions. The ethanol concentration was obtained from on-line measurements of the ethanol mole fraction in the exhaust gas. A computer model of the system was developed to optimize control performance. Productivities, biomass yields, and byproduct formation were evaluated. The feed rate control worked satisfactorily and maintained the ethanol concentration close to the setpoint during the cultivations. Biomass yields of 0.45 g/g were obtained on added hexoses during cultivation on hydrolysate and of 0.49 g/g during cultivation on a synthetic medium with glucose as the carbon source. Exponential growth was achieved with a specific growth rate of 0.18 h-1 during cultivation on hydrolysate and 0.22 h-1 during cultivation on glucose.     

Microbial conversion of sugars from plant biomass to lactic acid or ethanol

Concerns for our environment and unease with our dependence on foreign oil have renewed interest in converting plant biomass into fuels and 'green' chemicals. The volume of plant matter available makes lignocellulose conversion desirable, although no single isolated organism has been shown to depolymerize lignocellulose and efficiently metabolize the resulting sugars into a specific product. This work reviews selected chemicals and fuels that can be produced from microbial fermentation of plant-derived cell-wall sugars and directed engineering for improvement of microbial biocatalysts. Lactic acid and ethanol production are highlighted, with a focus on engineered Escherichia coli .     

Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids

Industrial microorganisms have been developed as biocatalysts to provide new or to optimize existing processes for the biotechnological production of chemicals from renewable plant biomass. Rational strain development by metabolic engineering is crucial to successful processes, and is based on efficient genetic tools and detailed knowledge of metabolic pathways and their regulation. This review summarizes recent advances in metabolic engineering of the industrial model bacteria Escherichia coli and Corynebacterium glutamicum that led to efficient recombinant biocatalysts for the production of acetate, pyruvate, ethanol, d- and l-lactate, succinate, l-lysine and l-serine.     

Ethanol production from xylose by Fusarium oxysporum and the optimization of culture conditions

Bioethanol is the most commonly used renewable biofuel as an alternative to fossil fuels. Many microbial strains can convert lignocellulosics into bioethanol. However, very few natural strains with a high capability of fermenting pentose sugars and simultaneously utilizing various sugars have been reported. In this study, fermentation of sugar by Fusarium oxysporum G was performed for the production of ethanol to improve the performance of the fermentation process. The influences of pH, substrate concentration, temperature, and rotation speed on ethanol fermentation are investigated. The three significant factors (pH, substrate concentration, and temperature) are further optimized by quadratic orthogonal rotation regression combination design and response surface methodology (RSM). The optimum conditions are pH 4, 40?g/L of xylose, 32?°C, and 110?rpm obtained through single factor experiment design. Finally, it is found that the maximum ethanol production (10.0?g/L) can be achieved after 7 d of fermentation under conditions of pH 3.87, 45.2?g/L of xylose, and 30.4?°C. Glucose is utilized preferentially for the glucose–xylose mixture during the initial fermentation stage, but glucose and xylose are synchronously consumed without preference in the second period. These findings are significant for the potential industrial application of this strain for bioethanol production.     

An enzyme coimmobilized with a microorganism: the conversion of cellobiose to ethanol using beta-glucosidase and Saccharomyces cerevisiae in calcium alginate gels

The efficiency of beta-glucosidase and Saccharomyces cerevisiae in directly converting cellobiose to ethanol was studied for various combinations of the two catalytic species, both free and immobilized, in order to elucidate the advantages of using a coimmobilized system. The coimmobilized preparation was superior to a combination of separately immobilized biocatalysts. However, in this preparation, one-half the enzyme activity was lost within a week when incubated at the operational temperature in the absence of substrate. In continuous experiments, an 80% conversion of cellobiose to ethanol was obtained using the coimmobilized preparation, compared to 40% using separately immobilized biocatalysts when applying a dilution rate of 0.1 h(-1) in a packedbed reactor. The immobilized biocatalysts showed no decline in productivity during two weeks of continuous operation.     

Bio-catalytic asymmetric synthesis of β-adrenergic receptor blocker precursor: (R)-2-bromo-1-(naphthalen-2-yl)ethanol

Abstract

Aromatic α-halohydrins, particularly 2-haloethanols as significant precursor of drugs, can easily be converted to chiral β-adrenergic receptor blockers. Eight strains of Lactobacillus curvatus were tested as biocatalysts for asymmetric reduction of 2-bromo-1-(naphthalen-2-yl)ethanone 1 to 2-bromo-1- (naphthalen-2-yl) ethanol 2. The parameters of the bioreduction were optimized using L. curvatus N4, the best biocatalyst found. As a result, (R)-2-bromo-1-(naphthalen-2-yl)ethanol 2, which can be β-adrenergic receptor blocker precursor, was produced for the first time in high yield and enantiomerically pure form using biocatalysts. Moreover, the gram scale synthesis was performed and 7.54?g of (R)-2 was synthesized as enantiopure form (enantiomeric excess >99%) in 48?h. The important advantages of this process are that it produces of (R)-2 for the first time in enantiopure form, in excellent yield and under environmentally friendly and moderate reaction conditions. This system is of the potential to be applied at a commercial scale.     

Towards industrial pentose-fermenting yeast strains

Production of bioethanol from forest and agricultural products requires a fermenting organism that converts all types of sugars in the raw material to ethanol in high yield and with a high rate. This review summarizes recent research aiming at developing industrial strains of Saccharomyces cerevisiae with the ability to ferment all lignocellulose-derived sugars. The properties required from the industrial yeast strains are discussed in relation to four benchmarks: (1) process water economy, (2) inhibitor tolerance, (3) ethanol yield, and (4) specific ethanol productivity. Of particular importance is the tolerance of the fermenting organism to fermentation inhibitors formed during fractionation/pretreatment and hydrolysis of the raw material, which necessitates the use of robust industrial strain background. While numerous metabolic engineering strategies have been developed in laboratory yeast strains, only a few approaches have been realized in industrial strains. The fermentation performance of the existing industrial pentose-fermenting S. cerevisiae strains in lignocellulose hydrolysate is reviewed. Ethanol yields of more than 0.4 g ethanol/g sugar have been achieved with several xylose-fermenting industrial strains such as TMB 3400, TMB 3006, and 424A(LNF-ST), carrying the heterologous xylose utilization pathway consisting of xylose reductase and xylitol dehydrogenase, which demonstrates the potential of pentose fermentation in improving lignocellulosic ethanol production.     

Monitoring and control of Gluconacetobacter xylinus fed-batch cultures using in situ mid-IR spectroscopy

A partial least-squares calibration model, relating mid-infrared spectral features with fructose, ethanol, acetate, gluconacetan, phosphate and ammonium concentrations has been designed to monitor and control cultivations of Gluconacetobacter xylinus and production of gluconacetan, a food grade exopolysaccharide (EPS). Only synthetic solutions containing a mixture of the major components of culture media have been used to calibrate the spectrometer. A factorial design has been applied to determine the composition and concentration in the calibration matrix. This approach guarantees a complete and intelligent scan of the calibration space using only 55 standards. This calibration model allowed standard errors of validation (SEV) for fructose, ethanol, acetate, gluconacetan, ammonium and phosphate concentrations of 1.16 g/l, 0.36 g/l, 0.22 g/l, 1.54 g/l, 0.24 g/l and 0.18 g/l, respectively. With G. xylinus, ethanol is directly oxidized to acetate, which is subsequently metabolized to form biomass. However, residual ethanol in the culture medium prevents bacterial growth. On-line spectroscopic data were implemented in a closed-loop control strategy for fed-batch fermentation. Acetate concentration was controlled at a constant value by feeding ethanol into the bioreactor. The designed fed-batch process allowed biomass production on ethanol. This was not possible in a batch process due to ethanol inhibition of bacterial growth. In this way, the productivity of gluconacetan was increased from 1.8 x 10(-3) [C-mol/C-mol substrate/h] in the batch process to 2.9 x 10(-3) [C-mol/C-mol substrate/h] in the fed-batch process described in this study.     

Immobilization of yeast cells on various supports for ethanol production

An immobilization technique has been developed for a packed bed fermenter which is being considered as one stage of a process for the production of fuel-grade ethanol from sugar solutions. Relatively inexpensive beech wood chips have been successfully used as the support material and relatively high cell loadings of 188 mg DW cells/g DW support have been achieved for a test system of Saccharomyces cerevisiae cultures.No washout of adsorbed cells occurs below a superficial liquid velocity of 8.9 × 10^-2 cm/s which can be increased to 9.7 × 10^-2 cm/s by including up to 1% Hercofloc solution in the reactor medium during the immobilization procedure. The immobilization procedure is practically unaffected by pH and temperature in the range 3.5 to 5.0 and 22 °C to 37 °C respectively.Typical ethanol productivity of 21.8g/l·hr has been obtained with wood-chip-adsorbed cells, which compares well with optimal values of 18 to 32g/l·hr obtained using free-suspension cultures in stirred-tank fermenters with cell recycle.