Evaluating oleaginous yeasts for enhanced microbial lipid production using sweetwater as a sustainable feedstock.

BACKGROUND: Yeasts exhibit promising potential for the microbial conversion of crude glycerol, owing to their versatility in delivering a wide range of value-added products, particularly lipids. Sweetwater, a methanol-free by-product of the fat splitting process, has emerged as a promising alternative feedstock for the microbial utilization of crude glycerol. To further optimize sweetwater utilization, we compared the growth and lipid production capabilities of 21 oleaginous yeast strains under different conditions with various glycerol concentrations, sweetwater types and pH. RESULTS: We found that nutrient limitation and the unique carbon composition of sweetwater boosted significant lipid accumulation in several strains, in particular Rhodosporidium toruloides NRRL Y-6987. Subsequently, to decipher the underlying mechanism, the transcriptomic changes of R. toruloides NRRL Y-6987 were further analyzed, indicating potential sugars and oligopeptides in sweetwater supporting growth and lipid accumulation as well as exogenous fatty acid uptake leading to the enhanced lipid accumulation. CONCLUSION: Our comparative study successfully demonstrated sweetwater as a cost-effective feedstock while identifying R. toluroides NRRL Y-6987 as a highly promising microbial oil producer. Furthermore, we also suggested potential sweetwater type and strain engineering targets that could potentially enhance microbial lipid production.

Systematic Adaptation of Bacillus licheniformis to 2-Phenylethanol Stress.

The compound 2-phenylethanol (2-PE) is a bulk flavor and fragrance with a rose-like aroma that can be produced by microbial cell factories, but its cellular toxicity inhibits cellular growth and limits strain performance. Specifically, the microbe Bacillus licheniformis has shown a strong tolerance to 2-PE. Understanding these tolerance mechanisms is crucial for achieving the hyperproduction of 2-PE. In this report, the mechanisms of B. licheniformis DW2 resistance to 2-PE were studied by multi-omics technology coupled with physiological and molecular biological approaches. 2-PE induced reactive oxygen species formation and affected nucleic acid, ribosome, and cell wall synthesis. To manage 2-PE stress, the antioxidant and global stress response systems were activated; the repair system of proteins and homeostasis of the ion and osmotic were initiated. Furthermore, the tricarboxylic acid cycle and NADPH synthesis pathways were upregulated; correspondingly, scanning electron microscopy revealed that cell morphology was changed. These results provide deeper insights into the adaptive mechanisms of B. licheniformis to 2-PE and highlight the potential targets for genetic manipulation to enhance 2-PE resistance. IMPORTANCE The ability to tolerate organic solvents is essential for bacteria producing these chemicals with high titer, yield, and productivity. As exemplified by 2-PE, bioproduction of 2-PE represents a promising alternative to chemical synthesis and plant extraction approaches, but its toxicity hinders successful large-scale microbial production. Here, a multi-omics approach is employed to systematically study the mechanisms of B. licheniformis DW2 resistance to 2-PE. As a 2-PE-tolerant strain, B. licheniformis displays multifactorial mechanisms of 2-PE tolerance, including activating global stress response and repair systems, increasing NADPH supply, changing cell morphology and membrane composition, and remodeling metabolic pathways. The current work yields novel insights into the mechanisms of B. licheniformis resistance to 2-PE. This knowledge can also be used as a clue for improving bacterial performances to achieve industrial-scale production of 2-PE and potentially applied to the production of other relevant organic solvents, such as tyrosol and hydroxytyrosol.

The Engineering, Expression, and Immobilization of Epimerases for D-allulose Production.

The rare sugar D-allulose is a potential replacement for sucrose with a wide range of health benefits. Conventional production involves the employment of the Izumoring strategy, which utilises D-allulose 3-epimerase (DAEase) or D-psicose 3-epimerase (DPEase) to convert D-fructose into D-allulose. Additionally, the process can also utilise D-tagatose 3-epimerase (DTEase). However, the process is not efficient due to the poor thermotolerance of the enzymes and low conversion rates between the sugars. This review describes three newly identified DAEases that possess desirable properties for the industrial-scale manufacturing of D-allulose. Other methods used to enhance process efficiency include the engineering of DAEases for improved thermotolerance or acid resistance, the utilization of Bacillus subtilis for the biosynthesis of D-allulose, and the immobilization of DAEases to enhance its activity, half-life, and stability. All these research advancements improve the yield of D-allulose, hence closing the gap between the small-scale production and industrial-scale manufacturing of D-allulose.

New and efficient purification process for recombinant human insulin produced in Escherichia coli.

A new and efficient purification process for recombinant human insulin production was developed by exploring new resins and optimizing purification steps from E. coli inclusion body washing to insulin polishing. A combined additives inclusion body wash protocol drastically improved efficiency in clarifying ZZ-proinsulin samples. ZZ-proinsulin recovery increased three-fold under optimized solubilization and sulfitolysis incubation temperature and duration. Desalting with Bio-Gel P4 and P6 resulted in higher sample loading and product recovery compared to conventional resins. A higher recovery (96%) and purity (81%) of ZZ-proinsulin were achievable with the Nuvia S cation exchanger for proinsulin purification compared to a reported process using expensive affinity chromatography resin. As the first step for insulin purification, process scale-up is more economical and practical when Nuvia HR-S cation exchanger was used instead of commonly used reversed-phase chromatography. Nuvia HR-S was highly effective in removing ZZ fusion protein (90% removal) after enzymatic cleavage, although ZZ fusion protein has a very close theoretical pI to human insulin, which was supposedly challenging to be removed by cation exchange chromatography. Also, insulin can be eluted at a lower ethanol % using Nuvia HR-S compared to other reported processes and is thus more environmentally sustainable. Recombinant human insulin was obtained with over 98% purity in just a single reversed-phase polishing step, which is comparable to the reference standard. The process workflow presented here can be potentially applied for the development of purification workflow for insulin analogs or other peptide products derived from E. coli inclusion body.Key points• Drastic efficiency improvement for inclusion body wash with combined additives.• High recovery of proinsulin purification with high capacity cation exchange resin.• Effective removal of fusion tag at lower ethanol % with high-resolution resin.

Brewing with malted barley or raw barley: what makes the difference in the processes?

Malted barley is the main source for fermentable sugars used by yeasts in the traditional brewing of beers but its use has been increasingly substituted by unmalted barley and other raw grain adjuncts in recent years. The incorporation of raw grains is mainly economically driven, with the added advantage of improved sustainability, by reducing reliance on the malting process and its associated cost. The use of raw grains however, especially in high proportion, requires modifications to the brewing process to accommodate the lack of malt enzymes and the differences in structural and chemical composition between malted and raw grains. This review describes the traditional malting and brewing processes for the production of full malt beer, compares the modifications to these processes, namely milling and mashing, when raw barley or other grains are used in the production of wort-a solution of fermentable extracts metabolized by yeast and converted into beer, and discusses the activity of endogenous malt enzymes and the use of commercial brewing enzyme cocktails which enable high adjunct brewing.

A propeptide toolbox for secretion optimization of Flavobacterium meningosepticum endopeptidase in Lactococcus lactis.

BACKGROUND: Lactic acid bacteria are a family of "generally regarded as safe" organisms traditionally used for food fermentation. In recent years, they have started to emerge as potential chassis for heterologous protein production. And more recently, due to their beneficial properties in the gut, they have been examined as potential candidates for mucosal delivery vectors, especially for acid-sensitive enzymes. One such application would be the delivery of gluten-digesting endopeptidases for the treatment of celiac disease. To facilitate these applications, an efficient recombinant protein expression toolbox is required, especially for recombinant protein secretion. While current tools for enhancing protein secretion consist mainly of signal peptides, secretion propeptides have also been observed to play a crucial role for protein secretion and improved yields. RESULTS: To expand the propeptide library for secretion optimization, we have mined and characterized three naturally occurring propeptides from the sequenced genomes of 109 Lactococcus species. These newly-mined propeptides were introduced after the N-terminal USP45 secretion signal to characterize and compare their effects on the secretion of Escherichia coli thioredoxin (TRX) and Flavobacterium meningosepticum prolyl endopeptidase (Fm PEP) in Lactococcus lactis NZ9000. All three propeptides, along with the positive control LEISSTCDA, improved volumetric secretion yields by 1.4-2.3-folds. However, enhancement of secretion yield is dependent on protein of interest. For TRX, the optimal combination of USP45 signal peptide and LEISSTCDA produced a 2.3-fold increase in secretion yields. Whilst for Fm PEP, propeptide 1 with USP45 signal peptide improved volumetric secretion yields by 2.2-fold compared to a 1.4-fold increase by LEISSTCDA. Similar trends in Fm PEP activity and protein yield also demonstrated minimal effect of the negative charged propeptides on PEP activity and thus folding. CONCLUSIONS: Overall, we have characterized three new propeptides for use in L. lactis secretion optimization. From success of these propeptides for improvement of secretion yields, we anticipate this collection to be valuable to heterologous protein secretion optimisation in lactic acid bacteria. We have also demonstrated for the first time, secretion of Fm PEP in L. lactis for potential use as a therapy agent in celiac disease.

Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis.

OBJECTIVES: To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis. RESULTS: The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively. CONCLUSIONS: Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

Systems Biology of Gut Microbiota-Human Receptor Interactions: Toward Anti-inflammatory Probiotics.

The incidence and prevalence of inflammatory disorders have increased globally, and is projected to double in the next decade. Gut microbiome-based therapeutics have shown promise in ameliorating chronic inflammation. However, they are largely experimental, context- or strain-dependent and lack a clear mechanistic basis. This hinders precision probiotics and poses significant risk, especially to individuals with pre-existing conditions. Molecules secreted by gut microbiota act as ligands to several health-relevant receptors expressed in human gut, such as the G-protein coupled receptors (GPCRs), Toll-like receptor 4 (TLR4), pregnane X receptor (PXR), and aryl hydrocarbon receptor (AhR). Among these, the human AhR expressed in different tissues exhibits anti-inflammatory effects and shows activity against a wide range of ligands produced by gut bacteria. However, different AhR ligands induce varying host responses and signaling in a tissue/organ-specific manner, which remain mostly unknown. The emerging systems biology paradigm, with its powerful in silico tool repertoire, provides opportunities for comprehensive and high-throughput strain characterization. In particular, combining metabolic models with machine learning tools can be useful to delineate tissue and ligand-specific signaling and thus their causal mechanisms in disease and health. The knowledge of such a mechanistic basis is indispensable to account for strain heterogeneity and actualize precision probiotics.

Systematic evaluation of genome-wide metabolic landscapes in lactic acid bacteria reveals diet- and strain-specific probiotic idiosyncrasies.

Lactic acid bacteria (LAB) are well known to elicit health benefits in humans, but their functional metabolic landscapes remain unexplored. Here, we analyze differences in growth, intestinal persistence, and postbiotic biosynthesis of six representative LAB and their interactions with 15 gut bacteria under 11 dietary regimes by combining multi-omics and in silico modeling. We confirmed predictions on short-term persistence of LAB and their interactions with commensals using cecal microbiome abundance and spent-medium experiments. Our analyses indicate that probiotic attributes are both diet and species specific and cannot be solely explained using genomics. For example, although both Lacticaseibacillus casei and Lactiplantibacillus plantarum encode similarly sized genomes with diverse capabilities, L. casei exhibits a more desirable phenotype. In addition, "high-fat/low-carb" diets more likely lead to detrimental outcomes for most LAB. Collectively, our results highlight that probiotics are not "one size fits all" health supplements and lay the foundation for personalized probiotic design.

Creation of cavitation activity in a microfluidic device through acoustically driven capillary waves.

We present a study on achieving intense acoustic cavitation generated by ultrasonic vibrations in polydimethylsiloxane (PDMS) based microfluidic devices. The substrate to which the PDMS is bonded was forced into oscillation with a simple piezoelectric transducer attached at 5 mm from the device to a microscopic glass slide. The transducer was operated at 100 kHz with driving voltages ranging between 20 V and 230 V. Close to the glass surface, pressure and vibration amplitudes of up to 20 bar and 400 nm were measured respectively. It is found that this strong forcing leads to the excitation of nonlinear surface waves when gas-liquid interfaces are present in the microfluidic channels. Also, it is observed that nuclei leading to intense inertial cavitation are generated by the entrapment of gas pockets at those interfaces. Subsequently, cavitation bubble clusters with void fractions of more than 50% are recorded with high-speed photography at up to 250,000 frames/s. The cavitation clusters can be sustained through the continuous injection of gas using a T-junction in the microfluidic device.

Co-expression of Skp and FkpA chaperones improves cell viability and alters the global expression of stress response genes during scFvD1.3 production.

BACKGROUND: The overexpression of scFv antibody fragments in the periplasmic space of Escherichia coli frequently results in extensive protein misfolding and loss of cell viability. Although protein folding factors such as Skp and FkpA are often exploited to restore the solubility and functionality of recombinant protein products, their exact impact on cellular metabolism during periplasmic antibody fragment expression is not clearly understood. In this study, we expressed the scFvD1.3 antibody fragment in E. coli BL21 and evaluated the overall physiological and global gene expression changes upon Skp or FkpA co-expression. RESULTS: The periplasmic expression of scFvD1.3 led to a rapid accumulation of insoluble scFvD1.3 proteins and a decrease in cell viability. The co-expression of Skp and FkpA improved scFvD1.3 solubility and cell viability in a dosage-dependent manner. Through mutagenesis experiments, it was found that only the chaperone activity of FkpA, not the peptidyl-prolyl isomerase (PPIase) activity, is required for the improvement in cell viability. Global gene expression analysis of the scFvD1.3 cells over the chaperone-expressing cells showed a clear up-regulation of genes involved in heat-shock and misfolded protein stress responses. These included genes of the major HSP70 DnaK chaperone family and key proteases belonging to the Clp and Lon protease systems. Other metabolic gene expression trends include: (1) the differential regulation of several energy metabolic genes, (2) down-regulation of the central metabolic TCA cycle and transport genes, and (3) up-regulation of ribosomal genes. CONCLUSIONS: The simultaneous activation of multiple stress related and other metabolic genes may constitute the stress response to protein misfolding in the scFvD1.3 cells. These gene expression information could prove to be valuable for the selection and construction of reporter contructs to monitor the misfolded protein stress response during antibody fragment production.

A SH3_5 Cell Anchoring Domain for Non-recombinant Surface Display on Lactic Acid Bacteria.

Lactic acid bacteria (LAB) are a group of gut commensals increasingly recognized for their potential to deliver bioactive molecules in vivo. The delivery of therapeutic proteins, in particular, can be achieved by anchoring them to the bacterial surface, and various anchoring domains have been described for this application. Here, we investigated a new cell anchoring domain (CAD4a) isolated from a Lactobacillus protein, containing repeats of a SH3_5 motif that binds non-covalently to peptidoglycan in the LAB cell wall. Using a fluorescent reporter, we showed that C-terminal CAD4a bound Lactobacillus fermentum selectively out of a panel of LAB strains, and cell anchoring was uniform across the cell surface. Conditions affecting CAD4a anchoring were studied, including temperature, pH, salt concentration, and bacterial growth phase. Quantitative analysis showed that CAD4a allowed display of 105 molecules of monomeric protein per cell. We demonstrated the surface display of a functional protein with superoxide dismutase (SOD), an antioxidant enzyme potentially useful for treating gut inflammation. SOD displayed on cells could be protected from gastric digestion using a polymer matrix. Taken together, our results show the feasibility of using CAD4a as a novel cell anchor for protein surface display on LAB.

Characterizing Escherichia coli DH5alpha growth and metabolism in a complex medium using genome-scale flux analysis.

Genome-scale flux analysis of Escherichia coli DH5alpha growth in a complex medium was performed to investigate the relationship between the uptake of various nutrients and their metabolic outcomes. During the exponential growth phase, we observed a sequential consumption order of serine, aspartate and glutamate in the complex medium as well as the complete consumption of key carbohydrate nutrients, glucose and trehalose. Based on the consumption and production rates of the measured metabolites, constraints-based flux analysis of a genome-scale E. coli model was then conducted to elucidate their utilization in the metabolism. The in silico analysis revealed that the cell exploited biosynthetic precursors taken up directly from the complex medium, through growth-related anabolic pathways. This suggests that the cell could be functioning in an energetically more efficient manner by reducing the energy needed to produce amino acids. The in silico simulation also allowed us to explain the observed rapid consumption of serine: excessively consumed external serine from the complex medium was mainly converted into pyruvate and glycine, which in turn, led to the acetate accumulation. The present work demonstrates the application of an in silico modeling approach to characterizing microbial metabolism under complex medium condition. This work further illustrates the use of in silico genome-scale analysis for developing better strategies related to improving microbial growth and enhancing the productivity of desirable metabolites.

Enhancement of plasmid DNA yields during fed-batch culture of a fruR-knockout Escherichia coli strain.

Well-characterized derivatives of Escherichia coli K12 such as DH5alpha are the host strains commonly used for plasmid DNA production. Owing to the prospective clinical demand for large quantities of plasmid DNA for gene therapy and DNA vaccination, existing plasmid production processes need to be optimized to attain higher plasmid yields. To date, nearly all production optimization efforts are focused on media or fermentation process design. Although there has been a simple empirical evaluation of the available host strains, there is a lack of systematic effort at engineering these host strains for improved plasmid DNA production. In view of this, we engineered DH5alpha WT (wild-type) cells carrying a DNA vaccine plasmid by knocking out the fruR (fructose repressor) [also known as the Cra (catabolite repressor activator)] global regulator gene and evaluated the growth and plasmid yields of these P+ (plasmid-bearing) fruR cells (fruR-knockout cells) during fed-batch cultures with exponential feeding. The P+ fruR cells showed a more rapid accumulation of plasmid DNA towards the end of the fed-batch cultures compared with the P+ WT cells. As a result, the specific plasmid yield of the P+ fruR cells was 21% higher than that of the P+ WT cells [19.2 versus 15.9 mg/g DCW (dry cell weight)]. These results demonstrate that, from an initial high-yielding fermentation process, the knockout of the fruR global regulator gene in E. coli DH5alpha further improves plasmid yields during fed-batch culture.

HEXIM1 Peptide Exhibits Antimicrobial Activity Against Antibiotic Resistant Bacteria Through Guidance of Cell Penetrating Peptide.

The emergence of antibiotic resistant bacteria is one of the biggest threats to human health worldwide. In 2017, World Health Organization listed the world's most dangerous antibiotic-resistant bacteria or "superbugs," such as carbapenem-resistant Pseudomonas aeruginosa and Escherichia coli, indicating the highest priority needs for new antibiotics. The possibility that such infectious diseases may soon be untreatable, due to decreased antibiotic efficacy, creates an urgent need for novel and alternative antimicrobials. Antimicrobial peptides are naturally occurring small molecules found in the innate immunity of mammals, plants and bacteria, and are potentially therapeutic candidates against drug-resistant bacteria. In this study, we examine the antimicrobial activities of the cytotoxic peptides derived from the basic region (BR) of the human hexamethylene bisacetamide-inducible protein 1 (HEXIM1). We found that, when fused with a cell penetrating peptide, the HEXIM1 BR peptide and its derivative, BR-RRR12, exhibited inhibitory activities against selected "superbugs." Negligible effects on the viability of human keratinocyte cell line were observed when the bactericidal dosages of HEXIM1 BR peptides were used. Different killing kinetics were observed between the membrane permeabilizing antimicrobial peptides and HEXIM1 BR peptides, suggesting that a different antimicrobial mechanism might be utilized by the HEXIM1 BR peptides. Using an in vitro translation system based on E. coli lysates, we found that HEXIM1 BR peptides blocked bacterial translation. Taken together, we identify the HEXIM1 BR peptide as a novel antimicrobial peptide with potent inhibitory activity against antibiotic-resistant "superbugs."

Inactivating FruR global regulator in plasmid-bearing Escherichia coli alters metabolic gene expression and improves growth rate.

The introduction of plasmids into Escherichia coli is known to impose a metabolic burden, which diminishes the growth rate. This effect could arise from perturbation of the central metabolic pathways, which supply precursors and energy for macromolecule synthesis. We knocked out a global regulator of central metabolism, FruR (also called Cra), to assess its phenotypic effect in E. coli carrying plasmids. During bioreactor runs, a higher specific growth rate of 0.91h(-1) was observed for the plasmid-bearing fruR knockout (P+ fruR) cells compared to its parental plasmid-bearing wildtype (P+ WT) cells (0.75h(-1)), while both the plasmid-free cells displayed similar growth rates (1.0h(-1), respectively). To investigate gene expression changes possibly related to the growth rate recovery, quantitative reverse transcriptase PCR and 2DE proteomic studies were performed. In P+ fruR cells, expression of enzymes involved in sugar catabolism, glycolysis and transcription/translation processes were upregulated, while those related to gluconeogenesis, tricarboxylic acid cycle and stress response were downregulated. Our findings demonstrate that the inactivation of FruR global regulator in recombinant E. coli alters metabolic gene expression and significantly reduces growth retardation from the burden of maintaining a plasmid. This study represents the first attempt to explore the role of a global regulatory gene on plasmid metabolic burden.

Antibody engineering of a cytotoxic monoclonal antibody 84 against human embryonic stem cells: Investigating the effects of multivalency on cytotoxicity.

Antibody fragments have shown targeted specificity to their antigens, but only modest tissue retention times in vivo and in vitro. Multimerization has been used as a protein engineering tool to increase the number of binding units and thereby enhance the efficacy and retention time of antibody fragments. In this work, we explored the effects of valency using a series of self-assembling polypeptides based on the GCN4 leucine zipper multimerization domain fused to a single-chain variable fragment via an antibody upper hinge sequence. Four engineered antibody fragments with a valency from one to four antigen-binding units of a cytotoxic monoclonal antibody 84 against human embryonic stem cells (hESC) were constructed. We hypothesized that higher cytotoxicity would be observed for fragments with increased valency. Flow cytometry analysis revealed that the trimeric and tetrameric engineered antibody fragments resulted in the highest degree of cytotoxicity to the undifferentiated hESC, while the engineered antibody fragments were observed to have improved tissue penetration into cell clusters. Thus, a trade off was made for the trimeric versus tetrameric fragment due to improved tissue penetration. These results have direct implications for antibody-mediated removal of undifferentiated hESC during regenerative medicine and cell therapy.

Quantitative real-time polymerase chain reaction for determination of plasmid copy number in bacteria.

A method for determination of plasmid copy number (PCN) in bacteria by real-time quantitative polymerase chain reaction (QPCR) was developed as an alternative to current PCN assays. Conventional methods for PCN estimation are generally not of high throughput, laborious, have low reproducibility, require large amounts of biological samples and are applicable only for a narrow dynamic range. Real-time QPCR, using the ABI Prism 7000, was able to sensitively detect the quantity of the pUC ori based plasmid, NS3, transformed into Escherichia coli host, DH5alpha, to be 411+/-6.1. The PCN of pBR322 plasmid DNA in DH5alpha was estimated to be 40+/-0.6 which is within its previously reported PCN range of approximately 30 to 70. QPCR was found to show good reproducibility and high sensitivity in detecting a two fold difference in template concentration, and a wide linear dynamic range covering 0.5 pg to 50 ng of DNA. PCNs of DH5alpha bearing plasmids pBR322 and NS3 computed from real-time QPCR assay were validated by that of agarose gel assay, and a marginal difference of only 13.0% and 10.7% was found for the two plasmids respectively. The QPCR assay was able to detect changes in PCN of plasmid producing DH5alpha during the course of a 2 l batch fermentation.

Effect of linker flexibility and length on the functionality of a cytotoxic engineered antibody fragment.

Engineered antibody fragments often contain natural or synthetic linkers joining the antigen-binding domain and multimerization regions, and the roles of these linkers have largely been overlooked. To investigate linker effects on structural properties and functionality, six bivalent cytotoxic antibody fragments with of linkers of varying flexibility and length were constructed: (1) 10-AA mouse IgG3 upper hinge region, (2) 20-AA mouse IgG3 upper hinge region repeat, (3) 10-AA glycine and serine linker, (4) 20-AA glycine and serine linker repeat, (5) 21-AA artificial linker, and (6) no-linker control. Interestingly, a higher cytotoxicity was observed for fragments bearing the rigid short linkers compared to the flexible longer linkers. More importantly, amino acid composition related to the rigidity/flexibility was found to be of greater importance upon cytotoxicity than linker length alone. To further study the structure-function relationship, molecular modelling and dynamics simulation were exploited. Resultantly, the rigid mouse IgG3 upper hinge region was predicted to enhance structural stability of the protein during the equilibrium state, indicating the improved cytotoxicity over other combinations of fragments. This prediction was validated by measuring the thermal stability of the mouse IgG3 upper hinge as compared to the artificial linker, and shown to have a higher melting temperature which coincides with a higher structural stability. Our findings clearly suggest that appropriate linker design is required for enhancing the structural stability and functionality of engineered antibody fragments.