A gradient phage-assisted continuous evolution method for screening suppressor tRNAs in Escherichia coli.

Suppressor tRNAs, notable for their capability of reading through the stop codon while maintaining normal peptide synthesis, are promising in treating diseases caused by premature termination codons (PTC). However, the lack of effective engineering methods for suppressor tRNAs has curtailed their application potential. Here, we introduce a directed evolution technology that employs phage-assisted continuous evolution (PACE), combined with gradient biosensors featuring various PTCs in the M13 gene III. Utilizing this novel methodology, we have successfully evolved tRNATrp (UGG) reading through the UGA stop codon in Escherichia coli. Massively parallel sequencing revealed that these mutations predominantly occurred in the anticodon loop. Finally, two suppressor tRNATrp (UGA) mutants exhibited over fivefold increases in readthrough efficiency.

Directed Evolution of Escherichia coli Nissle 1917 to Utilize Allulose as Sole Carbon Source.

Sugar substitutes are popular due to their akin taste and low calories. However, excessive use of aspartame and erythritol can have varying effects. While D-allulose is presently deemed a secure alternative to sugar, its excessive consumption is not devoid of cellular stress implications. In this study, the evolution of Escherichia coli Nissle 1917 (EcN) is directed to utilize allulose as sole carbon source through a combination of adaptive laboratory evolution (ALE) and fluorescence-activated droplet sorting (FADS) techniques. Employing whole genome sequencing (WGS) and clustered regularly interspaced short palindromic repeats interference (CRISPRi) in conjunction with compensatory expression displayed those genetic mutations in sugar and amino acid metabolic pathways, including glnP, glpF, gmpA, nagE, pgmB, ybaN, etc., increased allulose assimilation. Enzyme-substrate dynamics simulations and deep learning predict enhanced substrate specificity and catalytic efficiency in nagE A247E and pgmB G12R mutants. The findings evince that these mutations hold considerable promise in enhancing allulose uptake and facilitating its conversion into glycolysis, thus signifying the emergence of a novel metabolic pathway for allulose utilization. These revelations bear immense potential for the sustainable utilization of D-allulose in promoting health and well-being.

Efficient isopropanol-butanol-ethanol (IBE) fermentation by a gene-modified solventogenic Clostridium species under the co-utilization of Fe(III) and butyrate.

To elevate the efficiency of acetone-butanol-ethanol (ABE) fermentation by the wild-type strain WK, an optimal co-utilization system (20 mM Fe3+ and 5 g/L butyrate) was established to bring about a 22.22% increment in the yield of ABE mixtures with a significantly enhanced productivity (0.32 g/L/h). With the heterologous introduction of the secondary alcohol dehydrogenase encoded gene (adh), more than 95% of acetone was eliminated to convert 4.5 g/L isopropanol with corresponding increased butanol and ethanol production by 21.08% and 65.45% in the modified strain WK::adh. Under the optimal condition, strain WK::adh was capable of producing a total of 25.46 g/L IBE biosolvents with an enhanced productivity of 0.35 g/L/h by 45.83% over the original conditions. This work for the first time successfully established a synergetic system of co-utilizing Fe(III) and butyrate to demonstrate a feasible and efficient manner for generating the value-added biofuels through the metabolically engineered solventogenic clostridial strain.

Elimination of carbon catabolite repression through gene-modifying a solventogenic Clostridium sp. strain WK to enhance butanol production from the galactose-rich red seaweed.

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.

Extracellular expression of agarolytic enzymes in Clostridium sp. strain and its application for butanol production from Gelidium amansii.

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.

Unraveling the unique butyrate re-assimilation mechanism of Clostridium sp. strain WK and the application of butanol production from red seaweed Gelidium amansii through a distinct acidolytic pretreatment.

Exploration of the algae-derived biobutanol synthesis has become one of the hotspots due to its highly cost-effective and environment-friendly features. In this study, a solventogenic strain Clostridium sp. strain WK produced 13.96 g/L butanol with a maximal yield of 0.41 g/g from glucose in the presence of 24 g/L butyrate. Transcriptional analysis indicated that the acid re-assimilation of this strain was predominantly regulated by genes buk-ptb rather than ctfAB, explaining its special phenotypes including high butyrate tolerance and the pH-independent fermentation. In addition, a butyric acid-mediated hydrolytic system was established for the first time to release a maximal yield of 0.35 g/g reducing sugars from the red algal biomass (Gelidium amansii). Moreover, 4.48 g/L of butanol was finally achieved with a significant enhancement by 29.9 folds. This work reveals an unconventional metabolic pathway for butanol synthesis in strain WK, and demonstrates the feasibility to develop renewable biofuels from marine resources.

Microbial debromination of hexabromocyclododecanes.

Hexabromocyclododecanes (HBCDs), a new sort of brominated flame retardants (BFRs), are globally prevalent and recalcitrant toxic environmental pollutants. HBCDs have been found in many environmental media and even in the human body, leading to serious health concerns. HBCDs are biodegradable in the environment. By now, dozens of bacteria have been discovered with the ability to transform HBCDs. Microbial debromination of HBCDs is via HBr-elimination, HBr-dihaloelimination, and hydrolytic debromination. Biotic transformation of HBCDs yields many hydroxylated and lower brominated compounds which lack assessment of ecological toxicity. Bioremediation of HBCD pollution has only been applied in the laboratory. Here, we review the current knowledge about microbial debromination of HBCDs, aiming to promote the bioremediation applied in HBCD contaminated sites. KEY POINTS: • Microbial debromination of HBCDs is via hydrolytic debromination, HBr-elimination, and HBr-dihaloelimination. • Newly occurred halogenated contaminants such as HBCDs hitch the degradation pathway tamed by previously discharged anthropogenic organohalides. • Strategy that combines bioaugmentation with phytoremediation for bioremediation of HBCD pollution is promising.

Emerging technologies for genetic modification of solventogenic clostridia: From tool to strategy development.

Solventogenic clostridia has been considered as one of the most potential microbial cell factories for biofuel production in the biorefinery industry. However, the inherent shortcomings of clostridia strains such as low productivity, by-products formation and toxic tolerance still strongly restrict the large-scale application. Therefore, concerns regarding the genetic modification of solventogenic clostridia have spurred interests into the development of modern gene-editing tools. In this review, we summarize the latest advances of genetic tools involved in modifying solventogenic clostridia. Following a systematic comparison on their respective characteristics, we then review the corresponding strategies for overcoming the obstacles to the enhanced production. Discussing the progress of other microbial cell factories for solventogenesis, we finally describe the key challenges and trends with valuable recommendations for future large-scale biosolvent industrial application.

Emerging technologies for conversion of sustainable algal biomass into value-added products: A state-of-the-art review.

Concerns regarding high energy demand and gradual depletion of fossil fuels have attracted the desire of seeking renewable and sustainable alternatives. Similar to but better than the first- and second-generation biomass, algae derived third-generation biorefinery aims to generate value-added products by microbial cell factories and has a great potential due to its abundant, carbohydrate-rich and lignin-lacking properties. However, it is crucial to establish an efficient process with higher competitiveness over the current petroleum industry to effectively utilize algal resources. In this review, we summarize the recent technological advances in maximizing the bioavailability of different algal resources. Following an overview of approaches to enhancing the hydrolytic efficiency, we review prominent opportunities involved in microbial conversion into various value-added products including alcohols, organic acids, biogas and other potential industrial products, and also provide key challenges and trends for future insights into developing biorefineries of marine biomass.

Acidolysis as a biorefinery approach to producing advanced bioenergy from macroalgal biomass: A state-of-the-art review.

Facing fossil fuels consumption and its accompanying environmental pollution, macroalgae, as a major part of the third-generation (3G) biomass, has great potential for bioenergy development due to its species-abundant, renewable and carbohydrate-rich properties. Diluted acid treatment is one of the most effective approaches to releasing fermentable sugars from macroalgal biomass in a short period, but the optimal conditions need to be explored to maximize the hydrolytic yield for the subsequent microbial conversion. Therefore, this review aims to summarize the latest advances in various acids and other auxiliary methods adopted to increase the hydrolytic efficiency of macroalgae. Following an overview of the strategies of different algal types, methods involved in the bioconversion of biofuels and microbial fuel cells (MFC) from algal hydrolysates are also described. For the 3G biorefinery development, the review further discusses key challenges and trends for future utilizing marine biomass to achieve the large-scale industrial production.

Biobutanol production from sulfuric acid-pretreated red algal biomass by a newly isolated Clostridium sp. strain WK.

Marine biomass, especially the algal biomass, is currently considered to be one of the most potential candidates for biofuels conversion during the development of biomass utilization. In this study, a diluted sulfuric acid pretreatment method was established for biobutanol from red algal biomass Gelidium amansii using a newly isolated Clostridium sp. strain WK. Under the optimal condition of 2% sulfuric acid treated in 20 Min at 131 °C, the maximal hydrolysis percentage of biomass can reach up to 80.95%, and the biobutanol production was obtained to be 3.46 g/L with a yield of 0.20 g/g after the fermentation of biomass hydrolysate. This result demonstrated a 12.5-fold enhancement of conversion efficiency compared with the untreated control, which provides a new and efficient way to develop the biobutanol industry by utilizing the abundant, low cost, and carbohydrate-rich algal biomass.

Enhanced bioconversion of hemicellulosic biomass by microbial consortium for biobutanol production with bioaugmentation strategy.

As a renewable and sustainable source for next-generation biofuel production, lignocellulosic biomass can be effectively utilized in environmentally friendly manner. In this study, a stable, xylan-utilizing, anaerobic microbial consortium MC1 enriched from mangrove sediments was established, and it was taxonomically identified that the genera Ruminococcus and Clostridium from this community played a crucial role in the substrate utilization. In addition, a butanol-producing Clostridium sp. strain WST was introduced via the bioaugmentation process, which resulted in the conversion of xylan to biobutanol up to 10.8 g/L, significantly improving the butanol yield up to 0.54 g/g by 98-fold. When this system was further applied to other xylan-rich biomass, 1.09 g/L of butanol could be achieved from 20 g/L of corn cob. These results provide another new method to efficiently convert xylan, the main hemicellulose from lignocellulosic biomass into biofuels through a low-cost and eco-friendly manner.

The Draft Genome Sequence of a Novel High-Efficient Butanol-Producing Bacterium Clostridium Diolis Strain WST.

A wild-type solventogenic strain Clostridium diolis WST, isolated from mangrove sediments, was characterized to produce high amount of butanol and acetone with negligible level of ethanol and acids from glucose via a unique acetone-butanol (AB) fermentation pathway. Through the genomic sequencing, the assembled draft genome of strain WST is calculated to be 5.85 Mb with a GC content of 29.69% and contains 5263 genes that contribute to the annotation of 5049 protein-coding sequences. Within these annotated genes, the butanol dehydrogenase gene (bdh) was determined to be in a higher amount from strain WST compared to other Clostridial strains, which is positively related to its high-efficient production of butanol. Therefore, we present a draft genome sequence analysis of strain WST in this article that should facilitate to further understand the solventogenic mechanism of this special microorganism.

High-efficient production of biobutanol by a novel Clostridium sp. strain WST with uncontrolled pH strategy.

A novel Clostridium sp. strain WST isolated from mangrove sediments demonstrated its unique characteristics of producing high titer of biobutanol from low concentration of substrates via anaerobic fermentation. The strain is able to convert glucose and galactose to high amount of biobutanol up to 16.62 and 12.11 g/L, respectively, and the yields of 0.54 and 0.55 g/g were determined to be much higher than those from the previous reports on Clostridial batch fermentation. Moreover, the inherent strong regulatory system of strain WST also prompts itself to perform the fermentation process without any requirement of pH control. In addition to tolerance of high butanol concentration and negligible production of by-products (e.g., ethanol or acids), this strain has immense potential for the sustainable industry-scale production of biobutanol.

Genomic comparison of Clostridium species with the potential of utilizing red algal biomass for biobutanol production.

BACKGROUND: Sustainable biofuels, which are widely considered as an attractive alternative to fossil fuels, can be generated by utilizing various biomass from the environment. Marine biomass, such as red algal biomass, is regarded as one potential renewable substrate source for biofuels conversion due to its abundance of fermentable sugars (e.g., galactose). Previous studies focused on the enhancement of biofuels production from different Clostridium species; however, there has been limited investigation into their metabolic pathways, especially on the conversion of biofuels from galactose, via whole genomic comparison and evolutionary analysis. RESULTS: Two galactose-utilizing Clostridial strains were examined and identified as Clostridium acetobutylicum strain WA and C. beijerinckii strain WB. Via the genomic sequencing of both strains, the comparison of the whole genome together with the relevant protein prediction of 33 other Clostridium species was established to reveal a clear genome profile based upon various genomic features. Among them, five representative strains, including C. beijerinckii NCIMB14988, C. diolis DSM 15410, C. pasteurianum BC1, strain WA and WB, were further discussed to demonstrate the main differences among their respective metabolic pathways, especially in their carbohydrate metabolism. The metabolic pathways involved in the generation of biofuels and other potential products (e.g., riboflavin) were also reconstructed based on the utilization of marine biomass. Finally, a batch fermentation process was performed to verify the fermentative products from strains WA and WB using 60 g/L of galactose, which is the main hydrolysate from algal biomass. It was observed that strain WA and WB could produce up to 16.98 and 12.47 g/L of biobutanol, together with 21,560 and 10,140 mL/L biohydrogen, respectively. CONCLUSIONS: The determination of the production of various biofuels by both strains WA and WB and their genomic comparisons with other typical Clostridium species on the analysis of various metabolic pathways was presented. Through the identification of their metabolic pathways, which are involved in the conversion of galactose into various potential products, such as biobutanol, the obtained results extend the current insight into the potential capability of utilizing marine red algal biomass and provide a systematic investigation into the relationship between this genus and the generation of sustainable bioenergy.

Catalytic hydrolysis of starch for biohydrogen production by using a newly identified amylase from a marine bacterium Catenovulum sp. X3.

An identified cold-adaptive, organic solvents-tolerant alkaline α-amylase (HP664) from Catenovulum sp. strain X3 was heterologously expressed and characterized in E. coli, and it was further applied to starch saccharification for biohydrogen production. The recombinant HP664 belongs to a member of glycoside hydrolase family 13 (GH13), with a molecular weight of 69.6kDa without signal peptides, and also shares a relatively low similarity (49%) to other reported amylases. Biochemical characterization demonstrated that the maximal enzymatic activity of HP664 was observed at 35°C and pH 9.0. Most metal ions inhibited its activity; however, low polar organic solvents (e.g., benzene and n-hexane) could enhance the activity by 35-50%. Additionally, HP664 also exhibited the catalytic capability on various polysaccharides, including potato starch, amylopectin, dextrin and agar. In order to increase the bioavailability of starch for H2 production, HP664 was utilized to elevate fermentable oligosaccharide level, and the results revealed that the maximal hydrolytic percentage of starch was up to 44% with 12h of hydrolysis using 5.63U of HP664. Biohydrogen fermentation of the starch hydrolysate by Clostridium sp. strain G1 yielded 297.7mL of H2 after 84h of fermentation, which is 3.73-fold higher than the control without enzymatic treatment of HP664.

Synergistic enzymatic saccharification and fermentation of agar for biohydrogen production.

Nowadays, marine biomass is gradually considered as another utilizable material for the sustainable bioenergy development. In the present study, galactose, the main component of agar polysaccharide, was utilized for the biohydrogen production by Enterobacter sp. CN1. The highest hydrogen yield of 303.2mL/g was obtained in the cultivation media containing 5.87g/L of galactose, together with initial pH of 7.3 and incubation temperature of 36°C, after the response surface methodology (RSM) analysis. After the saccharification process by the agarase (AgaXa) and neoagarobiose hydrolase (NH852), the agar hydrolysate obtained was further applied to generate biohydrogen by strain CN1. Under the synergistic enzymatic saccharification and fermentation process, the production of biohydrogen was obtained to be 5047±228mL/L from 50g/L of agar, resulting in 3.86-fold higher than the control without enzymatic pretreatment.

Characterization of a xylanase-producing Cellvibrio mixtus strain J3-8 and its genome analysis.

Cellvibrio mixtus strain J3-8 is a gram-negative, xylanase-producing aerobic soil bacterium isolated from giant snails in Singapore. It is able to produce up to 10.1 U ml(-1) of xylanase, which is comparable to xylanase production from known bacterial and fungal strains. Genome sequence analysis of strain J3-8 reveals that the assembled draft genome contains 5,171,890 bp with a G + C content of 46.66%, while open reading frame (ORF) annotations indicate a high density of genes encoding glycoside hydrolase (GH) families involved in (hemi)cellulose hydrolysis. On the basis of 15 identified putative xylanolytic genes, one metabolic pathway in strain J3-8 is constructed for utilization of xylan. In addition, a 1,083 bp xylanase gene from strain J3-8 represents a new member of GH11 family. This gene is verified to be novel via phylogenetic analysis. To utilize this novel gene for hydrolysis of xylan to xylose, it is expressed in recombinant E. coli and characterized for its hydrolytic activity. This study shows that strain J3-8 is a potential candidate for hydrolysis of lignocellulosic materials.

Draft Genome Sequence of a Xylanase-Producing Bacterial Strain, Cellvibrio mixtus J3-8.

The xylanase-producing bacterial strain Cellvibrio mixtus J3-8 was isolated from grassland giant snails. The draft genome of strain J3-8 comprises 5,171,890 bp in 152 contigs with a G+C content of 46.66%. This is the first genome report about this bacterial species.