Bench-Scale Production of Heterologous Proteins from Extremophiles- Escherichia coli and Pichia pastoris based expression systems

Over the past few years considerable research attention has been assigned to extremophiles as sources of extremozymes due to their applicability in industrial processes, and the development of eco-friendly technologies. The establishment of efficient production strategies for heterologous proteins is an empirical process requiring broad background knowledge on available expression systems together with their major advantages and shortcomings. The studies conducted during the course of this thesis has included four enzymes originating from thermophiles namely, thermostable glycoside hydrolases, xylanase and cellulase from Rhodothermus marinus, cyclomaltodextrinase from Anoxybacillus flavithermus and a phospholipase from alkaliphilic Bacillus halodurans. Batch cultivation of R. marinus in the presence of xylan allowed low production of the native xylanase in sufficient amounts to probe cell-attachment studies by enzymatic and immunological techniques. Higher levels of the target proteins were achieved by intracellular and extracellular heterologous production using an Escherichia coli and Pichia pastoris based expression system respectively. The production of a functional enzyme is intimately related to the host's cellular machinery furthermore, as a prerequisite, the establishment of efficient bioprocess strategies is crucial for attaining optimum enzyme production yields. The results presented include bench-scale production strategies employing high cell density fed-batch cultivations with E. coli as a host. Also efficient extracellular production of thermostable xylanase and alkaliphilic phospholipase production using the methylotrophic yeast P. pastoris as a host is reported

Evaluation of teixobactin derivatives as potential antimicrobial agents.

Maters degree. University of KwaZulu-Natal, Durban.Methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE) are on the World Health Organization (WHO) high priority list of pathogens that require serious attention. Therefore, the need for novel class of compounds is vital in overcoming this problem. Teixobactin is a new class of antibiotic that has exhibited antimicrobial activity against resistant bacteria. In this study we are expanding the investigation of teixobactin derivatives against clinically relevant bacterial isolates from South African patients. The minimum inhibitory concentration (MIC), the minimal bactericidal concentration (MBC), the serum effect on the MIC’s and the time kill kinetics studies of three of our synthesized teixobactin derivatives 3, 4 and 5 were ascertained by broth microdilution according to the CLSI 2017 guidelines. Haemolysis on red blood cells (RBCs) and cytotoxicity on peripheral blood mononuclear cells (PBMCs) were performed to investigate the safety of these derivatives. MIC’s of the teixobactin derivatives against ATCC reference strains were between 4-64 µg/ml (3), 2-64 µg/ml (4) and 0.5-64 µg/ml (5). The MIC’s for MRSA were 32 µg/ml for (3), 2-4 µg/ml for (4) and 2-4 µg/ml for (5) whilst the MIC’s obtained for VRE’s were 8-16 μg/ml for (3), 4 μg/ml for (4) and 2-16 μg/ml for (5). 50% human serum had no effect on the MIC’s. All these derivatives did not show any effect on cell viability at their effective concentration. Teixobactin derivatives (3, 4 and 5) were capable of inhibiting bacterial growth in drug resistant bacteria and thus serve as potential antimicrobial agents

Investigation of the N-terminus amino functional of Arg10-Teixobactin

Teixobactin is a recently described antimicrobial peptide that shows high activity against gram-positive bacteria as well as mycobacterium tuberculosis. Due to both its structure as a head-to-side chain cyclodepsipeptide and its activity, it has attracted the attention of several research groups. In this regard, a large number of analogs with substitutions in both the cycle and the tail has been described. Here, we report the contribution of the N-terminus residue, N-Me-d-Phe, to the activity of Arg10-teixobactin. On the basis of our findings, we conclude that the N-terminus accepts minimum changes but not the presence of long alkyl chains. The presence of a positive charge is a requirement for the activity of the peptide. Furthermore, acylation of the N-terminus leads to total loss of activity

Super-Cationic Peptide Dendrimers¿Synthesis and Evaluation as Antimicrobial Agents

Microbial infections are a major public health concern. Antimicrobial peptides (AMPs) have been demonstrated to be a plausible alternative to the current arsenal of drugs that has become inefficient due to multidrug resistance. Herein we describe a new AMP family, namely the supercationic peptide dendrimers (SCPDs). Although all members of the series exert some antibacterial activity, we propose that special attention should be given to (KLK)2KLLKLL-NH2 (G1KLK-L2KL2), which shows selectivity for Gram-negative bacteria and virtually no cytotoxicity in HepG2 and HEK293. These results reinforce the validity of the SCPD family as a valuable class of AMP and support G1KLK-L2KL2 as a strong lead candidate for the future development of an antibacterial agent against Gram-negative bacteria

A novel Bacillus based multi-strain probiotic improves growth performance and intestinal properties of Clostridium perfringens challenged broilers

There is a necessity for the implementation of in-feed probiotics in the poultry production industry, following strict regulations around the use of antibiotic growth promoters (AGP). Bacillus spp. are becoming an attractive alternative because of their functionality and stability. This study aims to evaluate the effect of a novel multi-strain Bacillus based probiotic on growth performance and gut health in male Ross 308 broiler chickens challenged with Clostridium perfringens Type A. Broilers on a 4 phase feeding program were fed diets containing either a standard metabolizable energy (ME) (100%) or a reduced ME (98%) level. The test probiotic was compared to an un-supplemented negative control and a commercial benchmark product as positive control over a 35 D feeding trial, using a 2 × 3 factorial experimental design. Chicks were inoculated with a once-off dose of C. perfringens on day 14. Growth performance was measured weekly to calculate body weight (BW), feed intake (FI) and feed conversion ratio (FCR). Villi histomorphology, gut lesions, and liver weight were assessed at day 35. Broilers fed the reduced ME diet with the test probiotic achieved higher final BWs (P = 0.037) and FCR (P = 0.014) than the negative control. Broilers fed the standard ME diet with the test probiotic showed improved (P = 0.001) FCR than the negative control from day 21 onwards. Increased duodenal villi height (P = 0.012) and villi height to crypt depth ratio in the duodenum (P < 0.0001) and jejunum (P = 0.0004) were observed in broilers fed the reduced ME diet containing the test probiotic. Additionally, the test probiotic resulted in significantly reduced relative liver weights in both ME groups. Consequently, the results suggest that the novel multi-strain Bacillus based probiotic enhanced broiler performance and improved gut health and is thus attractive as an alternative to AGP’s in broiler production.The Department of Science and Technology, the CSIR (Pretoria, South Africa) and Ceva Animal Health (Pty) Ltd (Johannesburg, South Africa).http://ps.oxfordjournals.orgam2020Animal and Wildlife SciencesBiochemistryGeneticsMicrobiology and Plant Patholog

Lysine Scanning of Arg10-Teixobactin. Deciphering the Role of Hydrophobic and Hydrophilic Residues.

Teixobactin is a recently discovered antimicrobial cyclodepsipeptide with good activity against Gram positive bacteria. Taking Arg10-teixobactin as a reference, where the nonproteinogenic residue l-allo-enduracididine was substituted by arginine, a lysine scan was performed to identify the importance of keeping the balance between hydrophilic and hydrophobic amino acids for the antimicrobial activities of this peptide family. Thus, the substitution of four isoleucine residues present in the natural sequence by lysine led to a total loss of activity. On the other hand, the substitution of the polar noncharged residues and alanine by lysine allowed us to keep and in some cases to improve the antimicrobial activity

Potential opportunities to convert waste to bio-based chemicals at an industrial scale in South Africa

DATA AVAILABILITY STATEMENT : The datasets used during the research study are available from the corresponding author on reasonable request.Globally, greater than 30% of waste is disposed of in some form of landfill, and it is estimated that annual waste-related emissions will increase by up to 76% by 2050. Emissions arising from fossil fuel-derived products and waste disposal in landfills have prompted the development of alternative technologies that utilize renewable resources. Biomass feedstock is being investigated globally to produce renewable fuels and chemicals. Globally, crop-based biomass and waste biomass are the major feedstocks for chemical production, and the market value of crop-based biomass is expected to increase at the fastest rate. South America, Europe, and North America are currently the global leaders in renewable or bio-based chemical production. In South Africa (SA), the country is still heavily reliant on landfilling as a waste solution. Wastes from agricultural production processes in SA are considered promising feedstocks for beneficiation opportunities to produce bio-based chemicals. The second-generation (2G) agricultural feedstocks that can be used in SA include fruit waste; sugarcane by-products and waste; forestry, timber, pulp, and paper waste; and invasive alien plants. Fermentation, or “green chemistry” technologies, can be used to convert various feedstocks into bio-based chemicals. Bio-based chemicals may be used as drop-in substitutes for existing petrochemical products, for use in end-user industries such as automotive and transportation, textiles, pharmaceuticals, consumer and home appliances, healthcare, and food and beverages. Bioethanol, specifically, can be used in transport fuel, as feedstock for power generation, as an energy source for fuel cells along with hydrogen, and as feedstock in the chemicals industry. Bio-butanol, an olefin derivative, can be used as a drop-in replacement for petroleum-based butanol in all its applications. Different monomers of bio-based chemicals can be used to produce biopolymers, polyhydroxyalkanoates (PHAs), and polylactic acid (PLA), which are subsequently used to produce bioplastics. A total of 25 bio-based chemicals and the technology used to produce them are summarized in this paper. Overall, bioethanol remains the dominant sugar platform product globally. Drawing on global trends, the potential options for the South African market include bioethanol, n-butanol, acetic acid, and lactic acid. It is estimated that the conversion of 70% of the lignocellulosic biomass available in SA would meet 24% of the country’s liquid fuel requirement as a bioethanol equivalent. The most feasible sources of lignocellulosic biomass or waste for beneficiation in SA are generated by the agricultural sector, including sugarcane by-products and waste. Taking into consideration the abundance of lignocellulosic biomass, adequate market segment sizes, and socio-economic factors, it is apparent that there are potential opportunities to investigate the co-production of bioethanol with lactic acid or other bio-based chemicals on an industrial scale.Parliamentary Grant funding.http://www.mdpi.com/journal/fermentationhj2023Graduate School of Technology Management (GSTM)SDG-09: Industry, innovation and infrastructureSDG-12:Responsible consumption and productio

The methylotrophic yeast Pichia pastoris as a host for the expression and production of thermostable xylanase from the bacterium Rhodothermus marinus

A thermostable glycoside hydrolase family-10 xylanase originating from Rhodothermus marinus was cloned and expressed in the methylotrophic yeast Pichia pastoris (SMD1168H). The DNA sequence from Rmxyn10A encoding the xylanase catalytic module was PCR-amplified and cloned in frame with the Saccharomyces cerevisiae alpha-factor secretion signal under the control of the alcohol oxidase (AOX1) promotor. Optimisation of enzyme production in batch fermentors, with methanol as a sole carbon source, enabled secretion yields up to 3 g l(-1) xylanase with a maximum activity of 3130 U l(-1) to be achieved. N-terminal sequence analysis of the heterologous xylanase indicated that the secretion signal was correctly processed in P. pastoris and the molecular weight of 37 kDa was in agreement with the theoretically calculated molecular mass. Introduction of a heat-pretreatment step was however necessary in order to fold the heterologous xylanase to an active state, and at the conditions used this step yielded a 200-fold increase in xylanase activity. Thermostability of the produced xylanase was monitored by differential-scanning calorimetry, and the transition temperature (T-m) was 78 degrees C. R. marinus xylanase is the first reported thermostable gram-negative bacterial xylanase efficiently secreted by P. pastoris. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved

Effect of postinduction nutrient feed composition and use of lactose as inducer during production of thermostable xylanase in Escherichia coli glucose-limited fed-batch cultivations

Escherichia coli is a microorganism routinely used in the production of heterologous proteins. The overexpression of a xylanase (Xyn10A Delta NC), which originated from the thermophile Rhodo-thermus marinus cloned under the control of the strong T7/lac promoter in a defined medium (mAT) using a substrate-limited feed strategy, was however shown to impose a significant metabolic burden on host cells. This resulted in a decreased cell growth rate and ultimately also a decreased target protein production. The investigation hence centers on the effect of some selected nutrient feed additives (amino acid [Cys] or TCA-intermediates [citrate, succinate, malate]) used to relieve the metabolic burden imposed during the feeding and postinduction phases of these glucose-limited fed-batch cultivations. The use of either succinic acid or malic acid as feed-additives resulted in an increase in production of approximately 40% of the heterologous thermostable xylanase. Furthermore, use of lactose as an alternative inducer of the T7/lac promoter was also proven to be a suitable strategy that significantly prolonged the heterologous protein production phase as compared with induction using isopropyl P-D-thiogalactopyranoside (IPTG)