Advances and prospects in microbial production of biotin.

Biotin, serving as a coenzyme in carboxylation reactions, is a vital nutrient crucial for the natural growth, development, and overall well-being of both humans and animals. Consequently, biotin is widely utilized in various industries, including feed, food, and pharmaceuticals. Despite its potential advantages, the chemical synthesis of biotin for commercial production encounters environmental and safety challenges. The burgeoning field of synthetic biology now allows for the creation of microbial cell factories producing bio-based products, offering a cost-effective alternative to chemical synthesis for biotin production. This review outlines the pathway and regulatory mechanism involved in biotin biosynthesis. Then, the strategies to enhance biotin production through both traditional chemical mutagenesis and advanced metabolic engineering are discussed. Finally, the article explores the limitations and future prospects of microbial biotin production. This comprehensive review not only discusses strategies for biotin enhancement but also provides in-depth insights into systematic metabolic engineering approaches aimed at boosting biotin production.

Ultrafast Dynamics of Extraordinary Optical Transmission through Two-Slit Plasmonic Antenna.

We have theoretically investigated the spatial-temporal dynamics of extraordinary optical transmission (EOT) through a two-slit plasmonic antenna under femtosecond laser dual-beam irradiation. The dynamic interference of the crossed femtosecond laser dual-beam with the transiently excited surface plasmon polariton waves are proposed to characterize the particular spatial-temporal evolutions of EOT. It is revealed that the dynamic EOT can be flexibly switched with tunable symmetry through the respective slit of a two-slit plasmonic antenna by manipulating the phase correlation of the crossed femtosecond laser dual-beam. This is explained as tunable interference dynamics by phase control of surface plasmon polariton waves, allowing the dynamic modulation of EOT at optimized oblique incidences of dual-beams. Furthermore, we have obtained the unobserved traits of symmetry-broken transient spectra of EOT from the respective up- and down-slit of the antenna under crossed femtosecond laser dual-beam irradiation. This study can provide fundamental insights into the ultrafast dynamics of EOT in two-slit plasmonic antennas, which can be helpful to advance a wide range of applications, such as ultrafast plasmonic switch, ultrahigh resolution imaging, the transient amplification of non-linear effects, etc.

Molecular-Scale Plasmon Trapping via a Graphene-Hybridized Tip-Substrate System.

We theoretically investigated the plasmon trapping stability of a molecular-scale Au sphere via designing Au nanotip antenna hybridized with a graphene sheet embedded Silica substrate. A hybrid plasmonic trapping model is self-consistently built, which considers the surface plasmon excitation in the graphene-hybridized tip-substrate system for supporting the scattering and gradient optical forces on the optical diffraction-limit broken nanoscale. It is revealed that the plasmon trapping properties, including plasmon optical force and potential well, can be unprecedentedly adjusted by applying a graphene sheet at proper Fermi energy with respect to the designed tip-substrate geometry. This shows that the plasmon potential well of 218 kBT at room temperature can be determinately achieved for trapping of a 10 nm Au sphere by optimizing the surface medium film layer of the designed graphene-hybridized Silica substrate. This is explained as the crucial role of graphene hybridization participating in plasmon enhancement for generating the highly localized electric field, in return augmenting the trapping force acting on the trapped sphere with a deepened potential well. This study can be helpful for designing the plasmon trapping of very small particles with new routes for molecular-scale applications for molecular-imaging, nano-sensing, and high-sensitive single-molecule spectroscopy, etc.

Effects of orphan histidine kinases on clostridial sporulation progression and metabolism.

Solventogenesis and sporulation of clostridia are the main responsive adaptations to the acidic environment during acetone-butanol-ethanol (ABE) fermentation. It was hypothesized that five orphan histidine kinases (HKs) including Cac3319, Cac0323, Cac0903, Cac2730, and Cac0437 determined the cell fates between sporulation and solventogenesis. In this study, the comparative genomic analysis revealed that a mutation in cac0437 appeared to contribute to the nonsporulating feature of ATCC 55025. Hence, the individual and interactive roles of five HKs in regulating cell growth, metabolism, and sporulation were investigated. The fermentation results of mutants with different HK expression levels suggested that cac3319 and cac0437 played critical roles in regulating sporulation and acids and butanol biosynthesis. Morphological analysis revealed that cac3319 knockout abolished sporulation (Stage 0) whereas cac3319 overexpression promoted spore development (Stage VII), and cac0437 knockout initiated but blocked sporulation before Stage II, indicating the progression of sporulation was altered through engineering HKs. By combinatorial HKs knockout, the interactive effects between two different HKs were investigated. This study elucidated the regulatory roles of HKs in clostridial differentiation and demonstrated that HK engineering can be effectively used to control sporulation and enhance butanol biosynthesis.

Laser Fabrication of Nanoholes on Silica through Surface Window Assisted Nano-Drilling (SWAN).

Nano-structures have significant applications in many fields such as chip fabrications, nanorobotics, and solar cells. However, realizing nanoscale structures on hard and brittle materials is still challenging. In this paper, when processing the silica surface with a tightly focused Bessel beam, the smallest nanohole with ~20 nm diameter has been realized by precisely controlling the interior and superficial interaction of the silica material. An effective surface window assisted nano-drilling (SWAN) mechanism is proposed to explain the generation of such a deep subwavelength structure, which is supported by the simulation results of energy depositions.

Theoretical Study on Symmetry-Broken Plasmonic Optical Tweezers for Heterogeneous Noble-Metal-Based Nano-Bowtie Antennas.

Plasmonic optical tweezers with a symmetry-tunable potential well were investigated based on a heterogeneous model of nano-bowtie antennas made of different noble substances. The typical noble metals Au and Ag are considered as plasmonic supporters for excitation of hybrid plasmonic modes in bowtie dimers. It is proposed that the plasmonic optical trapping force around a quantum dot exhibits symmetry-broken characteristics and becomes increasingly asymmetrical with increasing applied laser electric field. Here, it is explained by the dominant plasmon hybridization of the heterogeneous Au-Ag dimer, in which the plasmon excitations can be inconsistently modified by tuning the applied laser electric field. In the spectrum regime, the wavelength-dependent plasmonic trapping potential exhibits a two-peak structure for the heterogeneous Au-Ag bowtie dimer compared to a single-peak trapping potential of the Au-Au bowtie dimer. In addition, we comprehensively investigated the influence of structural parameter variables on the plasmonic potential well generated from the heterogeneous noble nano-bowtie antenna with respect to the bowtie edge length, edge/tip rounding, bowtie gap, and nanosphere size. This work could be helpful in improving our understanding of wavelength and laser field tunable asymmetric nano-tweezers for flexible and non-uniform nano-trapping applications of particle-sorting, plasmon coloring, SERS imaging, and quantum dot lighting.

A high-efficient strategy for combinatorial engineering paralogous gene family: A case study on histidine kinases in Clostridium.

Microorganisms harbor bulks of functionally similar or undefined genes, which belong to paralogous gene family. There is a necessity of exploring combinatorial or interactive functions of these genes, but conventional loss-of-function strategy with one-by-one rounds suffers extremely low efficiency for generating mutant libraries with all gene permutations. Here, taking histidine kinases (HKs) in Clostridium acetobutylicum as a proof-of-concept, we developed a multi-plasmid cotransformation strategy for generating all theoretical HKs combinations in one round. For five HKs with 31 theoretical combinations, the library containing 22 mutants within all the possible HKs-inactivated combinations was constructed with 11 days compared to 242 days by conventional strategy, while the other 9 combinations cannot survive. Six mutants with the enhanced butanol production and tolerance were obtained with changes of cell development during fermentation, one of which could produce 54.2% more butanol (56.4% more solvents), while the butanol production of other mutants was unchanged or decreased. The cotransformation strategy demonstrated potentials for fast exploring pleiotropic function of paralogous family genes in cell survival, cell development, and target product metabolism.

Metabolic Engineering of Histidine Kinases in Clostridium beijerinckii for Enhanced Butanol Production.

Clostridium beijerinckii, a promising industrial microorganism for butanol production, suffers from low butanol titer and lack of high-efficiency genetical engineering toolkit. A few histidine kinases (HKs) responsible for Spo0A phosphorylation have been demonstrated as functionally important components in regulating butanol biosynthesis in solventogenic clostridia such as C. acetobutylicum, but no study about HKs has been conducted in C. beijerinckii. In this study, six annotated but uncharacterized candidate HK genes sharing partial homologies (no less than 30%) with those in C. acetobutylicum were selected based on sequence alignment. The encoding region of these HK genes were deleted with CRISPR-Cas9n-based genome editing technology. The deletion of cbei2073 and cbei4484 resulted in significant change in butanol biosynthesis, with butanol production increased by 40.8 and 17.3% (13.8 g/L and 11.5 g/L vs. 9.8 g/L), respectively, compared to the wild-type. Faster butanol production rates were observed, with butanol productivity greatly increased by 40.0 and 20.0%, respectively, indicating these two HKs are important in regulating cellular metabolism in C. beijerinckii. In addition, the sporulation frequencies of two HKs inactivated strains decreased by 96.9 and 77.4%, respectively. The other four HK-deletion (including cbei2087, cbei2435, cbei4925, and cbei1553) mutant strains showed few phenotypic changes compared with the wild-type. This study demonstrated the role of HKs on sporulation and solventogenesis in C. beijerinckii, and provided a novel engineering strategy of HKs for improving metabolite production. The hyper-butanol-producing strains generated in this study have great potentials in industrial biobutanol production.

Tunable potential well for plasmonic trapping of metallic particles by bowtie nano-apertures.

In this paper, the tunable optical trapping dependence on wavelength of incident beam is theoretically investigated based on numerical simulations. The Monte Carlo method is taken into account for exploring the trapping characteristics such as average deviation and number distribution histogram of nanoparticles. It is revealed that both the width and the depth of potential well for trapping particles can be flexibly adjusted by tuning the wavelength of the incident beam. In addition, incident wavelengths for the deepest potential well and for the strongest stiffness at bottom are separated. These phenomena are explained as the strong plasmon coupling between tweezers and metallic nanoparticles. In addition, required trapping fluence and particles' distributions show distinctive properties through carefully modifying the incident wavelengths from 1280 nm to 1300 nm. Trapping with lowest laser fluence can be realized with 1280 nm laser and trapping with highest precision can be realized with 1300 nm laser. This work will provide theoretical support for advancing the manipulation of metallic particles and related applications such as single-molecule fluorescence and surface enhanced Raman spectroscopy.

Lens-on-lens microstructures.

Microlenses with multiple focal lengths play an important role in three-dimensional imaging and the real-time detection of unconfined or fluctuating targets. In this Letter, we present a novel method of fabricating lens-on-lens microstructures (LLMs) using a two-step femtosecond laser wet etching process. A 3×3 LLM array was made with a diameter of 129.0 µm. The fabricated LLM has two focal lengths, 80.4 and 188.7 µm, showing excellent two-level focusing and imaging abilities. Its size and focal length can be controlled by adjusting laser power and etching time. Its surface roughness remains about 61 nm. This simple and efficient method for large-scale production of LLMs has potential applications in diverse optical systems.

Evaluation of hydrophobic micro-zeolite-mixed matrix membrane and integrated with acetone-butanol-ethanol fermentation for enhanced butanol production.

BACKGROUND: Butanol is regarded as an advanced biofuel that can be derived from renewable biomass. However, the main challenge for microbial butanol production is low butanol titer, yield and productivity, leading to intensive energy consumption in product recovery. Various alternative separation technologies such as extraction, adsorption and gas stripping, etc., could be integrated with acetone-butanol-ethanol (ABE) fermentation with improving butanol productivity, but their butanol selectivities are not satisfactory. The membrane-based pervaporation technology is recently attracting increasing attention since it has potentially desirable butanol selectivity. RESULTS: The performance of the zeolite-mixed polydimethylsiloxane (PDMS) membranes were evaluated to recover butanol from butanol/water binary solution as well as fermentation broth in the integrated ABE fermentation system. The separation factor and butanol titer in permeate of the zeolite-mixed PDMS membrane were up to 33.0 and 334.6 g/L at 80°C, respectively, which increased with increasing zeolite loading weight in the membrane as well as feed temperature. The enhanced butanol separation factor was attributed to the hydrophobic zeolites with large pore size providing selective routes preferable for butanol permeation. In fed-batch fermentation incorporated with pervaporation, 54.9 g/L ABE (34.5 g/L butanol, 17.0 g/L acetone and 3.4 g/L ethanol) were produced from 172.3 g/L glucose. The overall butanol productivity and yield increased by 16.0 and 11.1%, respectively, which was attributed to the alleviated butanol inhibition by pervaporation and reassimilation of acids for ABE production. The zeolite-mixed membrane produced a highly concentrated condensate containing 169.6 g/L butanol or 253.3 g/L ABE, which after phase separation easily gave the final product containing >600 g/L butanol. CONCLUSIONS: Zeolite loading in the PDMS matrix was attributed to improving the pervaporative performance of the membrane, showing great potential to recover butanol with high purity. Therefore, this zeolite-mixed PDMS membrane had the potential to improve biobutanol production when integrating with ABE fermentation.

A carbon nanotube filled polydimethylsiloxane hybrid membrane for enhanced butanol recovery.

The carbon nanotubes (CNTs) filled polydimethylsiloxane (PDMS) hybrid membrane was fabricated to evaluate its potential for butanol recovery from acetone-butanol-ethanol (ABE) fermentation broth. Compared with the homogeneous PDMS membrane, the CNTs filled into the PDMS membrane were beneficial for the improvement of butanol recovery in butanol flux and separation factor. The CNTs acting as sorption-active sites with super hydrophobicity could give an alternative route for mass transport through the inner tubes or along the smooth surface. The maximum total flux and butanol separation factor reached up to 244.3 g/m(2)·h and 32.9, respectively, when the PDMS membrane filled with 10 wt% CNTs was used to separate butanol from the butanol/water solution at 80°C. In addition, the butanol flux and separation factor increased dramatically as temperature increased from 30°C to 80°C in feed solution since the higher temperature produced more free volumes in polymer chains to facilitate butanol permeation. A similar increase was also observed when butanol titer in solution increased from 10 g/L to 25 g/L. Overall, the CNTs/PDMS hybrid membrane with higher butanol flux and selectivity should have good potential for pervaporation separation of butanol from ABE fermentation broth.

Evaluation of asymmetric polydimethylsiloxane-polyvinylidene fluoride composite membrane and incorporated with acetone-butanol-ethanol fermentation for butanol recovery.

The polydimethylsiloxane-polyvinylidene fluoride (PDMS-PVDF) composite membrane was studied for its pervaporation performance to removal of butanol from butanol/ABE solution, fermentation broth as well as incorporated with acetone-butanol-ethanol (ABE) fermentation. The total flux and butanol titer in permeate through the PDMS-PVDF membrane were up to 769.6 g/m(2)h and 323.5 g/L at 80 °C, respectively. The butanol flux and total flux increased with increasing the feed temperature as well as the feed butanol titer. The butanol separation factor and butanol titer in permeate decreased slightly in the presence of acetone and ethanol in the feed due to their preferential dissolution and competitive permeation through the membrane. In fed-batch fermentation incorporated with pervaporation, butanol titer and flux in permeate maintained at a steady level with the range of 139.9-154.0 g/L and 13.3-16.3 g/m(2)h, respectively, which was attributed to the stable butanol titer in fermentation broth as well as the excellent hydrophobic nature of the PDMS-PVDF matrix. Therefore, the PDMS-PVDF composite membrane had a great potential in the in situ product recovery with ABE fermentation, enabling the economic production of biobutanol.

Rapid fabrication of a large-area close-packed quasi-periodic microlens array on BK7 glass.

Large-area close-packed microlens arrays (MLAs) are highly desirable for structured light and integrated optical applications. However, efficient realization of ultralarge area MLAs with a high fill factor is still technically challenging, especially on glass material. In this Letter we propose a high-efficiency MLA fabrication method using single-pulsed femtosecond laser wet etch and close-packed quasi-periodic concave MLAs consisting of three million units fabricated on silica glass within an hour. The fabricated MLAs are demonstrated to have extreme optical smoothness (∼8.5 nm) by an atomic force microscope. It has also been demonstrated that the profile of the quasi-periodic concave structures could be easily tuned by changing the laser scanning speed or the pulse energy. Additionally, the optical performances of the MLA diffusers were investigated by using sharp focusing, high-resolution imaging, and flat-top illumination.

Rapid fabrication of large-area concave microlens arrays on PDMS by a femtosecond laser.

A fast and single-step process is developed for the fabrication of low-cost, high-quality, and large-area concave microlens arrays (MLAs) by the high-speed line-scanning of femtosecond laser pulses. Each concave microlens can be generated by a single laser pulse, and over 2.78 million microlenses were fabricated on a 2 × 2 cm(2) polydimethylsiloxane (PDMS) sheet within 50 min, which greatly enhances the processing efficiency compared to the classical laser direct writing method. The mechanical pressure induced by the expansion of the laser-induced plasmas as well as a long resolidifing time is the reason for the formation of smooth concave spherical microstructures. We show that uniform microlenses with different diameters and depths can be controlled by adjusting the power of laser pulses. Their high-quality optical performance is also demonstrated in this work.

Bioinspired wetting surface via laser microfabrication.

Bioinspired special wettibilities including superhydrophobicity and tunable adhesive force have drawn considerable attention because of their significant potential for fundamental research and practical applications. This review summarizes recent progress in the development of bioinspired wetting surfaces via laser microfabrication, with a focus on controllable, biomimetic, and switchable wetting surfaces, as well as their applications in biology, microfluidic, and paper-based devices, all of which demonstrate the ability of laser microfabrication in producing various multiscale structures and its adaptation in a great variety of materials. In particular, compared to other techniques, laser microfabrication can realize special modulation ranging from superhydrophilic to superhydrophobic without the assistance of fluorination, allowing much more freedom to achieve complex multiple-wettability integration. The current challenges and future research prospects of this rapidly developing field are also being discussed. These approaches open the intriguing possibility of the development of advanced interfaces equipped with the integration of more functionalities.

Scalable shape-controlled fabrication of curved microstructures using a femtosecond laser wet-etching process.

Materials with curvilinear surface microstructures are highly desirable for micro-optical and biomedical devices. However, realization of such devices efficiently remains technically challenging. This paper demonstrates a facile and flexible method to fabricate curvilinear microstructures with controllable shapes and dimensions. The method composes of femtosecond laser exposures and chemical etching process with the hydrofluoric acid solutions. By fixed-point and step-in laser irradiations followed by the chemical treatments, concave microstructures with different profiles such as spherical, conical, bell-like and parabola were fabricated on silica glasses. The convex structures were replicated on polymers by the casting replication process. In this work, we used this technique to fabricate high-quality microlens arrays and high-aspect-ratio microwells which can be used in 3D cell culture. This approach offers several advantages such as high-efficient, scalable shape-controllable and easy manipulations.

Controllable adhesive superhydrophobic surfaces based on PDMS microwell arrays.

This paper presents a one-step method to fabricate superhydrophobic surfaces with extremely controllable adhesion based on PDMS microwell arrays. The microwell array structures are rapidly produced on PDMS films by a point-by-point femtosecond laser scanning process. The as-prepared superhydrophobic surfaces show water controllable adhesion that ranges from ultrahigh to ultralow by adjusting the extent of overlap of the adjacent microwells, on which the sliding angle can be controlled from 180° (a water droplet can not slide down even when the as-prepared surface is turned upside down) to 3°. A "micro-airbag effect" is introduced to explain the adhesion transition phenomenon of the microwell array structures. This work provides a facile and promising strategy to fabricate superhydrophobic surfaces with controllable adhesion.

A facile preparation route for netlike microstructures on a stainless steel using an ethanol-mediated femtosecond laser irradiation.

Netlike or porous microstructures are highly desirable in metal implants and biomedical monitoring applications. However, realization of such microstructures remains technically challenging. Here, we report a facile and environmentally friendly method to prepare netlike microstructures on a stainless steel by taking the full advantage of the liquid-mediated femtosecond laser ablation. An unordered netlike structure and a quasi-ordered array of holes can be fabricated on the surface of stainless steel via an ethanol-mediated femtosecond laser line-scan method. SEM analysis of the surface morphology indicates that the porous netlike structure is in the micrometer scale and the diameter of the quasi-ordered holes ranges from 280 nm to 320 nm. Besides, we find that the obtained structures are tunable by altering the laser processing parameters especially scanning speed.

Direct fabrication of seamless roller molds with gapless and shaped-controlled concave microlens arrays.

This Letter demonstrates the direct fabrication of gapless concave microlenses on glass cylinders, which can be used as seamless roller molds for the continuous imprinting of large-area microlens arrays. The method involves femtosecond laser exposures followed by a chemical wet-etching process. A honeycomb-like concave microlens array was fabricated on a glass cylinder with a diameter of 3 mm. We demonstrated the flexibility of the method in tuning the shape and depth of the concave structures by the arrangements of the laser exposure spots and laser powers, and examined the replicating ability of the roller mold by the polymer castling method.