Mohab A. Al-Hinai

Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations.

Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)‐based downregulation of CoA transferase (CoAT, the first enzyme in the acetone‐formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl‐CoA and acetyl‐CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl‐CoA. In order to increase then the flux towards butyryl‐CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl‐CoA to acetoacetyl‐CoA, the first step of the pathway from acetyl‐CoA to butyryl‐CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl‐CoA pool and reduce the acetyl‐CoA pool in order to achieve improved butanol titers and selectivity. Biotechnol. Bioeng. 2009;102: 38–49.

Novel System for Efficient Isolation of Clostridium Double-Crossover Allelic Exchange Mutants Enabling Markerless Chromosomal Gene Deletions and DNA Integration

ABSTRACT Isolation of Clostridium mutants based on gene replacement via allelic exchange remains a major limitation for this important genus. Use of a heterologous counterselection marker can facilitate the identification of the generally rare allelic exchange events. We report on the development of an inducible counterselection marker and describe its utility and broad potential in quickly and efficiently generating markerless DNA deletions and integrations at any genomic locus without the need for auxotrophic mutants or the use of the mobile group II introns. This system is based on a codon-optimized mazF toxin gene from Escherichia coli under the control of a lactose-inducible promoter from Clostridium perfringens. This system is potentially applicable to almost all members of the genus Clostridium due to their similarly low genomic GC content and comparable codon usage. We isolated all allelic-exchange-based gene deletions (ca_p0167, sigF, and sigK) or disruptions (ca_p0157 and sigF) we attempted and integrated a 3.6-kb heterologous DNA sequence (made up of a Clostridium ljungdahlii 2.1-kb formate dehydrogenase [fdh] gene plus a FLP recombination target [FRT]-flanked thiamphenicol resistance marker) into the Clostridium acetobutylicum chromosome. Furthermore, we report on the development of a plasmid system with inducible segregational instability, thus enabling efficient deployment of the FLP-FRT system to generate markerless deletion or integration mutants. This enabled expeditious deletion of the thiamphenicol resistance marker from the fdh integrant strain as well as the sigK deletion strain. More generally, our system can potentially be applied to other organisms with underdeveloped genetic tools.

Synthetic tolerance: three noncoding small RNAs, DsrA, ArcZ and RprA, acting supra-additively against acid stress

Synthetic acid tolerance, especially during active cell growth, is a desirable phenotype for many biotechnological applications. Natively, acid resistance in Escherichia coli is largely a stationary-phase phenotype attributable to mechanisms mostly under the control of the stationary-phase sigma factor RpoS. We show that simultaneous overexpression of noncoding small RNAs (sRNAs), DsrA, RprA and ArcZ, which are translational RpoS activators, increased acid tolerance (based on a low-pH survival assay) supra-additively up to 8500-fold during active cell growth, and provided protection against carboxylic acid and oxidative stress. Overexpression of rpoS without its regulatory 5′-UTR resulted in inferior acid tolerance. The supra-additive effect of overexpressing the three sRNAs results from the impact their expression has on RpoS-protein levels, and the beneficial perturbation of the interconnected RpoS and H-NS networks, thus leading to superior tolerance during active growth. Unlike the overexpression of proteins, overexpression of sRNAs imposes hardly any metabolic burden on cells, and constitutes a more effective strain engineering strategy.

The Clostridium Sporulation Programs: Diversity and Preservation of Endospore Differentiation

SUMMARY Bacillus and Clostridium organisms initiate the sporulation process when unfavorable conditions are detected. The sporulation process is a carefully orchestrated cascade of events at both the transcriptional and posttranslational levels involving a multitude of sigma factors, transcription factors, proteases, and phosphatases. Like Bacillus genomes, sequenced Clostridium genomes contain genes for all major sporulation-specific transcription and sigma factors (spo0A, sigH, sigF, sigE, sigG, and sigK) that orchestrate the sporulation program. However, recent studies have shown that there are substantial differences in the sporulation programs between the two genera as well as among different Clostridium species. First, in the absence of a Bacillus-like phosphorelay system, activation of Spo0A in Clostridium organisms is carried out by a number of orphan histidine kinases. Second, downstream of Spo0A, the transcriptional and posttranslational regulation of the canonical set of four sporulation-specific sigma factors (σF, σE, σG, and σK) display different patterns, not only compared to Bacillus but also among Clostridium organisms. Finally, recent studies demonstrated that σK, the last sigma factor to be activated according to the Bacillus subtilis model, is involved in the very early stages of sporulation in Clostridium acetobutylicum, C. perfringens, and C. botulinum as well as in the very late stages of spore maturation in C. acetobutylicum. Despite profound differences in initiation, propagation, and orchestration of expression of spore morphogenetic components, these findings demonstrate not only the robustness of the endospore sporulation program but also the plasticity of the program to generate different complex phenotypes, some apparently regulated at the epigenetic level.

σK of Clostridium acetobutylicum is the first known sporulation-specific sigma factor with two developmentally separated roles, one early and one late in sporulation

Sporulation in the model endospore-forming organism Bacillus subtilis proceeds via the sequential and stage-specific activation of the sporulation-specific sigma factors, σ(H) (early), σ(F), σ(E), σ(G), and σ(K) (late). Here we show that the Clostridium acetobutylicum σ(K) acts both early, prior to Spo0A expression, and late, past σ(G) activation, thus departing from the B. subtilis model. The C. acetobutylicum sigK deletion (ΔsigK) mutant was unable to sporulate, and solventogenesis, the characteristic stationary-phase phenomenon for this organism, was severely diminished. Transmission electron microscopy demonstrated that the ΔsigK mutant does not develop an asymmetric septum and produces no granulose. Complementation of sigK restored sporulation and solventogenesis to wild-type levels. Spo0A and σ(G) proteins were not detectable by Western analysis, while σ(F) protein levels were significantly reduced in the ΔsigK mutant. spo0A, sigF, sigE, sigG, spoIIE, and adhE1 transcript levels were all downregulated in the ΔsigK mutant, while those of the sigH transcript were unaffected during the exponential and transitional phases of culture. These data show that σ(K) is necessary for sporulation prior to spo0A expression. Plasmid-based expression of spo0A in the ΔsigK mutant from a nonnative promoter restored solventogenesis and the production of Spo0A, σ(F), σ(E), and σ(G), but not sporulation, which was blocked past the σ(G) stage of development, thus demonstrating that σ(K) is also necessary in late sporulation. sigK is expressed very early at low levels in exponential phase but is strongly upregulated during the middle to late stationary phase. This is the first sporulation-specific sigma factor shown to have two developmentally separated roles.

Current scenario of catalysts for biodiesel production: A critical review

Abstract Due to increasing concerns about global warming and dwindling oil supplies, the world’s attention is turning to green processes that use sustainable and environmentally friendly feedstock to produce renewable energy such as biofuels. Among them, biodiesel, which is made from nontoxic, biodegradable, renewable sources such as refined and used vegetable oils and animal fats, is a renewable substitute fuel for petroleum diesel fuel. Biodiesel is produced by transesterification in which oil or fat is reacted with short chain alcohol in the presence of a catalyst. The process of transesterification is affected by the mode of reaction, molar ratio of alcohol to oil, type of alcohol, nature and amount of catalysts, reaction time, and temperature. Various studies have been carried out using different oils as the raw material; different alcohols (methanol, ethanol, butanol); different catalysts; notably homogeneous catalysts such as sodium hydroxide, potassium hydroxide, sulfuric acid, and supercritical fluids; or, in some cases, enzymes such as lipases. This article focuses on the application of heterogeneous catalysts for biodiesel production because of their environmental and economic advantages. This review contains a detailed discussion on the advantages and feasibility of catalysts for biodiesel production, which are both environmentally and economically viable as compared to conventional homogeneous catalysts. The classification of catalysts into different categories based on a catalyst’s activity, feasibility, and lifetime is also briefly discussed. Furthermore, recommendations have been made for the most suitable catalyst (bifunctional catalyst) for low-cost oils to valuable biodiesel and the challenges faced by the biodiesel industry with some possible solutions.

Bio-Oil Upgrading by Catalytic Cracking Over Different Solid Catalysts

Fossil fuel crises along with global environmental issues, due to combustion of fossil fuel, lead to focus on biomass derived fuels. Bio-oil nowadays is seriously considered to be one of the favorable, renewable and alternative energy sources to replace fossil fuel and has become a significant energy carrier for transportation, industrial and commercial applications. In this study, bio-oil was upgraded by catalytic cracking in a fixed bed reactor in the presence of three different catalysts HY, H-mordenite and HZSM-5.All of the experimental runs were carried out at 500 °C, 0.3MPa and 15:1 oil to catalyst ratio. Catalysts characterization revealed that HZSM-5 with uniform pore and TPD analysis shows the presence of large number of acidic sites as compared to HY and H-mordenite. HZSM-5 proved its effectiveness in terms of deoxygenation and converting oxygenating compounds to hydrocarbons. The amount of hydrocarbons formed was 16.27 wt % OLP for HZSM-5, 15.16 wt% for HY and 14.954 wt % for H-mordenite. HZSM-5 possessed a strong acidity, uniform pore size and high activities which tended to permit the transformation of the oxygenated compounds present in the bio-oil to hydrocarbons. The upgraded bio-oil obtained posses improved physiochemical properties such pH which was increased from 2.21 to 3.56 while density was decreased upto 0.82 kg/m. The calorific value also increased upto 31.65 kJ/kg. The improved bio-oil by HZSM-5 catalyst can be considered as a potential for to be used as direct fuel.