Safety and efficacy of a feed additive consisting of l-threonine (produced with Escherichia coliCGMCC 7.455) for all animal species (Kempex Holland B.V.).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of the feed additive consisting of l-threonine produced by fermentation with Escherichia coli CGMCC 7.455 when used as a nutritional additive in feed and water for drinking for all animal species and categories. The production strain is genetically modified. None of the introduced genetic modifications raised a safety concern. Viable cells of the production strain and its DNA were not detected in the final additive. Therefore, the final product does not give raise to any safety concern regarding the genetic modification of the production strain. The use of l-threonine (≥ 98.5%) produced with E. coli CGMCC 7.455 to supplement feed is safe for the target species. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) has concerns on the safety of the simultaneous oral administration of l-threonine via water for drinking and feed due to possible amino acid imbalances and hygienic reasons. The use of l-threonine produced with E. coli CGMCC 7.455 in animal nutrition raises no safety concerns to consumers of animal products and to the environment. In the absence of data, the FEEDAP Panel cannot conclude on the potential of the additive to be irritant to skin or eyes, or on its potential to be a dermal sensitiser. The endotoxin activity in the additive does not pose a risk for the user via inhalation. The additive l-threonine is regarded as an effective source of the amino acid l-threonine for all non-ruminant species. In order to be as efficacious in ruminants as in non-ruminants, it should be protected from ruminal degradation.

Reconstruction the feedback regulation of amino acid metabolism to develop a non-auxotrophic L-threonine producing Corynebacterium glutamicum.

L-Threonine is an important feed additive with the third largest market size among the amino acids produced by microbial fermentation. The GRAS (generally regarded as safe) industrial workhorse Corynebacterium glutamicum is an attractive chassis for L-threonine production. However, the present L-threonine production in C. glutamicum cannot meet the requirement of industrialization due to the relatively low production level of L-threonine and the accumulation of large amounts of by-products (such as L-lysine, L-isoleucine, and glycine). Herein, to enhance the L-threonine biosynthesis in C. glutamicum, releasing the aspartate kinase (LysC) and homoserine dehydrogenase (Hom) from feedback inhibition by L-lysine and L-threonine, respectively, and overexpressing four flux-control genes were performed. Next, to reduce the formation of by-products L-lysine and L-isoleucine without the cause of an auxotrophic phenotype, the feedback regulation of dihydrodipicolinate synthase (DapA) and threonine dehydratase (IlvA) was strengthened by replacing the native enzymes with heterologous analogues with more sensitive feedback inhibition by L-lysine and L-isoleucine, respectively. The resulting strain maintained the capability of synthesizing enough amounts of L-lysine and L-isoleucine for cell biomass formation but exhibited almost no extracellular accumulation of these two amino acids. To further enhance L-threonine production and reduce the by-product glycine, L-threonine exporter and homoserine kinase were overexpressed. Finally, the rationally engineered non-auxotrophic strain ZcglT9 produced 67.63 g/L (17.2% higher) L-threonine with a productivity of 1.20 g/L/h (108.0% higher) in fed-batch fermentation, along with significantly reduced by-product accumulation, representing the record for L-threonine production in C. glutamicum. In this study, we developed a strategy of reconstructing the feedback regulation of amino acid metabolism and successfully applied this strategy to de novo construct a non-auxotrophic L-threonine producing C. glutamicum. The main end by-products including L-lysine, L-isoleucine, and glycine were almost eliminated in fed-batch fermentation of the engineered C. glutamicum strain. This strategy can also be used for engineering producing strains for other amino acids and derivatives.

Self-regulated efficient production of L-threonine via an artificial quorum sensing system in engineered Escherichia coli.

Imbalance in carbon flux distribution is one of the most important factors affecting the further increase in the yield of high value-added natural products in microbial metabolic engineering. Meanwhile, the most common inducible expression systems are difficult to achieve industrial-scale production due to the addition of high-cost or toxic inducers during the fermentation process. Quorum sensing system, as a typical model for density-dependent induction of gene expression, has been widely applied in synthetic biology. However, there are currently few reports for efficient production of microbial natural products by using quorum sensing system to self-regulate carbon flux distribution. Here, we designed an artificial quorum sensing system to achieve efficient production of L-threonine in engineered Escherichia coli by altering the carbon flux distribution of the central metabolic pathways at specific periods. Under the combination of switch module and production module, the system was applied to divide the microbial fermentation process into two stages including growth and production, and improve the production of L-threonine by self-inducing the expression of pyruvate carboxylase and threonine extracellular transporter protease after a sufficient amount of cell growth. The final strain TWF106/pST1011, pST1042pr could produce 118.2 g/L L-threonine with a yield of 0.57 g/g glucose and a productivity of 2.46 g/(L· h). The establishment of this system has important guidance and application value for the production of other high value-added chemicals in microorganisms by self-regulation.

An artificial biocatalytic cascade for efficient synthesis of norepinephrine by combination of engineered L-threonine transaldolase with multi-enzyme expression fine-tuning.

Norepinephrine, a kind of ß-adrenergic receptor agonist, is commonly used for treating shocks and hypotension caused by a variety of symptoms. The development of a straightforward, efficient and environmentally friendly biocatalytic route for manufacturing norepinephrine remains a challenge. Here, we designed and realized an artificial biocatalytic cascade to access norepinephrine starting from 3, 4-dihydroxybenzaldehyde and L-threonine mediated by a tailored-made L-threonine transaldolase PsLTTA-Mu1 and a newly screened tyrosine decarboxylase ErTDC. To overcome the imbalance of multi-enzymes in a single cell, engineering of PsLTTA for improved activity and fine-tuning expression mode of multi-enzymes in single E.coli cells were combined, leading to a robust whole cell biocatalyst ES07 that could produce 100 mM norepinephrine with 99% conversion, delivering a highest time-space yield (3.38 g/L/h) ever reported. To summarized, the current study proposed an effective biocatalytic approach for the synthesis of norepinephrine from low-cost substrates, paving the way for industrial applications of enzymatic norepinephrine production.

Enhanced synthesis of chloramphenicol intermediate L-threo-p-nitrophenylserine using engineered L-threonine transaldolase and by-product elimination.

L-threo-p-nitrophenylserine (component 2) is an important intermediate during synthesis of chloramphenicol. However, its biosynthesis is limited by enzyme activity and stereoselectivity. In this study, we achieved a breakthrough in the high-efficiency production of 2 by employing engineered Chitiniphilus shinanonensis L-threonine transaldolase (ChLTTA) in conjunction with a by-product elimination system within a one-pot reaction. Notably, a novel visual stepwise high-throughput screening method was developed for the directed evolution of ChLTTA, leveraging its characteristic color. The engineered mutant F70D/F59A (Mu6 variant) emerged as a star performer, exhibiting a remarkable 2.6-fold increase in catalytic efficiency over the wild-type ChLTTA, coupled with an outstanding 91.5 % diastereoisomer excess (de). Molecular dynamics (MD) simulations unraveled the mechanism responsible for the enhanced catalytic performance observed in the Mu6 variant. Meanwhile, the Mu6 variant was coupled with Saccharomyces cerevisiae ethanol dehydrogenase (ScADH) and Candida boidinii formate dehydrogenase (CbFDH) to create a high-efficiency cascade system (E.coli/pRSF-Mu6-ScADH-CbFDH). Under optimized conditions, this cascade system demonstrated unparalleled performance, yielding 201.5 mM of 2 with an impressive conversion of 95.9 % and a de value of 94.5 %. This achievement represents the highest reported yield to date. This study offers a novel insight into the sustainable and efficient production of chloramphenicol intermediate.

Metabolic Engineering of Escherichia coli for Production of 2,5-Dimethylpyrazine.

2,5-Dimethylpyrazine (2,5-DMP) is a high-value-added alkylpyrazine compound with important applications in both the food and pharmaceutical fields. In response to the increasing consumer preference for natural products over chemically synthesized ones, efforts have been made to develop efficient microbial cell factories for the production of 2,5-DMP. However, the previously reported recombinant strains have exhibited low yields and relied on expensive antibiotics and inducers. In this study, we employed metabolic engineering strategies to develop an Escherichia coli strain capable of producing 2,5-DMP at high levels without the need for inducers or antibiotics. Initially, the biosynthesis pathway of 2,5-DMP was constructed that realized 2,5-DMP production from glucose. Subsequently, efforts focused on enhancing 2,5-DMP production by improving the availability of the cofactor NAD+ and precursor l-threonine. Additionally, the supply and conversion of l-threonine were balanced by optimizing the copy number of the key gene tdh on the chromosome and by modifying the l-threonine transport system. The final engineering strain D19 produced 3.1 g/L of 2,5-DMP, which is the highest titer for fermentative production of 2,5-DMP using glucose as the carbon source up to date. The strategies used in this study lay a good foundation for the production of 2,5-DMP on a large scale.

Design of a dual-responding genetic circuit for high-throughput identification of L-threonine-overproducing Escherichia coli.

L-threonine is a crucial amino acid that is extensively employed in the realms of food, animal feed and pharmaceuticals. Unfortunately, the lack of an appropriate biosensor has hindered the establishment of a robust high-throughput screening (HTS) system for the identification of the desired strains from random mutants. In this study, a dual-responding genetic circuit that capitalizes on the L-threonine inducer-like effect, the L-threonine riboswitch, and a signal amplification system was designed for the purpose of screening L-threonine overproducers. This platform effectively enhanced the performance of the enzyme and facilitated the identification of high L-threonine-producing strains from a random mutant library. Consequently, pathway optimization and directed evolution of the key enzyme enhanced L-threonine production by 4 and 7-fold, respectively. These results demonstrate the potential of biosensor design for dynamic metabolite detection and offer a promising tool for HTS and metabolic regulation for the development of L-threonine-hyperproducing strains.

Metabolic Engineering of Escherichia coli for De Novo Production of 1,2-Butanediol.

1,2-Butanediol (1,2-BDO) is an important platform chemical widely utilized in the synthesis of polyester polyols, plasticizers, cosmetics, and pharmaceuticals. However, no natural metabolic pathway for its biosynthesis has been identified, and biological production of 1,2-BDO from renewable bioresources has not been reported so far. In this study, we designed and experimentally verified a feasible non-natural synthesis pathway for the de novo production of 1,2-BDO from renewable carbohydrates for the first time. This pathway extends the l-threonine synthesis pathway by introducing two artificial metabolic modules to sequentially convert l-threonine into 2-hydroxybutyric acid and 1,2-BDO. Following key enzyme screening and enhancement of l-threonine synthesis module in the chassis microorganism, the best engineered Escherichia coli strain was able to produce 0.15 g/L 1,2-BDO using glucose as the sole carbon source. This work lays the foundation for the bioproduction of 1,2-BDO from renewable resources.

Engineering a Novel Acetyl-CoA Pathway for Efficient Biosynthesis of Acetyl-CoA-Derived Compounds.

Acetyl-CoA is an essential central metabolite in living organisms and a key precursor for various value-added products as well. However, the intracellular availability of acetyl-CoA limits the efficient production of these target products due to complex and strict regulation. Here, we proposed a new acetyl-CoA pathway, relying on two enzymes, threonine aldolase and acetaldehyde dehydrogenase (acetylating), which can convert one l-threonine into one acetyl-CoA, one glycine, and generate one NADH, without carbon loss. Introducing the acetyl-CoA pathway could increase the intracellular concentration of acetyl-CoA by 8.6-fold compared with the wild-type strain. To develop a cost-competitive and genetically stable acetyl-CoA platform strain, the new acetyl-CoA pathway, driven by the constitutive strong promoter, was integrated into the chromosome of Escherichia coli. We demonstrated the practical application of this new acetyl-CoA pathway by high titer production of ß-alanine, mevalonate, and N-acetylglucosamine. At the same time, this pathway achieved a high-yield production of glycine, a value-added commodity chemical for the synthesis of glyphosate and thiamphenicol. This work shows the potential of this new acetyl-CoA pathway for the industrial production of acetyl-CoA-derived compounds.

Exploring solubility and energetics: Dissolution of biologically important l-threonine in diverse aqueous organic mixtures across the temperature range of 288.15 K to 308.15 K.

This research provides a thorough investigation into the solubility behavior and solution thermodynamics of l-threonine in significant organic solvent systems. The work was done on measuring the actual solubility and subsequently calculating overall transfer solvation free energetics (∆Genergetic0i) and transfer entropies (∆St0i) at a temperature of 298.15 K. These measurements were performed as l-threonine transitioned from water to different water-organic mixed solvents systems. The saturated solubilities of l-threonine were determined using the 'gravimetric method' at five equidistant temperatures namely 288.15 K, 293.15 K, 298.15 K, 303.15 K and 308.15 K. By analyzing the data on solubility, we further obtained the different energies involved in solvation related issues. In the case of single solvents, the nature of solubility of l-threonine was observed like: dimethylsulfoxide (DMSO) < acetonitrile (ACN) < N, N-dimethylformamide (DMF) < ethylene glycol (EG) < water (H2O), irrespective of the experimental conditions. Specifically, at 298.15 K, the solubilities of l-threonine in single solvents were found to be as follows: 0.8220 mol per kg of water, 0.3101 mol per kg of EG, 0.1337 mol per kg of DMF, 0.1107 mol per kg DMSO and 0.1188 mol per kg of ACN. This research critically examines the relationship between the experimental saturated solubility of l-threonine and the complex properties influencing its solvation energy in diverse aqueous organic solvent systems.

Rational Design of l-Threonine Transaldolase-Mediated System for Enhanced Florfenicol Intermediate Production.

l-threo-p-methylsulfonylphenylserine (compound 1b) is the main intermediate of florfenicol, and its efficient synthesis has been the subject of current research. Herein, Burkholderia diffusa l-threonine transaldolase (BuLTTA) was rationally designed based on the sequence-structure-function relationship. A mutant M4 (Asn35Ser/Thr352Asn) could produce 35.5 mM 1b with 88.8% conversion and 93.8% diastereoselectivity, 314 and 129% of the values observed for wild-type BuLTTA. Molecular dynamics simulations indicated that the shortened distance between key active site residues and the transition state (PLP-1b) and the improved hydrogen bond force enhanced the catalytic performance of the M4 variant. Then, the mutant M4 was combined with K. kurtzmanii alcohol dehydrogenase (KkADH) to eliminate the BuLTTA-inhibiting byproduct acetaldehyde, and a cosubstrate was added to regenerate the ADH cofactor NADH. Under optimized conditions, the yield of 1b reached 115.2 mM with a conversion of 96% and a diastereoselectivity of 95.5%. This work provides a new strategy for the efficient and sustainable production of 1b.

Creating Polyploid Escherichia Coli and Its Application in Efficient L-Threonine Production.

Prokaryotic genomes are generally organized in haploid. In synthetic biological research, efficient chassis cells must be constructed to produce bio-based products. Here, the essential division of the ftsZ gene to create functional polyploid E. coli is regulated. The artificial polyploid E. coli containing 2-4 chromosomes is confirmed through PCR amplification, terminator localization, and flow cytometry. The polyploid E. coli exhibits a larger cell size, and its low pH tolerance and acetate resistance are stronger than those of haploid E. coli. Transcriptome analysis shows that the genes of the cell's main functional pathways are significantly upregulated in the polyploid E. coli. These advantages of the polyploid E. coli results in the highest reported L-threonine yield (160.3 g L-1 ) in fed-batch fermentation to date. In summary, an easy and convenient method for constructing polyploid E. coli and demonstrated its application in L-threonine production is developed. This work provides a new approach for creating an excellent host strain for biochemical production and studying the evolution of prokaryotes and their chromosome functions.

Exogenous Threonine-Induced Conversion of Threonine-Xylose Amadori Compound to Heyns Compound for Efficiently Promoting the Formation of Pyrazines.

The involvement of exogenous threonine during the degradation of l-threonine-d-xylose Amadori rearrangement product (Thr-ARP) was found to promote the formation of pyrazines. A model including Thr-ARP and 15N-labeled l-threonine was applied to reveal the role of free threonine in Thr-ARP conversion to pyrazines. Quantitative analyses of pyrazines in the model of Thr-ARP/15N-labeled threonine showed a precedence of the endogenous threonine (formed by the degradation of Thr-ARP) over the exogenous threonine in pyrazines formation, and the ratio of 15N to 14N content in pyrazines increased significantly over time. According to the observed occurrence of the Heyns rearrangement products (HRP) derived from 15N-threonine, as well as the sharp decrease of 15N-threonine content and a rapid increase of 14N endogenous threonine at the initial stage of heat treatment, it was proposed that aldimine condensation between exogenous threonine and Thr-ARP followed by the hydrolysis led to the endogenous threonine and the generation of HRP. Then, the HRP underwent dehydration followed by hydrolysis to form exogenous threonine and deoxyxyosones, and the dehydration and hydrolysis of deoxyxyosones to form organic acids was inhibited, but the retro-aldolization of deoxyxyosones was promoted, facilitating the generation of reactive α-dicarbonyl compounds. In this way, exogenous threonine accelerated the release of endogenous threonine and α-dicarbonyl compounds and the pH decline was slowed down, which was favorable for the formation of pyrazines.

A study on L-threonine and L-serine uptake in Escherichia coli K-12.

In the current study, we report the identification and characterization of the yifK gene product as a novel amino acid carrier in E. coli K-12 cells. Both phenotypic and biochemical analyses showed that YifK acts as a permease specific to L-threonine and, to a lesser extent, L-serine. An assay of the effect of uncouplers and composition of the reaction medium on the transport activity indicates that YifK utilizes a proton motive force to energize substrate uptake. To identify the remaining threonine carriers, we screened a genomic library prepared from the yifK-mutant strain and found that brnQ acts as a multicopy suppressor of the threonine transport defect caused by yifK disruption. Our results indicate that BrnQ is directly involved in threonine uptake as a low-affinity but high-flux transporter, which forms the main entry point when the threonine concentration in the external environment reaches a toxic level. By abolishing YifK and BrnQ activity, we unmasked and quantified the threonine transport activity of the LIV-I branched chain amino acid transport system and demonstrated that LIV-I contributes significantly to total threonine uptake. However, this contribution is likely smaller than that of YifK. We also observed the serine transport activity of LIV-I, which was much lower compared with that of the dedicated SdaC carrier, indicating that LIV-I plays a minor role in the serine uptake. Overall, these findings allow us to propose a comprehensive model of the threonine/serine uptake subsystem in E. coli cells.

Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine.

L-threonine is an essential amino acid used widely in food, cosmetics, animal feed and medicine. The thrABC operon plays an important role in regulating the biosynthesis of L-theronine. In this work, we systematically analyzed the effects of separating thrAB and thrC in different proportions on strain growth and L-threonine production in Escherichia coli firstly. The results showed that higher expression of thrC than thrAB enhanced cell growth and L-threonine production; however, L-threonine production decreased when the thrC proportion was too high. The highest L-threonine production was achieved when the expression intensity ratio of thrAB to thrC was 3:5. Secondly, a stationary phase promoter was also used to dynamically regulate the expression of engineered thrABC. This strategy improved cell growth and shortened the fermentation period from 36 h to 24 h. Finally, the acetate metabolic overflow was reduced by deleting the ptsG gene, leading to a further increase in L-threonine production. With these efforts, the final strain P 2.1 -2901ΔptsG reached 40.06 g/L at 60 h fermentation, which was 96.85% higher than the initial control strain TH and the highest reported titer in shake flasks. The maximum L-threonine yield and productivity was obtained in reported fed-batch fermentation, and L-threonine production is close to the highest titer (127.30 g/L). In this work, the expression ratio of genes in the thrABC operon in E. coli was studied systematically, which provided a new approach to improve L-threonine production and its downstream products.

Heat Capacities of L-Cysteine, L-Serine, L-Threonine, L-Lysine, and L-Methionine.

In an effort to establish reliable thermodynamic data for amino acids, heat capacity and phase behavior are reported for L-cysteine (CAS RN: 52-90-4), L-serine (CAS RN: 56-45-1), L-threonine (CAS RN: 72-19-5), L-lysine (CAS RN: 56-87-1), and L-methionine (CAS RN: 63-68-3). Prior to heat capacity measurements, initial crystal structures were identified by X-ray powder diffraction, followed by a thorough investigation of the polymorphic behavior using differential scanning calorimetry in the temperature range from 183 K to the decomposition temperature determined by thermogravimetric analysis. Crystal heat capacities of all five amino acids were measured by Tian-Calvet calorimetry in the temperature interval (262-358) K and by power compensation DSC in the temperature interval from 215 K to over 420 K. Experimental values of this work were compared and combined with the literature data obtained with adiabatic calorimetry. Low-temperature heat capacities of L-threonine and L-lysine, for which no or limited literature data was available, were measured using the relaxation (heat pulse) calorimetry. As a result, reference heat capacities and thermodynamic functions for the crystalline phase from near 0 K to over 420 K were developed.

Overexpression of the key genes in the biosynthetic pathways of lipid A and peptidoglycan in Escherichia coli.

Gram-negative bacterium Escherichia coli has a tripartite cell envelope with a cytoplasmic membrane, a peptidoglycan layer, and an asymmetric outer membrane containing lipopolysaccharide in its outer leaflet. The biogenesis of peptidoglycan and lipopolysaccharide shares the same substrate UDP-GlcNAc. From UDP-GlcNAc, MurA catalyzes the first reaction for peptidoglycan biosynthesis, while LpxA catalyzes the first reaction for lipopolysaccharide biosynthesis. This study demonstrates that murA overexpression in E. coli MG1655 inhibited the cell growth and increased the cell length, whereas lpxA overexpression in MG1655 neither inhibited the cell growth nor increased the cell length. Further study showed that individual overexpression of the other eight genes encoding the enzymes to catalyze the initial reactions in the biosynthetic pathway of lipopolysaccharide did not inhibit the cell growth. When MG1655/pBad-lpxA, MG1655/pBad-lpxD, and MG1655/pBad-lpxH were transformed with pFW01-thrA*BC-rhtC that contains the key genes for L-threonine biosynthesis and transport, the L-threonine production was increased. The L-threonine production in MG1655/pFW01-thrA*BC-rhtC/pBad-lpxH increased 46.1% as compared to the control MG1655/pFW01-thrA*BC-rhtC/pBad.

Mutability-Landscape-Guided Engineering of l-Threonine Aldolase Revealing the Prelog Rule in Mediating Diastereoselectivity of C-C Bond Formation.

l-threonine aldolase (LTA) catalyzes C-C bond synthesis with moderate diastereoselectivity. In this study, with LTA from Cellulosilyticum sp (CpLTA) as an object, a mutability landscape was first constructed by performing saturation mutagenesis at substrate access tunnel amino acids. The combinatorial active-site saturation test/iterative saturation mutation (CAST/ISM) strategy was then used to tune diastereoselectivity. As a result, the diastereoselectivity of mutant H305L/Y8H/V143R was improved from 37.2 %syn to 99.4 %syn . Furthermore, the diastereoselectivity of mutant H305Y/Y8I/W307E was inverted to 97.2 %anti . Based on insight provided by molecular dynamics simulations and coevolution analysis, the Prelog rule was employed to illustrate the diastereoselectivity regulation mechanism of LTA, holding that the asymmetric formation of the C-C bond was caused by electrons attacking the carbonyl carbon atom of the substrate aldehyde from the re or si face. The study would be useful to expand LTA applications and guide engineering of other C-C bond-forming enzymes.

Production of natural flavor compounds using Bacillus subtilis-fermented soybean meal extract and their biological potential: a comprehensive in vitro study.

This study aims to investigate the production of natural flavor compounds through the utilization of Bacillus subtilis-fermented soybean meal extract and evaluate their biological potential. The experiment involved a comprehensive in vitro investigation to assess the capabilities and effects of the produced flavor compounds. The resulting flavor compounds were subjected to various in vitro tests to assess their properties, including cytotoxicity, antioxidant activity, anticancer potential, antiviral activity, and antimicrobial activity. To enhance the fermentation process, soybean meal extract was fortified with a combination of L-Lysine and L-Threonine. Gas chromatography-mass spectrometry (GC/MS) analysis was conducted on the fermented soybean meal using two strains of Bacillus subtilis, namely NRCH123 and NRCZ144. This analysis revealed the presence of various volatile compounds in all extracts, including Butylated hydroxytoluene. The fermented soybean extract with bacillus subtilis NRCZ144 (B2) fortified with a combination of 2.5% (w/w) L-Lysine and 2.5% w/w L-threonine (SLT2) exhibited a rich profile of flavor compounds, with Eucalyptol being identified as the predominant compound. The antioxidant activity of the SLT2 extract was found to be 72.04% at a concentration of 100 µg/mL, indicating significant antioxidant potential. Furthermore, when tested against the human liver cancer cell line HepG2, the extract demonstrated anticancer activity with an IC50 value of 2.26 µg/mL. The extract exhibited potent cytotoxicity, with an IC50 value of 1.02 µg/mL. Importantly, the SLT2 extract displayed strong antibacterial and antifungal activity, even at very low concentrations. The extract's antimicrobial properties indicate its potential for inhibiting the growth of bacteria and fungi.

Type I fimbriae subunit fimA enhances Escherichia coli biofilm formation but affects L-threonine carbon distribution.

The biofilm (BF) provides favorable growth conditions to cells, which has been exploited in the field of industrial biotechnology. Based on our previous research works on type I fimbriae for the biosynthesis of L-threonine (LT) in Escherichia coli, in this study, a fimA-overexpressing strain was engineered, which improved BF formation under industrial fermentation conditions. The morphological observation and characterization of BF formation were conducted to verify the function of the subunit FimA. However, it was not suitable for repeated-batch immobilized fermentation as the LT titer was not elevated significantly. The underlying molecular mechanisms of BF formation and the LT carbon flux were explored by transcriptomic analysis. The results showed that fimA regulated E. coli BF formation but affected LT carbon distribution. This study will stimulate thoughts about how the fimbriae gene regulated biofilms and amino acid excretion and will bring some consideration and provide a reference for the development of BF-based biomanufacturing processes in E. coli.