Genome-Scale Imaging of the 3D Organization and Transcriptional Activity of Chromatin.

The 3D organization of chromatin regulates many genome functions. Our understanding of 3D genome organization requires tools to directly visualize chromatin conformation in its native context. Here we report an imaging technology for visualizing chromatin organization across multiple scales in single cells with high genomic throughput. First we demonstrate multiplexed imaging of hundreds of genomic loci by sequential hybridization, which allows high-resolution conformation tracing of whole chromosomes. Next we report a multiplexed error-robust fluorescence in situ hybridization (MERFISH)-based method for genome-scale chromatin tracing and demonstrate simultaneous imaging of more than 1,000 genomic loci and nascent transcripts of more than 1,000 genes together with landmark nuclear structures. Using this technology, we characterize chromatin domains, compartments, and trans-chromosomal interactions and their relationship to transcription in single cells. We envision broad application of this high-throughput, multi-scale, and multi-modal imaging technology, which provides an integrated view of chromatin organization in its native structural and functional context.

Enhancing the Catalytic Efficiency of D-lactonohydrolase through the Synergy of Tunnel Engineering, Evolutionary Analysis, and Force-Field Calculations.

Computational design advances enzyme evolution and their use in biocatalysis in a faster and more efficient manner. In this study, a synergistic approach integrating tunnel engineering, evolutionary analysis, and force-field calculations has been employed to enhance the catalytic activity of D-lactonohydrolase (D-Lac), which is a pivotal enzyme involved in the resolution of racemic pantolactone during the production of vitamin B5. The best mutant, N96S/A271E/F274Y/F308G (M3), was obtained and its catalytic efficiency (kcat/KM) was nearly 23-fold higher than that of the wild-type. The M3 whole-cell converted 20 % of DL-pantolactone into D-pantoic acid (D-PA, >99 % e.e.) with a conversion rate of 47 % and space-time yield of 107.1 g L-1 h-1, demonstrating its great potential for industrial-scale D-pantothenic acid production. Molecular dynamics (MD) simulations revealed that the reduction in the steric hindrance within the substrate tunnel and conformational reconstruction of the distal loop resulted in a more favourable"catalytic" conformation, making it easier for the substrate and enzyme to enter their pre-reaction state. This study illustrates the potential of the distal residue on the pivotal loop at the entrance of the D-Lac substrate tunnel as a novel modification hotspot capable of reshaping energy patterns and consequently influencing the enzymatic activity.

4-Hydroxyphenylacetate 3-Hydroxylase (4HPA3H): A Vigorous Monooxygenase for Versatile O-Hydroxylation Applications in the Biosynthesis of Phenolic Derivatives.

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) is a long-known class of two-component flavin-dependent monooxygenases from bacteria, including an oxygenase component (EC 1.14.14.9) and a reductase component (EC 1.5.1.36), with the latter being accountable for delivering the cofactor (reduced flavin) essential for o-hydroxylation. 4HPA3H has a broad substrate spectrum involved in key biological processes, including cellular catabolism, detoxification, and the biosynthesis of bioactive molecules. Additionally, it specifically hydroxylates the o-position of the C4 position of the benzene ring in phenolic compounds, generating high-value polyhydroxyphenols. As a non-P450 o-hydroxylase, 4HPA3H offers a viable alternative for the de novo synthesis of valuable natural products. The enzyme holds the potential to replace plant-derived P450s in the o-hydroxylation of plant polyphenols, addressing the current significant challenge in engineering specific microbial strains with P450s. This review summarizes the source distribution, structural properties, and mechanism of 4HPA3Hs and their application in the biosynthesis of natural products in recent years. The potential industrial applications and prospects of 4HPA3H biocatalysts are also presented.

Purification and characterization of aspartic protease from Aspergillus niger and its efficient hydrolysis applications in soy protein degradation.

BACKGROUND: Adding acid protease to feed can enhance protein digestibility, boost feed utilization, and stimulate the growth of animals in breading industry. In order to obtain an acid protease with high hydrolysis efficiency to plant protein, in this study, an aspartic protease from Aspergillus niger was heterologous expressed in Pichia pastoris (P. pastoris). The enzymatic properties and application in soybean protein degradation were also studied. RESULTS: In our investigation, the high aspartic protease (Apa1) activity level of 1500 U/mL was achieved in 3 L bioreactor. After dialysis and anion exchange chromatography, the total enzyme activity and specific enzyme activity were 9412 U and 4852 U/mg, respectively. The molecular weight of the purified protease was 50 kDa, while the optimal pH and temperature were 3.0 and 50 °C, respectively. It was stable at pH 2.0-5.0 and 30-60 °C. Apa1 was used to hydrolyze soybean isolate protein (SPI) at 40 °C and pH 3.0, and a high hydrolysis degree (DH) of 61.65% was achieved. In addition, the molecular weight distribution of SPI hydrolysis products was studied, the result showed that the hydrolysis products were primarily oligopeptides with molecular weights of 189 Da or below. CONCLUSIONS: In this study, Apa1 was successfully expressed in P. pastoris and high expression level was obtained. In addition, the highest protein hydrolysis rate to SPI degradation so far was achieved. The acid protease in this study provides a new protease that is suitable for the feed industry, which will be very helpful to improve the feed utilization and promote the development of the breeding industry.

Immobilization of Actinobacillus succinogenes on nano- and micro-fiber membranes for efficient and robust production of succinic acid.

This work aimed to study the efficiency of nano- and micro- fiber membranes in immobilizing Actinobacillus succinogenes CCTCC M2012036 for succinic acid production. Among the four kinds of electrospun nanofiber membranes of cellulose acetate, chitosan, poly(vinyl alcohol) (PVA) and chitosan-PVA, the cellulose acetate nanofiber membrane-immobilized cells performed the best with a succinic acid concentration and yield to be 27.3 ± 3.5 g/L and 70.9 ± 5.8%. The cell-immobilized viscose microfiber membrane presented good reuse stability, and 17 batches of fermentation without activity loss were realized with the highest succinic acid yield of 83.20%. A microfiber membrane bioreactor was further constructed with the cell-immobilized viscose microfiber membrane to perform fermentation on a larger scale, and the concentration, yield and productivity of succinic acid were 73.20 g/L, 86.50% and 1.49 g/(L⋅h) using a fed-batch strategy, which were 124.30%, 127.60% and 124.2% of those obtained in the traditional fermenter. This study provided an approach for improving the practicality of biological succinic acid production.

Rtt105 functions as a chaperone for replication protein A to preserve genome stability.

Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA-ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA-importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA-ssDNA complex. Single-molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.

Human Arm Motion Prediction for Collision Avoidance in a Shared Workspace.

Industry 4.0 transforms classical industrial systems into more human-centric and digitized systems. Close human-robot collaboration is becoming more frequent, which means security and efficiency issues need to be carefully considered. In this paper, we propose to equip robots with exteroceptive sensors and online motion generation so that the robot is able to perceive and predict human trajectories and react to the motion of the human in order to reduce the occurrence of the collisions. The dataset for training is generated in a real environment in which a human and a robot are sharing their workspace. An Encoder-Decoder based network is proposed to predict the human hand trajectories. A Model Predictive Control (MPC) framework is also proposed, which is able to plan a collision-free trajectory in the shared workspace based on this human motion prediction. The proposed framework is validated in a real environment that ensures collision free collaboration between humans and robots in a shared workspace.

The clinical value of long - term electroencephalogram (EEG) in seizure - free populations: implications from a cross-sectional study.

BACKGROUD: This study aimed to explore the clinical value of long - term electroencephalogram (LTM EEG) in seizure-free individuals taking antiepileptic drugs (AEDs) for more than 2 years. We try to look for clinical factors associated with epileptiform activity on LTM EEG in seizure free patients. We hope that the detection of epileptiform activity by the LTM EEG recording can develop the better treatment strategy. METHODS: The LTM EEG recordings of 770 individuals with a definite diagnosis of epilepsy were assessed. Two hundred sixty-two individuals accorded with the inclusion criteria and exclusion criteria. We collect the demographic and clinical information and LTM EEG data of these 262 individuals. We analysed the data by one-way analysis of variance and Cox proportional hazards models. RESULTS: We found that more epileptiform activity were found with LTM EEG recording than regular EEG recording in seizure-free individuals. We found several clinical factors could be associated with epileptiform activity on LTM EEG in seizure free patients by a one-way analysis: symptomatic or cryptogenic epilepsy [hazard ratio (HR) = 2.6], history of cerebral trauma (HR = 7.5), and abnormal imaging findings (HR = 3.1). The following factors suggested a correlation between history of cerebral trauma (HR = 2.4) and history of cerebral surgery (HR = 3.4) with epileptiform activity on LTM EEG presentation by multivariate logistic regression analysis. CONCLUSIONS: The study indicated a correlation of a number of factors with abnormal LTM EEG presentation: symptomatic or cryptogenic epilepsy, history of cerebral trauma, history of cerebral surgery, and abnormal imaging findings. The LTM EEG recording may help find epileptiform activity in high risk seizure-free individuals. The individuals need be reevaluated the therapeutic strateagies, and increase the hope to reach real seizure-free.

Engineering Corynebacterium glutamicum for the de novo biosynthesis of tailored poly-γ-glutamic acid.

γ-Polyglutamic acid (γ-PGA) is a biodegradable polymer naturally produced by Bacillus spp. that has wide applications. Fermentation of γ-PGA using Bacillus species often requires the supplementation of L-glutamic acid, which greatly increases the overall cost. Here, we report a metabolically engineered Corynebacterium glutamicum capable of producing γ-PGA from glucose. The genes encoding γ-PGA synthase complex from B. subtilis (pgsB, C, and A) or B. licheniformis (capB, C, and A) were expressed under inducible promoter Ptac in a L-glutamic acid producer C. glutamicum ATCC 13032, which led to low levels of γ-PGA production. Subsequently, C. glutamicum F343 with a strong L-glutamic acid production capability was tested. C. glutamicum F343 carrying capBCA produced γ-PGA up to 11.4 g/L, showing a higher titer compared with C. glutamicum F343 expressing pgsBCA. By introducing B. subtilis glutamate racemase gene racE under Ptac promoter mutants with different expression strength, the percentage of L-glutamic acid units in γ-PGA could be adjusted from 97.1% to 36.9%, and stayed constant during the fermentation process, while the γ-PGA titer reached 21.3 g/L under optimal initial glucose concentrations. The molecular weight (Mw) of γ-PGA in the engineered strains ranged from 2000 to 4000 kDa. This work provides a foundation for the development of sustainable and cost-effective de novo production of γ-PGA from glucose with customized ratios of L-glutamic acid in C. glutamicum.

Construction of synthetic pathways for raspberry ketone production in engineered Escherichia coli.

Raspberry ketone is an important ingredient in the flavor and fragrance industries. Due to its low content in fruits and vegetables, the production of natural raspberry ketone using heterologous synthesis in microbial strains is recently attracting increased attention. In this work, a heterologous pathway to produce raspberry ketone from p-coumaric acid, including 4-coumarate: CoA ligase (4CL), benzalacetone synthase (BAS), and raspberry ketone/zingerone synthase (RZS1) from plants, was successfully assembled in Escherichia coli. When the RZS1 gene was introduced into E. coli and co-expressed with two other genes, the intermediate 4-hydroxybenzylidene acetone in the pathway was almost completely transformed into a raspberry ketone. Substituting TB medium for M9 medium increased raspberry ketone titers by 3-4 times. Furthermore, the heterologous pathway was partitioned into two modules; module one produced p-coumaroyl-CoA from p-coumaric acid by 4CL, and module two produced raspberry ketone from coumaroyl-CoA by the action of BAS and RZS1. Optimizing the balanced expression of the two modules, it was shown that moderate expression of module one and high expression of module two was the best combination to enhance raspberry ketone production. The engineered strain CZ-8 reached 90.97 mg/l of raspberry ketone, which was 12 times higher than previously reported. In addition, the preferred approach of the heterologous pathway was related to the heterologous genes from different sources; for example, 4CL from Arabidopsis thaliana seemed to be more suitable for raspberry ketone production than that from Petroselinum crispum. This work paves an alternative way for future economic production of natural raspberry ketone.

Construction of fibrous bed bioreactor for enhanced succinic acid production using wastewater of dextran fermentation.

A new applicability of wastewater of dextran fermentation (WWDF) for biological production of succinic acid with A. succinogenes CCTCC M2012036 was reported in this work for the first time. Notably, K2CO3 was used instead of MgCO3 in the pH regulating process for operational feasibility and a cell immobilization methodology by attaching cells on cotton fibrous matrix was adopted for cell recycle. The initial sugar concentration as well as matrix usage was optimized by investigating the cell growth, succinic acid concentration and yield. A rotated fibrous bed bioreactor was designed and constructed in order to increase the total cell amount and facilitate mass transportation in the fermentation system, and an average succinic acid yield, concentration and productivity of 0.82 g/g, 56.5 g/L and 1.28 g/L/h were realized in the repeated fed-batch fermentation, respectively. This research gave light to the optimization of succinic acid production towards a more cost-effective and operable direction.

Muconic acid production from glucose using enterobactin precursors in Escherichia coli.

Muconic acid (MA) is a promising bulk chemical due to its extensive industrial applications in the production of adipic acid and other valuable, biodegradable intermediates. MA is heretofore mainly produced from petrochemicals by organic reactions which are not environmentally friendly or renewable. Biological production processes provide a promising alternative for MA production. We designed an artificial pathway in Escherichia coli for the biosynthesis of MA using the catechol group of 2,3-dihydroxybenzoate, an intermediate in the enterobactin biosynthesis pathway. This approach consists of two heterologous microbial enzymes, including 2,3-dihydroxybenzoate decarboxylase and catechol 1,2-dioxygenase. The metabolic flow of carbon into the heterologous pathway was optimized by increasing the flux from chorismate through the enterobactin biosynthesis pathway and by regulating the shikimate pathway. Metabolic optimization enabled a concentration of 605.18 mg/L of MA from glucose in a shaking flask culture, a value nearly 484-fold higher than that of the initial recombinant strain. The results indicated that the production of MA from this pathway has the potential for further improvement.

Ultra-long high-sensitivity Φ-OTDR for high spatial resolution intrusion detection of pipelines.

An ultra-long phase-sensitive optical time domain reflectometry (Φ-OTDR) that can achieve high-sensitivity intrusion detection over 131.5km fiber with high spatial resolution of 8m is presented, which is the longest Φ-OTDR reported to date, to the best of our knowledge. It is found that the combination of distributed Raman amplification with heterodyne detection can extend the sensing distance and enhances the sensitivity substantially, leading to the realization of ultra-long Φ-OTDR with high sensitivity and spatial resolution. Furthermore, the feasibility of applying such an ultra-long Φ-OTDR to pipeline security monitoring is demonstrated and the features of intrusion signal can be extracted with improved SNR by using the wavelet detrending/denoising method proposed.

Microstructure and Properties of Hot Pressing Sintered SiC/Y3Al5O12 Composite Ceramics for Dry Gas Seals.

Silicon carbide (SiC) ceramics with high bending strength were prepared by hot pressing sintering (HPS) with yttrium aluminum garnet (Y3Al5O12, YAG) as sintering additive, and the effects of YAG content and sintering temperature on the sintering behavior, microstructure and mechanical properties of SiC ceramics were investigated in detail. The uniform distribution of YAG to form a liquid phase and the driving force provided by hot pressing sintering decrease the sintering temperature, improve the densification of SiC ceramics, and refine the crystal size. By means of suitable sintering conditions with the additional amount of YAG of 5 wt%, the sintering temperature of 1950 °C and a pressure of 30 MPa, the resultant SiC/YAG composite ceramics possesses high sintering and mechanical properties with the relative density of 98.53%, the bending strength of 675 MPa, the Vickers hardness of up to 17.92 GPa, and the elastic modulus of 386 GPa. The as-prepared SiC/YAG composite ceramics are promisingly used as the dry gas seal materials in the centrifugal compressors.

Computer-driven Evolution of Myrosinase from the Cabbage Aphid for Efficient Production of (R)-Sulforaphane.

Myrosinase (Myr) catalyzes the hydrolysis of glucosinolates, yielding biologically active metabolites. In this study, glucoraphanin (GRA) extracted from broccoli seeds was effectively hydrolyzed using a Myr-obtained cabbage aphid (Brevicoryne brassicae) (BbMyr) to produce (R)-sulforaphane (SFN). The gene encoding BbMyr was successfully heterologously expressed in Escherichia coli, resulting in the production of 1.6 g/L (R)-SFN, with a remarkable yield of 20.8 mg/gbroccoli seeds, achieved using recombination E. coli whole-cell catalysis under optimal conditions (pH 4.5, 45 °C). Subsequently, BbMyr underwent combinatorial simulation-driven mutagenesis, yielding a mutant, DE9 (N321D/Y426S), showing a remarkable 2.91-fold increase in the catalytic efficiency (kcat/KM) compared with the original enzyme. Molecular dynamics simulations demonstrated that the N321D mutation in loopA of mutant DE9 enhanced loopA stability by inducing favorable alterations in hydrogen bonds, while the Y426S mutation in loopB decreased spatial resistance. This research lays a foundation for the environmentally sustainable enzymatic (R)-SFN synthesis.

Engineered Zea mays phenylalanine ammonia-lyase for improve the catalytic efficiency of biosynthesis trans-cinnamic acid and p-coumaric acid.

Phenylalanine ammonia-lyase (PAL) plays a pivotal role in the biosynthesis of phenylalanine. PAL from Zea mays (ZmPAL2) exhibits a bi-function of direct deamination of L-phenylalanine (L-Phe) or L-tyrosine(-L-Tyr) to form trans-cinnamic acid or p-coumaric acid. trans-Cinnamic acid and p-coumaric acid are mainly used in flavors and fragrances, food additives, pharmaceutical and other fields. Here, the Activity of ZmPAL2 toward L-Phe or L-Tyr was improved by using semi-rational and rational designs. The catalytic efficiency (kcat/Km) of mutant PT10 (V258I/I459V/Q484N) against L-Phe was 30.8 µM-1 s-1, a 4.5-fold increase compared to the parent, and the catalytic efficiency of mutant PA1 (F135H/I459L) to L-tyrosine exhibited 8.6 µM-1 s-1, which was 1.6-fold of the parent. The yield of trans-cinnamic acid in PT10 reached 30.75 g/L with a conversion rate of 98%. Meanwhile, PA1 converted L-Tyr to yield 3.12 g/L of p-coumaric acid with a conversion rate of 95%. Suggesting these two engineered ZmPAL2 to be valuable biocatalysts for the synthesis of trans-cinnamic acid and p-coumaric acid. In addition, MD simulations revealed that the underlying mechanisms of the increased catalytic efficiency of both mutant PT10 and PA1 are attributed to the substrate remaining stable within the pocket and closer to the catalytically active site. This also provides a new perspective on engineered PAL.

Engineering of 4-hydroxyphenylacetate 3-hydroxylase derived from Pseudomonas aeruginosa for the ortho-hydroxylation of ferulic acid.

Polyphenolic compounds have natural antioxidant properties, and their antioxidant activity is usually related to the number and position of hydroxyls. Here, we successfully applied the engineered 4-hydroxyphenylacetate 3-hydroxylases (4HPA3Hs) derived from Pseudomonas aeruginosa to catalyze ferulic acid (FA) synthesis of ortho-hydroxyferulic acid (5-hydroxyferulic acid, 5-OHFA). Through optimization of co-expression, the oxygenase component (PaHpaB) and the reductase component (PaHpaC) in E. coli, and optimization of whole-cell catalytic conditions, the engineered strain BC catalyzed ortho-hydroxylation of 2 g/L of FA with a yield of 75 % from 39 %. Through tunnel engineering of PaHpaB, the obtained mutants F301A and Q376A almost completely transformed 2 g/L of FA. Further, a multiple mutant L214A/F301A/Q376A converted 4 g/L FA into 5-OHFA within 12 h, and the yield reached 99.9 %, which was approximately 2.39-fold of the wild type. The kcat/Km value of L214A/F301A/Q376A was about 307 times greater than that of the wide type. Analysis of three-dimensional structural models showed that L214, F301, and Q376 mutated into Ala, which greatly shortened the side chain and broadened the tunnel size, thereby significantly improving the catalytic efficiency of L214A/F301A/Q376A. This biosynthesis of 5-OHFA is simple, efficient, and green, suggesting that it is useful for efficient biosynthesis of polyphenolic compounds.

Spirit-based distillers' grain as a promising raw material for succinic acid production.

Spirit-based distillers' grain (SDG) is the main by-product of the Chinese liquor industry, with an annual output of approx. 100 million tons. The economical potential of fermentative production of succinic acid from SDG was investigated using Actinobacillus succinogens. Use of pretreated SDG (PSDG) as the sole source of C and N yielded succinic acid at 35.5 g l(-1) with a yield of 19.7 % (g per 100 g PSDG) after 48 h in a 3 l stirred bioreactor. SDG is thus a promising feedstock for the economical production of succinic acid.

Enhanced succinic acid production by Actinobacillus succinogenes after genome shuffling.

Succinic acid is an important platform chemical for synthesis of C4 compounds. We applied genome shuffling to improve fermentative production of succinic acid by A. succinogenes. Using a screening strategy composed of selection in fermentation broth, cultured in 96-deep-well plates, and condensed HPLC screening, a starting population of 11 mutants producing a higher succinic acid concentration was selected and subjected to recursive protoplasts fusion. After three rounds of genome shuffling, strain F3-II-3-F was obtained, producing succinic acid at 1.99 g/l/h with a yield of 95.6 g/l. The genome shuffled strain had about a 73 % improvement in succinic acid production compared to the parent strain after 48 h in fed-batch fermentation. The genomic variability of F3-II-3-F was confirmed by amplified fragment-length polymorphism. The activity levels of key enzymes involved in end-product formation from glucose and metabolic flux distribution during succinic acid production were compared between A. succinogenes CGMCC 1593 and F3-II-3-F. Increased activity of glucokinase, fructose-1,6-bisphosphate aldolase, PEP carboxykinase and fumarase, as well as decreased activity of pyruvate kinase, pyruvate formate-lyase, and acetate kinase explained the enhanced succinic acid production and decreased acetic acid formation. Metabolic flux analysis suggested that increased flux to NADH was the main reason for increased activity of the C4 pathway resulting in increased yields of succinic acid. The present work will be propitious to the development of a bio-succinic acid fermentation industry.

Effects of down-regulation of ackA expression by CRISPR-dCpf1 on succinic acid production in Actinobacillus succinogenes.

Succinic acid (SA), a key intermediate in the cellular tricarboxylic acid cycle (TCA), is a 4-carbon dicarboxylic acid of great industrial value. Actinobacillus succinogenes can ferment various carbon sources and accumulate relatively high concentrations of SA, but few reliable genetic engineering tools exist for A. succinogenes and this has hindered strain improvement to increase SA production for industrial application. Two different repressors, endonuclease-deactivated Cas9 (dCas9) from Streptococcus pyogenes and Cpf1 (dCpf1) from Francisella tularensis, were applied to construct a CRISPRi system in A. succinogenes. Codon-optimized Cas9 and native Cpf1 were successfully expressed in A. succinogenes, and the corresponding sgRNA and crRNA expression elements, promoted by the fumarate reductase promoter, frd, were introduced into the CRISPRi plasmid. The highest repression of the ackA gene (encoding acetate kinase) and thereby acetic acid production (~ eightfold) was achieved by the dCpf1-based CRISPRi system, in which the mutation site, E1006A acted at the start of the coding region of ackA, the gene which regulates acetic acid biosynthesis. Compared with the ackA gene knockout mutant, cell growth was moderately improved and SA production increased by 6.3%. Further, the SA titer and productivity in a 3 L fermenter reached 57.06 g/L and 1.87 g/L/h, and there was less acetic acid production. A dCpf1-based CRISPRi-mediated gene repression system was successfully established for the first time, providing a simple and effective tool for studying functional genomics in A. succinogenes and optimizing SA production.