An Insight Into Risk Assessment and Reformulation of Drug Products Manufactured Using Benzene Grade Carbomer: A Regulatory Perspective.

Cancer has been an enormous pain point for patients and regulatory bodies across the globe. In Dec. 2023, the US FDA released guidance on benzene-grade carbomer formulations, which triggered pharmaceutical manufacturers to assess risk, test finished products, and reformulate drug products with benzene-grade carbomer. The immediate implementation of the stoppage of finished products with benzene-grade carbomers has threatened pharmaceutical excipients and finished product manufacturers. The gravity of this situation prompted the US Pharmacopeia to extend the deadline for discontinuation from August 1, 2025, to August 1, 2026, allowing manufacturers ample time for reformulation and regulatory compliance.There is an immediate need to understand the guidance and to learn how manufacturers should do the risk assessment and approach reformulation. This review provides an in-depth analysis of the risk assessment and reformulation processes involved in various dosage forms utilizing benzene-grade carbomer, supported by specific case studies.This review offers insights into navigating the USFDA guidelines to ensure formulation safety and compliance, thus enabling pharmaceutical practitioners to uphold the highest standards of patient care and tackle life cycle management challenges.The decision of the USFDA to restrict the usage of high benzene content of carbomer in the formulation is a welcome move. This article has shown a way for researchers to see opportunities in the path and provide best-in-class medicines to patients with a better formulation safety profile.

Prospects of chloroplast metabolic engineering for developing nutrient-dense food crops.

Addressing nutritional deficiencies in food crops through biofortification is a sustainable approach to tackling malnutrition. Biofortification is continuously being attempted through conventional breeding as well as through various plant biotechnological interventions, ranging from molecular breeding to genetic engineering and genome editing for enriching crops with various health-promoting metabolites. Genetic engineering is used for the rational incorporation of desired nutritional traits in food crops and predominantly operates through nuclear and chloroplast genome engineering. In the recent past, chloroplast engineering has been deployed as a strategic tool to develop model plants with enhanced nutritional traits due to the various advantages it offers over nuclear genome engineering. However, this approach needs to be extended for the nutritional enhancement of major food crops. Further, this platform could be combined with strategies, such as synthetic biology, chloroplast editing, nanoparticle-mediated rapid chloroplast transformation, and horizontal gene transfer through grafting for targeting endogenous metabolic pathways for overproducing native nutraceuticals, production of biopharmaceuticals, and biosynthesis of designer nutritional compounds. This review focuses on exploring various features of chloroplast genome engineering for nutritional enhancement of food crops by enhancing the levels of existing metabolites, restoring the metabolites lost during crop domestication, and introducing novel metabolites and phytonutrients needed for a healthy daily diet.

Deciphering the role of miRNA in reprogramming plant responses to drought stress.

Drought is the most prevalent environmental stress that affects plants' growth, development, and crop productivity. However, plants have evolved adaptive mechanisms to respond to the harmful effects of drought. They reprogram their: transcriptome, proteome, and metabolome that alter their cellular and physiological processes and establish cellular homeostasis. One of the crucial regulatory processes that govern this reprogramming is post-transcriptional regulation by microRNAs (miRNAs). miRNAs are small non-coding RNAs, involved in the downregulation of the target mRNA via translation inhibition/mRNA degradation/miRNA-mediated mRNA decay/ribosome drop off/DNA methylation. Many drought-inducible miRNAs have been identified and characterized in plants. Their main targets are regulatory genes that influence growth, development, osmotic stress tolerance, antioxidant defense, phytohormone-mediated signaling, and delayed senescence during drought stress. Overexpression of drought-responsive miRNAs (Osa-miR535, miR160, miR408, Osa-miR393, Osa-miR319, and Gma-miR394) in certain plants has led to tolerance against drought stress indicating their vital role in stress mitigation. Similarly, knock down (miR166/miR398c) or deletion (miR169 and miR827) of miRNAs has also resulted in tolerance to drought stress. Likewise, engineered Arabidopsis plants with miR165, miR166 using short tandem target mimic strategy, exhibited drought tolerance. Since miRNAs regulate the expression of an array of drought-responsive genes, they can act as prospective targets for genetic manipulations to enhance drought tolerance in crops and achieve sustainable agriculture. Further investigations toward functional characterization of diverse miRNAs, and understanding stress-responses regulated by these miRNAs and their utilization in biotechnological applications is highly recommended.

Transgenic chickpea (Cicer arietinum L.) harbouring AtDREB1a are physiologically better adapted to water deficit.

BACKGROUND: Chickpea (Cicer arietinum L.) is the second most widely grown pulse and drought (limiting water) is one of the major constraints leading to about 40-50% yield losses annually. Dehydration responsive element binding proteins (DREBs) are important plant transcription factors that regulate the expression of many stress-inducible genes and play a critical role in improving the abiotic stress tolerance. Transgenic chickpea lines harbouring transcription factor, Dehydration Responsive Element-Binding protein 1A from Arabidopsis thaliana (AtDREB1a gene) driven by stress inducible promoter rd29a were developed, with the intent of enhancing drought tolerance in chickpea. Performance of the progenies of one transgenic event and control were assessed based on key physiological traits imparting drought tolerance such as plant water relation characteristics, chlorophyll retention, photosynthesis, membrane stability and water use efficiency under water stressed conditions. RESULTS: Four transgenic chickpea lines harbouring stress inducible AtDREB1a were generated with transformation efficiency of 0.1%. The integration, transmission and regulated expression were confirmed by Polymerase Chain Reaction (PCR), Southern Blot hybridization and Reverse Transcriptase polymerase chain reaction (RT-PCR), respectively. Transgenic chickpea lines exhibited higher relative water content, longer chlorophyll retention capacity and higher osmotic adjustment under severe drought stress (stress level 4), as compared to control. The enhanced drought tolerance in transgenic chickpea lines were also manifested by undeterred photosynthesis involving enhanced quantum yield of PSII, electron transport rate at saturated irradiance levels and maintaining higher relative water content in leaves under relatively severe soil water deficit. Further, lower values of carbon isotope discrimination in some transgenic chickpea lines indicated higher water use efficiency. Transgenic chickpea lines exhibiting better OA resulted in higher seed yield, with progressive increase in water stress, as compared to control. CONCLUSIONS: Based on precise phenotyping, involving non-invasive chlorophyll fluorescence imaging, carbon isotope discrimination, osmotic adjustment, higher chlorophyll retention and membrane stability index, it can be concluded that AtDREB1a transgenic chickpea lines were better adapted to water deficit by modifying important physiological traits. The selected transgenic chickpea event would be a valuable resource that can be used in pre-breeding or directly in varietal development programs for enhanced drought tolerance under parched conditions.

Gene network modules associated with abiotic stress response in tolerant rice genotypes identified by transcriptome meta-analysis.

Abiotic stress tolerance is a complex trait regulated by multiple genes and gene networks in plants. A range of abiotic stresses are known to limit rice productivity. Meta-transcriptomics has emerged as a powerful approach to decipher stress-associated molecular network in model crops. However, retaining specificity of gene expression in tolerant and susceptible genotypes during meta-transcriptome analysis is important for understanding genotype-dependent stress tolerance mechanisms. Addressing this aspect, we describe here "abiotic stress tolerant" (ASTR) genes and networks specifically and differentially expressing in tolerant rice genotypes in response to different abiotic stress conditions. We identified 6,956 ASTR genes, key hub regulatory genes, transcription factors, and functional modules having significant association with abiotic stress-related ontologies and cis-motifs. Out of the 6956 ASTR genes, 73 were co-located within the boundary of previously identified abiotic stress trait-related quantitative trait loci. Functional annotation of 14 uncharacterized ASTR genes is proposed using multiple computational methods. Around 65% of the top ASTR genes were found to be differentially expressed in at least one of the tolerant genotypes under different stress conditions (cold, salt, drought, or heat) from publicly available RNAseq data comparison. The candidate ASTR genes specifically associated with tolerance could be utilized for engineering rice and possibly other crops for broad-spectrum tolerance to abiotic stresses.

Mature embryo-derived wheat transformation for tolerance to moisture stress.

We have developed marker-free transgenic wheat using a transcription factor, AtDREB1A cloned from Arabidopsis. Southern hybridization confirmed a transgenic event with a single copy insertion. PCR analysis of the T1 plants showed four were positive only for AtDREB1A. A T1 plant (HRCB3#17-37) was marker-free and had good expression of drought tolerance in comparison with untransformed plants. The leaf relative water content of this T1 transgenic plant was 12-15% higher than that of the wild type during stress with an 8% higher yield under water deficit conditions compared with wild type plants.

Chloroplast transformation in new cultivars of tomato through particle bombardment.

A protocol has been established for genetic transformation of the chloroplasts in two new cultivars of tomato (Solanum lycopersicum L.) grown in India and Australia: Pusa Ruby and Yellow Currant. Tomato cv. Green Pineapple was also used as a control that has previously been used for establishing chloroplast transformation by other researchers. Selected tomato cultivars were finalized from ten other tested cultivars (Green Pineapple excluded) due to their high regeneration potential and better response to chloroplast transformation. This protocol was set up using a chloroplast transformation vector (pRB94) for tomatoes that is made up of a synthetic gene operon. The vector has a chimeric aadA selectable marker gene that is controlled by the rRNA operon promoter (Prrn). This makes the plant or chloroplasts resistant to spectinomycin and streptomycin. After plasmid-coated particle bombardment, leaf explants were cultured in 50 mg/L selection media. Positive explant selection from among all the dead-appearing (yellow to brown) explants was found to be the major hurdle in the study. Even though this study was able to find plastid transformants in heteroplasmic conditions, it also found important parameters and changes that could speed up the process of chloroplast transformation in tomatoes, resulting in homoplasmic plastid-transformed plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03954-3.

Cicer super-pangenome provides insights into species evolution and agronomic trait loci for crop improvement in chickpea.

Chickpea (Cicer arietinum L.)-an important legume crop cultivated in arid and semiarid regions-has limited genetic diversity. Efforts are being undertaken to broaden its diversity by utilizing its wild relatives, which remain largely unexplored. Here, we present the Cicer super-pangenome based on the de novo genome assemblies of eight annual Cicer wild species. We identified 24,827 gene families, including 14,748 core, 2,958 softcore, 6,212 dispensable and 909 species-specific gene families. The dispensable genome was enriched for genes related to key agronomic traits. Structural variations between cultivated and wild genomes were used to construct a graph-based genome, revealing variations in genes affecting traits such as flowering time, vernalization and disease resistance. These variations will facilitate the transfer of valuable traits from wild Cicer species into elite chickpea varieties through marker-assisted selection or gene-editing. This study offers valuable insights into the genetic diversity and potential avenues for crop improvement in chickpea.

Genome-wide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis.

BACKGROUND: The MYB gene family comprises one of the richest groups of transcription factors in plants. Plant MYB proteins are characterized by a highly conserved MYB DNA-binding domain. MYB proteins are classified into four major groups namely, 1R-MYB, 2R-MYB, 3R-MYB and 4R-MYB based on the number and position of MYB repeats. MYB transcription factors are involved in plant development, secondary metabolism, hormone signal transduction, disease resistance and abiotic stress tolerance. A comparative analysis of MYB family genes in rice and Arabidopsis will help reveal the evolution and function of MYB genes in plants. RESULTS: A genome-wide analysis identified at least 155 and 197 MYB genes in rice and Arabidopsis, respectively. Gene structure analysis revealed that MYB family genes possess relatively more number of introns in the middle as compared with C- and N-terminal regions of the predicted genes. Intronless MYB-genes are highly conserved both in rice and Arabidopsis. MYB genes encoding R2R3 repeat MYB proteins retained conserved gene structure with three exons and two introns, whereas genes encoding R1R2R3 repeat containing proteins consist of six exons and five introns. The splicing pattern is similar among R1R2R3 MYB genes in Arabidopsis. In contrast, variation in splicing pattern was observed among R1R2R3 MYB members of rice. Consensus motif analysis of 1kb upstream region (5' to translation initiation codon) of MYB gene ORFs led to the identification of conserved and over-represented cis-motifs in both rice and Arabidopsis. Real-time quantitative RT-PCR analysis showed that several members of MYBs are up-regulated by various abiotic stresses both in rice and Arabidopsis. CONCLUSION: A comprehensive genome-wide analysis of chromosomal distribution, tandem repeats and phylogenetic relationship of MYB family genes in rice and Arabidopsis suggested their evolution via duplication. Genome-wide comparative analysis of MYB genes and their expression analysis identified several MYBs with potential role in development and stress response of plants.

The 3366 chickpea genomes for research and breeding.

Genome sequences provide an unprecedented resource to rapidly develop modern crops. A recent paper by Varshney et al. provides genome variation maps of 3366 chickpea accessions. Here, we highlight how this breakthrough research can fundamentally change breeding practices of chickpea and potentially other crops.

Wheat Biofortification: Utilizing Natural Genetic Diversity, Genome-Wide Association Mapping, Genomic Selection, and Genome Editing Technologies.

Alleviating micronutrients associated problems in children below five years and women of childbearing age, remains a significant challenge, especially in resource-poor nations. One of the most important staple food crops, wheat attracts the highest global research priority for micronutrient (Fe, Zn, Se, and Ca) biofortification. Wild relatives and cultivated species of wheat possess significant natural genetic variability for these micronutrients, which has successfully been utilized for breeding micronutrient dense wheat varieties. This has enabled the release of 40 biofortified wheat cultivars for commercial cultivation in different countries, including India, Bangladesh, Pakistan, Bolivia, Mexico and Nepal. In this review, we have systematically analyzed the current understanding of availability and utilization of natural genetic variations for grain micronutrients among cultivated and wild relatives, QTLs/genes and different genomic regions regulating the accumulation of micronutrients, and the status of micronutrient biofortified wheat varieties released for commercial cultivation across the globe. In addition, we have also discussed the potential implications of emerging technologies such as genome editing to improve the micronutrient content and their bioavailability in wheat.

Comparative analysis of drought-responsive transcriptome in Indica rice genotypes with contrasting drought tolerance.

Genetic improvement in drought tolerance in rice is the key to save water for sustainable agriculture. Drought tolerance is a complex trait and involves interplay of a vast array of genes. Several genotypes of rice have evolved features that impart tolerance to drought and other abiotic stresses. Comparative analysis of drought stress-responsive transcriptome between drought-tolerant (DT) landraces/genotypes and drought-sensitive modern rice cultivars will unravel novel genetic regulatory mechanisms involved in stress tolerance. Here, we report transcriptome analysis in a highly DT rice landrace, Nagina 22 (N22), versus a high-yielding but drought-susceptible rice variety IR64. Both genotypes exhibited a diverse global transcriptional response under normal and drought conditions. Gene ontology (GO) analysis suggested that drought tolerance of N22 was attributable to the enhanced expression of several enzyme-encoding genes. Drought susceptibility of IR64 was attributable to significant down-regulation of regulatory components that confer drought tolerance. Pathway analysis unravelled significant up-regulation of several components of carbon fixation, glycolysis/gluconeogenesis and flavonoid biosynthesis and down-regulation of starch and sucrose metabolism in both the cultivars under drought. However, significant up-regulation of α-linolenic acid metabolic pathway observed in N22 under drought appears to be in good agreement with high drought tolerance of this genotype. Consensus cis-motif profiling of drought-induced co-expressed genes led to the identification of novel cis-motifs. Taken together, the results of the comparative transcriptome analysis led to the identification of specific genotype-dependent genes responsible for drought tolerance in the rice landrace N22.

Precise plant genome editing using base editors and prime editors.

The development of CRISPR-Cas systems has sparked a genome editing revolution in plant genetics and breeding. These sequence-specific RNA-guided nucleases can induce DNA double-stranded breaks, resulting in mutations by imprecise non-homologous end joining (NHEJ) repair or precise DNA sequence replacement by homology-directed repair (HDR). However, HDR is highly inefficient in many plant species, which has greatly limited precise genome editing in plants. To fill the vital gap in precision editing, base editing and prime editing technologies have recently been developed and demonstrated in numerous plant species. These technologies, which are mainly based on Cas9 nickases, can introduce precise changes into the target genome at a single-base resolution. This Review provides a timely overview of the current status of base editors and prime editors in plants, covering both technological developments and biological applications.

Isolation and functional characterization of lycopene beta-cyclase (CYC-B) promoter from Solanum habrochaites.

BACKGROUND: Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor beta-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene beta-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both alpha-and beta-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene beta-cyclase (CYC-B) promoter from a green fruited S. habrochaites genotype EC520061. RESULTS: A 908 bp region upstream to the initiation codon of the Lycopene beta-cyclase gene was cloned and identified as full-length promoter. To identify promoter region necessary for regulating developmental expression of the ShCYC-B gene, the full-length promoter and its three different 5' truncated fragments were cloned upstream to the initiation codon of GUS reporter cDNA in binary vectors. These four plant transformation vectors were separately transformed in to Agrobacterium. Agrobacterium-mediated transient and stable expression systems were used to study the GUS expression driven by the full-length promoter and its 5' deletion fragments in tomato. The full-length promoter showed a basal level activity in leaves, and its expression was upregulated > 5-fold in flowers and fruits in transgenic tomato plants. Deletion of -908 to -577 bp 5' to ATG decreases the ShCYC-B promoter strength, while deletion of -908 to -437 bp 5' to ATG led to significant increase in the activity of GUS in the transgenic plants. Promoter deletion analysis led to the identification of a short promoter region (-436 bp to ATG) that exhibited a higher promoter strength but similar developmental expression pattern as compared with the full-length ShCYC-B promoter. CONCLUSION: Functional characterization of the full-length ShCYC-B promoter and its deletion fragments in transient expression system in fruto as well as in stable transgenic tomato revealed that the promoter is developmentally regulated and its expression is upregulated in chromoplast-rich flowers and fruits. Our study identified a short promoter region with functional activity and developmental expression pattern similar to that of the full-length ShCYC-B promoter. This 436 bp promoter region can be used in promoter::reporter fusion molecular genetic screens to identify mutants impaired in CYC-B expression, and thus can be a valuable tool in understanding carotenoid metabolism in tomato. Moreover, this short promoter region of ShCYC-B may be useful in genetic engineering of carotenoid content and other agronomic traits in tomato fruits.

A Short History and Perspectives on Plant Genetic Transformation.

Plant genetic transformation is an important technological advancement in modern science, which has not only facilitated gaining fundamental insights into plant biology but also started a new era in crop improvement and commercial farming. However, for many crop plants, efficient transformation and regeneration still remain a challenge even after more than 30 years of technical developments in this field. Recently, FokI endonuclease-based genome editing applications in plants offered an exciting avenue for augmenting crop productivity but it is mainly dependent on efficient genetic transformation and regeneration, which is a major roadblock for implementing genome editing technology in plants. In this chapter, we have outlined the major historical developments in plant genetic transformation for developing biotech crops. Overall, this field needs innovations in plant tissue culture methods for simplification of operational steps for enhancing the transformation efficiency. Similarly, discovering genes controlling developmental reprogramming and homologous recombination need considerable attention, followed by understanding their role in enhancing genetic transformation efficiency in plants. Further, there is an urgent need for exploring new and low-cost universal delivery systems for DNA/RNA and protein into plants. The advancements in synthetic biology, novel vector systems for precision genome editing and gene integration could potentially bring revolution in crop-genetic potential enhancement for a sustainable future. Therefore, efficient plant transformation system standardization across species holds the key for translating advances in plant molecular biology to crop improvement.

Abiotic stress and ABA-inducible Group 4 LEA from Brassica napus plays a key role in salt and drought tolerance.

Late-embryogenesis abundant (LEA) proteins are a family of hydrophilic proteins that form an integral part of desiccation tolerance of seeds. LEA proteins have been also postulated to play a protective role under different abiotic stresses. Their role in abiotic stress tolerance has been well documented for Group 1, 2 and 3 LEAs among the nine different groups. The present study evaluates the functional role of a Group 4 LEA protein, LEA4-1 from Brassica napus. Expression analysis revealed that abscisic acid, salt, cold and osmotic stresses induce expression of LEA4-1 gene in leaf tissues in Brassica species. Conversely, reproductive tissues such as flowers and developing seeds showed constitutive expression of LEA4, which was up-regulated in flowers under salt stress. For functional evaluation of LEA4-1 with regard to stress tolerance, LEA4-1 cDNA was cloned from B. napus, and overexpressed in both Escherichia coli and transgenic Arabidopsis plants. Overexpression of BnLEA4-1 cDNA in E. coli conferred salt and extreme temperature tolerance to the transformed cells. Furthermore, transgenic Arabidopsis plants overexpressing BnLEA4-1 either under constitutive CaMV35S or abiotic stress inducible RD29A promoter showed enhanced tolerance to salt and drought stresses. These results demonstrate that LEA4-1 plays a crucial role in abiotic stress tolerance during vegetative stage of plant development.

Genome-Wide Identification and Analysis of Biotic and Abiotic Stress Regulation of C4 Photosynthetic Pathway Genes in Rice.

Photosynthetic fixation of CO2 is more efficient in C4 than in C3 plants. Rice is a C3 plant and a potential target for genetic engineering of the C4 pathway. It is known that genes encoding C4 enzymes are present in C3 plants. However, no systematic analysis has been conducted to determine if these C4 gene family members are expressed in diverse rice genotypes. In this study, we identified 15 genes belonging to the five C4 gene families in rice genome through BLAST search using known maize C4 photosynthetic pathway genes. Phylogenetic relationship of rice C4 photosynthetic pathway genes and their isoforms with other grass genomes (Brachypodium, maize, Sorghum and Setaria), showed that these genes were highly conserved across grass genomes. Spatiotemporal, hormone, and abiotic stress specific expression pattern of the identified genes revealed constitutive as well as inductive responses of the C4 photosynthetic pathway in different tissues and developmental stages of rice. Expression levels of C4 specific gene family members in flag leaf during tillering stage were quantitatively analyzed in five rice genotypes covering three species, viz. Oryza sativa, ssp. japonica (cv. Nipponbare), Oryza sativa, ssp. indica (cv IR64, Swarna), and two wild species Oryza barthii and Oryza australiensis. The results showed that all the identified genes expressed in rice and exhibited differential expression pattern during different growth stages, and in response to biotic and abiotic stress conditions and hormone treatments. Our study concludes that C4 photosynthetic pathway genes present in rice play a crucial role in stress regulation and might act as targets for C4 pathway engineering via CRISPR-mediated breeding.

Heterologous expression of rice RNA-binding glycine-rich (RBG) gene OsRBGD3 in transgenic Arabidopsis thaliana confers cold stress tolerance.

Imparting cold stress tolerance to crops is a major challenge in subtropical agriculture. New genes conferring cold tolerance needs to be identified and characterised for sustainable crop production in low-temperature stress affected areas. Here we report functional characterisation of OsRBGD3, classified previously as a class D glycine-rich RNA recognition motif (RRM) containing proteins from a drought-tolerant Indica rice cultivar N22. The gene was isolated by screening yeast one-hybrid library using the minimal promoter region of the OsMYB38 that is necessary for cold stress-responsive expression. OsRBGD3 exhibited cold, drought and salt stress inductive expression in a drought tolerant N22 rice cultivar as compared with susceptible variety IR64. OsRBGD3 was found to be localised to both nuclear and cytoplasmic subcellular destinations. Constitutive overexpression of the OsRBGD3 in transgenic Arabidopsis conferred tolerance to cold stress. ABA sensitivity was also observed in transgenic lines suggesting the regulatory role of this gene in the ABA signalling pathway. OsRBGD3 overexpression also attributed to significant root development and early flowering in transgenics. Hence, OsRBGD3 could be an important target for developing cold tolerant early flowering rice and other crops' genotypes for increasing production in low temperature affected areas.

Characterization and phylogenetic analysis of ectoine biosynthesis genes from Bacillus halodurans.

Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), is a natural compound, which serves as a protective substance in many bacterial cells. In this study, the putative ectABC gene cluster from Bacillus halodurans was heterologously expressed in E. coli and the production of ectoine was confirmed by HPLC analysis. The activity of the enzymes coded by the ectA, B and C genes were found to be higher in induced transgenic cells compared to the uninduced cells. Phylogenetic analysis revealed sequence identities ranging from 36-73% for ectA gene, 55-81% for ectB gene and 55-80% for ectC gene indicating that the enzymes are evolutionarily well conserved.