New regulatory role of Znf1 in transcriptional control of pentose phosphate pathway and ATP synthesis for enhanced isobutanol and acid tolerance.

To develop a cost-effective microbial cell factory for the production of biofuels and biochemicals, an understanding of tolerant mechanisms is vital for the construction of robust host strains. Here, we characterized a new function of a key metabolic transcription factor named Znf1 and its involvement in stress response in Saccharomyces cerevisiae to enhance tolerance to advanced biofuel, isobutanol. RNA-sequencing analysis of the wild-type versus the znf1Δ deletion strains in glucose revealed a new role for transcription factor Znf1 in the pentose phosphate pathway (PPP) and energy generation. The gene expression analysis confirmed that isobutanol induces an adaptive cell response, resulting in activation of ATP1-3 and COX6 expression. These genes were Znf1 targets that belong to the electron transport chain, important to produce ATPs. Znf1 also activated PPP genes, required for the generation of key amino acids, cellular metabolites, and maintenance of NADP/NADPH redox balance. In glucose, Znf1 also mediated the upregulation of valine biosynthetic genes of the Ehrlich pathway, namely ILV3, ILV5, and ARO10, associated with the generation of key intermediates for isobutanol production. Using S. cerevisiae knockout collection strains, cells with deleted transcriptional regulatory gene ZNF1 or its targets displayed hypersensitivity to isobutanol and acid inhibitors; in contrast, overexpression of ZNF1 enhanced cell survival. Thus, the transcription factor Znf1 functions in the maintenance of energy homeostasis and redox balance at various checkpoints of yeast metabolic pathways. It ensures the rapid unwiring of gene transcription in response to toxic products/by-products generated during biofuel production. Importantly, we provide a new approach to enhance strain tolerance during the conversion of glucose to biofuels.

[1-11C]-Butanol Positron Emission Tomography reveals an impaired brain to nasal turbinates pathway in aging amyloid positive subjects.

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 [1-11C]-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 [1-11C]-Butanol. Imaging with either [11C]-PiB or [18F]-FBB identified 8 amyloid PET positive (Aß+) and 16 Aß- 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 Aß+ 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.

Metabolically engineer Clostridium saccharoperbutylacetonicum for comprehensive conversion of acid whey into valuable biofuels and biochemicals.

As a byproduct of dairy production, the disposal of acid whey poses severe environmental challenges. Herein, an innovative solution involving metabolically engineering Clostridium saccharoperbutylacetonicum to convert all carbon sources in acid whey into sustainable biofuels and biochemicals was presented. By introducing several heterologous metabolic pathways relating to metabolisms of lactose, galactose, and lactate, the ultimately optimized strain, LM-09, exhibited exceptional performance by producing 15.1 g/L butanol with a yield of 0.33 g/g and a selectivity of 89.9%. Through further overexpression of alcohol acyl transferase, 2.7 g/L butyl acetate along with 6.4 g/L butanol was generated, resulting in a combined yield of 0.37 g/g. This study achieves the highest reported butanol titer and yield using acid whey as substrate in clostridia and marks pioneering production of esters using acid whey. The findings demonstrate an innovative bioprocess that enhances renewable feedstock biotransformation, thereby promoting economic viability and environmental sustainability of biomanufacturing.

Continuous H-B-E fermentation by Clostridium carboxidivorans: CO vs syngas.

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 CO2 + H2 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 (DL) 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 DL 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 gDM/L) was obtained with pure CO at DL 0.05 h-1; the highest solvent productivity (60 mg/L*h of total solvent) was obtained at DL 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.

Base editor-mediated large-scale screening of functional mutations in bacteria for industrial phenotypes.

Base editing, the targeted introduction of point mutations into cellular DNA, holds promise for improving genome-scale functional genome screening to single-nucleotide resolution. Current efforts in prokaryotes, however, remain confined to loss-of-function screens using the premature stop codons-mediated gene inactivation library, which falls far short of fully releasing the potential of base editors. Here, we developed a base editor-mediated functional single nucleotide variant screening pipeline in Escherichia coli. We constructed a library with 31,123 sgRNAs targeting 462 stress response-related genes in E. coli, and screened for adaptive mutations under isobutanol and furfural selective conditions. Guided by the screening results, we successfully identified several known and novel functional mutations. Our pipeline might be expanded to the optimization of other phenotypes or the strain engineering in other microorganisms.

Genomic insights into clostridia in bioenergy production: Comparison of metabolic capabilities and evolutionary relationships.

Bacteria from diverse genera, including Acetivibrio, Bacillus, Cellulosilyticum, Clostridium, Desulfotomaculum, Lachnoclostridium, Moorella, Ruminiclostridium, and Thermoanaerobacterium, have attracted significant attention due to their versatile metabolic capabilities encompassing acetogenic, cellulolytic, and C1-metabolic properties, and acetone-butanol-ethanol fermentation. Despite their biotechnological significance, a comprehensive understanding of clostridial physiology and evolution has remained elusive. This study reports an extensive comparative genomic analysis of 48 fully sequenced bacterial genomes from these genera. Our investigation, encompassing pan-genomic analysis, central carbon metabolism comparison, exploration of general genome features, and in-depth scrutiny of Cluster of Orthologous Groups genes, has established a holistic whole-genome-based phylogenetic framework. We have classified these strains into acetogenic, butanol-producing, cellulolytic, CO2-fixating, chemo(litho/organo)trophic, and heterotrophic categories, often exhibiting overlaps. Key outcomes include the identification of misclassified species and the revelation of insights into metabolic features, energy conservation, substrate utilization, stress responses, and regulatory mechanisms. These findings can provide guidance for the development of efficient microbial systems for sustainable bioenergy production. Furthermore, by addressing fundamental questions regarding genetic relationships, conserved genomic features, pivotal enzymes, and essential genes, this study has also contributed to our comprehension of clostridial biology, evolution, and their shared metabolic potential.

Adaptive laboratory evolution and transcriptomics-guided engineering of Escherichia coli for increased isobutanol tolerance.

As a renewable energy from biomass, isobutanol is considered as a promising alternative to fossil fuels. To biotechnologically produce isobutanol, strain development using industrial microbial hosts, such as Escherichia coli, has been conducted by introducing a heterologous isobutanol synthetic pathway. However, the toxicity of produced isobutanol inhibits cell growth, thereby restricting improvements in isobutanol titer, yield, and productivity. Therefore, the development of robust microbial strains tolerant to isobutanol is required. In this study, isobutanol-tolerant mutants were isolated from two E. coli parental strains, E. coli BL21(DE3) and MG1655(DE3), through adaptive laboratory evolution (ALE) under high isobutanol concentrations. Subsequently, 16 putative genes responsible for isobutanol tolerance were identified by transcriptomic analysis. When overexpressed in E. coli, four genes (fadB, dppC, acs, and csiD) conferred isobutanol tolerance. A fermentation study with a reverse engineered isobutanol-producing E. coli JK209 strain showed that fadB or dppC overexpression improved isobutanol titers by 1.5 times, compared to the control strain. Through coupling adaptive evolution with transcriptomic analysis, new genetic targets utilizable were identified as the basis for the development of an isobutanol-tolerant strain. Thus, these new findings will be helpful not only for a fundamental understanding of microbial isobutanol tolerance but also for facilitating industrially feasible isobutanol production.

Model construction and theoretical evaluation of the performance improvement of acetone-butanol-ethanol extractive fermentation by adding surfactant.

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.

Synthesis of isobutanol using acetate as sole carbon source in Escherichia coli.

BACKGROUND: With concerns about depletion of fossil fuel and environmental pollution, synthesis of biofuels such as isobutanol from low-cost substrate by microbial cell factories has attracted more and more attention. As one of the most promising carbon sources instead of food resources, acetate can be utilized by versatile microbes and converted into numerous valuable chemicals. RESULTS: An isobutanol synthetic pathway using acetate as sole carbon source was constructed in E. coli. Pyruvate was designed to be generated via acetyl-CoA by pyruvate-ferredoxin oxidoreductase YdbK or anaplerotic pathway. Overexpression of transhydrogenase and NAD kinase increased the isobutanol titer of recombinant E. coli from 121.21 mg/L to 131.5 mg/L under batch cultivation. Further optimization of acetate supplement concentration achieved 157.05 mg/L isobutanol accumulation in WY002, representing the highest isobutanol titer by using acetate as sole carbon source. CONCLUSIONS: The utilization of acetate as carbon source for microbial production of valuable chemicals such as isobutanol could reduce the consumption of food-based substrates and save production cost. Engineering strategies applied in this study will provide a useful reference for microbial production of pyruvate derived chemical compounds from acetate.

Preliminary evidence that Merkel cells exert chemosensory functions in human epidermis.

The mechanotransduction of light-touch sensory stimuli is considered to be the main physiological function of epidermal Merkel cells (MCs). Recently, however, MCs have been demonstrated to be also thermo-sensitive, suggesting that their role in skin physiologically extends well beyond mechanosensation. Here, we demonstrate that in healthy human skin epidermal MCs express functional olfactory receptors, namely OR2AT4, just like neighbouring keratinocytes. Selective stimulation of OR2AT4 by topical application of the synthetic odorant, Sandalore®, significantly increased Piccolo protein expression in MCs, as assessed by quantitative immunohistomorphometry, indicating increased vesicle trafficking and recycling, and significantly reduced nerve growth factor (NGF) immunoreactivity within MCs, possibly indicating increased neurotrophin release upon OR2AT4 activation. Live-cell imaging showed that Sandalore® rapidly induces a loss of FFN206-dependent fluorescence in MCs, suggesting OR2AT4-dependent MC depolarization and subsequent vesicle secretion. Yet, in contrast to keratinocytes, OR2AT4 stimulation by Sandalore® altered neither the number nor the proliferation status of MCs. These preliminary ex vivo findings demonstrate that epidermal MCs also exert OR-dependent chemosensory functions in human skin, and invite one to explore whether these newly identified properties are dysregulated in selected skin disorders, for example, in pruritic dermatoses, and if these novel MC functions can be therapeutically targeted to maintain/promote skin health.

Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a.

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.

Main genome characteristics of butanol-producing Clostridium sp. UCM В-7570 strain.

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.

Enhancement of biohydrogen production in Clostridium acetobutylicum ATCC 824 by overexpression of glyceraldehyde-3-phosphate dehydrogenase gene.

In the dark fermentation of hydrogen, development of production host is crucial as bacteria act on substrates and produce hydrogen. The present study aimed to improve hydrogen production through the development of Clostridium acetobutylicum as a superior biohydrogen producer. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which produces NADH/NADPH for metabolites and energy in primary pathways, was introduced to enhance hydrogen production. The strain CAC824-G containing gapC that encodes GAPDH showed a 66.3 % higher hydrogen production than the wild-type strain, with increased NADH and NADPH pools. Glucose consumption and other byproducts, such as acetone, butanol, and ethanol, were also high in CAC824-G. Overexpression of gapC resulted in increased hydrogen production with sugars obtained from different biomass, even in the presence of inhibitors such as vanillin, 5-hydroxymethylfufural, acetic acid, and formic acid. Our results imply that overexpression of gapC in Clostridium is possible to expand the production of the reported biochemicals to produce hydrogen.

The effect of Alnus incana (L.) Moench extracts in ameliorating iron overload-induced hepatotoxicity in male albino rats.

Iron overload causes multiorgan dysfunction and serious damage. Alnus incana from the family Betulaceae, widely distributed in North America, is used for treating diseases. In this study, we investigated the iron chelating, antioxidant, anti-inflammatory, and antiapoptotic activities of the total and butanol extract from Alnus incana in iron-overloaded rats and identified the bioactive components in both extracts using liquid chromatography-mass spectrometry. We induced iron overload in the rats via six intramuscular injections of 12.5 mg iron dextran/100 g body weight for 30 days. The rats were then administered 60 mg ferrous sulfate /kg body weight once daily using a gastric tube. The total and butanol extracts were given orally, and the reference drug (deferoxamine) was administered subcutaneously for another month. After two months, we evaluated the biochemical, histopathological, histochemical, and immunohistochemical parameters. Iron overload significantly increased the serum iron level, liver biomarker activities, hepatic iron content, malondialdehyde, tumor necrosis factor-alpha, and caspase-3 levels. It also substantially (P < 0.05) reduced serum albumin, total protein, and total bilirubin content, and hepatic reduced glutathione levels. It caused severe histopathological alterations compared to the control rats, which were markedly (P < 0.05) ameliorated after treatment. The total extract exhibited significantly higher anti-inflammatory and antiapoptotic activities but lower antioxidant and iron-chelating activities than the butanol extract. Several polyphenolic compounds, including flavonoids and phenolic acids, were detected by ultraperformance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) analysis. Our findings suggest that both extracts might alleviate iron overload-induced hepatoxicity and other pathological conditions characterized by hepatic iron overload, including thalassemia and sickle-cell anemia.

Efforts to install a heterologous Wood-Ljungdahl pathway in Clostridium acetobutylicum enable the identification of the native tetrahydrofolate (THF) cycle and result in early induction of solvents.

Here, we report the construction of a Clostridium acetobutylicum strain ATCC 824 (pCD07239) by heterologous expression of carbonyl branch genes (CD630_0723∼CD630_0729) from Clostridium difficile, aimed at installing a heterologous Wood-Ljungdahl pathway (WLP). As part of this effort, in order to validate the methyl branch of the WLP in the C. acetobutylicum, we performed 13C-tracing analysis on knockdown mutants of four genes responsible for the formation of 5-methyl-tetrahydrofolate (5-methyl-THF) from formate: CA_C3201, CA_C2310, CA_C2083, and CA_C0291. While C. acetobutylicum 824 (pCD07239) could not grow autotrophically, in heterotrophic fermentation, it began producing butanol at the early growth phase (OD600 of 0.80; 0.162 g/L butanol). In contrast, solvent production in the parent strain did not begin until the early stationary phase (OD600 of 7.40). This study offers valuable insights for future research on biobutanol production during the early growth phase.

A dynamic kinetic model captures cell-free metabolism for improved butanol production.

Cell-free systems are useful tools for prototyping metabolic pathways and optimizing the production of various bioproducts. Mechanistically-based kinetic models are uniquely suited to analyze dynamic experimental data collected from cell-free systems and provide vital qualitative insight. However, to date, dynamic kinetic models have not been applied with rigorous biological constraints or trained on adequate experimental data to the degree that they would give high confidence in predictions and broadly demonstrate the potential for widespread use of such kinetic models. In this work, we construct a large-scale dynamic model of cell-free metabolism with the goal of understanding and optimizing butanol production in a cell-free system. Using a combination of parameterization methods, the resultant model captures experimental metabolite measurements across two experimental conditions for nine metabolites at timepoints between 0 and 24 h. We present analysis of the model predictions, provide recommendations for butanol optimization, and identify the aldehyde/alcohol dehydrogenase as the primary bottleneck in butanol production. Sensitivity analysis further reveals the extent to which various parameters are constrained, and our approach for probing valid parameter ranges can be applied to other modeling efforts.

Minicell-forming Escherichia coli mutant with increased chemical production capacity and tolerance to toxic compounds.

Minicell, a small spherical form of bacterium produced by abnormal fission, possesses cytoplasmic constituents similar to those of the parental cell, except for genomic DNA. E. coli strains were engineered to produce minicells and value-added chemicals. Minicell-forming mutants showed enhanced tolerance to toxic chemicals and a higher intracellular NADH/NAD+ ratio than the wild-type. When toxic chemicals such as isobutanol, isobutyraldehyde, and isobutyl acetate were produced in this mutant, the titers increased by 67 %, 175 %, and 214 %, respectively. In addition, morphological changes and membrane dispersion mechanisms in minicell-forming mutants improved lycopene production by 259 %. This increase in production capacity was more pronounced when biomass hydrolysate was used as the substrate. Isobutanol and lycopene production also increased by 92 % and 295 %, respectively, on using the substrate in the mutant. It suggests that minicell-forming mutants are an excellent platform for biochemical production.

Ameliorative Effects of Thunbergia erecta L. Leaves Against the Initiation of Hepatocarcinogenesis Induced by Diethylnitrosamine in the Rat Model.

Thunbergia erecta L. contains cytotoxic and liver-protective compounds. Thunbergia erecta L. leaves were macerated in 70% aqueous ethanol, then fractionated with ethyl acetate (9.3 g) and butanol (12.7 g), and attenuated Den-induced liver cancer in a Wistar rat experimental model. Ethyl acetate and butanol fractions were chromatographed using column chromatography and solid-phase extraction (SPE); Vicenin-II (1), kaempferol (2), biochanin A, sissotrin 7-O-ß-glucopyranoside (3), gentianose (4), acacetin 7-O-ß-glucopyranoside (5), apigenin 7-O-ß-glucopyranoside (6), and rosmarinic acid (7) were extracted, and their structures were determined using NMR spectroscopy and ESI-mass spectrometry. Sixty rats were divided into six groups (ten each): control group, Den group, doxorubicin/Den-treated group, butanol fraction/Den-treated group, and isolated acacetin 7-O-ß-glucopyranoside/Den-treated group. The liver enzymes and proinflammatory biomarkers were used to estimate the liver function. In addition, liver tissues were collected for analysis of oxidative stress markers, gene expression, and histopathology. There is a significant increase in the levels of liver enzymes, AFP, and TNF-ἁ. This was conveyed by a significant increase of IL-1 and caspase-3, elevation of MDA and reduction of GSH, and suppression of Bcl2 and elevation of Bax expression. All parameters in butanol, ethyl acetate fractions, and isolated acacetin 7-O-ß-glucopyranoside (major constituents) of T. erecta L. were significantly improved to values close to those of the control group.

Production of biofuels from C1 -gases with Clostridium and related bacteria-Recent advances.

Clostridium spp. are suitable for the bioconversion of C1 -gases (e.g., CO2 , 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 C4 or C6 fatty acids, previously produced by a chain elongating microorganism (commonly Clostridium kluyveri), into the corresponding bioalcohol.

Bacterial metabolic engineering for the production of second-generation (2 G) bioethanol and biobutanol; a review.

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.