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Although the industrial production of butanol has been carried out for decades by bacteria of the Clostridium species, recent studies have shown the use of the yeast Saccharomyces cerevisiae as a promising alternative. While the production of n-butanol by this yeast is still very far from its tolerability (up to 2% butanol), the improvement in the tolerance can lead to an increase in butanol production. The aim of the present work was to evaluate the adaptive capacity of the laboratory strain X2180-1B and the Brazilian ethanol-producing strain CAT-1 when submitted to two strategies of adaptive laboratory Evolution (ALE) in butanol. The strains were submitted, in parallel, to ALE with successive passages or with UV irradiation, using 1% butanol as selection pressure. Despite initially showing greater tolerance to butanol, the CAT-1 strain did not show great improvements after being submitted to ALE. Already the laboratory strain X2180-1B showed an incredible increase in butanol tolerance, starting from a condition of inability to grow in 1% butanol, to the capacity to grow in this same condition. With emphasis on the X2180_n100#28 isolated colony that presented the highest maximum specific growth rate among all isolated colonies, we believe that this colony has good potential to be used as a model yeast for understanding the mechanisms that involve tolerance to alcohols and other inhibitory compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Leveraging renewable carbon-based resources for energy and chemical production is a promising approach to decrease reliance on fossil fuels. This entails a thermo/biotechnological procedure wherein bacteria, notably Clostridia, ferment syngas, converting CO or CO 2 + H 2 into Hexanol, Butanol and Ethanol (H-B-E fermentation). This work reports of Clostridium carboxidivorans performance in a stirred tank reactor continuously operated with respect to the gas and the cell/liquid phases. The primary objective was to assess acid and solvent production at pH 5.6 by feeding pure CO or synthetic syngas under gas flow differential conditions. Fermentation tests were conducted at four different dilution rates (D L ) of the fresh medium in the range 0.034-0.25 h -1 . The fermentation pathways of C. carboxidivorans were found to be nearly identical for both CO and syngas, with consistent growth and metabolite production at pH 5.6 within a range of dilution rates. Wash-out conditions were observed at a D L of 0.25 h -1 regardless of the carbon source. Ethanol was the predominant solvent produced, but a shift towards butanol production was observed with CO as the substrate and towards hexanol production with synthetic syngas. In particular, the maximum cell concentration (0.5 g DM /L) was obtained with pure CO at D L 0.05 h -1 ; the highest solvent productivity (60 mg/L*h of total solvent) was obtained at D L 0.17 h -1 by using synthetic syngas as C-source. The findings highlight the importance of substrate composition and operating conditions in syngas fermentation processes. These insights contribute to the optimization of syngas fermentation processes for biofuel and chemical production., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Biofuel production by Clostridium acetobutylicum is compromised by strain degeneration due to loss of its pSOL1 megaplasmid. Here we used engineering biology to stably integrate pSOL1 into the chromosome together with a synthetic isopropanol pathway. In a membrane bioreactor continuously fed with glucose mineral medium, the final strain produced advanced biofuels, n-butanol and isopropanol, at high yield (0.31 g g -1 ), titre (15.4 g l -1 ) and productivity (15.5 g l -1  h -1 ) without degeneration., (© 2024. The Author(s).)

Background: Reduced clearance of cerebrospinal fluid (CSF) has been suggested as a pathological feature of Alzheimer's disease (AD). With extensive documentation in non-human mammals and contradictory human neuroimaging data it remains unknown whether the nasal mucosa is a CSF drainage site in humans. Here, we used dynamic PET with [1- 11 C]-Butanol, a highly permeable radiotracer with no appreciable brain binding, to test the hypothesis that tracer drainage from the nasal pathway reflects CSF drainage from brain. As a test of the hypothesis, we examined whether brain and nasal fluid drainage times were correlated and affected by brain amyloid., Methods: 24 cognitively normal subjects (≥ 65 years) were dynamically PET imaged for 60 min. using [1- 11 C]-Butanol. Imaging with either [ 11 C]-PiB or [ 18 F]-FBB identified 8 amyloid PET positive (Aβ+) and 16 Aβ- subjects. MRI-determined regions of interest (ROI) included: the carotid artery, the lateral orbitofrontal (LOF) brain, the cribriform plate, and an All-turbinate region comprised of the superior, middle, and inferior turbinates. The bilateral temporalis muscle and jugular veins served as control regions. Regional time-activity were used to model tracer influx, egress, and AUC., Results: LOF and All-turbinate 60 min AUC were positively associated, thus suggesting a connection between the brain and the nose. Further, the Aβ+ subgroup demonstrated impaired tracer kinetics, marked by reduced tracer influx and slower egress., Conclusion: The data show that tracer kinetics for brain and nasal turbinates are related to each other and both reflect the amyloid status of the brain. As such, these data add to evidence that the nasal pathway is a potential CSF drainage site in humans. These data warrant further investigation of brain and nasal contributions to protein clearance in neurodegenerative disease., (© 2024. The Author(s).)

2-Methyl-1-butanol (2MB) and 3-Methyl-1-butanol (3MB) are microbial volatile organic compounds (VOCs) and found in indoor air. Here, we applied rice as a bioindicator to investigate the effects of these indoor microbial volatile pollutants. A remarkable decrease in germination percentage, shoot and root elongation, as well as lateral root numbers were observed in 3MB. Furthermore, ROS production increased by 2MB and 3MB, suggesting that pentanol isomers could induce cytotoxicity in rice seedlings. The enhancement of peroxidase (POD) and catalase (CAT) activity provided evidence that pentanol isomers activated the enzymatic antioxidant scavenging systems, with a more significant effect observed in 3MB. Furthermore, 3MB induced higher activity levels of glutathione (GSH), oxidized glutathione (GSSG), and the GSH/GSSG ratio in rice compared to the levels induced by 2MB. Additionally, qRT-PCR analysis showed more up-regulation in the expression of glutaredoxins (GRXs), peroxiredoxins (PRXs), thioredoxins (TRXs), and glutathione S-transferases (GSTUs) genes in 3MB. Taking the impacts of pentanol isomers together, the present study suggests that 3MB exhibits more cytotoxic than 2MB, as such has critical effects on germination and the early seedling stage of rice. Our results provide molecular insights into how isomeric indoor microbial volatile pollutants affect plant growth through airborne signals., Competing Interests: Declaration of Competing Interest The authors declare no potential conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Candida albicans is the causative agent of invasive fungal infections. Its hyphae-forming ability is regarded as one of the important virulence factors. To unravel the impact of butanol on Candida albicans , it was placed in O +ve complete human serum with butanol (1% v / v ). The Candida transcriptome under butanol stress was then identified by mRNA sequencing. Studies including electron microscopy demonstrated the inhibition of hyphae formation in Candida under the influence of butanol, without any significant alteration in growth rate. The numbers of genes upregulated in the butanol in comparison to the serum alone were 1061 (20 min), 804 (45 min), and 537 (120 min). Candida cells exhibited the downregulation of six hypha-specific transcription factors and the induction of four repressor/regulator genes. Many of the hypha-specific genes exhibited repression in the medium with butanol. The genes related to adhesion also exhibited repression, whereas, among the heat-shock genes, three showed inductions in the presence of butanol. The fungal-specific genes exhibited induction as well as repression in the butanol-treated Candida cells. Furthermore, ten upregulated genes formed the core stress gene set in the presence of butanol. In the gene ontology analysis, enrichment of the processes related to non-coding RNA, ribosome biosynthesis, and metabolism was observed in the induced gene set. On the other side, a few GO biological process terms, including biofilm formation and filamentous growth, were enriched in the repressed gene set. Taken together, under butanol stress, Candida albicans is unable to extend hyphae and shows growth by budding. Many of the genes with perturbed expression may have fitness or virulence attributes and may provide prospective sites of antifungal targets against C. albicans.

Severe butanol toxicity to the metabolism of solventogenic clostridia significantly impede the application of fermentative butanol as a biofuel. Liquid-liquid extraction is an efficient method to reduce the butanol toxicity by in-situ removing it in the extractant phase. Butanol mass transfer into extractant phase in static acetone-butanol-ethanol (ABE) extractive fermentation with biodiesel as the extractant could be enhanced by adding a tiny amount of surfactant such as tween-80. In the case of corn-based ABE extractive fermentation by Clostridium acetobutylicum ATCC 824 using biodiesel originated from waste cooking oil as extractant, addition of 0.14% (w/v) tween-80 could increase butanol production in biodiesel and total solvents production by 21% and 17%, respectively, compared to those of control under non-surfactant existence. Furthermore, a mathematical model was developed to elucidate the mechanism of enhanced ABE extractive fermentation performance. The results indicated that the mass transfer improvement was obtained by effectively altering the physical properties of the self-generated bubbles during ABE extractive fermentation, such as reducing bubble size and extending its retention time in extractant phase, etc. Overall, this study provided an efficient approach for enhancing biobutanol production by integration of bioprocess optimization and model interpretation., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The rapid development of new molecular methods and approaches, sequencing technologies, has provided new insights into genetic and structural features of bacterial genomes. Information about the genetic organization of metabolic pathways and their regulatory elements has greatly contributed to the increase in the number of studies related to the construction of new bacterial strains with improved characteristics. In this study, the entire genome of the producing strain Clostridium sp. UCM В-7570 from the "Collection of producing strains of microorganisms and plant lines for food and agricultural biotechnology" of Institute of Food Biotechnology and Genomics of the National Academy of Sciences of Ukraine was sequenced and characterized. The genome was assembled into the scaffold with a total size of 4,470,321 bp and a GC content of 29.7%. The total number of genes identified was 4262, of which 4057 encoded proteins, 10 were rRNA operons, and 80 were tRNA genes. The genes of the sequenced genome encoding enzymes involved in butanol fermentation were found and analyzed. They were organized into cluster structures, and their protein sequences were found to be similar to the corresponding strains of C. acetobutylicum, C. beijerinckii, and C. pasteurianum type strains with the highest similarity to the latter. Thus, Clostridium sp. UCM В-7570 producing strain was identified as C. pasteurianum and suggested for metabolic engineering purposes., (© 2023. The Author(s), under exclusive licence to Institute of Plant Genetics Polish Academy of Sciences.)

Clostridium species are re-emerging as biotechnological workhorses for industrial acetone-butanol-ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25-100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the spo0A and upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome., (© 2023. The Author(s).)

Butyl butyrate has broad applications in foods, cosmetics, solvents, and biofuels. Microbial synthesis of bio-based butyl butyrate has been regarded as a promising approach recently. Herein, we engineered Clostridium tyrobutyricum ATCC 25755 to achieve de novo biosynthesis of butyl butyrate from fermentable sugars. Through introducing the butanol synthetic pathway (enzyme AdhE2), screening alcohol acyltransferases (AATs), adjusting transcription of VAAT and adhE2 (i.e., optimizing promoter), and efficient supplying butyryl-CoA, an excellent engineered strain, named MUV3, was obtained with ability to produce 4.58 g/L butyl butyrate at 25 °C with glucose in serum bottles. More NADH is needed for butyl butyrate synthesis, thus mannitol (the more reduced substrate) was employed to produce butyl butyrate. Ultimately, 62.59 g/L butyl butyrate with a selectivity of 95.97%, and a yield of 0.21 mol/mol was obtained under mannitol with fed-batch fermentation in a 5 L bioreactor, which is the highest butyl butyrate titer reported so far. Altogether, this study presents an anaerobic fermentative platform for de novo biosynthesis of butyl butyrate in one step, which lays the foundation for butyl butyrate biosynthesis from renewable biomass feedstocks., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2023 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Clostridium spp. are suitable for the bioconversion of C 1 -gases (e.g., CO 2 , CO and syngas) into different bioproducts. These products can be used as biofuels and are reviewed here, focusing on ethanol, butanol and hexanol, mainly. The production of higher alcohols (e.g., butanol and hexanol) has hardly been reviewed. Parameters affecting the optimization of the bioconversion process and bioreactor performance are addressed as well as the pathways involved in these bioconversions. New aspects, such as mixotrophy and sugar versus gas fermentation, are also reviewed. In addition, Clostridia can also produce higher alcohols from the integration of the Wood-Ljungdahl pathway and the reverse ß-oxidation pathway, which has also not yet been comprehensively reviewed. In the latter process, the acetogen uses the reducing power of CO/syngas to reduce C 4 or C 6 fatty acids, previously produced by a chain elongating microorganism (commonly Clostridium kluyveri), into the corresponding bioalcohol., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Although strain tolerance to high product concentrations is a barrier to the economically viable biomanufacturing of industrial chemicals, chemical tolerance mechanisms are often unknown. To reveal tolerance mechanisms, an automated platform was utilized to evolve Escherichia coli to grow optimally in the presence of 11 industrial chemicals (1,2-propanediol, 2,3-butanediol, glutarate, adipate, putrescine, hexamethylenediamine, butanol, isobutyrate, coumarate, octanoate, hexanoate), reaching tolerance at concentrations 60%-400% higher than initial toxic levels. Sequencing genomes of 223 isolates from 89 populations, reverse engineering, and cross-compound tolerance profiling were employed to uncover tolerance mechanisms. We show that: 1) cells are tolerized via frequent mutation of membrane transporters or cell wall-associated proteins (e.g., ProV, KgtP, SapB, NagA, NagC, MreB), transcription and translation machineries (e.g., RpoA, RpoB, RpoC, RpsA, RpsG, NusA, Rho), stress signaling proteins (e.g., RelA, SspA, SpoT, YobF), and for certain chemicals, regulators and enzymes in metabolism (e.g., MetJ, NadR, GudD, PurT); 2) osmotic stress plays a significant role in tolerance when chemical concentrations exceed a general threshold and mutated genes frequently overlap with those enabling chemical tolerance in membrane transporters and cell wall-associated proteins; 3) tolerization to a specific chemical generally improves tolerance to structurally similar compounds whereas a tradeoff can occur on dissimilar chemicals, and 4) using pre-tolerized starting isolates can hugely enhance the subsequent production of chemicals when a production pathway is inserted in many, but not all, evolved tolerized host strains, underpinning the need for evolving multiple parallel populations. Taken as a whole, this study provides a comprehensive genotype-phenotype map based on identified mutations and growth phenotypes for 223 chemical tolerant isolates., Competing Interests: Declaration of competing interest All authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

With the determination of the Leloir pathway in a solventogenic wild-type strain WK through the transcriptional analysis, two pivotal genes (galK and galT) were systematically co-expressed to demonstrate a significantly enhanced galactose utilization for butanol production with the elimination of carbon catabolite repression (CCR). The gene-modified strain WK-Gal-4 could effectively co-utilize galactose and glucose by directly using an ultrasonication-assisted butyric acid-pretreated Gelidium amansii hydrolysate (BAU) as the substrate, exhibiting the optimal sugar consumption and butanol production from BAU of 20.31 g/L and 7.8 g/L with an increment by 62.35 % and 61.49 % over that by strain WK, respectively. This work for the first time develops a feasible approach to utilizing red algal biomass for butanol fermentation through exploring the metabolic regulation of carbohydrate catabolism, also offering a novel route to develop the future biorefinery using the cost-effective and sustainable marine feedstocks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The second generation (2 G) biofuels were introduced to solve the issues associated with first-generation biofuel (dependency on food materials) and fossil fuels, such as reservoirs diminution, high demand, price fluctuation, and lethal greenhouse gases emission. Butanol and ethanol are the main 2 G biofuels. They are used as a disinfectant, antiseptic, and chemical solvent in the pharmaceutical, plastic, textiles, cosmetics, and fuel industries. Currently, their bacterial biological production from lignocellulosic material at the industrial level with primitive microorganisms is under development and not economical and qualitative compatible as compared to that of fossil origin, due to the slow growth rate, low titer, recalcitrant nature of lignocellulose, strain intolerance to a higher amount of butanol and ethanol, and strain inability to tolerate inhibitors accumulated during pretreatment of lignocellulosic materials. Therefore, metabolic engineering strategies such as redirection of carbon flux, knocking out competing pathways, enhancing strain robustness and wide range of substrate utilization ability, and overexpression of enzymes involved in their biological synthesis have been applied to bacteria for enhancing their ability for 2 G ethanol and butanol production in a highly cost-effective amount from lignocellulosic materials. Herein, we summarized and reviewed the progress in metabolic engineering of bacterial species such as Clostridium spp,Escherichia coli, and Zymomonas mobilis for the synthesis of 2 G butanol and ethanol, especially from lignocellulosic materials., (© The Author(s) 2022. Published by Oxford University Press on behalf of Applied Microbiology International.)

A system for co-cultivation of anaerobic fungi with anaerobic bacteria was established based on lactate cross-feeding to produce butyrate and butanol from plant biomass. Several co-culture formulations were assembled that consisted of anaerobic fungi (Anaeromyces robustus, Neocallimastix californiae, or Caecomyces churrovis) with the bacterium Clostridium acetobutylicum. Co-cultures were grown simultaneously (e.g., 'one pot'), and compared to cultures where bacteria were cultured in fungal hydrolysate sequentially. Fungal hydrolysis of lignocellulose resulted in 7-11 mM amounts of glucose and xylose, as well as acetate, formate, ethanol, and lactate to support clostridial growth. Under these conditions, one-stage simultaneous co-culture of anaerobic fungi with C. acetobutylicum promoted the production of butyrate up to 30 mM. Alternatively, two-stage growth slightly promoted solventogenesis and elevated butanol levels (∼4-9 mM). Transcriptional regulation in the two-stage growth condition indicated that this cultivation method may decrease the time required to reach solventogenesis and induce the expression of cellulose-degrading genes in C. acetobutylicum due to relieved carbon-catabolite repression. Overall, this study demonstrates a proof of concept for biobutanol and bio-butyrate production from lignocellulose using an anaerobic fungal-bacterial co-culture system., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

In the last decades, fermentative production of n-butanol has regained substantial interest mainly owing to its use as drop-in-fuel. The use of lignocellulose as an alternative to traditional acetone-butanol-ethanol fermentation feedstocks (starchy biomass and molasses) can significantly increase the economic competitiveness of biobutanol over production from non-renewable sources (petroleum). However, the low cost of lignocellulose is offset by its high recalcitrance to biodegradation which generally requires chemical-physical pre-treatment and multiple bioreactor-based processes. The development of consolidated processing (i.e., single-pot fermentation) can dramatically reduce lignocellulose fermentation costs and promote its industrial application. Here, strategies for developing microbial strains and consortia that feature both efficient (hemi)cellulose depolymerization and butanol production will be depicted, that is, rational metabolic engineering of native (hemi)cellulolytic or native butanol-producing or other suitable microorganisms; protoplast fusion of (hemi)cellulolytic and butanol-producing strains; and co-culture of (hemi)cellulolytic and butanol-producing microbes. Irrespective of the fermentation feedstock, biobutanol production is inherently limited by the severe toxicity of this solvent that challenges process economic viability. Hence, an overview of strategies for developing butanol hypertolerant strains will be provided., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The determination of various volatiles in postmortem blood samples has been reported in many previous studies. The presence of some of them in postmortem specimens reflects microbial activity in the sample while others are detected mainly after consumption of alcoholic beverages or due to antemortem metabolic processes. This contribution aims to determine in 1954 postmortem blood samples, from respective number of unnatural deaths autopsy cases, the frequency of detection of some common volatile compounds, including acetaldehyde, acetone, 2-propanol, ethyl acetate, methanol, as well as, the higher alcohols 1-propanol, 1-butanol, isobutanol and 2-methyl-1-butanol and 3-methyl-1-butanol; moreover, their patterns in respect to the ethanol and 1-propanol concentrations and the putrefaction state of the corpse at autopsy. Acetone was the most frequently detected volatile (82 %), followed by acetaldehyde (44 %) and 2-propanol (34 %). Methanol was detected in 12 % of the samples and only in the presence of ethanol. The most frequently detected higher alcohol was 1-propanol (51 %), followed by isobutanol (8.5 %), 1-butanol (3.6 %) and methyl-butanols (2.0%); the latter three higher alcohols were detected in the presence of 1-propanol indicating possibly a common origin. Samples from cases with putrefaction had higher 1-propanol concentrations, than those without putrefaction, and, moreover, they were significantly correlated with 1-butanol concentrations., Competing Interests: Conflict of interest The authors declare no conflict of interest for the submitted manuscript entitled: “The frequency of ethanol, higher alcohols and other low molecular weight volatiles in postmortem blood samples from unnatural deaths”., (Copyright © 2022. Published by Elsevier B.V.)

In this study, Clostridium sp. strain WK-AN1 carrying both genes of agarase (Aga0283) and neoagarobiose hydrolase (NH2780) were successfully constructed to convert agar polysaccharide directly into butanol, contributing to overcome the lack of algal hydrolases in solventogenic clostridia. Through the optimization by the Plackett-Burman design (PBD) and response surface methodology (RSM), a maximal butanol production of 6.42 g/L was achieved from 17.86 g/L agar. Further application of utilizing the butyric acid pretreated Gelidium amansii hydrolysate demonstrated the modified strain obtained the butanol production of 7.83 g/L by 1.63-fold improvement over the wild-type one. This work for the first time establishes a novel route to utilize red algal polysaccharides for butanol fermentation by constructing a solventogenic clostridia-specific secretory expression system for heterologous agarases, which will provide insights for future development of the sustainable third-generation biomass energy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Serine/threonine protein kinases (STKs) are important for signal transduction and involved in multiple physiological processes, including cell growth, central metabolism, and sporulation in bacteria. However, the role of STKs in solventogenic clostridia remains unclear. Here, we identified and comprehensively investigated six STK candidates in Clostridium beijerinckii. These STKs were classified into four groups with distinct characteristics via analysis of genetic organizations, prediction of protein domains, and multiple sequence alignment. Cbei0566 is a member of the PrkA family with 41% identity to PrkA from Bacillus subtilis, and both Cbei0666 and Cbei0813 are two-component-like STKs. Cbei1151 and Cbei1929 belong to the Hanks family STKs and consist of a cytoplasmic catalytic domain, a transmembrane region, and extracellular sensor domains. In-frame deletion mutants of cbei0566, cbei0666, cbei1929, and cbei2661 displayed similar cell growth with wild type. Both Δcbei0666 and Δcbei2661 improved acetone-butanol-ethanol (ABE) production by 14.3% (19.2 g/L vs. 16.8 g/L), and the sporulation frequencies of Δcbei0566, Δcbei1929, and Δcbei2661 significantly decreased to 35.5%, 55.1% and 44.8%, respectively. The restored phenotypes after genetic complementation demonstrated their direct link to STKs deletion. Remarkably, overexpressing cbei0566 contributed to 41.5% more spore formation and cbei1929 overexpression enhanced ABE production from 19.3 to 24.2 g/L, along with 25% less acids. These results revealed that Cbei0566 and Cbei1929 had prominent regulatory functions. This study expands the current knowledge of the existence and functions of STKs in prokaryotes and highlights the importance of STK-mediated signaling networks in developing superior strains. KEY POINTS: • First reported serine/threonine protein kinases in solventogenic clostridia • Six STKs with distinct properties possessed diverse functions in C. beijerinckii • Cbei1929 and Cbei0566 remarkably regulated solventogenesis and sporulation., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The growing population increases the need to develop advanced biological methods for utilizing renewable and sustainable resources to produce environmentally friendly biofuels. Currently, energy resources are limited for global demand and are constantly depleting and creating environmental problems. Some higher chain alcohols, like butanol and ethanol, processing similar properties to gasoline, can be alternate sources of biofuel. However, the industrial production of these alcohols remains challenging because they cannot be efficiently produced by microbes naturally. Therefore, butanol is the most interesting biofuel candidate with a higher octane number produced naturally by microbes through Acetone-Butanol-Ethanol fermentation. Feedstock selection as the substrate is the most crucial step in biobutanol production. Lignocellulosic biomass has been widely used to produce cellulosic biobutanol using agricultural wastes and residue. Specific necessary pretreatments, fermentation strategies, bioreactor designing and kinetics, and modeling can also enhance the efficient production of biobutanol. The recent genetic engineering approaches of gene knock in, knock out, and overexpression to manipulate pathways can increase the production of biobutanol in a user friendly host organism. So far various genetic manipulation techniques like antisense RNA, TargeTron Technology and CRISPR have been used to target Clostridium acetobutylicum for biobutanol production. This review summarizes the recent research and development for the efficient production of biobutanol in various aspects., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study sought to provide a theoretical basis for effectively controlling the content of higher alcohols and esters in fermented foods. In this work, isoleucine (Ile) or leucine (Leu) at high levels was used as the sole nitrogen source for a BAT2 mutant and its parental Saccharomyces. cerevisiae 38 to investigate the effects of the addition of amounts of Ile or Leu and BAT2 on the aroma components in the flavor profile using gas chromatography mass spectrometer (GC-MS). The results showed that 2-methyl-butyraldehyde, 2-methyl-1-butanol, and 2-methylbutyl-acetate were the products positively correlated with the Ile addition amount. In addition, 3-methyl-butyraldehyde, 3-methyl-1-butanol, and 3-methylbutyl-acetate were the products positively correlated with Leu addition amount. BAT2 deletion resulted in a significant decline in the yields of 2-methyl-butyraldehyde, 3-methyl-butyraldehyde,2-methyl-1-butanol, and 3-methyl-1-butanol, but also an increase in the yields of 2-methylbutyl-acetate and 3-methylbutyl-acetate. We speculated that BAT2 regulated the front and end of this metabolite chain in a feedback manner. Improved metabolic chain analyses, including the simulated energy metabolism of Ile or Leu, indicated that reducing the added amount of branched-chain amino acids, BAT mutation, and eliminating the role of energy cofactors such as NADH/NAD+ were three important ways to control the content of high alcohols and esters in fermented foods.

Background: Lignocellulosic biomass is recognized as an effective potential substrate for biobutanol production. Though many pretreatment and detoxification methods have been set up, the fermentability of detoxicated lignocellulosic substrate is still far lower than that of starchy feedstocks. On the other hand, the number of recent efforts on rational metabolic engineering approaches to increase butanol production in Clostridium strains is also quite limited, demonstrating the physiological complexity of solventogenic clostridia. In fact, the strain performance is greatly impacted by process control. developing efficient process control strategies could be a feasible solution to this problem., Results: In this study, oxidoreduction potential (ORP) controlling was applied to increase the fermentability of enzymatically hydrolyzed steam-exploded corn stover (SECS) for butanol production. When ORP of detoxicated SECS was controlled at - 350 mV, the period of fermentation was shortened by 6 h with an increase of 27.5% in the total solvent (to 18.1 g/L) and 34.2% in butanol (to 10.2 g/L) respectively. Silico modeling revealed that the fluxes of NADPH, NADH and ATP strongly differed between the different scenarios. Quantitative analysis showed that intracellular concentrations of ATP, NADPH/NADP + , and NADH/NAD + were increased by 25.1%, 81.8%, and 62.5%. ORP controlling also resulted in a 2.1-fold increase in butyraldehyde dehydrogenase, a 1.2-fold increase in butanol dehydrogenase and 29% increase in the cell integrity., Conclusion: ORP control strategy effectively changed the intracellular metabolic spectrum and significantly improved Clostridium cell growth and butanol production. The working mechanism can be summarized into three aspects: First, Glycolysis and TCA circulation pathways were strengthened through key nodes such as pyruvate carboxylase [EC: 6.4.1.1], which provided sufficient NADH and NADPH for the cell. Second, sufficient ATP was provided to avoid "acid crash". Third, the key enzymes activities regulating butanol biosynthesis and cell membrane integrity were improved., (© 2022. The Author(s).)

In this study, continuous cultivations of C.carboxidivorans to study heterotrophic and mixotrophic conversion of glucose and H 2 , CO 2 , and CO were established. Glucose fermentations at pH 6 showed a high ratio of alcohol-to-acid production of 2.79 mol mol -1 . While H 2 or CO 2 were not utilized together with glucose, CO feeding drastically increased the combined alcohol titer to 9.1 g l -1 . Specifically, CO enhanced acetate (1.9-fold) and ethanol (1.7-fold) production and triggered chain elongation to butanol (1.5-fold) production but did not change the alcohol:acid ratio. Flux balance analysis showed that CO served both as a carbon and energy source, and CO mixotrophy displayed a carbon and energy efficiency of 45 and 77%, respectively. This study expands the knowledge on physiology and metabolism of C.carboxidivorans and can serve as the starting point for rational engineering and process intensification to establish efficient production of alcohols and acids from carbon waste., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The replacement of fossil fuels and petrochemicals with sustainable alternatives is necessary to mitigate the effects of climate change and also to counteract diminishing fossil resources. Acetogenic microorganisms such as Clostridium spp. are promising sources of fuels and basic chemical precursors because they efficiently utilize CO and CO 2 as carbon source. However the conversion into high titers of butanol and hexanol is challenging., Results: Using a metabolic engineering approach we transferred a 17.9-kb gene cluster via conjugation, containing 13 genes from C. kluyveri and C. acetobutylicum for butanol and hexanol biosynthesis, into C. ljungdahlii. Plasmid-based expression resulted in 1075 mg L -1 butanol and 133 mg L -1 hexanol from fructose in complex medium, and 174 mg L -1 butanol and 15 mg L -1 hexanol from gaseous substrate (20% CO 2 and 80% H 2 ) in minimal medium. Product formation was increased by the genomic integration of the heterologous gene cluster. We confirmed the expression of all 13 enzymes by targeted proteomics and identified potential rate-limiting steps. Then, we removed the first-round selection marker using CRISPR/Cas9 and integrated an additional 7.8 kb gene cluster comprising 6 genes from C. carboxidivorans. This led to a significant increase in the hexanol titer (251 mg L -1 ) at the expense of butanol (158 mg L -1 ), when grown on CO 2 and H 2 in serum bottles. Fermentation of this strain at 2-L scale produced 109 mg L -1 butanol and 393 mg L -1 hexanol., Conclusions: We thus confirmed the function of the butanol/hexanol biosynthesis genes and achieved hexanol biosynthesis in the syngas-fermenting species C. ljungdahlii for the first time, reaching the levels produced naturally by C. carboxidivorans. The genomic integration strain produced hexanol without selection and is therefore suitable for continuous fermentation processes., (© 2022. The Author(s).)

Unlike gaseous C 1 feedstocks for acetogenic bacteria, there has been less attention on liquid C 1 feedstocks, despite benefits in terms of energy efficiency, mass transfer and integration within existing fermentation infrastructure. Here, we present growth of Eubacterium limosum ATCC8486 using methanol and formate as substrates, finding evidence for the first time of native butanol production. We varied ratios of methanol-to-formate in batch serum bottle fermentations, showing butyrate is the major product (maximum specific rate 220 ± 23 mmol-C gDCW -1 day -1 ). Increasing this ratio showed methanol is the key feedstock driving the product spectrum towards more reduced products, such as butanol (maximum titre 2.0 ± 1.1 mM-C). However, both substrates are required for a high growth rate (maximum 0.19 ± 0.011 h -1 ) and cell density (maximum 1.2 ± 0.043 gDCW l -1 ), with formate being the preferred substrate. In fact, formate and methanol are consumed in two distinct growth phases - growth phase 1, on predominately formate and growth phase 2 on methanol, which must balance. Because the second growth varied according to the first growth on formate, this suggests butanol production is due to overflow metabolism, similar to 2,3-butanediol production in other acetogens. However, further research is required to confirm the butanol production pathway in E. limosum, particularly given, unlike other substrates, methanol likely results in mostly NADH generation, not reduced ferredoxin., (© 2021 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Biobutanol is gaining much attention as a potential biofuel due to its superior properties over ethanol. Butanol has been naturally produced via acetone-butanol-ethanol (ABE) fermentation by many Clostridium species, which are not very user-friendly bacteria. Therefore, to improve butanol titers and yield, various butanol synthesis pathways have been engineered in Escherichia coli, a much more robust and convenient host than Clostridium species. This review mainly focuses on the biosynthesis of n-butanol in engineered E. coli with an emphasis on efficient enzymes for butanol production in E. coli, butanol competing pathways, and genome engineering of E. coli for butanol production. In addition, the use of alternate strategies for butanol biosynthesis/enhancement, alternate substrates for the low cost of butanol production, and genetic improvement for butanol tolerance in E. coli have also been discussed., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Objectives: This study aimed to investigate the effect of the n-butanol fraction of the methanol leaf extract of Artemisia campestris (BFAC), growing wild in the arid zone of Tunisia, on induced obesity in male Wistar rats., Methods: The total phenolic content and antioxidant capacity of the BFAC were estimated. The main phenolic composition of the BFAC was determined using the high-performance chromatography system coupled with a diode array detector technics. Five groups of rats received either a standard diet (SD group), a high-fat diet (HFD group), or an HFD supplemented with oral administration of BFAC for eight weeks., Results: The BFAC showed higher phenolic content and antioxidant potential than the total leaf methanol extract. Chlorogenic acid, rutin, and dicaffeoylquinic acids were identified in the BFAC. HFD increased body and relative liver weights, as well as serum and hepatic levels of triglycerides and total cholesterol, compared to SD. HFD generated significant oxidative stress in the liver by increasing lipid peroxidation and reducing glutathione-S-transferase, catalase, and glutathione peroxidase activities, compared to SD. These HFD-altered parameters were restored to normal values by oral treatment with the BFAC., Conclusions: These findings give first evidence about the antiobesity efficacy of A. campestris . Such a study would enhance existing information and promote the use of this species., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

To reveal the mechanism of anti-renal fibrosis effects of an n -butanol extract from Amygdalus mongolica, renal fibrosis was induced with unilateral ureteral obstruction (UUO) and then treated with an n -butanol extract (BUT) from Amygdalus mongolica (Rosaceae). Sixty male Sprague-Dawley rats were randomly divided into the sham-operated, renal fibrosis (RF) model, benazepril hydrochloride-treated model (1.5 mg kg -1 ) and BUT-treated (1.75, 1.5 and 1.25 g kg -1 ) groups and the respective drugs were administered intragastrically for 21 days. Related biochemical indices in rat serum were determined and histopathological morphology observed. Serum metabolomics was assessed with HPLC-Q-TOF-MS. The BUT reduced levels of blood urea nitrogen, serum creatinine and albumin and lowered the content of malondialdehyde and hydroxyproline in tissues. The activity of superoxide dismutase in tissues was increased and an improvement in the severity of RF was observed. Sixteen possible biomarkers were identified by metabolomic analysis and six key metabolic pathways, including the TCA cycle and tyrosine metabolism, were analyzed. After treatment with the extract, 8, 12 and 9 possible biomarkers could be detected in the high-, medium- and low-dose groups, respectively. Key biomarkers of RF, identified using metabolomics, were most affected by the medium dose. A. mongolica BUT extract displays a protective effect on RF in rats and should be investigated as a candidate drug for the treatment of the disease., (© 2022 Chen Gao et al., published by Sciendo.)

Butyrate is produced by chemical synthesis based on crude oil, produced by microbial fermentation, or extracted from animal fats (M. Dwidar, J.-Y. Park, R. J. Mitchell, and B.-I. Sang, The Scientific World Journal, 2012:471417, 2012, https://doi.org/10.1100/2012/471417). Butyrate production by anaerobic bacteria is highly favorable since waste or sustainable resources can be used as the substrates. For this purpose, the native hyper-butanol producer Clostridium saccharoperbutylacetonicum N1-4(HMT) was used as a chassis strain due to its broad substrate spectrum. BLASTp analysis of the predicted proteome of C. saccharoperbutylacetonicum N1-4(HMT) resulted in the identification of gene products potentially involved in acetone-butanol-ethanol (ABE) fermentation. Their participation in ABE fermentation was either confirmed or disproven by the parallel production of acids or solvents and the respective transcript levels obtained by transcriptome analysis of this strain. The genes encoding phosphotransacetylase ( pta ) and butyraldehyde dehydrogenase ( bld ) were deleted to reduce acetate and alcohol formation. The genes located in the butyryl-CoA synthesis ( bcs ) operon encoding crotonase, butyryl-CoA dehydrogenase with electron-transferring protein subunits α and β, and 3-hydroxybutyryl-CoA dehydrogenase were overexpressed to channel the flux further towards butyrate formation. Thereby, the native hyper-butanol producer C. saccharoperbutylacetonicum N1-4(HMT) was converted into the hyper-butyrate producer C. saccharoperbutylacetonicum Δ bld Δ pta [pMTL83151_BCS_P bgaL ]. The transcription pattern following deletion and overexpression was characterized by a second transcriptomic study, revealing partial compensation for the deletion. Furthermore, this strain was characterized in pH-controlled fermentations with either glucose or Excello, a substrate yielded from spruce biomass. Butyrate was the main product, with maximum butyrate concentrations of 11.7 g·L -1 and 14.3 g·L -1 , respectively. Minimal amounts of by-products were detected. IMPORTANCE Platform chemicals such as butyrate are usually produced chemically from crude oil, resulting in the carry-over of harmful compounds. The selective production of butyrate using sustainable resources or waste without harmful by-products can be achieved by bacteria such as clostridia. The hyper-butanol producer Clostridium saccharoperbutylacetonicum N1-4(HMT) was converted into a hyper-butyrate producer. Butyrate production with very small amounts of by-products was established with glucose and the sustainable lignocellulosic sugar substrate Excello extracted from spruce biomass by the biorefinery Borregaard (Sarpsborg, Norway).

Background: Butyl acetate is a versatile compound that is widely used in the chemical and food industry. The conventional butyl acetate synthesis via Fischer esterification of butanol and acetic acid using catalytic strong acids under high temperature is not environmentally benign. Alternative lipase-catalyzed ester formation requires a significant amount of organic solvent which also presents another environmental challenge. Therefore, a microbial cell factory capable of producing butyl acetate through fermentation of renewable resources would provide a greener approach to butyl acetate production., Result: Here, we developed a metabolically engineered strain of Escherichia coli that efficiently converts glucose to butyl acetate. A modified Clostridium CoA-dependent butanol production pathway was used to synthesize butanol which was then condensed with acetyl-CoA through an alcohol acetyltransferase. Optimization of alcohol acetyltransferase expression and redox balance with auto-inducible fermentative controlled gene expression led to an effective titer of 22.8 ± 1.8 g/L butyl acetate produced in a bench-top bioreactor., Conclusion: Building on the well-developed Clostridium CoA-dependent butanol biosynthetic pathway, expression of an alcohol acetyltransferase converts the butanol produced into butyl acetate. The results from this study provided a strain of E. coli capable of directly producing butyl acetate from renewable resources at ambient conditions., (© 2022. The Author(s).)

Cyanobacteria are natural photosynthetic microbes which can be engineered for sustainable conversion of solar energy and carbon dioxide into chemical products. Attempts to improve target production often require an improved understanding of the native cyanobacterial host system. Valuable insights into cyanobacterial metabolism, biochemistry and physiology have been steadily increasing in recent years, stimulating key advancements of cyanobacteria as cell factories for biochemical, including biofuel, production. In the present review, we summarize the current progress in engineering cyanobacteria and discuss the achieved and potential utilization of these advances in cyanobacteria for the production of the bulk chemical butanol, specifically isobutanol and 1-butanol., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cyanobacteria hold promise for renewable chemical production due to their photosynthetic nature, but engineered strains frequently display poor production characteristics. These difficulties likely arise in part due to the distinctive photoautotrophic metabolism of cyanobacteria. In this work, we apply a genome-scale metabolic model of the cyanobacteria Synechococus sp. PCC 7002 to identify strain designs accounting for this unique metabolism that are predicted to improve the production of various biofuel alcohols (e.g. 2-methyl-1-butanol, isobutanol, and 1-butanol) synthesized via an engineered biosynthesis pathway. Using the model, we identify that the introduction of a large, non-native NADH-demand into PCC 7002's metabolic network is predicted to enhance production of these alcohols by promoting NADH-generating reactions upstream of the production pathways. To test this, we construct strains of PCC 7002 that utilize a heterologous, NADH-dependent nitrite reductase in place of the native, ferredoxin-dependent enzyme to create an NADH-demand in the cells when grown on nitrate-containing media. We find that photosynthetic production of both isobutanol and 2-methyl-1-butanol is significantly improved in the engineered strain background relative to that in a wild-type background. We additionally identify that the use of high-nutrient media leads to a substantial prolongment of the production curve in our alcohol production strains. The metabolic engineering strategy identified and tested in this work presents a novel approach to engineer cyanobacterial production strains that takes advantage of a unique aspect of their metabolism and serves as a basis on which to further develop strains with improved production of these alcohols and related products., (Copyright © 2021 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Anthropogenic carbon dioxide (CO 2 ) release in the atmosphere from fossil fuel combustion has inspired scientists to study CO 2 to biofuel conversion. Oxygenic phototrophs such as cyanobacteria have been used to produce biofuels using CO 2 . However, oxygen generation during oxygenic photosynthesis adversely affects biofuel production efficiency. To produce n-butanol (biofuel) from CO 2 , here we introduce an n-butanol biosynthesis pathway into an anoxygenic (non-oxygen evolving) photoautotroph, Rhodopseudomonas palustris TIE-1 (TIE-1). Using different carbon, nitrogen, and electron sources, we achieve n-butanol production in wild-type TIE-1 and mutants lacking electron-consuming (nitrogen-fixing) or acetyl-CoA-consuming (polyhydroxybutyrate and glycogen synthesis) pathways. The mutant lacking the nitrogen-fixing pathway produce the highest n-butanol. Coupled with novel hybrid bioelectrochemical platforms, this mutant produces n-butanol using CO 2 , solar panel-generated electricity, and light with high electrical energy conversion efficiency. Overall, this approach showcases TIE-1 as an attractive microbial chassis for carbon-neutral n-butanol bioproduction using sustainable, renewable, and abundant resources., (© 2021. The Author(s).)

n-Butanol is an essential chemical intermediate produced through microbial fermentation. However, its toxicity to microbial cells has limited its production to a great extent. The anaerobe lactic acid bacteria (LAB) are the most resistant to n-butanol, so it should be the first choice for improving n-butanol production. The present article aims to review the following aspects of n-butanol production by LAB: (1) the tolerance of LAB to n-butanol, including its tolerance level and potential tolerance mechanisms; (2) genome editing tools in the n-butanol-resistant LAB; (3) methods of LAB modification for n-butanol production and the production levels after modification. This review will provide a theoretical basis for further research on n-butanol production by LAB., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Cellulosic n-butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans, a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adhE2 from C. acetobutylicum for n-butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl-CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adhE2 would enhance n-butanol production. In this study, thiolase (thlA CA ) from C. acetobutylicum and 3-hydroxybutyryl-CoA dehydrogenase (hbd CT ) from C. tyrobutyricum were overexpressed in C. cellulovorans adhE2 to increase the flux from acetyl-CoA to butyryl-CoA. In addition, ferredoxin-NAD(P) + oxidoreductase (fnr), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl-CoA, which resulted in increased production of butyrate and butanol. The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n-butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adhE2-fnr CA -thlA CA -hbd CT produced n-butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n-butanol production from cellulosic biomass in consolidated bioprocessing., (© 2021 Wiley Periodicals LLC.)

In previous research, we modeled the ethanol production by certain bacteria under controlled experimental conditions in an attempt to quantify the production of microbial postmortem ethanol in cases where other alcohols were co-detected. This contribution on the modeling of postmortem ethanol production by Candida albicans is complementary to these previous studies. Τhis work aimed to study ethanol, higher alcohols (1-propanol, isobutanol, 2-methyl-1-butanol and 3-methyl-1-butanol), and 1-butanol production by Candida albicans: (i) in different culture media (Brain Heart Infusion, BHI and, Sabouraud Dextrose Broth, SDB), (ii) under mixed aerobic/anaerobic or strict anaerobic conditions, and (iii) at different temperatures (37 °C, 25 °C and, 4 °C), and develop simple mathematical models, resulted from fungal cultures at 25 °C, to predict the microbially produced ethanol in correlation with the other alcohols. The applicability of the models was tested in the C. albicans cultures in BHI and SDB media at 37 °C, in denatured human blood at 25 °C, acidic and neutral with different concentrations of additional glucose, in acidic denatured blood diluted with dextrose solution and in blood from autopsy cases. The received results indicated that the C. albicans models could apply in cases where yeasts have been activated in blood with elevated glucose levels. Overall, the in vitro ethanol production by C. albicans in blood depended on temperature, time, glucose (or carbohydrate) content, pH of the medium and endogenous changes in the medium composition through time. Our results showed that methyl-butanol is the most significant indicator of fungal ethanol production, followed by the equally important isobutanol and 1-propanol in qualitative and quantitative terms., Competing Interests: Conflict of interest The authors declare that there is no conflict of interest for this contribution., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: The aim of the study was to evaluate the possibility of using Y. lipolytica biomass as a whole-cell catalyst in the synthesis of lipophilic antioxidants, with the example of esterification of five phenolic acids with 1-butanol., Results: Freeze-dried Y. lipolytica biomass was successfully applied as a biocatalyst in the synthesis of esters of phenylpropanoic acid derivatives with 75-98% conversion. However, in the case of phenylacetic acid derivatives, results below 10% were obtained. The biological activity of phenolic acid esters was strongly associated with their chemical structures. Butyl 3-(4-hydroxyphenyl)propanoate showed an IC 50 value of 19 mg/ml (95 mM) and TEAC value of 0.427. Among the compounds tested, butyl esters of 3-(4-hydroxyphenyl)propanoic and 4-hydroxyphenylacetic acids exhibited the highest antifungal activity., Conclusions: Lipophilization of phenolic acids achieved by enzymatic esterification creates prospects for using these compounds as food additives with antioxidant properties in lipid-rich food matrices.

1-Butanol (1-BD) is a promising fuel additive which can be biosynthesized via reversed β-oxidation pathway in bacteria. However, heterologous reversed β-oxidation pathway is a carbon chain prolongation process with several genes overexpressed in most of bacterial hosts, leading to low titer of 1-BD and high cost for production. Here we displayed a forward β-oxidation pathway for 1-BD production in a kitchen waste oil (KWO) degrading Pseudomonas aeruginosa PA-3, and we proved that aldehyde dehydrogenase (ALDH) is a checkpoint for diverting metabolic flux into 1-BD biosynthesis. With nitrogen source supplied, titer of 1-BD was increased accompanied with 12 ALDH coding genes transcriptionally promoted to different degrees. At the same time, binding energies of these ALDHs with different length of acyl-CoAs in β-oxidation were calculated to identify their specificities. Based on the above information, ALDH deletions were conducted. We certified that deletion of ALDH8 and ALDH9 led to significant decreased titers of 1-BD. Finally, these two ALDHs were separately overexpressed in PA-3, and titer of 1-BD was promoted to 1.36 g/L at 72 h in shake flask. Totally in this work, we provided a forward β-oxidation pathway for 1-BD production from KWO, and the roles of ALDHs were confirmed.

The soil bacterium Bacillus subtilis forms beneficial biofilms that induce plant defences and prevent the growth of pathogens. It is naturally found in the rhizosphere, where microorganisms coexist in an extremely competitive environment, and thus have evolved a diverse arsenal of defence mechanisms. In this work, we found that volatile compounds produced by B. subtilis biofilms inhibited the development of competing biofilm colonies, by reducing extracellular matrix gene expression, both within and across species. This effect was dose-dependent, with the structural defects becoming more pronounced as the number of volatile-producing colonies increased. This inhibition was mostly mediated by organic volatiles, and we identified the active molecules as 3-methyl-1-butanol and 1-butanol. Similar results were obtained with biofilms formed by phylogenetically distinct bacterium sharing the same niche, Escherichia coli, which produced the biofilm-inhibiting 3-methyl-1-butanol and 2-nonanon. The ability of established biofilms to inhibit the development and spreading of new biofilms from afar might be a general mechanism utilized by bacterial biofilms to protect an occupied niche from the invasion of competing bacteria.

N-butanol is an important chemical and can be naturally synthesized by Clostridium species; however, the poor n-butanol tolerance of Clostridium impedes the further improvement in titer. In this study, Lactobacillus brevis, which possesses a higher butanol tolerance, was selected as host for heterologous butanol production. The Clostridium acetobutylicum genes thl, hbd, and crt which encode thiolase, β-hydroxybutyryl-CoA dehydrogenase, and crotonase, and the Treponema denticola gene ter, which encodes trans-enoyl-CoA reductase were cloned into a single plasmid to express the butanol synthesis pathway in L. brevis. A titer of 40 mg/L n-butanol was initially achieved with plasmid pLY15-opt, in which all pathway genes are codon-optimized. A titer of 450 mg/L of n-butanol was then synthesized when ter was further overexpressed in this pathway. The role of metabolic flux was reinforced with pLY15, in which only the ter gene was codon-optimized, which greatly increased the n-butanol titer to 817 mg/L. Our strategy significantly improved n-butanol synthesis in L. brevis and the final titer is the highest achieved amongst butanol-tolerant lactic acid bacteria.

n-Butanol is a renewable resource with a wide range of applications. Its physicochemical properties make it a potential substitute for gasoline. Saccharomyces cerevisiae can produce n-butanol via amino acid catabolic pathways, but the use of pure amino acids is economically unfeasible for large-scale production. The aim of this study was to optimize the production of n-butanol by S. cerevisiae from protein-rich agro-industrial by-products (sunflower and poultry offal meals). By-products were characterized according to their total protein and free amino acid contents and subjected to enzymatic hydrolysis. Protein hydrolysates were used as nitrogen sources for the production of n-butanol by S. cerevisiae, but only poultry offal meal hydrolysate (POMH) afforded detectable levels of n-butanol. Under optimized conditions (carbon/nitrogen ratio of 2 and working volume of 60%), 59.94 mg/L of n-butanol was produced using POMH and glucose as substrates. The low-cost agro-industrial by-product showed great potential to be used in the production of n-butanol by S. cerevisiae. Other protein-rich residues may also find application in biofuel production by yeasts.

Propene is one of the most important starting materials in the chemical industry. Herein, we report an enzymatic cascade reaction for the biocatalytic production of propene starting from n-butanol, thus offering a biobased production from glucose. In order to create an efficient system, we faced the issue of an optimal cofactor supply for the fatty acid decarboxylase OleT JE , which is said to be driven by either NAD(P)H or H 2 O 2 . In the first system, we used an alcohol and aldehyde dehydrogenase coupled to OleT JE by the electron-transfer complex putidaredoxin reductase/putidaredoxin, allowing regeneration of the NAD + cofactor. With the second system, we intended full oxidation of n-butanol to butyric acid, generating one equivalent of H 2 O 2 that can be used for the oxidative decarboxylation. As the optimal substrate is a long-chain fatty acid, we also tried to create an improved variant for the decarboxylation of butyric acid by using rational protein design. Within a mutational study with 57 designed mutants, we generated the mutant OleT V292I , which showed a 2.4-fold improvement in propene production in our H 2 O 2 -driven cascade system and reached total turnover numbers >1000., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

In this study, the role of CaCO 3 in n-butanol production was further investigated using corn straw hydrolysate (CSH) media by Clostridium acetobutylicum CICC 8016. CaCO 3 addition stimulated sugars utilization and butanol production. Further study showed that calcium salts addition to CSH media led to the increase in Ca 2+ concentration both intracellularly and extracellularly. Interestingly, without calcium salts addition, intracellular Ca 2+ concentration in the synthetic P2 medium was much higher than that in the CSH medium despite the lower extracellular Ca 2+ concentrations in the P2 medium. These results indicated that without additional calcium salts, Ca 2+ uptake by C. acetobutylicum CICC 8016 in the CSH medium may be inhibited by non-sugar biomass degradation compounds, such as furans, phenolics and organic acids. Comparative proteomics analysis results showed that most enzymes involved in glycolysis, redox balance and amino acids metabolism were up-regulated with CaCO 3 addition. This study provides further insights into the role of CaCO 3 in n-butanol production using real biomass hydrolysate.

The sustainable production of solvents from above ground carbon is highly desired. Several clostridia naturally produce solvents and use a variety of renewable and waste-derived substrates such as lignocellulosic biomass and gas mixtures containing H 2 /CO 2 or CO. To enable economically viable production of solvents and biofuels such as ethanol and butanol, the high productivity of continuous bioprocesses is needed. While the first industrial-scale gas fermentation facility operates continuously, the acetone-butanol-ethanol (ABE) fermentation is traditionally operated in batch mode. This review highlights the benefits of continuous bioprocessing for solvent production and underlines the progress made towards its establishment. Based on metabolic capabilities of solvent producing clostridia, we discuss recent advances in systems-level understanding and genome engineering. On the process side, we focus on innovative fermentation methods and integrated product recovery to overcome the limitations of the classical one-stage chemostat and give an overview of the current industrial bioproduction of solvents.

n -Butanol is often considered a potential substitute for gasoline due to its physicochemical properties being closely related to those of gasoline. In this study, we extend our earlier work to convert endogenously producing butyrate via the FASII pathway using thioesterase TesBT to its corresponding alcohol, i.e., butanol. We first assembled pathway genes, i.e., car encoding carboxylic acid reductase from Mycobacterium marinum , sfp encoding phosphopantetheinyl transferase from Bacillus subtilis , and adh2 encoding alcohol dehydrogenase from S. cerevisiae , responsible for bioconversion of butyrate to butanol in three different configurations (Operon, Pseudo-Operon, and Monocistronic) to achieve optimum expression of each gene and compared with the clostridial solventogenic pathway for in vivo conversion of butyrate to butanol under aerobic conditions. An E. coli strain harboring car , sfp , and adh2 in pseudo-operon configuration was able to convert butyrate to butanol with 100% bioconversion efficiency when supplemented with 1 g/L of butyrate. Further, co-cultivation of an upstream strain (butyrate-producing) with a downstream strain (butyrate to butanol converting) at different inoculation ratios was investigated, and an optimized ratio of 1:4 (upstream strain: downstream strain) was found to produce ∼2 g/L butanol under fed-batch fermentation. Further, a mono-cultivation approach was applied by transforming a plasmid harboring tesBT gene into the downstream strain. This approach produced 0.42 g/L in a test tube and ∼2.9 g/L butanol under fed-batch fermentation. This is the first report where both mono- and co-cultivation approaches were tested and compared for butanol production, and butanol titers achieved using both strategies are the highest reported values in recombinant E. coli utilizing FASII pathway.

N-butanol, a valued solvent and potential fuel extender, could possibly be produced by fermentation using either native producers, i.e. solventogenic Clostridia, or engineered platform organisms such as Escherichia coli or Pseudomonas species, if the main process obstacle, a low final butanol concentration, could be overcome. A low final concentration of butanol is the result of its high toxicity to production cells. Nevertheless, bacteria have developed several mechanisms to cope with this toxicity and one of them is active butanol efflux. This review presents information about a few well characterized butanol efflux pumps from Gram-negative bacteria (P. putida and E. coli) and summarizes knowledge about putative butanol efflux systems in Gram-positive bacteria., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Acidogenic clostridia naturally producing acetic and butyric acids has attracted high interest as a novel host for butyrate and n-butanol production. Among them, Clostridium tyrobutyricum is a hyper butyrate-producing bacterium, which re-assimilates acetate for butyrate biosynthesis by butyryl-CoA/acetate CoA transferase (CoAT), rather than the phosphotransbutyrylase-butyrate kinase (PTB-BK) pathway widely found in clostridia and other microbial species. To date, C. tyrobutyricum has been engineered to overexpress a heterologous alcohol/aldehyde dehydrogenase, which converts butyryl-CoA to n-butanol. Compared to conventional solventogenic clostridia, which produce acetone, ethanol, and butanol in a biphasic fermentation process, the engineered C. tyrobutyricum with a high metabolic flux toward butyryl-CoA produced n-butanol at a high yield of > 0.30 g/g and titer of > 20 g/L in glucose fermentation. With no acetone production and a high C4/C2 ratio, butanol was the only major fermentation product by the recombinant C. tyrobutyricum, allowing simplified downstream processing for product purification. In this review, novel metabolic engineering strategies to improve n-butanol and butyrate production by C. tyrobutyricum from various substrates, including glucose, xylose, galactose, sucrose, and cellulosic hydrolysates containing the mixture of glucose and xylose, are discussed. Compared to other recombinant hosts such as Clostridium acetobutylicum and Escherichia coli, the engineered C. tyrobutyricum strains with higher butyrate and butanol titers, yields and productivities are the most promising hosts for potential industrial applications.

Background: Owing to the increase in energy consumption, fossil fuel resources are gradually depleting which has led to the growing environmental concerns; therefore, scientists are being urged to produce sustainable and ecofriendly fuels. Thus, there is a growing interest in the generation of biofuels from renewable energy resources using microbial fermentation., Main Text: Butanol is a promising biofuel that can substitute for gasoline; unfortunately, natural microorganisms pose challenges for the economical production of 1-butanol at an industrial scale. The availability of genetic and molecular tools to engineer existing native pathways or create synthetic pathways have made non-native hosts a good choice for the production of 1-butanol from renewable resources. Non-native hosts have several distinct advantages, including using of cost-efficient feedstock, solvent tolerant and reduction of contamination risk. Therefore, engineering non-native hosts to produce biofuels is a promising approach towards achieving sustainability. This paper reviews the currently employed strategies and synthetic biology approaches used to produce 1-butanol in non-native hosts over the past few years. In addition, current challenges faced in using non-native hosts and the possible solutions that can help improve 1-butanol production are also discussed., Conclusion: Non-native organisms have the potential to realize commercial production of 1- butanol from renewable resources. Future research should focus on substrate utilization, cofactor imbalance, and promoter selection to boost 1-butanol production in non-native hosts. Moreover, the application of robust genetic engineering approaches is required for metabolic engineering of microorganisms to make them industrially feasible for 1-butanol production.

The search for gasoline substitutes has grown in recent decades, leading to the increased production of ethanol as viable alternative. However, research in recent years has shown that butanol exhibits various advantages over ethanol as a biofuel. Furthermore, butanol can also be used as a chemical platform, serving as an intermediate product and as a solvent in industrial reactions. This alcohol is naturally produced by some Clostridium species; however, Clostridial fermentation processes still have inherent problems, which focuses the interest on Saccharomyces cerevisiae for butanol production, as an alternative organism for the production of this alcohol. S. cerevisiae exhibits great adaptability to industrial conditions and can be modified with a wide range of genetic tools. Although S. cerevisiae is known to naturally produce isobutanol, the n-butanol synthesis pathway has not been well established in wild S. cerevisiae strains. Two strategies are most commonly used for of S. cerevisiae butanol production: the heterologous expression of the Clostridium pathway or the amino acid uptake pathways. However, butanol yields produced from S. cerevisiae are lower than ethanol yield. Thus, there are still many challenges needed to be overcome, which can be minimized through genetic and evolutive engineering, for butanol production by yeast to become a reality.

The mitigation of end-product inhibition during the biosynthesis of n-butanol is demonstrated for an in-situ product recovery (ISPR) system employing a poly(ionic liquid) (PIL) absorbent. The thermodynamic affinity of poly(vinyldodecylimidazolium bromide) [P(VC 12 ImBr)] for n-butanol, acetone and ethanol versus water was measured at conditions experienced in a typical acetone-ethanol-butanol (ABE) fermentation. In addition to providing a high n-butanol partition coefficient (PC = 6.5) and selectivity (α BuOH/water = 46), P(VC 12 ImBr) is shown to be biocompatible with Saccharomyces cerevisiae and Clostridium acetobutylicum. Furthermore, the diffusivity of n-butanol in a hydrated PIL provides absorption rates that support ISPR applications. Using a 5 wt% PIL phase fraction relative to the aqueous phase mass, P(VC 12 ImBr) improved the volumetric productivity of a batch ABE ISPR process by 31% relative to a control fermentation. The concentration of n-butanol in the P(VC 12 ImBr) phase was sufficient to increase the alcohol concentration from 1.5 wt% in the fermentation medium to 25 wt% in the saturated PIL, thereby facilitating downstream n-butanol recovery., (© 2019 American Institute of Chemical Engineers.)