Acetolactate Synthase Research Articles

Sensitivity of Southcentral U.S. Johnsongrass Accessions to Selected Herbicides

Abstract New technologies in grain sorghum allow for the use of multiple acetyl CoA carboxylase- (ACCase) or acetolactate synthase- (ALS) inhibiting herbicides for johnsongrass control. With the growing issue of herbicide resistance, producers need to understand which herbicides will successfully control johnsongrass accessions. To determine the efficacy of herbicides recently registered or ones with potential to become available for use in grain sorghum, johnsongrass seeds were collected from 2017 to 2021 in Arkansas, Kansas, Texas, and Oklahoma and were screened for sensitivity to fluazifop, quizalofop, nicosulfuron, and imazamox. Additionally, glyphosate sensitivity was evaluated because of its use before planting or postharvest. Quizalofop resulted in 100% mortality of all johnsongrass accessions. Of the johnsongrass accessions evaluated, 89% were completely controlled with glyphosate. The ALS inhibitors nicosulfuron and imazamox resulted in 100% mortality of all Oklahoma accessions, but failures occurred on samples from other states. One accession from Kansas, 12 from Texas, and eight from Arkansas were found to have reduced sensitivity to nicosulfuron and imazamox. If producers plan to plant grain sorghum in areas with johnsongrass populations, an ACCase-inhibitor herbicide will most likely provide effective control. Imazamox and nicosulfuron, in conjunction with the appropriate trait, can be utilized in areas with sensitive johnsongrass populations or where other sensitive grass species are present.

Emergence of multiple resistance to EPSPS and ALS herbicides in smooth pigweed (Amaranthus hybridus): a growing concern in Brazil

Abstract Recently, farmers in Brazil have observed a decline in efficacy of glyphosate, chlorimuron, and imazethapyr control of smooth pigweed (Amaranthus hybridus L.). The objectives of this study were to quantify the resistance of Amaranthus in Brazil to glyphosate and acetolactate synthase (ALS)-inhibiting herbicides, elucidate the mechanism of resistance, and assess the frequency of sensitivity shifts to glyphosate and chlorimuron in Brazil. Dose–response assays were conducted in a greenhouse with glyphosate, chlorimuron, and imazethapyr. This was followed by sequencing of the EPSPS and ALS genes. Additionally , 740 Amaranthus populations across several Brazilian states were monitored over 4 yr, subjected to a single discriminatory dose of glyphosate and chlorimuron. The populations BR18Asp051 and BR21Asp205 were resistant to glyphosate, chlorimuron, and imazethapyr. The elevated resistance level to glyphosate in these populations is attributed to multiple amino acid substitutions (TAP-IVS) in the EPSPS gene; and cross-resistance to sulfonylureas and imidazolinones is conferred by the Trp-574-Leu substitution in the ALS gene in both populations. Overall, resistance distribution indicated that 88% of the sampled populations were considered sensitive to glyphosate, while 66% were sensitive to chlorimuron. Furthermore, 10% of the samples demonstrated multiple resistance to both active ingredients. A shift in glyphosate sensitivity was observed in four states in Brazil; however, sensitivity shifts to chlorimuron were more widely dispersed in Brazilian agricultural regions.

Effects of a Novel Tripyrasulfone Herbicide on Key Soil Enzyme Activities in Paddy Rice Soil

Weeds significantly impact paddy yields, and herbicides offer a cost-effective, rapid, and efficient solution compared to manual weeding, ensuring agricultural productivity. Tripyrasulfone, a novel 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor developed by Qingdao Kingagroot Chemicals Co., Ltd., has demonstrated high efficacy when applied post-emergence, causing characteristic foliar bleaching in susceptible weed species, distinct from conventional acetolactate synthase, acetyl-CoA carboxylase, and synthetic auxin herbicides. This study investigates the impact of tripyrasulfone on the activity of key soil enzymes (urease (UE), acid phosphatase (ACP), sucrase (SC), catalase (CAT), and dehydrogenase (DHA)) in paddy soils from Jilin Province and Shandong Province. Different doses of tripyrasulfone (0.1, 1.0, and 2.5 mg kg−1) were applied, and the enzymatic activities were measured. Results indicated that tripyrasulfone initially inhibited UE and ACP activities before activating them. On the 20th day after treatment, UE activity had returned to control levels, whereas ACP activity remained significantly higher, showing long-lasting activation. SC and CAT activities were inhibited but gradually recovered to control levels. Furthermore, DHA activity was activated with a sustained effect, remaining significantly higher than the control group even 20 days after treatment. Overall, the impact of tripyrasulfone on soil enzyme activities diminished over time, suggesting that tripyrasulfone posed minimal long-term ecological risk to soil health.

Transcriptomics-guided rational engineering in Bacillus licheniformis for enhancing poly-γ-glutamic acid biosynthesis using untreated molasses

The utilization of non-food raw materials for microbial synthesis of poly-γ-glutamic acid (γ-PGA) presents a promising alternative to conventional food-based biosynthesis. However, the complex carbon source and composition of molasses, a prevalent non-food raw material, may impose constraints on its conversion and utilization by microorganisms. This study aimed to enhance the capacity of Bacillus licheniformis to convert untreated molasses into γ-PGA through transcriptomic analysis to guide metabolic modifications. Initial results from the transcriptomic analysis indicated that the strain utilizing molasses exhibited decreased expression of genes associated with substrate utilization (Module 1) and by-product synthesis (Module 2), while upregulating genes related to precursor synthesis (Module 3). Furthermore, we performed a knockout of the acetolactate synthase (AlsS) to reduce the synthesis of metabolic by-products and a knockout of the global regulator (CcpA) to alleviate carbon catabolite repression (CCR) and then promote substrate utilization. Ultimately, following the tandem overexpression of precursor supplying key genes, the titer of γ-PGA reached 48.26 g/L with a productivity of 1.15 g/L/h, using untreated molasses as the sole carbon source, which was 3.12-fold of the starting strain. These findings offer significant insights into the cost-effective synthesis of γ-PGA by bioconversion of untreated molasses during fermentation.

Field-evolved cross-resistance to ALS inhibiting herbicides in redroot pigweed (Amaranthus retroflexus L.) populations and alternative chemical options for effective control

Abstract Twelve putative-resistant (R) redroot pigweed populations were collected in sunflower and soybean fields located in northeastern Greece, after repeated exposure to the acetolactate synthase (ALS)-inhibiting herbicides imazamox and tribenuron-methyl. Studies were conducted to determine the resistance status to these two ALS-inhibiting herbicides and evaluate alternative post- and preemergence herbicides for effective control. Two susceptible (S) populations were also included for comparison. Eleven out of the 12 putative-R populations studied in the whole plant dose response pot experiments were characterized as cross-resistant (R) to the imidazolinone imazamox and the sulfonylurea tribenuron-methyl. In contrast, the putative R5 and the two reference populations (S1, S2) populations were found susceptible. Sequencing of the ALS gene revealed that a point mutation (TGG to TTG at position 574) was selected in domain B, where in combination with domain A the majority of point mutations conferring resistance have been detected, resulting in an amino acid substitution from tryptophan (Trp) to leucine (Leu) in the 11 R populations. By contrast, all sequenced plants of the three susceptible populations were found with the wild-type allele encoding Trp574. The labeled rate of the postemergence herbicides tembotrione and dicamba provided fair to excellent control of the populations with ALS cross-resistance. In contrast, at this rate the preemergence herbicides S-metolachlor+terbuthylazine, isoxaflutole, aclonifen, metribuzin, and pendimethalin provided excellent control. These findings strongly suggest that 11 redroot pigweed populations have evolved cross-resistance to ALS-inhibiting herbicides, but viable options for chemical control of this weed still exist.

A Trp-574-Leu mutation in acetolactate synthase confers imazamox resistance in barnyardgrass (Echinochloa crus-galli) from China

Abstract Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is increasingly infesting imidazolinone-tolerant (IMI-T) rice fields in China, imazamox resistance of E. crus-galli has become the major concern for weed management in IMI-T rice fields. In this study, the susceptible population JLGY-3 (S) and the suspected resistant population JHXY-2 (R) collected from IMI-T rice fields were used as research subjects. When treated with imazamox, JHXY-2 (R) population showed a high level of herbicide resistance with a resistance index (RI) of 31.2. JHXY-2 (R) was cross-resistant to all five acetolactate synthase (ALS) inhibitors from different chemical families, but sensitive to herbicides inhibiting acetyl-CoA carboxylase (ACCase). In order to understand the reason why JHXY-2 (R) was resistant to imazamox, we performed experiments to characterize potential TSR and NTSR mechanisms. A trp-574-leu amino acid mutation in ALS and low imazamox ALS sensitivity were the main mechanism underlying imazamox resistance in this JHXY-2 (R) population. There was no significant difference in ALS gene expression and ALS protein abundance between R and S populations. High-performance liquid chromatography-tandem mass spectrometry analysis showed enhanced metabolism of imazamox in JHXY-2 (R), which was in contrast to the results of pretreatment with a metabolic enzyme inhibitor. Treatments with the P450/GST inhibitors did not alter the resistance level of JHXY-2 (R) against imazamox. To further clarify the NTSR mechanism of JHXY-2 (R), transcriptome sequencing showed that there was almost no significant difference in the expression of P450 and GST metabolic enzyme genes between R and S populations, and only GST-U1 showed a significant induction in R population. In conclusion, amino acid mutation and higher enzyme activity of ALS are the main causes of imazamox resistance in JHXY-2 (R). However, given the differences in imazamox residues in the leaves of the E. crus-galli, there may still be undetectable NTSR that are causing imazamox resistance in the R population.

Regional diversity of the ALS gene and hormesis due to tribenuron-methyl in Centaurea cyanus L.

Centaurea cyanus L. is a common field weed in Eastern Europe but only in Poland biotypes of this species with resistance to acetolactate synthase (ALS) inhibitors have been confirmed. This phenomenon is constantly developing and spreading to consecutive regions of Poland. This study aimed to assess the response of selected Polish C. cyanus populations to tribenuron-methyl and to analyse the genetic variability of the ALS gene of C. cyanus populations resistant to ALS inhibitors. Between 2017 and 2021, 13 seed samples were collected from eastern Poland and a dose-response study with tribenuron-methyl was performed. Eleven populations resistant to tribenuron-methyl were identified. All populations from this study as well as 6 additional resistant populations characterised in the previous dose-response studies were subjected to molecular analysis of the ALS gene. Target-site resistance due to mutations P197S, P197Q, P197T and P197A were identified in 8 populations from Warmia-Masuria and Podlaskie provinces. This is the first case of target-site resistance (TSR) in C. cyanus confirmed by sequencing of the ALS gene. Moreover in some resistant plants, ten changes in the amino acid ALS sequence were identified in comparison to those in the susceptible ones. In none of the populations were all mutations detected in the same individual. The highest frequency of mutations was detected in Warmia-Masuria province. Some C. cyanus populations resistant to ALS inhibitors showed hormesis effect concerning shoot fresh weight after tribenuron-methyl treatment. Stimulation due to half the recommended dose of tribenuron-methyl was the highest and the difference between untreated and treated plants was statistically significant in two populations from Warmia-Masuria and in one from Podlaskie province.

An easy and sensitive assay for acetohydroxyacid synthases based on the simultaneous detection of substrates and products in a single step.

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) catalyzes the first step in the synthesis of the branched-chain amino acids valine, leucine, and isoleucine, pathways being present in plants and microorganisms, but not in animals. Thus, AHAS is an important target for numerous herbicides and, more recently, for the development of antimicrobial agents. The need to develop new and optimized herbicides and pharmaceuticals requires a detailed understanding of the biochemistry of AHAS. AHAS transfers an activated two-carbon moiety derived from pyruvate to either pyruvate or 2-oxobutyrate as acceptor substrates, forming 2-acetolactate or 2-acetohydroxy-2-butyrate, respectively. Various methods have been described in the literature to biochemically characterize AHAS with respect to substrate preferences, substrate specificity, or kinetic parameters. However, the simultaneous detection and quantification of substrates and unstable products of the AHAS-catalyzed reaction have always been a challenge. Using AHAS isoform II from Escherichia coli, we have developed a sensitive assay for AHAS-catalyzed reactions that uses derivatization with ethyl chloroformate to stabilize and volatilize all reactants in the aqueous solution and detect them by gas chromatography coupled to flame ionization detection or mass spectrometry. This assay allows us to characterize the product formation in reactions in single and dual substrate reactions and the substrate specificity of AHAS, and to reinterpret previous biochemical observations. This assay is not limited to the AHAS-catalyzed reactions, but should be applicable to studies of many metabolic pathways.

Allelic interactions defining Raphanus raphanistrum AHAS resistance level: strong vs weak target-site AHAS resistance alleles.

Chlorsulfuron resistance and genetic dominance was evaluated in Raphanus raphanistrum genotypes homozygous (122-RR, 376-RR), heterozygous (122-RS, 376-RS) and compound heterozygous (122-R/376-R) for the target-site resistance mutations Ala-122-Tyr and Asp-376-Glu in the AHAS (acetohydroxyacid synthase) gene. At the AHAS level, 122-RR and 122-RS plants exhibited significantly higher I50 values than 376-RR and 376-RS plants, respectively. However, plants of the compound heterozygous genotype (122-R/376-R), showed no difference in AHAS activity compared to the 122-RS genotype but lower activity than the 122-RR genotype, and showed a nearly 400-fold greater I50 value than both genotypes (376-RR and 376-RS) carrying the 376-Glu allele. At the whole-plant level, 100% survival was observed for 122-RR plants at the highest chlorsulfuron dose of 640 g ha-1, yet 376-RR plants showed no survival at 380 g ha-1. Thus, this survival difference resulted in different median lethal dose (LD50) estimates [>640 (122-RR) versus 330 g ha-1(376-RR)]. The effect of chlorsulfuron in reducing aboveground growth of surviving plants also was markedly lower for the homozygous 122-RR (GR50 = 566 g ha-1) than for 376-RR plants (GR50 = 66). Heterozygous plants carrying the 122-Tyr allele (122-RS) exhibited two- and five-fold higher LD50 values than both homozygous and heterozygous plants carrying the 376-Glu allele (376-RR, 376-RS), respectively. Along the difference in plant survival, 122-RS plants also showed four-fold higher GR50 than both 376-RR and 376-RS plants. Survival of plants with the compound heterozygous genotype (122-R/376-R) under increasing chlorsulfuron doses was similar to 122-RR or 122-RS genotypes. However, this compound heterozygous genotype showed two- and six-fold higher LD50 values than 376-RR or 376-RS genotypes, respectively. However, both resistance 122-Tyr and 376-Glu alleles were dominant or nearly dominant over the wild-type susceptible alleles (ALA-122 and ASP-376), and the resistance 122-Tyr allele was dominant over the 376-Glu allele. These results broaden our understanding of AHAS target-site resistance in R. raphanistrum and strengthens the hypothesis that the AHAS 122-Tyr allele corresponds to a stronger target-site resistance allele than the 376-Glu allele. © 2024 Society of Chemical Industry.

Development of Brassica carinata A. Braun resistant to acetolactate synthase–inhibiting herbicides

AbstractBrassica carinata A. Braun (carinata) has become an important oil crop for biofuel production in subtropical regions. Carinata is highly sensitive to acetolactate synthase (ALS)‐inhibiting herbicides, limiting its introduction into existing crop rotations. The objective of the study was to develop carinata lines resistant to ALS‐inhibiting herbicides. A susceptible carinata line was crossed with a resistant Brassica napus L. line. Lines derived from those crosses were screened at high doses of imidazolinones, which allowed identifying five lines with high levels of resistance. Doses to reduce plant growth 50% (GR50) and cause 50% injury (ID50) were four to nine times greater than susceptible lines. Resistant lines exhibited cross resistance with halosulfuron (sulfonylurea). Resistance was confirmed under field conditions with doses 2X and 4X for imazethapyr and 4X–8X for halosulfuron of their respective label doses. While susceptible lines died, resistant lines exhibited no injury or growth reductions compared with nontreated controls. Sequencing of the ALS gene indicated that all resistant lines carried a Trp574Leu amino acid substitution, a mutation responsible for resistance in other species. Crosses between resistant lines and a susceptible line demonstrated that the inheritance of the mutation corresponded with the resistance phenotype in the F2. The resistance trait behaved as a single, fully dominant allele, which makes it easier to transfer it to carinata lines with desirable agronomic traits. The resistant lines developed here provide flexibility for use in multiple crop rotations and opens the possibility to use ALS‐inhibiting herbicides for weed control within this crop's growing season.

Downy brome (Bromus tectorum) management and herbicide resistance in dryland wheat production across northeastern Oregon

Abstract Downy brome (Bromus tectorum L.) is a difficult species to control in the dryland wheat (Triticum aestivum L.) production areas of northeastern Oregon. The selection of herbicide-resistant B. tectorum populations has further complicated B. tectorum management. A survey of wheat growers was conducted in 2021 and 2022 to understand B. tectorum management practices. The survey included four questions based on the growing seasons from 2017 to 2022 related to crop rotation, tillage versus no-tillage, irrigation versus dryland, and herbicide programs. To determine the extent of herbicide resistance, seeds were collected from 49 B. tectorum populations in wheat fields in northeastern Oregon and tested for resistance. Herbicides tested were clethodim, glyphosate, imazamox, mesosulfuron, metribuzin, propoxycarbazone, pyroxasulfone, pyroxsulam, quizalofop, and sulfosulfuron. Winter wheat–summer fallow rotation was the most predominant cropping system in the region. Most of the fields were in no-tillage systems, and none were irrigated. Pyroxasulfone applied preemergence and acetolactate synthase (ALS) inhibitors applied postemergence were the most often used herbicides for B. tectorum control in winter wheat. Glyphosate was the most frequently used herbicide for B. tectorum control in summer fallow. Resistance screenings confirmed that 46 of the 49 B. tectorum populations were resistant to ALS inhibitors with different cross-resistance patterns. Two populations were resistant to metribuzin and exhibited multiple resistance to ALS inhibitors. All populations were susceptible to clethodim, glyphosate, pyroxasulfone, and quizalofop. The widespread occurrence of ALS inhibitor–resistant B. tectorum populations limits effective postemergence herbicide options in winter wheat.

Strategic lead compound design and development utilizing computer-aided drug discovery (CADD) to address herbicide-resistant Phalaris minor in wheat fields.

Wheat (Triticum aestivum) is a vital cereal crop and a staple food source worldwide. However, wheat grain productivity has significantly declined as a consequence of infestations by Phalaris minor. Traditional weed control methods have proven inadequate owing to the physiological similarities between P. minor and wheat during early growth stages. Consequently, farmers have turned to herbicides, targeting acetyl-CoA carboxylase (ACCase), acetolactate synthase (ALS) and photosystem II (PSII). Isoproturon targeting PSII was introduced in mid-1970s, to manage P. minor infestations. Despite their effectiveness, the repetitive use of these herbicides has led to the development of herbicide-resistant P. minor biotypes, posing a significant challenge to wheat productivity. To address this issue, there is a pressing need for innovative weed management strategies and the discovery of novel herbicide molecules. The integration of computer-aided drug discovery (CADD) techniques has emerged as a promising approach in herbicide research, that facilitates the identification of herbicide targets and enables the screening of large chemical libraries for potential herbicide-like molecules. By employing techniques such as homology modelling, molecular docking, molecular dynamics simulation and pharmacophore modelling, CADD has become a rapid and cost-effective medium to accelerate the herbicide discovery process significantly. This approach not only reduces the dependency on traditional experimental methods, but also enhances the precision and efficacy of herbicide development. This article underscores the critical role of bioinformatics and CADD in developing next-generation herbicides, offering new hope for sustainable weed management and improved wheat cultivation practices. © 2024 Society of Chemical Industry.

Chemical Synthesis, Herbicidal Activity, Crop Safety, and Molecular Basis of ortho-Fluoroalkoxy Substituted Sulfonylureas as Novel Acetohydroxyacid Synthase Inhibitors.

In the face of increasing resistance to the currently used commercial herbicides and the lack of success in identifying new herbicide targets, alternative herbicides need to be developed to control unwanted monocotyledon grasses in food crops. Here, a panel of 29 novel sulfonylurea-based compounds with ortho-fluoroalkoxy substitutions at the phenyl ring were designed and synthesized. Pot assays demonstrated that two of these compounds, 6d and 6u, have strong herbicidal activities against Echinochloa crus-galli, Eleusine indica, Alopecurus aequalis, and Alopecurus japonicus Steudel at a dosage of 15 g ha-1. Furthermore, these two compounds exhibited <5% inhibition against wheat at a dosage of 30 g ha-1 under post-emergence conditions. 6u also exhibited <5% inhibition against rice at a dosage of 30 g ha-1 under both post-emergence and pre-emergence conditions. A kinetics study demonstrated that 6d and 6u are potent inhibitors of Arabidopsis thaliana acetohydroxyacid synthase (AHAS; EC 2.2.1.6) with potent Ki values of 18 ± 1.1 and 11.9 ± 4.0 nM, respectively. The crystal structure of 6u in complex with A. thaliana (At)AHAS has also been determined at 2.7 Å resolution. These new compounds represent new alternative herbicide choices to protect wheat or rice from invading grasses.

Crystal Structures of Arabidopsis thaliana Acetohydroxyacid Synthase in Complex with the Herbicide Triasulfuron and Two Analogues with Herbicidal Activity in Field Trials.

Triasulfuron is a commercial herbicide of the sulfonylurea family. This compound targets acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6), the first enzyme in the branched chain amino acid biosynthesis pathway. Here, we have determined crystal structures of Arabidopsis thaliana AHAS (AtAHAS) in complex with triasulfuron and two newly designed herbicidal compounds, identified as FMO and CMO, showing that their binding modes are subtly different. Kinetic studies showed all three compounds exhibit varying Ki values, 0.192 ± 0.013 μM for triasulfuron, 0.086 ± 0.013 μM for FMO, and 1.448 ± 0.058 μM for CMO, but all are strong time-dependent accumulative inhibitors of AtAHAS. Apart from triasulfuron being a powerful herbicide with application rates of 10-15 g/ha in wheat fields, CMO and FMO are also herbicidal at 7.5-30 g/ha for barnyard grass. This study emphasizes that accumulative inhibition is an important factor that contributes to herbicidal activity.

Mutations in target gene confers resistance to acetolactate synthase inhibitors in Echinochloa phyllopogon

Echinochloa phyllopogon is a noxious weed that can harm rice over prolonged periods. Recently, a penoxsulam-resistant variant of E. phyllopogon with a mutation in the acetolactate synthase (ALS) gene was collected in Northeastern China. In the present study, the molecular mechanism underlying herbicide resistance in mutant populations was evaluated. The GR50 and IC50 values of the herbicide-resistant mutant 1-11 were 27.0- and 21.4-fold higher than those of the susceptible population 2-31, respectively. In addition, pre-application of malathion reduced the GR50 value of the resistant population. Additionally, mutant populations developed cross-resistance to other ALS inhibitors. E. phyllopogon ALS sequencing showed a Trp-574-Leu mutation in ALS2 variant 1-11. Molecular docking showed that the Trp-574-Leu substitution reduced the number of hydrogen bonds and altered the interaction between penoxsulam and ALS2. Transgenic Arabidopsis plants harboring the ALS2 mutant gene also showed resistance to penoxsulam and other ALS inhibitors. Overall, our study demonstrated that the Trp-574-Leu mutation and P450-mediated metabolic resistance lead to the cross-resistance of E. phyllopogon to ALS inhibitors.

Wild mustard (Sinapis arvensis L.) resistance to tribenuron methyl in wheat fields of Diyarbakır province, Türkiye

Chemical weed control is unavoidable in the fields where weeds cause problems in wheat production. However, resistance problems in weeds arise a few years later especially using herbicides with the same mode of action continuously. Frequent and inappropriate use of herbicides leads to various environmental, health and economic issues, as well as labour losses. Recently, growers complained that tribenuron-methyl (acetolactate synthase - ALS), commonly used to control wild mustard (Sinapis arvensis L.-SINAR), which is causing problems in the wheat fields of Diyarbakır province, is ineffective. To determine the extent of the problem, wild mustard seeds were collected from 56 suspected wheat fields in 16 districts of Diyarbakır. As a result of applications carried out with the method under controlled conditions, resistance was suspected in the population of 28 fields. The results of the dose-response experiments indicated that no resistance (RI

Efficient expression regulation of acetohydroxyacid synthase for production of branched-chain amino acids in Corynebacterium glutamicum

Corynebacterium glutamicum is a major workhorse in the industrial production of branched-chain amino acids (BCAAs). The acetohydroxyacid synthase (AHAS) encoded by ilvBN is a key enzyme in the biosynthesis of BCAAs. Enhancing AHAS expression is essential for engineering BCAA producers. However, at present, the available studies only used limited promoters to regulate AHAS expression, which is insufficient for achieving efficient regulation. Herein, we first employed a previously developed reporter system to screen out a strong constitutive promoter PgpmA* from six candidate promoters for expressing ilvBN. PgpmA* showcased the expression strength 23.3-fold that of the native promoter PilvBN. Moreover, three synthetic RBS libraries based on the promoter PgpmA* were constructed and evaluated by plate fluorescence imaging. The results revealed that "R(9)N(6)" was the best mutant library. A total of 36 RBS mutants with enhanced strength were further screened by evaluation in 96-deep-well plates, and the highest strength reached up to 62.3-fold that of PilvBN. Finally, the promoter PgpmA* was combined with three RBS mutants (WT, RBS18, and RBS36) to fine-tune the expression of ilvBNS155F for L-valine biosynthesis, respectively. Increased expression strength led to enhanced L-valine production, with titers of 1.17, 1.38, and 2.29 g/L, respectively. The combination of RBS18 strain with the further overexpression of ilvC produced 7.57 g/L L-valine. The regulatory elements obtained in this study can be utilized to modulate AHAS expression for BCAA production in C. glutamicum. Additionally, this strategy can guide the efficient expression regulation of other key enzymes.

Noxious Weed Species Monochoria vaginalis Resistant to Synthetic Auxin and Acetolactate Synthase Inhibitor Herbicides

&lt;em&gt;Monochoria vaginalis&lt;/em&gt; (Burm. f.) C. Presl is a dominant weed in paddy rice cultivation, significantly threatening productivity and farming sustainability. In Subang, Indonesia, synthetic auxin and acetolactate synthase (ALS) inhibitors are commonly used to control &lt;em&gt;M. vaginalis&lt;/em&gt;. However, farmers have currently reported declining efficacy of these herbicides. Therefore, this study aimed to confirm the resistance of &lt;em&gt;M. vaginalis&lt;/em&gt; to the early post-emergence application of synthetic auxin and ALS inhibitors. Plant bioassays were conducted using the pot test method to determine resistance level. Herbicides were applied 2 weeks after planting &lt;em&gt;M. vaginalis&lt;/em&gt; at 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 times the recommended dose, and untreated plants as control. Herbicides tested were 2,4-D, bensulfuron-methyl, bispyribac sodium, penoxulam, and sulfentrazone. The results obtained based on resistance index values (RI) showed that &lt;em&gt;M. vaginalis&lt;/em&gt; populations from Kediri and Gandasari, Subang, had moderately to high resistance to 2,4-D (RI: 8.15 and 13.0) and bensulfuron-methyl&lt;em&gt; &lt;/em&gt;(RI: 205.61 and 21.80). Weed was moderately resistant to bispyribac sodium&lt;em&gt; &lt;/em&gt;(RI of Kediri biotype: 8.79) and had low to moderate resistance to penoxulam herbicides (RI: 8.94 and 3.56). In contrast, &lt;em&gt;M. vaginalis-&lt;/em&gt;resistant biotypes remained susceptible to sulfentrazone (protoporphyrinogen oxidase/PPO inhibitors) herbicide. A significant increase in GR50 was observed between 28 and 56 days after herbicide application, signifying enhanced regrowth. The resistance of &lt;em&gt;M. vaginalis&lt;/em&gt; to synthetic auxin and ALS inhibitors presents the need for farmers to consider alternative herbicides, such as PPO inhibitors, to prevent the development of resistant weeds.

A205V, D376E, W574L, S653T, and S653N Substitutions in Acetohydroxy Acid Synthase from Amaranthus retroflexus L. Show Different Functional Impacts on Herbicide Resistance

Amaranthus retroflexus L. is a troublesome dicot weed in crop fields and has developed high resistance to nicosulfuron in China. The objective of this study was to determine the effects of specific resistance mutations (A205V, D376E, W574L, S653T, and S653N) of the acetohydroxy acid synthase enzyme (AHAS) on the resistance of A. retroflexus to nicosulfuron. The resistance mutations in A. retroflexus not only conferred 17.17- to 31.70-fold resistance to nicosulfuron but also greatly decreased AHAS sensitivity and increased AHAS binding affinity to substrate pyruvate, which mechanisms were primarily responsible for the observed A. retroflexus resistance. Molecular docking results indicated that these resistance mutations altered AHAS binding free energy with nicosulfuron. All the resistance mutations showed less sensitivity to feedback inhibition by branched-chain amino acids, but the mutations did not necessarily affect biosynthesis in A. retroflexus. This report to compares the various mutations of ArAHAS in vitro and contributes to understanding herbicide resistance in this field weed.

Multiple Effects of L-Leucine in Escherichia coli Lead to L-Leucine-Sensitive Growth in the Absence of Unphosphorylated PtsN.

In E. coli K-12, the absence of unphosphorylated PtsN (unphospho-PtsN) has been proposed to cause an L-leucine-sensitive growth phenotype (LeuS) by hyperactivated K+ uptake mediated impairment of the expression of the ilvBN operon, encoding subunits of the L-valine (Val)-sensitive acetohydroxyacid synthase I (AHAS I) that renders residual AHAS activity susceptible to inhibition by Leu and K+. This leads to AHAS insufficiency and a requirement for L-isoleucine (Ile). Herein, we provide an alternate mechanism for the LeuS of the ∆ptsN mutant. Genetic and physiological studies with suppressors of the LeuS indicate that impaired expression of the ilvBN operon jointly caused by the absence of unphospho-PtsN and the presence of Leu coupled to Leu-mediated repression of expression of AHAS III leads to AHAS insufficiency rendering residual AHAS activity susceptible to chronic Val stress that may be generated by exogenous Leu. Hyperactivated K+ uptake and an elevated α-ketobutyrate level mediate elevation of ilvBN expression and alleviate the LeuS. The requirement of unphospho-PtsN as a positive regulator of ilvBN expression may buffer Ile biosynthesis against Leu-mediated AHAS insufficiency and protect AHAS I function from chronic endogenous Val generated by Leu and could be realized in certain environments that impair AHAS function.