Confirmation and elimination of xylose metabolism bottlenecks in glucose phosphoenolpyruvate-dependent phosphotransferase system-deficient Clostridium acetobutylicum for simultaneous utilization of glucose, xylose, and arabinose

Efficient cofermentation of D-glucose, D-xylose, and L-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacterium Clostridium acetobutylicum, known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predicted glcG gene, encoding enzyme II of the D-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strain Clostridium acetobutylicum ATCC 824, resulting in greatly improved D-xylose and L-arabinose consumption in the presence of D-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermented D-glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result of glcG disruption. Furthermore, the inherent rate-limiting steps of the D-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of the D-xylose proton-symporter (cac1345), D-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integrating glcG disruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures of D-glucose, D-xylose, and L-arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels.     

Simultaneous bioconversion of glucose and xylose to ethanol by Saccharomyces cerevisiae in the presence of xylose isomerase

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of a glucose/xylose mixture was carried out by Saccharomyces cerevisiae in the presence of xylose isomerase. The SIF of 50 g l⁻¹ xylose gave an ethanol concentration and metabolic yield of 7.5 g l⁻¹ and 0.36 g (g xylose consumed)⁻¹. These parameters improved to 13.4 g l⁻¹ and 0.40 respectively, when borate was added to the medium. The SICF of a mixture of 50 g l⁻¹ glucose and 50 g l⁻¹ xylose gave an ethanol concentration and metabolic yield of 29.8 g l⁻¹ and 0.42 respectively, in the presence of borate. Temperature modulation from 30 °C to 35 °C during fermentation further enhanced the above parameters to 39 g l⁻¹ and 0.45 respectively. The approach was extended to the bioconversion of sugars present in a real lignocellulose hydrolysate (peanut-shell hydrolysate) to ethanol, with a fairly good yield. Received: 14 May 1999 / Received revision: 27 September 1999 / Accepted: 2 October 1999     

Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.     

Continuous production of ethanol from a glucose,xylose and arabinose mixture by a flocculent strain ofPichia stipitis

Summary Enhanced rates of continuous ethanol production by a flocculent strain ofPichia stipitis from a sugar mixture (xylose 75%, glucose 20%, arabinose 5%) were attained using a single-stage gas lift tower fermentor. With a substrate feed of 50g/l, the biomass accumulated at a level near 50g/l, showed a maximum and stable ethanol productivity of 10.7 g/l.h, with a substrate conversion of 80%; the ethanol yield reached 0.41g/g. In these operating conditions, similar performances were obtained when D.xylose alone was supplied.     

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

Background

Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose.

Results

The specific aerobic arabinose growth rate was identical, 0.03 h^-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h^-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)^-1 h^-1 than the xylose isomerase strain, which only reached 0.03 h^-1 and 0.02 g (g cells)^-1h^-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g^-1 compared with 0.18 g g^-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g^-1 compared with 0.32 g g^-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l^-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l^-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)^-1 h^-1 compared with 0.01 g (g cells)^-1 h^-1 for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively.

Conclusion

The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.     

Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli.

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.     

Re-engineering Escherichia coli KJ122 to enhance the utilization of xylose and xylose/glucose mixture for efficient succinate production in mineral salt medium

Applied Microbiology and Biotechnology - Escherichia coli KJ122 was previously engineered to produce high concentration and yield of succinate in mineral salt medium containing glucose and sucrose...     

Control of the sequential utilization of glucose and fructose by Escherichia coli.

In Escherichia coli (ATCCI5224; ML308), glucose and fructose phosphotransferase systems (PT-systems) are constitutive but activities are increased five and 10-fold respectively by aerobic growth on their respective substrates in defined media. In mixtures, glucose is used preferentially and the fructose PT-system activity is kept at its minimum; but, on glucose exhaustion, it overshoots its steady-state level and growth continues on fructose without lag. Cyclic AMP prevents overshoot. Continuous cultures operating as turbidostats on mixtures of glucose and fructose do not use fructose if sufficient glucose is present to support growth. If less glucose is available, it is all used and sufficient fructose is metabolized concurrently to maintain the growth rate characteristic of glucose. Both PT-systems are inhibited by hexose phosphates. Presence of homologous substrate specifically sensitizes each PT-system to inactivation by N-ethylmaleimide (NEM). Glucose diminishes the ability of fructose to sensitize its PT-system to NEM. This effect parallels the inhibition of fructose utilization by glucose and suggests that glucose denies fructose access to the fructose-specific part of the PT-system.     

Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations

The growth characteristics and acetate production of several Escherichia coli strains were compared by using shake flasks, batch fermentations, and glucose-feedback-controlled fed-batch fermentations to assess the potential of each strain to grow at high cell densities. Of the E. coli strains tested, including JM105, B, W3110, W3100, HB101, DH1, CSH50, MC1060, JRG1046, and JRG1061, strains JM105 and B were found to have the greatest relative biomass accumulation, strain MC1060 accumulated the highest concentrations of acetic acid, and strain B had the highest growth rates under the conditions tested. In glucose-feedback-controlled fed-batch fermentations, strains B and JM105 produced only 2 g of acetate.liter-1 while accumulating up to 30 g of biomass.liter-1. Under identical conditions, strains HB101 and MC1060 accumulated less than 10 g of biomass.liter-1 and strain MC1060 produced 8 g of acetate.liter-1. The addition of various concentrations of sodium acetate to the growth medium resulted in a logarithmic decrease, with respect to acetate concentration, in the growth rates of E. coli JM105, JM105(pOS4201), and JRG1061. These data indicated that the growth of the E. coli strains was likely to be inhibited by the acetate they produced when grown on media containing glucose. A model for the inhibition of growth of E. coli by acetate was derived from these experiments to explain the inhibition of acetate on E. coli strains at neutral pH.     

High level expression of a thermostable Bacillus xylose (glucose) isomerase in Escherichia coli

Summary A thermophilic Bacillus sp. producing xylose (glucose) isomerase has been isolated. Its xy/A gene when cloned in Escherichia coli and expressed gave 37.5 and 12.8 units/ mg protein respectively for xylose and glucose isomerase activities at 85°C. A single heat treatment of the crude extract purified the enzyme further yielding the highest ever recorded activities of 150 and 49.02 units /mg protein.     

Simultaneous uptake and utilisation of glucose and xylose by Clostridium thermohydrosulfuricum

Abstract Growth studies of Clostridium thermohydrosulfuricum Rt8.B1 demonstrated that glucose and xylose were used simultaneously when supplied together at nonlimiting concentrations in pH-controlled batch culture. Under conditions of hyperbolic growth, both catabolite repression and inducer exclusion were absent. Glucose did not repress xylose metabolism (i.e. xylose permease and xylose isomerase genes were expressed in the presence of glucose and were not subject to catabolite inhibition when glucose was added to cultures growing on high concentrations of xylose). The kinetics of glucose and xylose utilisation indicated that separate systems were present for the uptake of these substrates when supplied together. Glucose utilisation was biphasic, indicating high- and low-affinity systems for glucose uptake. Xylose utilisation was directly proportional to the xylose concentration, suggesting a facilitated diffusion mechanism was operative for uptake.     

Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations.

The growth characteristics and acetate production of several Escherichia coli strains were compared by using shake flasks, batch fermentations, and glucose-feedback-controlled fed-batch fermentations to assess the potential of each strain to grow at high cell densities. Of the E. coli strains tested, including JM105, B, W3110, W3100, HB101, DH1, CSH50, MC1060, JRG1046, and JRG1061, strains JM105 and B were found to have the greatest relative biomass accumulation, strain MC1060 accumulated the highest concentrations of acetic acid, and strain B had the highest growth rates under the conditions tested. In glucose-feedback-controlled fed-batch fermentations, strains B and JM105 produced only 2 g of acetate.liter-1 while accumulating up to 30 g of biomass.liter-1. Under identical conditions, strains HB101 and MC1060 accumulated less than 10 g of biomass.liter-1 and strain MC1060 produced 8 g of acetate.liter-1. The addition of various concentrations of sodium acetate to the growth medium resulted in a logarithmic decrease, with respect to acetate concentration, in the growth rates of E. coli JM105, JM105(pOS4201), and JRG1061. These data indicated that the growth of the E. coli strains was likely to be inhibited by the acetate they produced when grown on media containing glucose. A model for the inhibition of growth of E. coli by acetate was derived from these experiments to explain the inhibition of acetate on E. coli strains at neutral pH.     

Construction of Escherichia coli strains producing L-serine from glucose

L-Serine is usually produced from glycine. We have genetically engineered Escherichia coli to produce L-serine from glucose intracellularly. D-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) in E. coli catalyzes the first committed step in L-serine formation but is inhibited by L-serine. To overcome this feedback inhibition, both the His(344) and Asn(346) residues of PGDH were converted to alanine and the mutated PGDH (PGDH(dr)) became insensitive to L-serine. However, overexpression of PGDH(dr) gave no significant increase of L-serine accumulation but, when L-serine deaminase genes (sdaA, sdaB and tdcG) were deleted, serine accumulated: (1) deletion of sdaA gave up to 0.03 mmol L-serine/g; (2) deletion of both sdaA and sdaB accumulated L-serine up to 0.09 mmol/g; and (3) deletion of sdaA, sdaB and tdcG gave up to 0.13 mmol L-serine/g cell dry wt.     

Evidence of a quinoprotein glucose dehydrogenase apoenzyme in several strains of Escherichia coli

Abstract When grown on glucose in K⁺-limited chemostat culture, or in batch culture with or without 2,4-dinitrophenol, several strains of Escherichia coli (including the type strain) were found to synthesize a quinoprotein glucose dehydrogenase apoenzyme. The pyridine nucleotides, NAD⁺ and NADP⁺, would not serve as cofactor, but activity could be demonstrated upon addition of 2,7,9-tricarboxy-1 H -pyrrolo(2,3- f )quinoline-4,5-dione (PQQ). Thus, in the presence of PQQ, but not in its absence, glucose was oxidized to gluconic acid. A mutant of E. coli PC 1000 was isolated that lacked Enzyme I of the phospho enol pyruvate phosphotransferase system (PTS) but still synthesized the glucose dehydrogenase apoenzyme. Whereas this mutant would not grow on glucose in the absence of PQQ, it would do so in the presence of low concentrations (1 μM) of this cofactor. On the basis of these observations, it is concluded that the protein (apoenzyme) formed is a genuine glucose dehydrogenase, but that it is not functional in growing cells due to their inability to synthesize the appropriate cofactor (PQQ), at least under these conditions.     

Simultaneous utilization of glucose and D-xylose by Candida shehatae in a chemostat

The kinetics of biomass formation, D-xylose utilization, and mixed substrate utilization were determined in a chemostat using the yeast Candida shehatae. The maximum growth rate of C. shehatae grown aerobically on D-xylose was 0.42 h⁻¹ and the Monod constant, K _s, was 0.06 g L⁻¹. The biomass yield, Y _{X/S}, ranged from 0.40 to 0.50 g g⁻¹ over a dilution rate range of 0.2–0.3 h⁻¹, when C. shehatae was grown on pure D-xylose. Mixtures of D-xylose and glucose (∼1 : 1) were simultaneously utilized over a dilution rate from 0.15 to 0.35 h⁻¹ at pH 3.5 and 4.5, but pH 3.5 reduced μ_max and reduced the dilution rate range over which D-xylose was utilized in the presence of glucose. At pH 4.5, μ_max was not reduced with the mixed sugar feed and the overall or lumped K _s value was not significantly increased (0.058 g L⁻¹ vs 0.06 g L⁻¹), when compared to a pure D-xylose feed. Kinetic data indicate that C. shehatae is an excellent candidate for chemostat production of value added products from renewable carbon sources, since simultaneous mixed substrate utilization was observed over a wide range of growth rates on a 1 : 1 mixture of glucose and D-xylose. Received 21 August 1997/ Accepted in revised form 28 May 1998