Bibliometric analysis and thematic review of Candida pathogenesis: Fundamental omics to applications as potential antifungal drugs and vaccines.

Invasive candidiasis caused by the pathogenic Candida yeast species has resulted in elevating global mortality. The pathogenicity of Candida spp. is not only originated from its primary invasive yeast-to-hyphal transition; virulence factors (transcription factors, adhesins, invasins, and enzymes), biofilm, antifungal drug resistance, stress tolerance, and metabolic adaptation have also contributed to a greater clinical burden. However, the current research theme in fungal pathogenicity could hardly be delineated with the increasing research output. Therefore, our study analysed the research trends in Candida pathogenesis over the past 37 years via a bibliometric approach against the Scopus and Web of Science databases. Based on the 3993 unique documents retrieved, significant international collaborations among researchers were observed, especially between Germany (Bernhard Hube) and the UK (Julian Naglik), whose focuses are on Candida proteinases, adhesins, and candidalysin. The prominent researchers (Neils Gow, Alistair Brown, and Frank Odds) at the University of Exeter and the University of Aberdeen (second top performing affiliation) UK contribute significantly to the mechanisms of Candida adaptation, tolerance, and stress response. However, the science mapping of co-citation analysis performed herein could not identify a hub representative of subsequent work since the clusters were semi-redundant. The co-word analysis that was otherwise adopted, revealed three research clusters; the cluster-based thematic analyses indicated the severeness of Candida biofilm and antifungal resistance as well as the elevating trend on molecular mechanism elucidation for drug screening and repurposing. Importantly, the in vivo pathogen adaptation and interactions with hosts are crucial for potential vaccine development.

International research collaborations have evident its significance in impactful work covering all aspects of Candida pathogenicity. Its current, diverse research was discussed thematically based on the comprehensive scientometric analysis with unidentified hub representatives for subsequent work.

Catalytically active inclusion bodies as a potential tool for biotechnology.

The initial assumption that viewed inclusion bodies as a hindrance to the efficient production of protein is no longer held due to the emergence of catalytically active inclusion bodies (CatIBs). Recent studies revealed their potential to be used in free form or immobilized as biocatalysts. The curiosity to acquire suitable catalysts has remained the measure of concern for researchers and industrialists. Numerous processes and production in various sectors of food industries, petroleum, pharmaceutical, cosmetics, and many others are still searching for a robust catalyst with outstanding features such as recyclability, resistance to pH, as well as temperature. CatIBs are forms of inclusion bodies that possess catalytic activity, which can improve catalysis efficiency, stability, and recyclability. One of the advantages of CatIBs is their potential to be used as catalysts for numerous bioprocesses when generated by an enzyme. These aggregates can efficiently be used as a replacement for traditional enzyme immobilization. This review tends to focus on the possibility of its application in various processes. The novelty of this review is that it considered the production of CatIBs both from artificial and natural perspectives, as well as how to improve it. Inclusion bodies' immobilization may provide an efficient alternative in the area of biocatalysis, and hence it will improve industrial sectors and substantially provide a means of achieving excellent performance in the near future.

Cloning, Expression and Functional Characterization of a Novel α-Humulene Synthase, Responsible for the Formation of Sesquiterpene in Agarwood Originating from Aquilaria malaccensis.

This study describes the cloning, expression and functional characterization of α-humulene synthase, responsible for the formation of the key aromatic compound α-humulene in agarwood originating from Aquilaria malaccensis. The partial sesquiterpene synthase gene from the transcriptome data of A. malaccensis was utilized for full-length gene isolation via a 3' RACE PCR. The complete gene, denoted as AmDG2, has an open reading frame (ORF) of 1671 bp and encodes for a polypeptide of 556 amino acids. In silico analysis of the protein highlighted several conserved motifs typically found in terpene synthases such as Asp-rich substrate binding (DDxxD), metal-binding residues (NSE/DTE), and cytoplasmic ER retention (RxR) motifs at their respective sites. The AmDG2 was successfully expressed in the E. coli:pET-28a(+) expression vector whereby an expected band of about 64 kDa in size was detected in the SDS-PAGE gel. In vitro enzyme assay using substrate farnesyl pyrophosphate (FPP) revealed that AmDG2 gave rise to two sesquiterpenes: α-humulene (major) and ß-caryophyllene (minor), affirming its identity as α-humulene synthase. On the other hand, protein modeling performed using AlphaFold2 suggested that AmDG2 consists entirely of α-helices with short connecting loops and turns. Meanwhile, molecular docking via AutoDock Vina (Version 1.5.7) predicted that Asp307 and Asp311 act as catalytic residues in the α-humulene synthase. To our knowledge, this is the first comprehensive report on the cloning, expression and functional characterization of α-humulene synthase from agarwood originating from A. malaccensis species. These findings reveal a deeper understanding of the structure and functional properties of the α-humulene synthase and could be utilized for metabolic engineering work in the future.

Molecular cloning and in silico analysis of chalcone isomerase from Polygonum minus.

BACKGROUND: Chalcone isomerase (CHI; EC 5.5.1.6) is one of the key enzymes in the flavonoid biosynthetic pathway that is responsible for the intramolecular cyclization of chalcones into specific 2S-flavanones. METHODS AND RESULTS: In this study, the open reading frame (ORF) of CHI was successfully isolated from the cDNA of Polygonum minus at 711-bp long, encoding for 236 amino acid residues, with a predicted molecular weight of 25.4 kDa. Multiple sequence alignment and phylogenetic analysis revealed that the conserved residues (Thr50, Tyr108, Asn115, and Ser192) in the cleft of CHI enzyme group active site are present in PmCHI protein sequence and classified as type I. PmCHI comprises more hydrophobic residues without a signal peptide and transmembrane helices. The three-dimensional (3D) structure of PmCHI predicted through homology modeling was validated by Ramachandran plot and Verify3D, with values within the acceptable range of a good model. PmCHI was cloned into pET-28b(+) plasmid, expressed in Escherichia coli BL21(DE3) at 16 °C and partially purified. CONCLUSION: These findings contribute to a deeper understanding of the PmCHI protein and its potential for further characterization of its functional properties in the flavonoid biosynthetic pathway.

Antimicrobial peptides from Bacillus spp. and strategies to enhance their yield.

Antibiotic resistance is a growing concern that is affecting public health globally. The search for alternative antimicrobial agents has become increasingly important. Antimicrobial peptides (AMPs) produced by Bacillus spp. have emerged as a promising alternative to antibiotics, due to their broad-spectrum antimicrobial activity against resistant pathogens. In this review, we provide an overview of Bacillus-derived AMPs, including their classification into ribosomal (bacteriocins) and non-ribosomal peptides (lipopeptides and polyketides). Additionally, we delve into the molecular mechanisms of AMP production and describe the key biosynthetic gene clusters involved. Despite their potential, the low yield of AMPs produced under normal laboratory conditions remains a challenge to large-scale production. This review thus concludes with a comprehensive summary of recent studies aimed at enhancing the productivity of Bacillus-derived AMPs. In addition to medium optimization and genetic manipulation, various molecular strategies have been explored to increase the production of recombinant antimicrobial peptides (AMPs). These include the selection of appropriate expression systems, the engineering of expression promoters, and metabolic engineering. Bacillus-derived AMPs offer great potential as alternative antimicrobial agents, and this review provides valuable insights on the strategies to enhance their production yield, which may have significant implications for combating antibiotic resistance. KEY POINTS: • Bacillus-derived AMP is a potential alternative therapy for resistant pathogens • Bacillus produces two main classes of AMPs: ribosomal and non-ribosomal peptides • AMP yield can be enhanced using culture optimization and molecular approaches.

Bacteria consortia enhanced hydrocarbon degradation of waxy crude oil.

Waxy crude oil is a problem to the oil and gas industry because wax deposition in pipelines reduces the quality of the crude oil. Currently, the industry uses chemicals to solve the problem but it is not environmentally friendly. As an alternative, the biodegradation approach is one of the options. Previously eleven thermophilic bacteria were isolated and exhibited high ability to degrade hydrocarbon up to 70% of waxy crude oil. However, despite the successful study on these single bacteria strains, it is believed that biodegradation of paraffin wax requires more than a single species. Five consortia were developed based on the biodegradation efficiency of 11 bacterial strains. Consortium 3 showed the highest biodegradation (77.77%) with more long-chain alkane degraded throughout the incubation compared to other consortia. Enhancement of hydrocarbon degradation was observed for all consortia especially in long chain alkane (C18-C40). Consortium 3 exhibited higher alkane monooxygenase, alcohol dehydrogenase, lipase, and esterase activities. Moreover, the dominant bacteria in the consortia were determined by denaturing gradient gel electrophoresis (DGGE), which showed the domination of genera Geobacillus, Parageobacillus, and Anoxybacillus. It can be concluded that the bacterial consortia showed higher biodegradation and improved degrading more long-chain hydrocarbon compared to a single isolate.

Feather-Degrading Bacillus cereus HD1: Genomic Analysis and Its Optimization for Keratinase Production and Feather Degradation.

Keratinase is an important enzyme that is used to degrade feather wastes produced by poultry industries and slaughterhouses that accumulate rapidly over time. The search for keratinase-producing microorganisms is important to potentially substitute physicochemical treatments of feather waste. In this study, the genome of Bacillus cereus HD1 and its keratinolytic prowess was investigated. The whole-genome shotgun size is 5,668,864 bp consisting of 6083 genes, 69 tRNAs, and 10 rRNAs. The genomic analyses revealed 15 potential keratinase genes and other enzymes that might assist keratin degradation, such as disulfide reductase and cysteine dioxygenase. The optimal conditions for feather degradation and keratinase production by B. cereus HD1 such as incubation time, pH, temperature, yeast extract, and glycerol concentrations were determined to be 5 days, pH 8, 37 °C, 0.05% (w/v), and 0.1% (v/v), respectively. Under optimized conditions, B. cereus HD1 exhibited feather degradation of 65%, with bacterial growth and maximum keratinase activity of 1.3 × 1011 CFU/mL and 41 U/mL, respectively, after 5 days of incubation in a feather basal medium. The findings obtained from this study may facilitate further research into utilizing B. cereus HD1 as a prominent keratinolytic enzymes production host and warrant potential biotechnological applications.

Opportunistic yeast pathogen Candida spp.: Secreted and membrane-bound virulence factors.

Candidiasis is a fungal infection caused by Candida spp. especially Candida albicans, C. glabrata, C. parapsilosis and C. tropicalis. Although the medicinal therapeutic strategies have rapidly improved, the mortality rate as candidiasis has continuously increased. The secreted and membrane-bound virulence factors (VFs) are responsible for fungal invasion, damage and translocation through the host enterocytes besides the evasion from host immune system. VFs such as agglutinin-like sequences (Als), heat shock protein 70, phospholipases, secreted aspartyl proteinases (Sap), lipases, enolases and phytases are mostly hydrolases which degrade or interact with the enterocyte membrane components. Candidalysin, however, acts as a peptide toxin to induce necrotic cell lysis. To date, structural studies of the VFs remain underexplored, hindering their functional analyses. Among the VFs, only Sap and Als have their structures deposited in Protein Data Bank (PDB). Therefore, this review scrutinizes the mechanisms of these VFs by discussing the VF-deficient studies of several Candida spp. and their abilities to produce these VFs. Nonetheless, their latest reported sequential and structural analyses are discussed to impart a wider perception of the host-pathogen interactions and potential vaccine or antifungal drug targets. This review signifies that more VFs structural investigations and mining in the emerging Candida spp. are required to decipher their pathogenicity and virulence mechanisms compared to the prominent C. albicans. LAY SUMMARY: Candida virulence factors (VFs) including mainly enzymes and proteins play vital roles in breaching the human intestinal barrier and causing deadly invasive candidiasis. Limited VFs' structural studies hinder deeper comprehension of their mechanisms and thus the design of vaccines and antifungal drugs against fungal infections.

Thermostable lipases and their dynamics of improved enzymatic properties.

Thermal stability is one of the most desirable characteristics in the search for novel lipases. The search for thermophilic microorganisms for synthesising functional enzyme biocatalysts with the ability to withstand high temperature, and capacity to maintain their native state in extreme conditions opens up new opportunities for their biotechnological applications. Thermophilic organisms are one of the most favoured organisms, whose distinctive characteristics are extremely related to their cellular constituent particularly biologically active proteins. Modifications on the enzyme structure are critical in optimizing the stability of enzyme to thermophilic conditions. Thermostable lipases are one of the most favourable enzymes used in food industries, pharmaceutical field, and actively been studied as potential biocatalyst in biodiesel production and other biotechnology application. Particularly, there is a trade-off between the use of enzymes in high concentration of organic solvents and product generation. Enhancement of the enzyme stability needs to be achieved for them to maintain their enzymatic activity regardless the environment. Various approaches on protein modification applied since decades ago conveyed a better understanding on how to improve the enzymatic properties in thermophilic bacteria. In fact, preliminary approach using advanced computational analysis is practically conducted before any modification is being performed experimentally. Apart from that, isolation of novel extremozymes from various microorganisms are offering great frontier in explaining the crucial native interaction within the molecules which could help in protein engineering. In this review, the thermostability prospect of lipases and the utility of protein engineering insights into achieving functional industrial usefulness at their high temperature habitat are highlighted. Similarly, the underlying thermodynamic and structural basis that defines the forces that stabilize these thermostable lipase is discussed. KEY POINTS: • The dynamics of lipases contributes to their non-covalent interactions and structural stability. • Thermostability can be enhanced by well-established genetic tools for improved kinetic efficiency. • Molecular dynamics greatly provides structure-function insights on thermodynamics of lipase.

Molecular strategies to increase keratinase production in heterologous expression systems for industrial applications.

Keratinase is an important enzyme that can degrade recalcitrant keratinous wastes to form beneficial recyclable keratin hydrolysates. Keratinase is not only important as an alternative to reduce environmental pollution caused by chemical treatments of keratinous wastes, but it also has industrial significance. Currently, the bioproduction of keratinase from native keratinolytic host is considered low, and this hampers large-scale usage of the enzyme. Straightforward approaches of cloning and expression of recombinant keratinases from native keratinolytic host are employed to elevate the amount of keratinase produced. However, this is still insufficient to compensate for the lack of its large-scale production to meet the industrial demands. Hence, this review aimed to highlight the various sources of keratinase and the strategies to increase its production in native keratinolytic hosts. Molecular strategies to increase the production of recombinant keratinase such as plasmid selection, promoter engineering, chromosomal integration, signal peptide and propeptide engineering, codon optimization, and glycoengineering were also described. These mentioned strategies have been utilized in heterologous expression hosts, namely, Escherichia coli, Bacillus sp., and Pichia pastoris, as they are most widely used for the heterologous propagations of keratinases to further intensify the production of recombinant keratinases adapted to better suit the large-scale demand for them. KEY POINTS: • Molecular strategies to enhance keratinase production in heterologous hosts. • Construction of a prominent keratinolytic host from a native strain. • Patent analysis of keratinase production shows rapid high interest in molecular field.

Biosurfactant Production and Growth Kinetics Studies of the Waste Canola Oil-Degrading Bacterium Rhodococcus erythropolis AQ5-07 from Antarctica.

With the progressive increase in human activities in the Antarctic region, the possibility of domestic oil spillage also increases. Developing means for the removal of oils, such as canola oil, from the environment and waste "grey" water using biological approaches is therefore desirable, since the thermal process of oil degradation is expensive and ineffective. Thus, in this study an indigenous cold-adapted Antarctic soil bacterium, Rhodococcus erythropolis strain AQ5-07, was screened for biosurfactant production ability using the multiple approaches of blood haemolysis, surface tension, emulsification index, oil spreading, drop collapse and "MATH" assay for cellular hydrophobicity. The growth kinetics of the bacterium containing different canola oil concentration was studied. The strain showed ß-haemolysis on blood agar with a high emulsification index and low surface tension value of 91.5% and 25.14 mN/m, respectively. Of the models tested, the Haldane model provided the best description of the growth kinetics, although several models were similar in performance. Parameters obtained from the modelling were the maximum specific growth rate (qmax), concentration of substrate at the half maximum specific growth rate, Ks% (v/v) and the inhibition constant Ki% (v/v), with values of 0.142 h-1, 7.743% (v/v) and 0.399% (v/v), respectively. These biological coefficients are useful in predicting growth conditions for batch studies, and also relevant to "in field" bioremediation strategies where the concentration of oil might need to be diluted to non-toxic levels prior to remediation. Biosurfactants can also have application in enhanced oil recovery (EOR) under different environmental conditions.

Recent advancement of engineering microbial hosts for the biotechnological production of flavonoids.

Flavonoids are polyphenols that are important organic chemicals in plants. The health benefits of flavonoids that result in high commercial values make them attractive targets for large-scale production through bioengineering. Strategies such as engineering a flavonoid biosynthetic pathway in microbial hosts provide an alternative way to produce these beneficial compounds. Escherichia coli, Saccharomyces cerevisiae and Streptomyces sp. are among the expression systems used to produce recombinant products, as well as for the production of flavonoid compounds through various bioengineering approaches including clustered regularly interspaced short palindromic repeats (CRISPR)-based genome engineering and genetically encoded biosensors to detect flavonoid biosynthesis. In this study, we review the recent advances in engineering model microbial hosts as being the factory to produce targeted flavonoid compounds.

Polyunsaturated fatty acids in marine bacteria and strategies to enhance their production.

Polyunsaturated fatty acids (PUFAs) play an important role in human diet. Despite the wide-ranging importance and benefits from heart health to brain functions, humans and mammals cannot synthesize PUFAs de novo. The primary sources of PUFA are fish and plants. Due to the increasing concerns associated with food security as well as issues of environmental contaminants in fish oil, there has been considerable interest in the production of polyunsaturated fatty acids from alternative resources which are more sustainable, safer, and economical. For instance, marine bacteria, particularly the genus of Shewanella, Photobacterium, Colwellia, Moritella, Psychromonas, Vibrio, and Alteromonas, are found to be one among the major microbial producers of polyunsaturated fatty acids. Recent developments in the area with a focus on the production of polyunsaturated fatty acids from marine bacteria as well as the metabolic engineering strategies for the improvement of PUFA production are discussed.

Functional Characterisation of New Sesquiterpene Synthase from the Malaysian Herbal Plant, Polygonum Minus.

Polygonum minus (syn. Persicaria minor) is a herbal plant that is well known for producing sesquiterpenes, which contribute to its flavour and fragrance. This study describes the cloning and functional characterisation of PmSTPS1 and PmSTPS2, two sesquiterpene synthase genes that were identified from P. minus transcriptome data mining. The full-length sequences of the PmSTPS1 and PmSTPS2 genes were expressed in the E. coli pQE-2 expression vector. The sizes of PmSTPS1 and PmSTPS2 were 1098 bp and 1967 bp, respectively, with open reading frames (ORF) of 1047 and 1695 bp and encoding polypeptides of 348 and 564 amino acids, respectively. The proteins consist of three conserved motifs, namely, Asp-rich substrate binding (DDxxD), metal binding residues (NSE/DTE), and cytoplasmic ER retention (RxR), as well as the terpene synthase family N-terminal domain and C-terminal metal-binding domain. From the in vitro enzyme assays, using the farnesyl pyrophosphate (FPP) substrate, the PmSTPS1 enzyme produced multiple acyclic sesquiterpenes of ß-farnesene, α-farnesene, and farnesol, while the PmSTPS2 enzyme produced an additional nerolidol as a final product. The results confirmed the roles of PmSTPS1 and PmSTPS2 in the biosynthesis pathway of P. minus, to produce aromatic sesquiterpenes.

The biology and the importance of Photobacterium species.

Photobacterium species are Gram-negative coccobacilli which are distributed in marine habitats worldwide. Some species are unique because of their capability to produce luminescence. Taxonomically, about 23 species and 2 subspecies are validated to date. Genomes from a few Photobacterium spp. have been sequenced and studied. They are considered a special group of bacteria because some species are capable of producing essential polyunsaturated fatty acids, antibacterial compounds, lipases, esterases and asparaginases. They are also used as biosensors in food and environmental monitoring and detectors of drown victim, as well as an important symbiont.

Humanizing glycosylation pathways in eukaryotic expression systems.

Glycosylation represents the most widespread posttranslational modifications, found in a broad spectrum of natural and therapeutic recombinant proteins. It highly affects bioactivity, site-specificity, stability, solubility, immunogenicity, and serum half-life of glycoproteins. Numerous expression hosts including yeasts, insect cells, transgenic plants, and mammalian cells have been explored for synthesizing therapeutic glycoproteins. However, glycosylation profile of eukaryotic expression systems differs from human. Glycosylation strategies have been proposed for humanizing the glycosylation pathways in expression hosts which is the main theme of this review. Besides, we also highlighted the glycosylation potential of protozoan parasites by emphasizing on the mammalian-like glycosylation potential of Leishmania tarentolae known as Leishmania expression system.

Features of the rare pathogen Meyerozyma guilliermondii strain SO and comprehensive in silico analyses of its adherence-contributing virulence factor agglutinin-like sequences.

Meyerozyma guilliermondii is a rare yeast pathogen contributing to the deadly invasive candidiasis. M. guilliermondii strain SO, as a promising protein expression host, showed 99% proteome similarity with the clinically isolated ATCC 6260 (type strain) in a recent comparative genomic analysis. However, their in vitro virulence features and in vivo pathogenicity were uncharacterized. This study aimed to characterize the in vitro and in vivo pathogenicity of M. guilliermondii strain SO and analyze its Als proteins (MgAls) via comprehensive bioinformatics approaches. M. guilliermondii strain SO showed lower and higher sensitivity towards ß-mercaptoethanol and lithium, respectively than the avirulent S. cerevisiae but exhibited the same tolerance towards cell wall-perturbing Congo Red with C. albicans. With 7.5× higher biofilm mass, M. guilliermondii strain SO also demonstrated 75% higher mortality rate in the zebrafish embryos with a thicker biofilm layer on the chorion compared to the avirulent S. cerevisiae. Being one of the most important Candida adhesins, sequence and structural analyses of four statistically identified MgAls showed that MgAls1056 was predicted to exhibit the most conserved amyloid-forming regions, tandem repeat domain and peptide binding cavity (PBC) compared to C. albicans Als3. Favoured from the predicted largest ligand binding site and druggable pockets, it showed the highest affinity towards hepta-threonine. Non-PBC druggable pockets in the most potent virulence contributing MgAls1056 provide new insights into developing antifungal drugs targeting non-albicans Candida spp. Virtual screening of available synthetic or natural bioactive compounds and MgAls1056 deletion from the fungal genome should be further performed and validated experimentally.Communicated by Ramaswamy H. Sarma.

Molecular control of sucrose utilization in Escherichia coli W, an efficient sucrose-utilizing strain.

Sucrose is an industrially important carbon source for microbial fermentation. Sucrose utilization in Escherichia coli, however, is poorly understood, and most industrial strains cannot utilize sucrose. The roles of the chromosomally encoded sucrose catabolism (csc) genes in E. coli W were examined by knockout and overexpression experiments. At low sucrose concentrations, the csc genes are repressed and cells cannot grow. Removal of either the repressor protein (cscR) or the fructokinase (cscK) gene facilitated derepression. Furthermore, combinatorial knockout of cscR and cscK conferred an improved growth rate on low sucrose. The invertase (cscA) and sucrose transporter (cscB) genes are essential for sucrose catabolism in E. coli W, demonstrating that no other genes can provide sucrose transport or inversion activities. However, cscK is not essential for sucrose utilization. Fructose is excreted into the medium by the cscK-knockout strain in the presence of high sucrose, whereas at low sucrose (when carbon availability is limiting), fructose is utilized by the cell. Overexpression of cscA, cscAK, or cscAB could complement the WΔcscRKAB knockout mutant or confer growth on a K-12 strain which could not naturally utilize sucrose. However, phenotypic stability and relatively good growth rates were observed in the K-12 strain only when overexpressing cscAB, and full growth rate complementation in WΔcscRKAB also required cscAB. Our understanding of sucrose utilization can be used to improve E. coli W and engineer sucrose utilization in strains which do not naturally utilize sucrose, allowing substitution of sucrose for other, less desirable carbon sources in industrial fermentations.

Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci.

BACKGROUND: Metabolic engineering projects often require integration of multiple genes in order to control the desired phenotype. However, this often requires iterative rounds of engineering because many current insertion approaches are limited by the size of the DNA that can be transferred onto the chromosome. Consequently, construction of highly engineered strains is very time-consuming. A lack of well-characterised insertion loci is also problematic. RESULTS: A series of knock-in/knock-out (KIKO) vectors was constructed for integration of large DNA sequences onto the E. coli chromosome at well-defined loci. The KIKO plasmids target three nonessential genes/operons as insertion sites: arsB (an arsenite transporter); lacZ (ß-galactosidase); and rbsA-rbsR (a ribose metabolism operon). Two homologous 'arms' target each insertion locus; insertion is mediated by λ Red recombinase through these arms. Between the arms is a multiple cloning site for the introduction of exogenous sequences and an antibiotic resistance marker (either chloramphenicol or kanamycin) for selection of positive recombinants. The resistance marker can subsequently be removed by flippase-mediated recombination. The insertion cassette is flanked by hairpin loops to isolate it from the effects of external transcription at the integration locus. To characterize each target locus, a xylanase reporter gene (xynA) was integrated onto the chromosomes of E. coli strains W and K-12 using the KIKO vectors. Expression levels varied between loci, with the arsB locus consistently showing the highest level of expression. To demonstrate the simultaneous use of all three loci in one strain, xynA, green fluorescent protein (gfp) and a sucrose catabolic operon (cscAKB) were introduced into lacZ, arsB and rbsAR respectively, and shown to be functional. CONCLUSIONS: The KIKO plasmids are a useful tool for efficient integration of large DNA fragments (including multiple genes and pathways) into E. coli. Chromosomal insertion provides stable expression without the need for continuous antibiotic selection. Three non-essential loci have been characterised as insertion loci; combinatorial insertion at all three loci can be performed in one strain. The largest insertion at a single site described here was 5.4 kb; we have used this method in other studies to insert a total of 7.3 kb at one locus and 11.3 kb across two loci. These vectors are particularly useful for integration of multigene cassettes for metabolic engineering applications.

Differentiation of High-Fructose Corn Syrup Adulterated Kelulut Honey Using Physicochemical, Rheological, and Antibacterial Parameters.

Kelulut (stingless bee) honey (KH) possesses a wide range of benefits for human consumption and can exhibit medical effects. Due to its high value, this premium honey is often adulterated with different types of cheaper sugars, causing low nutrients and potential food safety threats in the final product. This study aims to determine the physicochemical, rheological, and antibacterial properties of sugar-based adulterated KH from the stingless bee species Heterotrigona itama. Adulterated samples were prepared using pure honey mixed with different concentrations of high fructose corn syrup (HFCS), i.e., 10%, 20%, 30%, 40%, and 50%. Water activity, colour, total soluble solids, pH, turbidity, viscosity, and antimicrobial activity of KH were determined. In addition, the primary sugar composition (fructose, glucose, and trehalulose) was determined by high-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD). This study shows that the increasing percentage of HFCS addition in the KH samples significantly increases (p < 0.05) the total soluble solids, colour, pH, turbidity, viscosity, glucose, and fructose content; meanwhile, the water activity and trehalulose were reduced significantly (p < 0.05). Antimicrobial activity against S. aureus was reduced significantly (p = 0.006) by an increased percentage of HFCS compared to Control. Antimicrobial activity against P. aeruginosa was also found to be reduced significantly but showed non-significant effect from an increased percentage of HFCS in honey (p = 0.413). The bacterium S. aureus was more vulnerable to treatment with honey from both Control and adulterated groups compared to P. aeruginosa. In conclusion, HFCS-adulterated KH and authentic KH can be differentiated using all the parameters investigated. These data are vital for the governing bodies to ensure that KH sold in the markets is free from HFCS adulteration.