Transcription factor-based biosensor: A molecular-guided approach for advanced biofuel synthesis.

As a sustainable and renewable alternative to petroleum fuels, advanced biofuels shoulder the responsibility of energy saving, emission reduction and environmental protection. Traditional engineering of cell factories for production of advanced biofuels lacks efficient high-throughput screening tools and regulating systems, impeding the improvement of cellular productivity and yield. Transcription factor-based biosensors have been widely applied to monitor and regulate microbial cell factory products due to the advantages of fast detection and in-situ screening. This review updates the design and application of transcription factor-based biosensors tailored for advanced biofuels and related intermediates. The construction and genetic parts selection principle of biosensors are discussed. Strategies to enhance the performance of biosensor, including regulating promoter strength and RBS strength, optimizing plasmid copy number, implementing genetic amplifier, and modulating the structure of transcription factor, have also been summarized. We further review the application of biosensors in high-throughput screening of new metabolic engineering targets, evolution engineering, confirmation of protein function, and dynamic regulation of metabolic flux for higher production of advanced biofuels. At last, we discuss the current limitations and future trends of transcription factor-based biosensors.

Life-cycle analysis of biohydrogen production via dark-photo fermentation from wheat straw.

This study presents a life-cycle analysis using energy conversion characteristics as an evaluation index to assess the feasibility of this production method. The results indicate that for a system processing 1000 kg/h of wheat straw, the addition of 12000 kg/h of 2 wt% H2SO4 and 120 kg/h of CH3COONa yields 340,000 L/h of H2 and 348.6 kW of electricity. The energy conversion efficiency from the feedstock to the product is 21.4 %, while the efficiency from the hydrolysate to the product is 62.2 %. The total CO2 emission is 27.1 kg/h. Variations in the hydrolysate have the most significant impact on energy conversion efficiency. This study explores the feasibility of industrial-scale biohydrogen production via dark-photo fermentation from wheat straw and analyzes the energy characteristic indices and the sensitivity of these indices to key parameters.

Endopeptidase O promotes Streptococcus suis immune evasion by cleaving the host- defence peptide cathelicidins.

Streptococcus suis is a zoonotic Gram-positive bacterium that causes invasive infections such as sepsis and meningitis, threatening public health worldwide. For successful establishment of infection, the bacterium should subvert the innate effectors of immune defence, including the cathelicidin family of host-defence peptides that combat pathogenic bacteria by directly disrupting cell membranes and coordinating immune responses. Here, our study shows that an extracellular endopeptidase O (PepO) of S. suis contributes to assisting the bacterium to resist cathelicidin-mediated killing, as the deletion of the pepO gene makes S. suis more sensitive to the human cathelicidin LL-37, as well as its mouse equivalent, mCRAMP. This protease targets and cleaves both LL-37 and mCRAMP, degrading them into shorter peptides with only a few amino acids, thereby abrogating their ability to kill S. suis. By cleaving LL-37 and mCRAMP, PepO impairs their chemotactic properties for neutrophil migration and undermines their anti-apoptosis activity, which is required for prolonging neutrophil lifespan. Also, PepO inhibits the ability of LL-37 and mCRAMP to promote lysosome development in macrophages. Moreover, the loss of PepO attenuates organ injury and decreases bacterial burdens in a murine model of S. suis bacteraemia. Taken together, these data provide novel insights into the role of the intrinsic proteolytic characteristics of PepO in S. suis-host interaction. Our findings demonstrate that S. suis utilizes the PepO protease to cleave cathelicidins, which is an immunosuppressive strategy adopted by this bacterium to facilitate pathogenesis.

Boosting photo-induced antimicrobial activity of lignin nanoparticles with curcumin and zinc oxide.

Lignin nanoparticles have gained increasing attention as a potential antimicrobial agent due to their biocompatibility, biodegradability, and low toxicity. However, the limited ability of lignin to act as an antibacterial is a major barrier to its widespread use. Thus, it is crucial to develop novel approaches to amplify lignin's biological capabilities in order to promote its effective utilization. In this study, we modified lignin nanoparticles (LNPs) with photo-active curcumin (Cur), zinc oxide (ZnO), or a combination of both to enhance their antimicrobial properties. The successful modifications of LNPs were confirmed using comprehensive characterization techniques. The antimicrobial efficacy of the modified LNPs was assessed against both gram-positive and gram-negative bacterial strains. The results showed that the modification of LNPs with Cur and ZnO have much higher antibacterial and antibiofilm activities than unmodified LNPs. Moreover, photo illumination resulted in even higher antibacterial activity. Furthermore, atomic force microscopy revealed bacterial cells lysis and membrane damage by ZnO/Cur modified LNPs. Our research demonstrates that ZnO/Cur modified LNPs can serve as novel hybrid materials with enhanced antimicrobial capabilities. In addition, the photo-induced enhancement in antibacterial activity not only demonstrated the versatility of this hybrid material but also opened up interesting potential for bioinspired therapeutics agents.

High titer (>200 g/L) lactic acid production from undetoxified pretreated corn stover.

Lignocellulosic biomass is a reliable feedstock for lactic acid fermentation, low product titers hamper the scale production of cellulosic lactic acid. In this study, a Densifying Lignocellulosic biomass with Chemicals (sulfuric acid) pretreatment based cellulosic lactic acid biorefinery system was developed and demonstrated from multi-dimensions of producing bacteria, fermentation modes, corn stover solid loadings, fermentation vessels, and product purification. Results suggested that several lactic acid bacteria exhibited high fermentation activity in high solid loading corn stover hydrolysates. Remarkably, simultaneous saccharification co-fermentation performed in 100-mL flasks enabled 210.1 g/L lactic acid from 40% solid loading corn stover hydrolysate. When simultaneous saccharification co-fermentation was performed in 3-L bioreactors, 157.4 g/L lactic acid was obtained from 35% solid loading corn stover hydrolysate. These obtained lactic acid titers are the highest reports until now when lignocellulosic biomasses are used as substrates, making it efficient for scale production of cellulosic lactic acid.

Engineering co-utilization of glucose and xylose for chemical overproduction from lignocellulose.

Bio-refining lignocellulose could provide a sustainable supply of fuels and fine chemicals; however, the challenges associated with the co-utilization of xylose and glucose typically compromise the efficiency of lignocellulose conversion. Here we engineered the industrial yeast Ogataea polymorpha (Hansenula polymorpha) for lignocellulose biorefinery by facilitating the co-utilization of glucose and xylose to optimize the production of free fatty acids (FFAs) and 3-hydroxypropionic acid (3-HP) from lignocellulose. We rewired the central metabolism for the enhanced supply of acetyl-coenzyme A and nicotinamide adenine dinucleotide phosphate hydrogen, obtaining 30.0 g l-1 of FFAs from glucose, with productivity of up to 0.27 g l-1 h-1. Strengthening xylose uptake and catabolism promoted the synchronous utilization of glucose and xylose, which enabled the production of 38.2 g l-1 and 7.0 g l-1 FFAs from the glucose-xylose mixture and lignocellulosic hydrolysates, respectively. Finally, this efficient cell factory was metabolically transformed for 3-HP production with the highest titer of 79.6 g l-1 in fed-batch fermentation in mixed glucose and xylose.

Understanding acid hydrolysis of corn stover during densification pretreatment for quantitative predictions of enzymatic hydrolysis efficiency using modified pretreatment severity factor.

DLCA(sa) pretreatment (densifying lignocellulosic biomass with sulfuric acid followed by autoclave treatment), featured with low treatment temperature and densification, demonstrate high efficiency in biomass pretreatment. In this study, the effects of temperature, acid loading, time on the hydrolysis of xylan, cellulose and lignin during DLCA(sa) pretreatment were systematically investigated. It was shown that DLCA(sa) pretreatment can effectively solubilize xylan, achieving an 84% xylose recovery under mild conditions (130 °C, 30 min, and 0.125 g/g acid loading). The conventional pretreatment severity factor correlated and further modified to improve the accuracy in evaluating the xylan hydrolysis. Additionally, a mathematical model based on the xylan hydrolytic kinetics was proposed to predict the enzymatic hydrolysis. Kinetic model suggested that mechanical densification facilitates the penetration of acid into the biomass matrix, leading to increased accessibility of xylan to acid catalysis.

Adaptive laboratory evolution boosts Yarrowia lipolytica tolerance to vanillic acid.

Microbial tolerance to lignocellulose-derived inhibitors, such as aromatic acids, is critical for the economical production of biofuels and biochemicals. Here, adaptive laboratory evolution was applied to improve the tolerance of Yarrowia lipolytica to a representative aromatic acid inhibitor vanillic acid. The transcriptome profiling of evolved strain suggested that the tolerance could be related to the up-regulation of RNA processing and multidrug transporting pathways. Further analysis by reverse engineering confirmed that the amplification of YALI0_F13475g coding for transcriptional coactivator and YALI0_E25201g coding for multidrug transporter conferred tolerance not only to vanillic acid but also towards ferulic acid, p-coumaric acid, p-hydroxybenzoic acid and syringic acid. These findings suggested that regulation of RNA processing and multidrug transporting pathways may be important for enhanced aromatic acid tolerance in Y. lipolytica. This study provides valuable genetic information for robust strain construction for lignocellulosic biorefinery.

Big data mining, rational modification, and ancestral sequence reconstruction inferred multiple xylose isomerases for biorefinery.

The isomerization of xylose to xylulose is considered the most promising approach to initiate xylose bioconversion. Here, phylogeny-guided big data mining, rational modification, and ancestral sequence reconstruction strategies were implemented to explore new active xylose isomerases (XIs) for Saccharomyces cerevisiae. Significantly, 13 new active XIs for S. cerevisiae were mined or artificially created. Moreover, the importance of the amino-terminal fragment for maintaining basic XI activity was demonstrated. With the mined XIs, four efficient xylose-utilizing S. cerevisiae were constructed and evolved, among which the strain S. cerevisiae CRD5HS contributed to ethanol titers as high as 85.95 and 94.76 g/liter from pretreated corn stover and corn cob, respectively, without detoxifying or washing pretreated biomass. Potential genetic targets obtained from adaptive laboratory evolution were further analyzed by sequencing the high-performance strains. The combined XI mining methods described here provide practical references for mining other scarce and valuable enzymes.

Preparing Biosurfactant Glucolipids from Crude Sophorolipids via Chemical Modifications and Their Potential Application in the Food Industry.

This investigation developed a novel strategy for efficiently preparing glucolipids (GLs) by chemically modifying crude sophorolipids. Running this strategy, crude sophorolipids were effectively transformed into GLs through deglycosylation and de-esterification, with a yield of 54.1%. The acquired GLs were then purified via stepwise extractions, and 66.2% of GLs with 95% purity was recovered. GLs are more hydrophobic and present a stronger surface activity than acidic sophorolipids (ASLs). More importantly, these GLs displayed a superior antimicrobial activity to that of ASLs against the tested Gram-positive food pathogens, with a minimum inhibitory concentration of 32-64 mg/L, except against E. coli . This activity of GLs is pH-dependent and especially more powerful under acidic conditions. The mechanism involved is possibly associated with the more efficient adsorption of GLs, as demonstrated by the hydrophobicity of the cell membrane. These GLs could be used as antimicrobial agents for food preservation and health in the food industry.

A single-wavelength excited NIR fluorescence probe for distinguishing GSH/H2S and Cys/Hcy in living cells and zebrafish through separated dual-channels.

Biothiols and hydrogen sulfide, as critical sulfur-containing reactive substances, serve essential functions in various human pathological processes, making it challenging to simultaneously distinguish them due to their similar reactivity and structures (-SH). Here, we rationalized the development of a single-wavelength excitation near-infrared (NIR) fluorescence probe, FC-NBD, for distinguishing GSH/H2S and Cys/Hcy by separated fluorescence dual channels. In this probe, FC-NBD, composed of coumarin-benzopyrylium derivatives linked with nitro benzoxadiazole (NBD) via ether bonds, could quantitatively and selectively distinguish GSH/H2S and Cys/Hcy with a low limit of detection (LOD) of 0.199/0.177 µM and 0.106/0.076 µM, respectively. As expected, under single-wavelength excitation (470 nm), FC-NBD demonstrated distinctly separable green and NIR fluorescence emissions towards Cys/Hcy at 550 and 660 nm, but only exhibited a noticeable NIR fluorescence emission towards GSH/H2S at 660 nm. Moreover, FC-NBD could simultaneously visualize and discriminate GSH/H2S and Cys/Hcy in living cells as well as zebrafish through green and NIR channels at a single excitation wavelength.

Towards efficient enzymatic saccharification of pretreated lignocellulose: Enzyme inhibition by lignin-derived phenolics and recent trends in mitigation strategies.

Lignocellulosic biorefinery based on sugar-platform has been considered as an efficient strategy to replace fossil fuel-based refinery. In the bioconversion process, pretreatment is an essential step to firstly open up lignocellulose cell wall structure and enhance the accessibility of carbohydrates to hydrolytic enzymes. However, various lignin and/or carbohydrates degradation products (e.g. phenolics, 5-hydroxymethylfurfural, furfural) are also generated during pretreatment, which severely inhibit the following enzymatic hydrolysis and the downstream fermentation process. Among them, the lignin derived phenolics have been considered as the most inhibitory compounds and their inhibitory effects are highly dependent on the source of biomass and the type of pretreatment strategy. Although liquid-solid separation and subsequent washing can remove the lignin derived phenolics and other inhibitors, this is undesirable in the realistic industrial application where the whole slurry of pretreated biomass needs to be directly used in the hydrolysis process. This review summarizes the phenolics formation mechanism for various commonly applied pretreatment methods and discusses the key factors that affect the inhibitory effect of phenolics on cellulose hydrolysis. In addition, the recent achievements on the rational design of inhibition mitigation strategies to boost cellulose hydrolysis for sugar-platform biorefinery are also introduced. This review also provides guidance for rationally designing detoxification strategies to facilitate whole slurry hydrolysis which helps to realize the industrialization of lignocellulose biorefinery.

Densification pretreatment triggers efficient methanogenic performance and robust microbial community during anaerobic digestion of corn stover.

The recalcitrant characteristics of lignocellulosic waste and difficulties in biomass transportation and storage severely limit bioenergy production through anaerobic digestion (AD). In this study, Densifying Lignocellulosic biomass with Chemicals (DLC) pretreatment was developed to address these issues. The results showed that DLC treated corn stover (CS) reached a cumulative methane yield of as high as 224.30 mL/g VS (Volatile Solids), which was 59.27 % higher than that of un-treated. The reduced scum formation in the reactor, increased components consumption of solid phase, and higher organic biodegradability of liquid phase in AD of DLC treated CS enhanced methane yield. Microbial analysis indicated that DLC pretreatment affected the bacterial and methanogenic community structure, and a co-network with Comamonas and Methanobacterium, etc. as hub microbes was constructed. This study proposed a promising technology that could be potentially applied to industrial AD of lignocellulosic biomass.

Developing Rhodococcus opacus and Sphingobium sp. coculture systems for valorization of lignin-derived dimers.

Bioconversion is being regarded as a promising way for lignin valorization because it enables funneling diverse lignin components into single compounds, overcoming the heterogeneity of lignin. Although numerous lignin-derived aromatic monomers have been funneled to target compounds in previous studies, the bioconversion of low-molecular-weight lignin (LMW-lignin) fragments, for example, lignin-derived dimers, has been rarely systematically studied, impeding further conversion of lignin. In this study, coculture systems were designed and developed to funnel multiple lignin-derived dimers to cis, cis-muconate and gallate by combining lignin-derived dimers cleavage bacterium Sphingobium sp. and monomers conversion bacterium Rhodococcus opacus. With the developed coculture systems, ß-O-4 type dimer guaiacylglycerol-ß-guaiacyl ether, 4-O-5 type dimer 4,4'-dihydroxydiphenyl ether, ß-5 type dimer balanophonin, ß-ß type dimer pinoresinol, ß-1 type dimer 1,2-bis(4-hydroxy-3-methoxyphehyl)-1,3-propanediol and 5-5 type dimer 5,5'-dehydrodivanillate were converted to cis, cis-muconate. Additionally, the developed coculture systems also showed potential in conversion of lignin-derived dimers to gallate. The application of alkali lignin for cis, cis-muconate production further demonstrated the effectiveness of the designed coculture systems. Overall, the developed coculture systems are beneficial to lignin biological valorization, and also provide references for the valorization of other bio-resources.

A novel fermentation strategy for efficient xylose utilization and microbial lipid production in lignocellulosic hydrolysate.

The sugar utilization efficiency and the tolerance of microorganism to inhibitors are essential for lipid production from lignocellulosic biomass. In this study, the sugar consumption and inhibitor tolerance characteristics of Trichosporon dermatis 32,903 were investigated. The results showed that the lipid yield on xylose was much lower than that on glucose, while these substrates exhibited comparative efficiency for cell growth. High inoculum size improved the tolerance of T. dermatis 32,903 to inhibitors. Based on these characteristics, sugar-targeted-utilization and cyclic fermentation strategy was developed. The tolerance of high inoculum size to inhibitors was utilized, glucose was targeted for lipid fermentation and xylose was targeted for cell growth. As a result, the lipid production efficiency was greatly enhanced. The lipid titer in hydrolysate of DLCA (Densifying Lignocellulosic biomass with Chemicals followed by Autoclave) pretreated rice straw was improved to as high as 38.4 g/L with lipid yield of 0.207 g/g consumed sugar.

Rapid evolution and mechanism elucidation for efficient cellobiose-utilizing Saccharomyces cerevisiae through Synthetic Chromosome Rearrangement and Modification by LoxPsym-mediated Evolution.

Lack of cellobiose utilization capability for many microorganisms results in carbon source waste in lignocellulosic biorefinery. In this study, genes for cellobiose transport and hydrolysis were introduced to Saccharomyces cerevisiae synV, a semi-synthetic yeast with an inducible SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxPsym-mediated Evolution) system incorporated into its chromosome V, endowing cellobiose utilization capability to this strain. Thereafter, two evolved strains with 98.1% and 79.2% improvement, respectively, in cellobiose utilization rate were obtained through induced SCRaMbLE. Further studies suggested that the enhanced cellobiose utilization capability directly correlated with copy number increases of introduced genes and some chromosome structural variations. In particular, it was experimentally demonstrated for the first time that deletion of redox stress related gene MXR1 and ATP conversion related gene ADK2 contributed to enhanced cellobiose conversion. Thereafter, the effectiveness of MXR1 and ADK2 deletions was demonstrated in artificial hydrolysate and rice straw hydrolysate, respectively.

Establishing a novel 3D printing bioinks system with recombinant human collagen.

Bioinks are one of the key elements in realizing three-dimensional (3D) bioprinting. However, bioinks prepared from conventional collagen are hindered to their further applications due to concerns of collagen purity, unstable mechanical properties, and low solubility under neutralized conditions. This study aimed to develop a reliable UV-curable bioink system from a novel water-soluble recombinant human collagen (RHC). RHC was modified by methacrylic anhydride (MAA) and later crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) to obtain Pro-RHCMA. 1H nuclear magnetic resonance (1H NMR) confirmed the methacryloyl grafts, Fourier transform-infrared spectroscopy (FT-IR) illustrated the chemical crosslinking in producing the Pro-RHCMA. Internal morphology, mechanical properties and degradation of UV cured boinks were MAA and EDC/NHS modification-dependent. Photorheological properties and printability of the bioinks were determined. Cellular bioactivities were sustained within the printed bioinks, validating the bioinks biocompatibility in vitro. Finally, qRT-PCR revealed that the Pro-RHCMA bioinks provided a cell-friendly microenvironment for human umbilical vein endothelial cells (HUVECs) and human foreskin fibroblasts (HFFs), by supporting the expression of extracellular matrix (ECM) and angiogenesis-associated proteins, respectively. Taken together, this novel RHC-based bioink system shows great potential in tissue engineering and regenerative medicine.

Preparation of Chitosan/Recombinant Human Collagen-Based Photo-Responsive Bioinks for 3D Bioprinting.

Collagen and chitosan are frequently used natural biomaterials in tissue engineering. However, most collagen is derived from animal tissue, with inconsistent quality and pathogen transmittance risks. In this context, we aimed to use a reliable Type-III recombinant human collagen (RHC) as an alternative biomaterial together with chitosan to develop novel photo-responsive bioinks for three-dimensional (3D) bioprinting. RHC was modified with methacrylic anhydride to obtain the RHC methacryloyl (RHCMA) and mixed with acidified chitosan (CS) to form composites CS-RHCMA. The characterizations demonstrated that the mechanical properties and the degradation of the bioinks were tunable by introducing the CS. The printabilities improved by adding CS to RHCMA, and various structures were constructed via extrusion-based 3D printing successfully. Moreover, in vitro tests confirmed that these CS-RHCMA bioinks were biocompatible as human umbilical vein endothelial cells (HUVECs) were sustained within the constructs post-printing. The results from the current study illustrated a well-established bioinks system with the potential to construct different tissues through 3D bioprinting.

Understanding the toxicity of lignin-derived phenolics towards enzymatic saccharification of lignocellulose for rationally developing effective in-situ mitigation strategies to maximize sugar production from lignocellulosic biorefinery.

The lignin-derived phenolics are highly inhibitory toward lignocellulose enzymatic hydrolysis, while the relationship between phenolic structure and inhibitory effect is still not fully understood. In this study, the compositions of phenolics from dilute acid pretreated wheat straw were analyzed and their impact on cellulose hydrolysis was studied. With increase of pretreatment severity, more toxic phenolics were produced from lignin degradation reactions, which were the major contributor to the increased inhibitory effect of pretreatment hydrolysate towards cellulases. Through analyzing the relationship of phenolic structure and their inhibitory effect, a useful model was developed to predict the phenolics-caused inhibition by combining the indexes of electrophilicity and hydrophobicity. Further, through understanding the interactions between phenolics and cellulases, a novel biocomponent alleviator was rationally designed to block the phenolics-cellulase interactions, the degree of improvement of enzymatic hydrolysis reached as high as 135.8%. This study provides directions for developing more effective pretreatment and detoxification methods.

A highly selective turn-on fluorescence probe with large Stokes shift for detection of palladium and its applications in environment water and living cells.

Nowadays, palladium has been widely used in many fields, which facilitates all aspects of our life. However, it may cause water and soil pollution and bring irreversible damage to the environment and organisms. Developing a fluorescence probe for rapid, highly sensitive and selective detection of palladium is still a poser. In this work, we designed and synthesized a novel fluorescence probe (RHS) for specific detection of palladium. Based on Pd0-mediated Tsuji-Trost reaction, the fluorescence probe was constructed by a rhodol derivative as thefluorophore and an allyl carbonate moiety as the specific palladium reactive site. The probe displayed excellent properties for detecting palladium, such as high selectivity and sensitivity, rapid response (20 min) and large Stokes shift (155 nm). The detection limit was determined to be as low as 0.140 µM with a linear range from 20 to 80 µM. After addition of palladium in RHS solution, the color of the solution turned from yellow to blue, indicating palladium could be monitored by the naked eyes. Moreover, probe RHS was successfully applied to palladium detection in environmental water samples. Importantly, with low cytotoxicity and good biocompatibility, the probe could monitor palladium in living cells.