Ultrahigh-throughput screening of industrial enzyme-producing strains by droplet-based microfluidic system.

Droplet-based microfluidics has emerged as a powerful tool for single-cell screening with ultrahigh throughput, but its widespread application remains limited by the accessibility of a droplet microfluidic high-throughput screening (HTS) platform, especially to common laboratories having no background in microfluidics. Here, we first developed a microfluidic HTS platform based on fluorescence-activated droplet sorting technology. This platform allowed (i) encapsulation of single cells in monodisperse water-in-oil droplets; (ii) cell growth and protein production in droplets; and (iii) sorting of droplets based on their fluorescence intensities. To validate the platform, a model selection experiment of a binary mixture of Bacillus strains was performed, and a 45.6-fold enrichment was achieved at a sorting rate of 300 droplets per second. Furthermore, we used the platform for the selection of higher α-amylase-producing Bacillus licheniformis strains from a mutant library generated by atmospheric and room temperature plasma mutagenesis, and clones displaying over 50% improvement in α-amylase productivity were isolated. This droplet screening system could be applied to the engineering of other industrially valuable strains.

Development and Validation of a Tunable Diode Laser Absorption Spectroscopy System for Hot Gas Flow and Small-Scale Flame Measurement.

TDLAS (tunable diode laser absorption spectroscopy) is an important gas analysis method that can be employed to obtain characteristic parameters non-invasively by the infrared absorption spectra of tracer molecules such as CH4, H2O and O2. In this study, a portable H2O-based TDLAS system with a dual optical path was developed with the aim of assessing the combustion characteristics of flammable gases. Firstly, a calculation method of gas characteristics including temperature and velocity combining absorption spectra and a HITRAN database was provided. Secondly, to calibrate and validate this TDLAS system precisely, a pressure vessel and a shock tube were introduced innovatively to generate static or steady flow fields with preset constant temperatures, pressures, or velocities. Static tests within environment pressures up to 2 MPa and steady flow field tests with temperatures up to 1600 K and flow velocities up to 950 m/s were performed for verification. It was proved that this system can provide an accurate values for high temperature and velocity gas flows. Finally, an experimental investigation of CH4/air flames was conducted to test the effectiveness of the system when applied to small diffusion flames. This TDLAS system gave satisfactory flame temperature and velocity data owing to the dual optical path design and high frequency scanning, which compensated for scale effects and pulsation of the flame. This work demonstrates a valuable new approach to thermal hazard analysis in specific environments.

Recent advances in droplet microfluidics for enzyme and cell factory engineering.

Enzymes and cell factories play essential roles in industrial biotechnology for the production of chemicals and fuels. The properties of natural enzymes and cells often cannot meet the requirements of different industrial processes in terms of cost-effectiveness and high durability. To rapidly improve their properties and performances, laboratory evolution equipped with high-throughput screening methods and facilities is commonly used to tailor the desired properties of enzymes and cell factories, addressing the challenges of achieving high titer and the yield of the target products at high/low temperatures or extreme pH, in unnatural environments or in the presence of unconventional media. Droplet microfluidic screening (DMFS) systems have demonstrated great potential for exploring vast genetic diversity in a high-throughput manner (>106/h) for laboratory evolution and have been increasingly used in recent years, contributing to the identification of extraordinary mutants. This review highlights the recent advances in concepts and methods of DMFS for library screening, including the key factors in droplet generation and manipulation, signal sources for sensitive detection and sorting, and a comprehensive summary of success stories of DMFS implementation for engineering enzymes and cell factories during the past decade.

Genomic, transcriptomic, and metabolic characterizations of Escherichia coli adapted to branched-chain higher alcohol tolerance.

Microbial-produced branched-chain higher alcohols (BCHAs), such as isopropanol, isobutanol, and isopentanol in Escherichia coli, have emerged as promising alternative biofuels under development. Elucidating and improving the tolerance of E. coli to BCHAs are important issues for microbial production of BCHAs due to their physiological inhibitory effect. Previous works aimed at understanding the genetic basis of E. coli tolerance to BCHAs with a comparative genome, reverse engineering, or transcriptome approach have gained some important insights into the mechanism of tolerance. However, investigation on BCHA tolerance from the whole-genomic, transcriptomic, and metabolic levels via a systematic approach has not yet been completely elucidated. Here, in this study, genomic, transcriptomic, and 13C-metabolic flux analyses (13C-MFA) of an evolved E. coli strain adapted to BCHA tolerance were conducted. Genome mutation of negative regulation factor (rssB, acrB, and clpX) of RpoS level suggested upregulation of RpoS activity in BCHA tolerance of E. coli. From a more detailed perspective, enhanced energy metabolism was observed to be the main characteristic of E. coli strain tolerant to BCHAs. Enhanced energy metabolism has been achieved through several routes, which included redistribution of the central carbon metabolism, upregulation of the energy generation machinery, and facilitating the operation of electron transferring chain. Evidence of multiple solutions of genotype modification toward BCHA tolerance was also revealed through comparative analysis of previous works from different groups.

Enhanced production of d-psicose 3-epimerase in Bacillus subtilis by regulation of segmented fermentation.

d-Psicose 3-epimerase is an enzyme that catalyzes the synthesis of d-psicose from d-fructose. We cloned the d-psicose 3-epimerase from Ruminococcus sp. (RDPE) and expressed it in Bacillus subtilis A311. By a two-step pH regulation of segmented fermentation, we significantly improved the RDPE production and decreased the fermentation cost. The two-step regulation consisted of the first step maintained the pH value at 7.0 for 24 H and the second step adjusted the pH value up to 7.5 slowly for another 24 H. Finally, the RDPE production was increased to 74 U/mL, which was about 2.5-fold compared with the control. Our segmented fermentation strategy provides an important experimental basis for the industrial-scale production of RDPE.

Biosensor-enabled droplet microfluidic system for the rapid screening of 3-dehydroshikimic acid produced in Escherichia coli.

Genetically encoded biosensors are powerful tools used to screen metabolite-producing microbial strains. Traditionally, biosensor-based screening approaches also use fluorescence-activated cell sorting (FACS). However, these approaches are limited by the measurement of intracellular fluorescence signals in single cells, rather than the signals associated with populations comprising multiple cells. This characteristic reduces the accuracy of screening because of the variability in signal levels among individual cells. To overcome this limitation, we introduced an approach that combined biosensors with droplet microfluidics (i.e., fluorescence-activated droplet sorting, FADS) to detect labeled cells at a multi-copy level and in an independent droplet microenvironment. We used our previously reported genetically encoded biosensor, 3-dehydroshikimic acid (3-DHS), as a model with which to establish the biosensor-based FADS screening method. We then characterized and compared the effects of the sorting method on the biosensor-based screening system by subjecting the same mutant library to FACS and FADS. Notably, our developed biosensor-enabled, droplet microfluidics-based FADS screening system yielded an improved positive mutant enrichment rate and increased productivity by the best mutant, compared with the single-cell FACS system. In conclusion, the combination of a biosensor and droplet microfluidics yielded a more efficient screening method that could be applied to the biosensor-based high-throughput screening of other metabolites.

High-efficiency expression and secretion of human FGF21 in Bacillus subtilis by intercalation of a mini-cistron cassette and combinatorial optimization of cell regulatory components.

BACKGROUND: Recombinant human Fibroblast growth factor 21 (rhFGF21) is an endocrine hormone that has profound effects on treatment of metabolic diseases. However, rhFGF21 is prone to form inclusion body when expressed in bacteria, which results in, the downstream process of purification of bioactive rhFGF21 is time-consuming and labor intensive. The aim of this work is to explore a new method for improving the soluble expression and secretion level of rhFGF21 in B. subtilis. RESULTS: A codon optimized rhFGF21 gene was expressed under the control of a strong inducible promoter PmalA in B. subtilis. A mini-cistron cassette (from gsiB) was located upstream of rhFGF21 in expression vector (pMATEFc5), which could reduce the locally stabilized mRNA secondary structure of transcripts and enhance the efficiency of translation initiation. Then various chaperones were further overexpressed to improve the expression efficiency of rhFGF21. Results showed that overexpression of the chaperone DnaK contributed to the increase of solubility of rhFGF21. Moreover, an extracellular proteases deficient strain B. subtilis Kno6cf was used to accumulate the secreted rhFGF21 solidly. In addition, eleven signal peptides from B. subtilis were evaluated and the SPdacB appeared the highest secretion yield of rhFGF21 in B. subtilis. Finally, the combinatorial optimized strain achieved an about ninefold increase of the soluble rhFGF21 production after 24 h of flask fermentation in comparison with the initial production strain. CONCLUSION: This work provided a comprehensive strategy for secretory expressing the heterologous protein rhFGF21 in B. subtilis. To our knowledge, this is the first report of the highly efficient production of rhFGF21 in B. subtilis and this approach may provide some suggestions for heterologous proteins production in B. subtilis.

Ultra-high-throughput picoliter-droplet microfluidics screening of the industrial cellulase-producing filamentous fungus Trichoderma reesei.

The selection of improved producers among the huge number of variants in mutant libraries is a key issue in filamentous fungi of industrial biotechnology. Here, we developed a droplet-based microfluidic high-throughput screening platform for selection of high-cellulase producers from filamentous fungus Trichoderma reesei. The screening system used a fluorogenic assay to measure amount of cellulase and its activity. The key effectors such as cellulase-inducing medium, spore germination, droplet cultivation time, droplet fluorescence signal detection, and droplet cell sorting were studied. An artificial pre-mixed library of high- and low-cellulase-producing T. reesei strains was screened successfully to verify the feasibility of our method. Finally, two cellulase hyperproducers exhibiting improvements in cellulase activity of 27% and 46% were isolated from an atmospheric and room-temperature plasma (ARTP)-mutated library. This high-throughput screening system could be applied to the engineering of T. reesei strains and other industrially valuable protein-producing filamentous fungi.

A Novel Methodology for Series Arc Fault Detection by Temporal Domain Visualization and Convolutional Neural Network.

AC arc faults are one of the most important causes of residential electrical wiring fires, which may produce extremely high temperatures and easily ignite surrounding combustible materials. The global interest in machine learning-based methods for arc fault diagnosis applications is increasing due to continuous challenges in efficiency and accuracy. In this paper, a temporal domain visualization convolutional neural network (TDV-CNN) methodology is proposed. The current transformer and high-speed data acquisition system are used to collect the current of a series of arc faults, then the signal is filtered by a digital filter and converted into a gray image in time sequence before being fed into TDV-CNN. Five different electric loads were selected for experimental validation with various signal characteristics, including vacuum cleaner, fluorescent lamp, dimmer, heater, and desktop computer. The experimental results confirm that the classification accuracy of the five loads' work states in the ten categories could reach 98.7% or even higher by adjusting parameters perfectly. The methodology is believed to be reliable for series arc detection with relatively high accuracy and also has important potential applications in other fault diagnosis fields.

Protodioscin ameliorates oxidative stress, inflammation and histology outcome in Complete Freund's adjuvant induced arthritis rats.

Protective effect of protodioscin or methyl protodioscin against inflammation had been reported in various inflammation diseases. This study aimed to investigate the effect of protodioscin against Complete Freund's adjuvant (CFA)-induced arthritis rats. Rats randomly divided into model groups were injected with CFA, companied with different dose of protodioscin (50, 100, and 200 mg/kg body weight). The histology, changes in biochemical parameters and inflammatory cytokines expression were detected for anti-inflammation effect evaluation of protodioscin. CFA treatment induced arthritic rats with swelling paw, ankle inflammation, and area of lymphocyte infiltration, upregulated inflammatory cytokines (IL-1ß, TNF-α, cyclo-oxygenase 2, and IL-6 as well as prostaglandin E2), articular elastase, myeloperoxidase, lipid peroxidase and nitrite oxide levels, downregulated glutathione, catalase, and superoxide dismutase. In contrast, protodioscin ameliorated all the changes induced by CFA in rats, suggesting the anti-inflammatory effect of protodioscin. We concluded that protodioscin administration into CFA-induced arthritis rats protected against CFA-induced oxidative stress, neutrophil infiltration, and inflammation, suggesting the anti-inflammatory effect and the therapeutic potential of protodioscin for arthritis.

Temporal-spatial analysis of transportation CO2 emissions in China: Clustering and policy recommendations.

Reducing transportation-related carbon dioxide (CO2) emissions in China poses significant challenges due to the sector's growth potential and variations among provinces and transportation modes. This study utilizes the bottom-up approach and the Logarithmic Mean Divisia Index (LMDI) decomposition method to calculate transportation CO2 emissions and explores the temporal-spatial differences across Chinese provinces. The results reveal that national transportation CO2 emissions increased by 50.14% from 2010 to 2019, and emissions from private cars present the fastest growth among all transportation modes by 254% over the decade. Spatially, higher emissions are found in eastern provinces, and neighboring provinces notably distinguish from each other in terms of the emission proportion of different modes and the factor analysis from LMDI. Regarding the heterogeneity of the spatial emission characteristics, a cluster-based evaluation method is proposed for the 31 provinces according to the emission structure and the LMDI decomposition. Four clusters are derived, each featuring varied emission distribution and driving factors. Correspondingly, policy recommendations are proposed to address the characteristics of each cluster, such as controlling car ownership, promoting integrated transport modes, improving fuel economy, and electrifying urban transportation services. The cluster-based analysis method can provide more specific suggestions to province targeting its emission characteristics rather than its location, which is one of the major contributions of this study.

Numerical investigations of AC arcs' thermal characteristics in the short gap of copper-cored wires.

Excessive alternating current (AC) arcs generated in electric systems will accumulate heat and easily cause fire. This paper studies the thermal characteristics of different numbers of AC arc plasma generated in a short gap of copper-cored wires in the air. The number of AC arcs is controlled in the AC arc experiment and an infrared thermal imager measures the temperature change at the specified position. Based on magnetohydrodynamics (MHD), a two-dimensional axisymmetric AC arc discharge numerical simulation model is established. The volt-ampere characteristic of the AC arc is used to solve the MHD simulation model to obtain the same 'zero current' characteristics as the real AC arc in the experiment. A large amount of heat accumulates in the electrode gaps when the arc generation, and then the heat dissipates in the 'zero current' stage. The continuously generated arc makes the temperature higher. The volume of the space area with a temperature higher than 10,000 K increases with the arc current, but is unrelated to the number of arcs. The volume of the space area with a temperature higher than 524.15 K and the temperature on the electrode are both positively correlated with the number of AC arcs and arc current. The results of this study can provide a reference for the detection standard of AC arc faults and the prevention of electrical fire.

Increasing activity and thermal resistance of Bacillus gibsonii alkaline protease (BgAP) by directed evolution.

Bacillus gibsonii Alkaline Protease (BgAP) is a recently reported subtilisin protease exhibiting activity and stability properties suitable for applications in laundry and dish washing detergents. However, BgAP suffers from a significant decrease of activity at low temperatures. In order to increase BgAP activity at 15°C, a directed evolution campaign based on the SeSaM random mutagenesis method was performed. An optimized microtiter plate expression system in B. subtilis was established and classical proteolytic detection methods were adapted for high throughput screening. In parallel, the libraries were screened for increased residual proteolytic activity after incubation at 58°C. Three iterative rounds of directed BgAP evolution yielded a set of BgAP variants with increased specific activity (K(cat)) at 15°C and increased thermal resistance. Recombination of both sets of amino acid substitutions resulted finally in variant MF1 with a 1.5-fold increased specific activity (15°C) and over 100 times prolonged half-life at 60°C (224 min compared to 2 min of the WT BgAP). None of the introduced amino acid substitutions were close to the active site of BgAP. Activity-altering amino acid substitutions were from non-charged to non-charged or from sterically demanding to less demanding. Thermal stability improvements were achieved by substitutions to negatively charged amino acids in loop areas of the BgAP surface which probably fostered ionic and hydrogen bonds interactions.

Directed evolution of cytochrome P450 for sterol epoxidation.

16,17-Epoxysterol plays an important role in pharmaceutical steroid synthesis. To investigate the potential application of cytochrome P450 for epoxysterol synthesis, an approach to the epoxidation of 16,17-epoxysterol, based on directed evolution of cytochrome P450 BM-3, was developed. This comprised random gene mutagenesis for optimizing the activity of P450 BM-3 for epoxidation of hydrophobic sterol, followed by the 7-ethoxycoumarin de-ethylation assay for general enzyme activity detection and the modified picric acid assay for epoxidation activity screening. By the two-step screening, one mutant from 792 clones showed specific substrate activity of converting progesterone to 16,17-epoxysterol, which validated the possibility to evolve the cytochrome P450 for the synthesis of steroidal epoxides.

Fuzi polysaccharides improve immunity in immunosuppressed mouse models by regulating gut microbiota composition.

Rationale and objectives: Fuzi, the dried root of Aconitum carmichaelii Debx, is one of the widely used traditional Chinese medicines. Fuzi polysaccharides are considered the most bioactive compounds with immunomodulatory functions, however, the mechanisms have not been evaluated. This study aims to systematically investigate the effects of Fuzi polysaccharides on the gut microbiota and immune function using a mouse model immunosuppressed with cyclophosphamide. Methods: The short-chain fatty acid levels in cecal contents were measured by gas chromatography-mass spectrometry. The gut microbiota 16S rRNA gene were sequenced by next generation sequencing. The mRNA expression levels of NF-κB, IL-6, TNF-α, iNOS and COX-2 were measured using quantitative real-time polymerase chain reaction. The protein expression of occludin and zonula occludens-1 were analyzed by Western blot. The white blood cells were counted using automated hematology analyzer, and CD4+FOXP3+/CD4+ ratio was measured by flow cytometry. Results and Conclusions: Fuzi polysaccharides had the function of elevating the concentration of acetic acid, propionic acid, isobutyric acid, and n-butyric acid in the cecum. Meanwhile, Fuzi polysaccharides could decrease the relative abundance of Helicobacter, Anaerotruncus, Faecalibacterium, Lachnospira, Erysipelotrichaceae_UCG-003, Mucispirillum, and Mycoplasma, and increase the relative abundance of Rhodospirillales, Ruminococcaceae_UCG-013, Mollicutes_RF39, Ruminococcus_1, Christensenellaceae_R-7_group, and Muribaculaceae in the gut. Furthermore, Fuzi polysaccharides exhibited the function of increasing spleen and thymus indices and number of white blood cells and lymphocytes. Fuzi polysaccharides could reverse the decreased mRNA expression of NF-кB, IL-6, and iNOS, differentiation of CD4+FOXP3+ regulatory T cells as well as protein expression of occludin and zonula occludens-1 induced by cyclophosphamide. In addition, the mRNA and protein expression of cytokines were significantly correlated with the abundance of gut microbiota under Fuzi polysaccharides treatment. Collectively, the above results demonstrated that Fuzi polysaccharides could regulate inflammatory cytokines and gut microbiota composition of immunosuppressive mice to improve immunity, thereby shedding light on revealing the molecular mechanism of polysaccharides of traditional Chinese medicines in the future.

Characterization of a Riboflavin-Producing Mutant of Bacillus subtilis Isolated by Droplet-Based Microfluidics Screening.

Bacillus subtilis is one of the commonly used industrial strains for riboflavin production. High-throughput screening is useful in biotechnology, but there are still an insufficient number of articles focusing on improving the riboflavin production of B. subtilis by this powerful tool. With droplet-based microfluidics technology, single cells can be encapsulated in droplets. The screening can be carried out by detecting the fluorescence intensity of secreted riboflavin. Thus, an efficient and high-throughput screening method suitable for riboflavin production strain improvement could be established. In this study, droplet-based microfluidics screening was applied, and a more competitive riboflavin producer U3 was selected from the random mutation library of strain S1. The riboflavin production and biomass of U3 were higher than that of S1 in flask fermentation. In addition, the results of fed-batch fermentation showed that the riboflavin production of U3 was 24.3 g/L, an 18% increase compared with the parent strain S1 (20.6 g/L), and the yield (g riboflavin/100 g glucose) increased by 19%, from 7.3 (S1) to 8.7 (U3). Two mutations of U3 (sinRG89R and icdD28E) were identified through whole genome sequencing and comparison. Then they were introduced into BS168DR (parent of S1) for further analysis, which also caused riboflavin production to increase. This paper provides protocols for screening riboflavin-producing B. subtilis with droplet-based microfluidics technology and reveals mutations in riboflavin overproduction strains.

Repurposing conformational changes in ANL superfamily enzymes to rapidly generate biosensors for organic and amino acids.

Biosensors are powerful tools for detecting, real-time imaging, and quantifying molecules, but rapidly constructing diverse genetically encoded biosensors remains challenging. Here, we report a method to rapidly convert enzymes into genetically encoded circularly permuted fluorescent protein-based indicators to detect organic acids (GECFINDER). ANL superfamily enzymes undergo hinge-mediated ligand-coupling domain movement during catalysis. We introduce a circularly permuted fluorescent protein into enzymes hinges, converting ligand-induced conformational changes into significant fluorescence signal changes. We obtain 11 GECFINDERs for detecting phenylalanine, glutamic acid and other acids. GECFINDER-Phe3 and GECFINDER-Glu can efficiently and accurately quantify target molecules in biological samples in vitro. This method simplifies amino acid quantification without requiring complex equipment, potentially serving as point-of-care testing tools for clinical applications in low-resource environments. We also develop a GECFINDER-enabled droplet-based microfluidic high-throughput screening method for obtaining high-yield industrial strains. Our method provides a foundation for using enzymes as untapped blueprint resources for biosensor design, creation, and application.

Quantifying the Impact of Alternative Bus Stop Platforms on Vehicle Emissions and Individual Pollution Exposure at Bus Stops.

Due to stop-and-go events, bus stops are often treated as "hot spots" for air pollution. The design of bus stops should be optimized to reduce emissions and exposure for transit commuters. The objective of this study was to analyze the impact of bus stop platform types on vehicle emissions and individual pollution exposure. Second-by-second emissions data were first collected from one bus using a portable emission measurement system (PEMS). Microscopic traffic simulation was then used to estimate overall traffic emissions under six scenarios with different bus stop settings. Numerical simulation of pollutant dispersion was also conducted to calculate individual pollution exposure at bus stops. The results of PEMS tests showed no significant differences between bus emissions generated near two different types of stops. However, the effect of platform types on overall traffic emissions was revealed using traffic simulation. The results demonstrated that bus bays reduced the emissions of other heavy-duty vehicles. However, bus bays were not always effective during rush hours. The study also highlighted the importance of the location of bus stops, the number of bus lines, and the length of the platform, in addition to dynamic characteristics of traffic flows in the design of bus stop platforms. Bus stop platforms also affected individuals' exposure due to the changes in the pollutant flow field. The passenger's exposure at one bus stop was influenced by both the platform type and standing location. Results suggested that in a condition with a wind direction perpendicular to the bus stop shelter, the total exposure level to CO was lower at the bus bay stop if a passenger stood at the upstream of the station platform. However, the exposure was less at the downstream of the curbside bus stop.

Effective and Acceptable Eco-Driving Guidance for Human-Driving Vehicles: A Review.

Eco-driving guidance refers to courses, warnings, or suggestions provided to human drivers to improve driving behaviour to enable less energy use and emissions. This paper reviews existing eco-driving guidance studies and identifies challenges to tackle in the future. We summarize two categories of current guidance systems, static and dynamic, distinguished by whether real-world driving records are used to generate behaviour guidance or not. We find that influencing factors, such as the content of suggestions, the display methods, and drivers' socio-demographic characteristics, have varied effects on the guidance results across studies. Drivers are reported to have basic eco-driving knowledge, while the question of how to motivate the acceptance and practice of such behaviour, especially in the long term, is overlooked. Adaptive driving suggestions based on drivers' individual habits can improve the effectiveness and acceptance while this field is under investigation. In-vehicle assistance presents potential safety issues, and visualized in-vehicle assistance is reported to be most distractive. Given existing studies focusing on the operational level, a common agreement on the guidance design and associated influencing factors has yet to be reached. Research on the systematic and tactical design of eco-driving guidance and in-vehicle interaction is advised.

Microfluidic screening and genomic mutation identification for enhancing cellulase production in Pichia pastoris.

BACKGROUND: Pichia pastoris is a widely used host organism for heterologous production of industrial proteins, such as cellulases. Although great progress has been achieved in improving protein expression in P. pastoris, the potential of the P. pastoris expression system has not been fully explored due to unknown genomic impact factors. Recently, whole-cell directed evolution, employing iterative rounds of genome-wide diversity generation and high-throughput screening (HTS), has been considered to be a promising strategy in strain improvement at the genome level. RESULTS: In this study, whole-cell directed evolution of P. pastoris, employing atmospheric and room temperature plasma (ARTP) mutagenesis and droplet-based microfluidic HTS, was developed to improve heterogenous cellulase production. The droplet-based microfluidic platform based on a cellulase-catalyzed reaction of releasing fluorescence was established to be suitable for methanol-grown P. pastoris. The validation experiment showed a positive sorting efficiency of 94.4% at a sorting rate of 300 droplets per second. After five rounds of iterative ARTP mutagenesis and microfluidic screening, the best mutant strain was obtained and exhibited the cellulase activity of 11,110 ± 523 U/mL, an approximately twofold increase compared to the starting strain. Whole-genome resequencing analysis further uncovered three accumulated genomic alterations in coding region. The effects of point mutations and mutant genes on cellulase production were verified using reconstruction of point mutations and gene deletions. Intriguingly, the point mutation Rsc1G22V was observed in all the top-performing producers selected from each round, and gene deletion analysis confirmed that Rsc1, a component of the RSC chromatin remodeling complex, might play an important role in cellulase production. CONCLUSIONS: We established a droplet-based microfluidic HTS system, thereby facilitating whole-cell directed evolution of P. pastoris for enhancing cellulase production, and meanwhile identified genomic alterations by whole-genome resequencing and genetic validation. Our approaches and findings would provide guides to accelerate whole-cell directed evolution of host strains and enzymes of high industrial interest.