Induction of aldose reductase and xylitol dehydrogenase activities in Candida tenuis CBS 4435

In this study the ability of various sugars and sugar alcohols to induce aldose reductase (xylose reductase) and xylitol dehydrogenase (xylulose reductase) activities in the yeast Candida tenuis was investigated. Both enzyme activities were induced when the organism was grown on d-xylose or l-arabinose as well as on the structurally related sugars d-arabinose or d-lyxose. Mixtures of d-xylose with the more rapidly metabolizable sugar d-glucose resulted in a decrease in the levels of both enzymes formed. These results show that the utilization of d-xylose by C. tenuis is regulated by induction and catabolite repression. Furthermore, the different patterns of induction on distinct sugars suggest that the synthesis of both enzymes is not under coordinate control.     

Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass

Lignocellulosic biomass is an attractive carbon source for bio-based fuel and chemical production; however, its compositional heterogeneity hinders its commercial use. Since most microbes possess carbon catabolite repression (CCR), mixed sugars derived from the lignocellulose are consumed sequentially, reducing the efficacy of the overall process. To overcome this barrier, microbes that exhibit the simultaneous consumption of mixed sugars have been isolated and/or developed and evaluated for the lignocellulosic biomass utilization. Specific strains of Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis have been engineered for simultaneous glucose and xylose utilization via mutagenesis or introduction of a xylose metabolic pathway. Other microbes, such as Lactobacillus brevis, Lactobacillus buchneri, and Candida shehatae possess a relaxed CCR mechanism, showing simultaneous consumption of glucose and xylose. By exploiting CCR-negative phenotypes, various integrated processes have been developed that incorporate both enzyme hydrolysis of lignocellulosic material and mixed sugar fermentation, thereby enabling greater productivity and fermentation efficacy.     

Ethanol production from pentoses and sugar-cane bagasse hemicellulose hydrolysate by Mucor and Fusarium species

The fermentation of carbohydrates and hemicellulose hydrolysate by Mucor and Fusarium species has been investigated, with the following results. Both Mucor and Fusarium species are able to ferment various sugars and alditols, including d-glucose, pentoses and xylitol, to ethanol. Mucor is able to ferment sugar-cane bagasse hemicellulose hydrolysate to ethanol. Fusarium F5 is not able to ferment sugar-cane bagasse hemicellulose hydrolysate to ethanol. During fermentation of hemicellulose hydrolysates, d-glucose was utilized first, followed by d-xylose and l-arabinose. Small amounts of xylitol were produced by Mucor from d-xylose through oxidoreduction reactions, presumably mediated by the enzyme aldose reductase¹ (alditol: NADP⁺ 1-oxidoreductase, EC 1.1.1.21). For pentose fermentation, d-xylose was the preferred substrate. Only small amounts of ethanol were produced from l-arabinose and d-arabitol. No ethanol was produced from l-xylose, d-arabinose or l-arabitol.     

Enhanced butanol production in Clostridium acetobutylicum ATCC 824 by double overexpression of 6-phosphofructokinase and pyruvate kinase genes

This study elucidated the importance of two critical enzymes in the regulation of butanol production in Clostridium acetobutylicum ATCC 824. Overexpression of both the 6-phosphofructokinase (pfkA) and pyruvate kinase (pykA) genes increased intracellular concentrations of ATP and NADH and also resistance to butanol toxicity. Marked increases of butanol and ethanol production, but not acetone, were also observed in batch fermentation. The butanol and ethanol concentrations were 29.4 and 85.5 % higher, respectively, in the fermentation by double-overexpressed C. acetobutylicum ATCC 824/pfkA+pykA than the wild-type strain. Furthermore, when fed-batch fermentation using glucose was carried out, the butanol and total solvent (acetone, butanol, and ethanol) concentrations reached as high as 19.12 and 28.02 g/L, respectively. The reason for improved butanol formation was attributed to the enhanced NADH and ATP concentrations and increased tolerance to butanol in the double-overexpressed strain.     

Factors in acid treated bagasse inhibiting ethanol production from d-xylose by Pachysolen tannophilus

The fermentation of d-xylose, the major sugar-cane bagasse hemicellulose component, to ethanol by Pachysolen tannophilus is inhibited by various factors produced or released during the acid hydrolysis of the bagasse or during the fermentation process. These include ethanol, iron, chromium, copper, nickel, acetic acid and furfural. Ethanol production by P. tannophilus is inhibited by ethanol fconcentrations >24 g l^?1. Furfural and acetic acid concentrations as low as 0.3 and 7 g l^?1, respectively, and iron, chromium, nickel and copper at concentrations of 0.07, 0.01, 0.01 and 0.004 g l^?1, respectively. Similar concentrations may be found in acid-hydrolysed bagasse. The removal of these factors by treatment with ion-exchange resin resulted in the fermentation of the sugars to ethanol. The d-glucose was used rapidly and completely whereas d-xylose utilization was slow and incomplete. An ethanol concentration of 4.1 g l^?1 was produced and an ethanol yield of 0.32 was obtained. Xylitol in significant amounts was produced.     

Engineering Clostridium acetobutylicum for production of kerosene and diesel blendstock precursors

Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2–2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD^D485G variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad^D485G) ABE products resulted in a blend with nearly 50 wt%≤C₉ products, which are unsuitable for diesel. To selectively increase butanol production, C. beijerinckii aldehyde dehydrogenase and C. ljungdhalii butanol dehydrogenase were co-expressed (strain designate 824(Cb ald-Cl bdh)), which increased butanol titers by 27% to 16.9 g L⁻¹ while acetone and ethanol titers remained essentially unaffected. The solvent ratio from 824(Cb ald-Cl bdh) resulted in more than 80 wt% of catalysis products having a carbon chain length≥C₁₁ which amounts to 9.8 g L⁻¹ of products suitable as kerosene or diesel blendstock based on fermentation volume. To further increase solvent production, we investigated expression of both native and heterologous chaperones in C. acetobutylicum. Expression of a heat shock protein (HSP33) from Bacillus psychrosaccharolyticus increased the total solvent titer by 22%. Co-expression of HSP33 and aldehyde/butanol dehydrogenases further increased ABE formation as well as acetone and butanol yields. HSP33 was identified as the first heterologous chaperone that significantly increases solvent titers above wild type C. acetobutylicum levels, which can be combined with metabolic engineering to further increase solvent production.     

Selective reduction of xylose to xylitol from a mixture of hemicellulosic sugars

The biocatalytic reduction of d-xylose to xylitol requires separation of the substrate from l-arabinose, another major component of hemicellulosic hydrolysate. This step is necessitated by the innate promiscuity of xylose reductases, which can efficiently reduce l-arabinose to l-arabinitol, an unwanted byproduct. Unfortunately, due to the epimeric nature of d-xylose and l-arabinose, separation can be difficult, leading to high production costs. To overcome this issue, we engineered an E. coli strain to efficiently produce xylitol from d-xylose with minimal production of l-arabinitol byproduct. By combining this strain with a previously engineered xylose reductase mutant, we were able to eliminate l-arabinitol formation and produce xylitol to near 100% purity from an equiweight mixture of d-xylose, l-arabinose, and d-glucose.     

Induction of Xylose Reductase and Xylitol Dehydrogenase Activities in Pachysolen tannophilus and Pichia stipitis on Mixed Sugars

The induction of xylose reductase and xylitol dehydrogenase activities on mixed sugars was investigated in the yeasts Pachysolen tannophilus and Pichia stipitis. Enzyme activities induced on d-xylose served as the controls. In both yeasts, d-glucose, d-mannose, and 2-deoxyglucose inhibited enzyme induction by d-xylose to various degrees. Cellobiose, l-arabinose, and d-galactose were not inhibitory. In liquid batch culture, P. tannophilus utilized d-glucose and d-mannose rapidly and preferentially over d-xylose, while d-galactose consumption was poor and lagged behind that of the pentose sugar. In P. stipitis, all three hexoses were used preferentially over d-xylose. The results showed that the repressibility of xylose reductase and xylitol dehydrogenase may limit the potential of yeast fermentation of pentose sugars in hydrolysates of lignocellulosic substrates.     

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Synthetic microbial communities have become a focus of biotechnological research since they can overcome several of the limitations of single-specie cultures. A paradigmatic example is Clostridium cellulovorans DSM 743B, which can decompose lignocellulose but cannot produce butanol. Clostridium beijerinckii NCIMB 8052 however, is unable to use lignocellulose but can produce high amounts of butanol from simple sugars. In our previous studies, both organisms were cocultured to produce butanol by consolidated bioprocessing. However, such consolidated bioprocessing implementation strongly depends on pH regulation. Since low pH (pH 4.5–5.5) is required for butanol fermentation, C. cellulovorans cannot grow well and saccharify sufficient lignocellulose to feed both strains at a pH below 6.4. To overcome this bottleneck, this study engineered C. cellulovorans by adaptive laboratory evolution, inactivating cell wall lyases genes (Clocel_0798 and Clocel_2169), and overexpressing agmatine deiminase genes (augA, encoded by Cbei_1922) from C. beijerinckii NCIMB 8052. The generated strain WZQ36: 743B*6.0*3△lyt0798△lyt2169-(pXY1-P_thl-augA) can tolerate a pH of 5.5. Finally, the alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824 was introduced into the strain to enable butanol production at low pH, in coordination with solvent fermentation of C. beijerinckii in consortium. The engineered consortium produced 3.94 g/L butanol without pH control within 83 hr, which is more than 5-fold of the level achieved by wild consortia under the same conditions. This exploration represents a proof of concept on how to combine metabolic and evolutionary engineering to coordinate coculture of a synthetic microbial community.     

Metabolic engineering of d-xylose pathway in Clostridium beijerinckii to optimize solvent production from xylose mother liquid

Clostridium beijerinckii is an attractive butanol-producing microbe for its advantage in co-fermenting hexose and pentose sugars. However, this Clostridium strain exhibits undesired efficiency in utilizing d-xylose, one of the major building blocks contained in lignocellulosic materials. Here, we reported a useful metabolic engineering strategy to improve d-xylose consumption by C. beijerinckii. Gene cbei2385, encoding a putative d-xylose repressor XylR, was first disrupted in the C. beijerinckii NCIMB 8052, resulting in a significant increase in d-xylose consumption. A d-xylose proton-symporter (encoded by gene cbei0109) was identified and then overexpressed to further optimize d-xylose utilization, yielding an engineered strain 8052xylR-xylT(ptb) (xylR inactivation plus xylT overexpression driven by ptb promoter). We investigated the strain 8052xylR-xylT(ptb) in fermenting xylose mother liquid, an abundant by-product from industrial-scale xylose preparation from corncob and rich in d-xylose, finally achieving a 35% higher Acetone, Butanol and Ethanol (ABE) solvent titer (16.91g/L) and a 38% higher yield (0.29g/g) over those of the wild-type strain. The strategy used in this study enables C. beijerinckii more suitable for butanol production from lignocellulosic materials.     

Electron Spin Resonance Analysis of the Effect of Butanol on the Membrane Fluidity of Intact Cells of Clostridium acetobutylicum

Analysis of electron spin resonance spectra of 5-doxyl stearic acid in aqueous suspensions of Clostridium acetobutylicum ATCC 824 and the butanol-tolerant SA-2 derivative during a small-scale fermentation at three different butanol challenge levels indicated that the SA-2 strain is able to respond to the physical fluidizing effect of high (1.5%) butanol challenge by reducing its membrane fluidity at 12 and 30 h. The wild-type 824 strain was unable to so respond when challenged at the 1.5% level.     

Metabolic engineering of Clostridium acetobutylicum M5 for highly selective butanol production

To improve butanol selectivity, Clostridium acetobutylicum M5(pIMP1E1AB) was constructed by adhE1-ctfAB complementation of C. acetobutylicum M5, a derivative strain of C. acetobutylicum ATCC 824, which does not produce solvents due to the lack of megaplasmid pSOL1. The gene products of adhE1-ctfAB catalyze the formation of acetoacetate and ethanol/butanol with acid re-assimilation in solventogenesis. Effects of the adhE1-ctfAB complementation of M5 were studied by batch fermentations under various pH and glucose concentrations, and by flux balance analysis using a genome-scale metabolic model for this organism. The metabolically engineered M5(pIMP1E1AB) strain was able to produce 154 mM butanol with 9.9 mM acetone at pH 5.5, resulting in a butanol selectivity (a molar ratio of butanol to total solvents) of 0.84, which is much higher than that (0.57 at pH 5.0 or 0.61 at pH 5.5) of the wild-type strain ATCC 824. Unlike for C. acetobutylicum ATCC 824, a higher level of acetate accumulation was observed during fermentation of the M5 strain complemented with adhE1 and/or ctfAB. A plausible reason for this phenomenon is that the cellular metabolism was shifted towards acetate production to compensate reduced ATP production during the largely growth-associated butanol formation by the M5(pIMP1E1AB) strain.     

The transport and mediation mechanisms of the common sugars in Escherichia coli

Escherichia coli can uptake and utilize many common natural sugars to form biomass or valuable target bio-products. Carbon catabolite repression (CCR) will occur and hamper the efficient production of bio-products if E. coli strains are cultivated in a mixture of sugars containing some preferred sugar, such as glucose. Understanding the transport and metabolism mechanisms of the common and inexpensive sugars in E. coli is important for further improving the efficiency of sugar bioconversion and for reducing industrial fermentation costs using the methods of metabolic engineering, synthetic biology and systems biology. In this review, the transport and mediation mechanisms of glucose, fructose, sucrose, xylose and arabinose are discussed and summarized, and the hierarchical utilization principles of these sugars are elucidated.     

Genetic Engineering of Inhibitor-Tolerant Saccharomyces cerevisiae for Improved Xylose Utilization in Ethanol Production

For economical lignocellulose-to-ethanol production, a desirable biocatalyst should tolerate inhibitors derived from preteatment of lignocellulose and be able to utilize heterogeneous biomass sugars of hexoses and pentoses. Previously, we developed an inhibitor-tolerant Saccharomyces cerevisiae strain NRRL Y-50049 that is able to in situ detoxify common aldehyde inhibitors such as 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2-furaldehyde (HMF). In this study, we genetically engineered Y-50049 to enable and enhance its xylose utilization capability. A codon-optimized xylose isomerase gene for yeast (YXI) was synthesized and introduced into a defined chromosomal locus of Y-50049. Two newly identified xylose transport related genes XUT4 and XUT6, and previously reported xylulokinase gene (XKS1), and xylitol dehydrogenase gene (XYL2) from Scheffersomyces stipitis were also engineered into the yeast resulting in strain NRRL Y-50463. The engineered strain was able to grow on xylose as sole carbon source and a minimum ethanol production of 38.6?g?l^?1 was obtained in an anaerobic fermentation on mixed sugars of glucose and xylose in the presence of furfural and HMF.     

Butanol Production by a Butanol-Tolerant Strain of Clostridium acetobutylicum in Extruded Corn Broth

By employing serial enrichment, a derivative of Clostridium acetobutylicum ATCC 824 was obtained which grew at concentrations of butanol that prevented growth of the wild-type strain. The parent strain demonstrated a negative growth rate at 15 g of butanol/liter, whereas the SA-1 mutant was still able to grow at a rate which was 66% of the uninhibited control. SA-1 produced consistently higher concentrations of butanol (from 5 to 14%) and lower concentrations of acetone (12.5 to 40%) than the wild-type strain in 4 to 20% extruded corn broth (ECB). Although the highest concentration of butanol was produced by SA-1 and the wild-type strain in 14% ECB, the best solvent ratio with respect to optimizing butanol and decreasing acetone occurred between 4 and 8% ECB for SA-1. SA-1 demonstrated higher conversion efficiency to butanol than the wild-type strain at every concentration of ECB tested. Characterization of the wild-type and SA-1 strain in 6% ECB demonstrated the superiority of the latter in terms of growth rate, time of onset of butanol production, carbohydrate utilization, pH resistance, and final butanol concentration in the fermentation broth.