Critical physical parameters for optimum recombinant protein production in yeast systems

Yeasts become the most preferred expression system for the production of recombinant proteins which play an important role in the development of biopharmaceutical products, antibodies for disease treatment, and enzymes for the food industries. The ability to grow in simple media, and ease of genetic manipulation with the benefits of typical eukaryotic expression which include protein processing, folding, and posttranslational modifications, have pushed them as one of the emerging hosts for recombinant protein production. Furthermore, yeasts are additionally quicker, easy to use, and cost-effective with high yield production in comparison to higher expression hosts. The effective productivity of the recombinant proteins is also influenced by the external parameters. This paper reviews different optimization methods of the recombinant protein production for several factors such as pH, temperature, media, agitation rate, inducer, inoculum size and induction time using one factor at a time (OFAT), Response Surface Methodology (RSM) and Artificial Neural Network (ANN). This review highlights the current studies regarding the optimization of the recombinant proteins expressed in three different yeasts namely; Saccharomyces cerevisiae, Komagataella phaffii, and Yarrowia lipolytica. These are the critical parameters which can be used to optimize the recombinant protein in yeast systems. The purification methods used to purify the proteins are also discussed for each system

Development of an enzyme-based fiber optic biosensor for detection of haloalkanes

Purpose – The main purpose of this study is to demonstrate the development of an enzyme-based sensor for haloalkane detection. Haloalkane is a toxic compound that is found as genotoxic impurities in pharmaceutical products and contaminants in waste. The need to investigate the genotoxic level in pharmaceutical manufacturing is very crucial because of its toxicity effects on human health. The potential of mini protein as an alternative bioreceptor was explored with the aim to be more effective and stable under extreme conditions. Design/methodology/approach – Mini proteins of haloalkane dehalogenase (HLD) were computationally designed and experimentally validated. Tapered multimode fiber (TMMF) was bio-functionalized with a bioreceptor either native (positive control) or mini protein. The absorbance-based sensor resulting from the binding interaction of mini protein with haloalkane was monitored through a wavelength range of 200-1,300 nm. Findings – An increment of the UV absorption is observed at 325 nm when haloalkane interacted with the immobilized bioreceptors, native or mini protein. Both biosensors displayed a continuous response over the range of 5-250 mM haloalkane. They also had the capability to detect haloalkanes below 1 min and with an operational stability of up to seven days without significant loss of sensitivity. Practical implications – The results indicate the potential viability of the enzyme HLD-based sensor to monitor the existence of haloalkane in both pharmaceutical and environmental products. Originality/value – The paper describes an outcome of experimental work on TMMF-based biosensor coated with HLD for label-free haloalkane detection. Mini protein can be used as an effective bioreceptor with some structural modification to improve functionality and stability

Review on fatty acid desaturases and their roles in temperature acclimatisation

Carbohydrates and proteins are polymers of polysaccharides and polypeptide residues, respectively. Conversely, lipids are made up of a wide range of compounds with tremendous differences in structures and lack building blocks. Therefore, fatty acids constitute the major components of various lipid classes such as glycerides and sphingolipids. Fatty acids are organic compounds containing a carboxylic acid group mostly at the end of an aliphatic chain and are categorized into saturated and unsaturated. Most fatty acids especially the so-called polyunsaturated fatty acids (PUFAs) and their derivatives play key biological roles in inflammatory response, cell division, control of lipid metabolism, as signaling molecules, supply of energy and protecting the biological membranes structure and function. In this study, a general overview of fatty acids, their biosynthesis mechanisms as well as biological importance have been discussed. Fatty acid desaturase enzymes and their sources have also been reviewed. Recent studies on the functional expression of different types of desaturase enzymes have also been discussed

Heterologous expression of PA8FAD9 and functional characterization of a Δ9-fatty acid desaturase from a cold-tolerant Pseudomonas sp. A8

Fatty acid desaturase enzymes are capable of inserting double bonds between carbon atoms of saturated fatty acyl-chains to produce unsaturated fatty acids. A gene coding for a putative Δ9-fatty acid desaturase-like protein was isolated from a cold-tolerant Pseudomonas sp. A8, cloned and heterologously expressed in Escherichia coli. The gene named as PA8FAD9 has an open reading frame of 1185 bp and codes for 394 amino acids with a predicted molecular weight of 45 kDa. The enzyme showed high Δ9-fatty acid desaturase-like protein activity and increased overall levels of cellular unsaturated fatty acids in the recombinant E. coli cells upon expression at different temperatures. The results showed that the ratio of palmitoleic to palmitic acid in the recombinant E. coli cells increased by more than twice the amount observed in the control cells at 20 °C using 0.4 mM IPTG. GCMS analysis confirmed the ability of this enzyme to convert exogenous stearic acid to oleic acid incorporated into the recombinant E. coli membrane phospholipids. It may be concluded that the PA8FAD9 gene from Pseudomonas sp. A8 codes for a putative Δ9-fatty acid desaturase protein actively expressed in E. coli under the influence of temperature and an inducer

Comprehensive approaches for the detection of Burkholderia pseudomallei and diagnosis of melioidosis in human and environmental samples

Melioidosis is endemic in Southeast Asia and northern Australia. The causative agent of melioidosis is a Gram-negative bacterium, Burkholderia pseudomallei. Its invasion can be fatal if melioidosis is not treated promptly. It is intrinsically resistant to a variety of antibiotics. In this paper, we present a comprehensive overview of the current trends on melioidosis cases, treatments, B. pseudomallei virulence factors, and molecular techniques to detect the bacterium from different samples. The clinical and microbial diagnosis methods of identification and detection of B. pseudomallei are commonly used for the rapid diagnosis and typing of strains, such as polymerase chain reaction or multi-locus sequence typing. The genotyping strategies and techniques have been constantly evolving to identify genomic loci linked to or associated with this human disease. More research strategies for detecting and controlling melioidosis should be encouraged and conducted to understand the current situation. In conclusion, we review existing diagnostic methodologies for melioidosis detection and provide insights on prospective diagnostic methods for the bacterium

Haematococcus pluvialis as a potential source of Astaxanthin with diverse applications in industrial sectors: current research and future directions

Haematococcus pluvialis, a green microalga, appears to be a rich source of valuable bioactive compounds, such as astaxanthin, carotenoids, proteins, lutein, and fatty acids (FAs). Astaxanthin has a variety of health benefits and is used in the nutraceutical and pharmaceutical industries. Astaxanthin, for example, preserves the redox state and functional integrity of mitochondria and shows advantages despite a low dietary intake. Because of its antioxidant capacity, astaxanthin has recently piqued the interest of researchers due to its potential pharmacological effects, which include anti-diabetic, anti-inflammatory, and antioxidant activities, as well as neuro-, cardiovascular-, ocular, and skin-protective properties. Astaxanthin is a popular nutritional ingredient and a significant component in animal and aquaculture feed. Extensive studies over the last two decades have established the mechanism by which persistent oxidative stress leads to chronic inflammation, which then mediates the majority of serious diseases. This mini-review provides an overview of contemporary research that makes use of the astaxanthin pigment. This mini-review provides insight into the potential of H. pluvialis as a potent antioxidant in the industry, as well as the broad range of applications for astaxanthin molecules as a potent antioxidant in the industrial sector

Secretory expression of thermostable alkaline protease from Bacillus stearothermophilus FI by using native signal peptide and α-factor secretion signal in Pichia pastoris

The thermostable alkaline protease from Bacillus stearothermophilus F1 has high potential for industrial applications, and attempt to produce the enzyme in yeast for higher yield was undertaken. Secretory expression of F1 protease through yeast system could improve enzyme’s capability, thus simplifying the purification steps. Mature and full genes of F1 protease were cloned into Pichia pastoris expression vectors (pGAPZαB and pPICZαB) and transformed into P. pastoris strains (GS115 and SMD1168H) via electroporation method. Recombinant F1 protease under regulation constitutive GAP promoter revealed that the highest expression was achieved after 72 h cultivation. While inducible AOX promoter showed that 0.5% (v/v) methanol was the best to induce expression. It was proven that constitutive expression strategy was better than inducible system. The α-secretion signal from the plasmid demonstrated higher secretory expression level of F1 protease as compared to native Open Reading Frame (ORF) in GS115 strain (GE6GS). Production medium YPTD was found to be the best for F1 protease expression with the highest yield of 4.13 U/mL. The protein was expressed as His-tagged fusion protein with a size about 34 kDa

A newly isolated yeast as an expression host for recombinant lipase

Pichia guilliermondii strain SO isolated from spoiled orange was developed for use as an alternative expression host by using Pichia pastoris as the model of the experiment. This is the first study to report on the capability of P. guilliermondii SO as a host to express thermostable T1 lipase from Geobacillus zalihae. Alcohol oxidase and formaldehyde dehydrogenase promoters were present in the yeast genome. Interestingly, the recombinant yeast [SO/pPICZαB/T1-2 (SO2)] took only 30 h to reach optimal production with minimal methanol induction [1.5% (v/v)] in YPTM medium, as compared to P. pastoris, which took longer to reach its optimal condition. The purification yield of the His-tagged fusion lipase was 68.58%, with specific activity of 194.58 U/mg. The optimum temperature was 65°C at pH 9 in glycine-NaOH buffer, and it was stable up to 70°C in a wide pH range from pH 5 to 12. In conclusion, a newly isolated yeast from spoiled orange has been proven suitable for use as an expression host

Pichia pastoris as a host to overexpress the thermostable T1 lipase from Geobacillus zalihae

Pichia pastoris was known to be a good expression system in producing various heterologous proteins. The gene encoding thermostable Tl lipase from Geobacillus zalihae was cloned into pPICZαB and expressed in P. pastoris strains (GS115, X-33 and KM71H) under regulation of alcohol oxidase promoter. The expression of the gene in Escherichia coli system showed low expression level. Therefore, this study would highlight on the overexpression of the T1 lipase in yeast extracellularly. Recombinant X-33/pPICZαB/T1-2 (XPB2), GS115/pPICZαB/T1-5 (GPB5) and KM71H/pPICZαB/T1-7 (KPB7) were chosen for optimization in shake flask. Optimization strategies showed that these recombinants preferred YPTM medium with initial induction OD600nm = 7 cell biomass and 2% (v/v) methanol to provide optimal expression conditions. Hyper-resistant transformants at 3000 ug/mL zeocin gave better expression than 100 ug/mL zeocin selection. Time course study by using different inocula age showed that OD600nm = 7 expressed lipase at the highest level. The highest expression level was attained with GPB5 (88 U/ml), XPB2 (81 U/ml) and KPB7 (51 U/ml). Western Blot analysis confirmed that the molecular mass of recombinant Tl lipase was 45 kDa. In conclusion, thermostable Tl lipase was successfully overexpressed by using secretory P. pastoris system with two-fold higher than E. coli system

Bioprocess Strategy of Haematococcus lacustris for Biomass and Astaxanthin Production Keys to Commercialization: Perspective and Future Direction

Haematococcus lacustris (formerly called Haematococcus pluvialis) is regarded as the most promising microalgae for the production of natural astaxanthin, which is secondary metabolism used as a dietary supplement, also for cosmetic applications, due to its high antioxidant activity. Astaxanthin has a wide range of biological activities and high economic potential, and currently dominates the market in its synthetic form. Furthermore, because of the difficulty of bioprocess and the high cost of cultivation, astaxanthin extracted from this microalga is still expensive due to its low biomass and pigment productivities. Large-scale biomass production in biotechnological production necessitates the processing of a large number of cultures as well as the use of both indoor and outdoor systems, such as open pond raceway systems and photo-bioreactors (PBR). The photo-bioreactors systems are suitable for mass production because growth conditions can be controlled, and the risk of contamination can be reduced to a certain extent and under specific culture parameters. This review discusses current technologies being developed to improve cultivation and operation efficiency and profitability, as well as the effect of parameter factors associated with H. lacustris cultivation on biomass and astaxanthin bioproduction, and even strategies for increasing bioproduction and market potential for H. lacustris astaxanthin