Improvement of succinate production from methane by combining rational engineering and laboratory evolution in Methylomonas sp. DH-1.

Recently, methane has been considered a next-generation carbon feedstock due to its abundance and it is main component of shale gas and biogas. Methylomonas sp. DH-1 has been evaluated as a promising industrial bio-catalyst candidate.

Metabolic engineering of Caldicellulosiruptor bescii for hydrogen production.

Hydrogen is an alternative fuel for transportation vehicles because it is clean, sustainable, and highly flammable. However, the production of hydrogen from lignocellulosic biomass by microorganisms presents challenges. This microbial process involves multiple complex steps, including thermal, chemical, and mechanical treatment of biomass to remove hemicellulose and lignin, as well as enzymatic hydrolysis to solubilize the plant cell walls.

Hydrogen Production from Barley Straw and Miscanthus by the Hyperthermophilic Bacterium, .

This work aimed to evaluate the feasibility of biohydrogen production from Barley Straw and Miscanthus. The primary obstacle in plant biomass decomposition is the recalcitrance of the biomass itself. Plant cell walls consist of cellulose, hemicellulose, and lignin, which make the plant robust to decomposition.

Comprehensive Characterization of Mutant Co-Fermenting Cellobiose and Xylose through Genomic and Transcriptomic Analyses.

The development of a yeast strain capable of fermenting mixed sugars efficiently is crucial for producing biofuels and value-added materials from cellulosic biomass. Previously, a mutant YN14 strain capable of co-fermenting xylose and cellobiose was developed through evolutionary engineering of the wild-type CBS6054 strain, which was incapable of cofermenting xylose and cellobiose. In this study, through genomic and transcriptomic analyses, we sought to investigate the reasons for the improved sugar metabolic performance of the mutant YN14 strain in comparison with the parental CBS6054 strain.

Valorization of CO to β-farnesene in Rhodobacter sphaeroides.

The valorization of CO into valuable products is a sustainable strategy to help overcome the climate crisis. In particular, biological conversion is attractive as it can produce long-chain hydrocarbons such as terpenoids. This study reports the high yield of β-farnesene production from CO by expressing heterologous β-farnesene synthase (FS) into Rhodobacter sphaeroides.

Metabolite profiling and pathway prediction of laver (Porphyra dentata) kombucha during fermentation at different temperatures.

This study is aimed to explore the impact of fermentation temperature on laver kombucha by profiling the accumulation and degradation of metabolites and elucidating their related pathways of metabolism. Laver kombucha was produced through ultrasound-assisted extraction and fermentation using a biofilm called SCOBY at 25 and 30 °C (hereafter named K-25 and K-30, respectively) for 14 days. Overall, organic acids, soluble sugars, amino acids, and phenolic compounds were found to participate in the biosynthesis pathway.

Regulation of Reactive Oxygen Species Promotes Growth and Carotenoid Production Under Autotrophic Conditions in .

Industrial demand for capture and utilization using microorganisms to reduce CO, a major cause of global warming, is significantly increasing. is a suitable strain for the process of converting CO into high-value materials because it can accept CO and has various metabolic pathways. However, it has been mainly studied for heterotrophic growth that uses sugars and organic acids as carbon sources, not autotrophic growth.

Effects of Engineered Fermenting Cellobiose through Low-Energy-Consuming Phosphorolytic Pathway in Simultaneous Saccharification and Fermentation.

Until recently, four types of cellobiose-fermenting strains have been developed by introduction of a cellobiose metabolic pathway based on either intracellular β-glucosidase (GH1-1) or cellobiose phosphorylase (CBP), along with either an energy-consuming active cellodextrin transporter (CDT-1) or a non-energy-consuming passive cellodextrin facilitator (CDT-2). In this study, the ethanol production performance of two cellobiose-fermenting strains expressing mutant CDT-2 (N306I) with GH1-1 or CBP were compared with two cellobiose-fermenting strains expressing mutant CDT-1 (F213L) with GH1-1 or CBP in the simultaneous saccharification and fermentation (SSF) of cellulose under various conditions. It was found that, regardless of the SSF conditions, the phosphorolytic cellobiose-fermenting expressing mutant CDT-2 with CBP showed the best ethanol production among the four strains.

Observation of Cellodextrin Accumulation Resulted from Non-Conventional Secretion of Intracellular β-Glucosidase by Engineered Fermenting Cellobiose.

Although engineered fermenting cellobiose is useful for the production of biofuels from cellulosic biomass, cellodextrin accumulation is one of the main problems reducing ethanol yield and productivity in cellobiose fermentation with expressing cellodextrin transporter (CDT) and intracellular β-glucosidase (GH1-1). In this study, we investigated the reason for the cellodextrin accumulation and how to alleviate its formation during cellobiose fermentation using engineered fermenting cellobiose. From the series of cellobiose fermentation using expressing only GH1-1 under several culture conditions, it was discovered that small amounts of GH1-1 were secreted and cellodextrin was generated through trans-glycosylation activity of the secreted GH1-1.

An extra copy of the β-glucosidase gene improved the cellobiose fermentation capability of an engineered strain.

In a previously engineered recombinant, the cellobiose fermentation rate was significantly lower than the glucose fermentation rate. Thus, we implemented a genome-wide perturbation library to find gene targets for improving the cellobiose fermentation capability of the yeast strain. Unexpectedly, we discovered a transformant that contained an additional β-glucosidase gene (-), possibly through homologous recombination between the plasmids.

Enhanced cellobiose fermentation by engineered Saccharomyces cerevisiae expressing a mutant cellodextrin facilitator and cellobiose phosphorylase.

To efficiently ferment intermediate cellodextrins released during cellulose hydrolysis, Saccharomyces cerevisiae has been engineered by introduction of a heterologous cellodextrin utilizing pathway consisting of a cellodextrin transporter and either an intracellular β-glucosidase or a cellobiose phosphorylase. Among two types of cellodextrin transporters, the passive facilitator CDT-2 has not enabled better cellobiose fermentation than the active transporter CDT-1, which suggests that the CDT-2 might be engineered to provide energetic benefits over the active transporter in cellobiose fermentation. We attempted to improve cellobiose transporting activity of CDT-2 through laboratory evolution.

Evaluation of Ethanol Production Activity by Engineered Saccharomyces cerevisiae Fermenting Cellobiose through the Phosphorolytic Pathway in Simultaneous Saccharification and Fermentation of Cellulose.

In simultaneous saccharification and fermentation (SSF) for production of cellulosic biofuels, engineered capable of fermenting cellobiose has provided several benefits, such as lower enzyme costs and faster fermentation rate compared with wild-type fermenting glucose. In this study, the effects of an alternative intracellular cellobiose utilization pathway-a phosphorolytic pathway based on a mutant cellodextrin transporter (CDT-1 (F213L)) and cellobiose phosphorylase (SdCBP)-was investigated by comparing with a hydrolytic pathway based on the same transporter and an intracellular β-glucosidase (GH1-1) for their SSF performances under various conditions. Whereas the phosphorolytic and hydrolytic cellobiose-fermenting strains performed similarly under the anoxic SSF conditions, the hydrolytic performed slightly better than the phosphorolytic under the microaerobic SSF conditions.

Improved ethanol production by engineered Saccharomyces cerevisiae expressing a mutated cellobiose transporter during simultaneous saccharification and fermentation.

Although simultaneous saccharification and fermentation (SSF) of cellulosic biomass can offer efficient hydrolysis of cellulose through alleviating feed-back inhibition of cellulases by glucose, supplementation of β-glucosidase is necessary because most fermenting microorganisms cannot utilize cellobiose. Previously, we observed that SSF of cellulose by an engineered Saccharomyces cerevisiae expressing a cellobiose transporter (CDT-1) and an intracellular β-glucosidase (GH1-1) without β-glucosidase could not be performed as efficiently as the traditional SSF with extracellular β-glucosidase. However, we improved the ethanol production from SSF of cellulose by employing a further engineered S.

Optimization of Expression Conditions Enhances Production of Sepiapterin, a Precursor for Tetrahydrobiopterin Biosynthesis, in Recombinant Escherichia coli.

Sepiapterin is a precursor for the synthesis of tetrahydrobiopterin (BH4), which is a wellknown cofactor for aromatic amino acid hydroxylation and nitric oxide synthesis in higher mammals. In this study, a recombinant Escherichia coli BL21(DE3) strain harboring cyanobacterial guanosine 5'-triphosphate cyclohydrolase 1 (GCH1) and human 6- pyruvoyltetrahydropterin synthase (PTPS) genes was constructed to produce sepiapterin. The optimum conditions for T7 promoter-driven expression of GCH1 and PTPS were 30°C and 0.

Enhanced production of 2'-fucosyllactose in engineered Escherichia coli BL21star(DE3) by modulation of lactose metabolism and fucosyltransferase.

2'-Fucosyllactose (2-FL) is one of most abundant functional oligosaccharides in human milk, which is involved in many biological functions for human health. To date, most microbial systems for 2-FL production have been limited to use Escherichia coli JM strains since they cannot metabolize lactose. In this study, E.

Enhanced production of ε-caprolactone by coexpression of bacterial hemoglobin gene in recombinant Escherichia coli expressing cyclohexanone monooxygenase gene.

Baeyer-Villiger (BV) oxidation of cyclohexanone to epsilon-caprolactone in a microbial system expressing cyclohexanone monooxygenase (CHMO) can be influenced by not only the efficient regeneration of NADPH but also a sufficient supply of oxygen. In this study, the bacterial hemoglobin gene from Vitreoscilla stercoraria (vhb) was introduced into the recombinant Escherichia coli expressing CHMO to investigate the effects of an oxygen-carrying protein on microbial BV oxidation of cyclohexanone. Coexpression of Vhb allowed the recombinant E.

Analysis of cellodextrin transporters from Neurospora crassa in Saccharomyces cerevisiae for cellobiose fermentation.

Saccharomyces cerevisiae can be engineered to ferment cellodextrins produced by cellulases as a product of cellulose hydrolysis. Direct fermentation of cellodextrins instead of glucose is advantageous because glucose inhibits cellulase activity and represses the fermentation of non-glucose sugars present in cellulosic hydrolyzates. To facilitate cellodextrin utilization by S.

Simultaneous saccharification and fermentation by engineered Saccharomyces cerevisiae without supplementing extracellular β-glucosidase.

Simultaneous saccharification and fermentation (SSF) has been considered a promising and economical process for cellulosic ethanol production. Further cost savings could be gained by reducing enzyme loading and engineering host strain for ethanol production. In this study, we demonstrate efficient ethanol production by SSF without supplementation of β-glucosidase using an engineered Saccharomyces cerevisiae strain expressing a cellodextrin transporter and an intracellular β-glucosidase from Neurospora crassa.

Engineering of NADPH regenerators in Escherichia coli for enhanced biotransformation.

Efficient regeneration of NADPH is one of the limiting factors that constrain the productivity of biotransformation processes. In order to increase the availability of NADPH for enhanced biotransformation by engineered Escherichia coli, modulation of the pentose phosphate pathway and amplification of the transhydrogenases system have been conventionally attempted as primary solutions. Recently, other approaches for stimulating NADPH regeneration during glycolysis, such as replacement of native glyceradehdye-3-phosphate dehydrogenase (GAPDH) with NADP-dependent GAPDH from Clostridium acetobutylicum and introduction of NADH kinase catalyzing direct phosphorylation of NADH to NADPH from Saccharomyces cerevisiae, were attempted and resulted in remarkable impacts on NADPH-dependent bioprocesses.

Construction of an efficient xylose-fermenting diploid Saccharomyces cerevisiae strain through mating of two engineered haploid strains capable of xylose assimilation.

Saccharomyces cerevisiae can be engineered for xylose fermentation through introduction of wild type or mutant genes (XYL1/XYL1 (R276H), XYL2, and XYL3) coding for xylose metabolic enzymes from Scheffersomyces stipitis. The resulting engineered strains, however, often yielded undesirable phenotypes such as slow xylose assimilation and xylitol accumulation. In this study, we performed the mating of two engineered strains that exhibit suboptimal xylose-fermenting phenotypes in order to develop an improved xylose-fermenting diploid strain.

Two-stage acidic-alkaline hydrothermal pretreatment of lignocellulose for the high recovery of cellulose and hemicellulose sugars.

The focus of this work was to develop a combined acid and alkaline hydrothermal pretreatment of lignocellulose that ensures high recovery of both hexose and pentose. Dilute sulfuric acid and lime pretreatments were employed sequentially. Process performance was optimized in terms of catalyst concentration, retention time, and temperature using response surface methodology.

Enhanced xylitol production through simultaneous co-utilization of cellobiose and xylose by engineered Saccharomyces cerevisiae.

As Saccharomyces cerevisiae cannot utilize xylose as a carbon source, expression of XYL1 coding for xylose reductase (XR) from Scheffersomyces (Pichia) stipitis enabled production of xylitol from xylose with a high yield. However, insufficient supply of NAD(P)H for XR and inhibition of xylose uptake by glucose are identified as major constraints for achieving high xylitol productivity. To overcome these problems, we engineered S.

Effects of NADH kinase on NADPH-dependent biotransformation processes in Escherichia coli.

Sufficient supply of NADPH is one of the most important factors affecting the productivity of biotransformation processes. In this study, construction of an efficient NADPH-regenerating system was attempted using direct phosphorylation of NADH by NADH kinase (Pos5p) from Saccharomyces cerevisiae for producing guanosine diphosphate (GDP)-L-fucose and ε-caprolactone in recombinant Escherichia coli. Expression of Pos5p in a fed-batch culture of recombinant E.

Whole cell biosynthesis of a functional oligosaccharide, 2'-fucosyllactose, using engineered Escherichia coli.

Background: 2'-Fucosyllactose (2-FL) is a functional oligosaccharide present in human milk which protects against the infection of enteric pathogens. Because 2-FL can be synthesized through the enzymatic fucosylation of lactose with guanosine 5'-diphosphate (GDP)-l-fucose by α-1,2-fucosyltransferase (FucT2), an 2-FL producing Escherichia coli can be constructed through overexpressing genes coding for endogenous GDP- l-fucose biosynthetic enzymes and heterologous fucosyltransferase.

Results: The gene for FucT2 from Helicobacter pylori was introduced to the GDP-l-fucose producing recombinant E.