Host For Protein Production Research Articles

Knock-out of the major regulator Flo8 in Komagataella phaffii results in unique host strain performance for methanol-free recombinant protein production

Flo8 is a main transcriptional regulator of flocculation and pseudohyphal growth in yeast. Disruption of FLO8 in the popular recombinant protein production host Komagataella phaffii (Pichia pastoris) prevents pseudohyphal growth and reduces cell-to-surface adherence, making the mutant an interesting platform for research and industry. However, knowledge of the physiological impact of the mutation remained scarce. In-depth analysis of transcriptome data from FLO8-deficient K. phaffii revealed that Flo8 affects genes involved in cell cycle, mating, respiration, and catabolite repression additionally to flocculation targets. One gene with considerably increased expression in flo8 was GTH1, encoding a high-affinity glucose transporter in K. phaffii. Its promoter (PG1) was previously established as a strong, glucose-regulatable alternative to methanol-induced promoters. PG1 and its improved derivatives PG1-3, D-PGS4 and D-PGS5, proved to be promising candidates for controlling recombinant protein production in the FLO8-deficient background. In small-scale screenings, PG13-controlled intracellular EGFP levels were 2.8-fold higher, and yields of different secreted recombinant proteins were up to 4.8-fold increased. The enhanced productivity of the flo8 mutant in combination with the PG1 variants was transferrable to glucose-limited fed-batch processes and could largely be attributed to higher transcriptional activity of the promoter, leading to a much higher productivity per chromosomally integrated gene copy. K. phaffii flo8 has many advantageous characteristics, such as reduced surface growth and increased transcriptional strength of glucose-regulatable promoters. These features turn the flo8 strain into a valuable new base strain for various experimental designs and establish flo8 as an excellent strain background for methanol-free recombinant protein production processes.

Silencing of RDR1 and RDR6 genes by a single RNAi enhances lettuce's capacity to express recombinant proteins in transient assays.

Enhanced recombinant protein expression was achieved in Salinas lettuce and commercial lettuce by designing a unique RNAi that knockdown the gene-silencing mechanism in transient assays. Improved yields of recombinant proteins (RP) are necessary for protein-production efficiency and ease of purification. Achieving high yield in non-tobacco plants will enable diverse plants to be used as hosts in transient protein-expression systems. With improved protein yield, lettuce (Lactuca sativa) could take the lead as a plant host for RP production. Therefore, this study aimed to improve RP production in lettuce var. Salinas by designing a single RNA interference (RNAi) construct targeting LsRDR1 and LsRDR6 using the Tsukuba system vector. Two RNAi constructs, RNAi-1 and RNAi-2, targeting common regions of LsRDR1 and LsRDR6 with 75% and 76% similarity, respectively, were employed to evaluate simultaneous gene silencing. Quantitative transcription analysis demonstrated that both RNAi constructs effectively knocked down LsRDR6 and LsRDR1, but not LsRDR2, at both 3 and 5days post-infiltration (dpi), with RNAi-1 exhibited slightly higher efficiency. Based on the protein yield, co-expression of RNAi-1 with enhanced green fluorescent protein (EGFP) increased EGFP expression by approximately 4.9-fold and 3.7-fold at 3dpi and 5dpi, respectively, compared to control. A similar but slightly lower increase (2.4-fold and 2.33-fold) was observed in commercial lettuce at 3and 5 dpi, respectively. To confirm these results, co-infiltration with Bet v 1, a major allergen from birch pollen, resulted in a 2.5-fold increase in expression in Salinas lettuce at 5 dpi. This study marks a significant advancement in enhancing transient protein production in lettuce, elevating its potential as a host for recombinant protein production.

High-level production of patatin in Pichia pastoris and characterization of N-glycosylation modification in food processing properties

Patatin is an acidic protein found in potatoes that is commonly used in food and pharmaceutical industries due to its excellent emulsifying and gelation abilities. Pichia pastoris is widely used as a host for recombinant protein production because it can incorporate post-translational modifications. In this study, a patatin titre of 2189.8 mg/L was achieved in a 5 L bioreactor using P. pastoris GS115 with signal peptide mutation, dual promoter construction, co-expression of chaperone proteins and optimised fermentation. To enhance the application of recombinant patatin in the food processing field, the level of N-glycosylation was elevated by genetic engineering. Properties of natural patatin, recombinant patatin and patatinL109T (N-glycosylated modified patatin) were investigated including foaming, hydrophobicity and emulsifying abilities. The functional properties of recombinant patatin were enhanced by introducing N-glycosylation, which also improved the water-holding capacity of its gel. The patatinL109T gel exhibited superior elasticity and water retention properties. The findings provide valuable insight and serve as a reference for the utilisation of recombinant patatin. The established enhancement strategy could be applied to other recombinant proteins.

Chinese hamster ovary cell line engineering strategies for modular production of custom extracellular vesicles.

Continuously secreted by all cell types, extracellular vesicles (EVs) are small membrane-bound structures which shuttle bioactive cargo between cells across their external environment. Their central role as natural molecular messengers and ability to cross biological barriers has garnered significant attention in the use of EVs as therapeutic delivery vehicles. Still, harnessing the potential of EVs is faced with many obstacles. A cell line engineering approach can be used to exploit EVs to encapsulate a bespoke cargo of interest. However, full details regarding native EV-loading mechanisms remain under debate, making this a challenge. While Chinese hamster ovary (CHO) cells are well known to be the preferred host for recombinant therapeutic protein production, their application as an EV producer cell host has been largely overlooked. In this study, we engineered CHO DG44 cells to produce custom EVs with bespoke cargo. To this end, genetic constructs employing split green fluorescent protein technology were designed for tagging both CD81 and protein cargoes to enable EV loading via self-assembling activity. To demonstrate this, NanoLuc and mCherry were used as model reporter cargoes to validate engineered loading into EVs. Experimental findings indicated that our custom EV approach produced vesicles with up to 15-fold greater cargo compared with commonly used passive loading strategies. When applied to recipient cells, we observed a dose-dependent increase in cargo activity, suggesting successful delivery of engineered cargo via our custom CHO EVs.

Protein Engineering of Vip3A in a Selected Bacillus thuringiensis Host for Consistent High Protein Production.

This study aimed to obtain reliable high Vip3A production from Bacillus thuringiensis (Bt) by modifying Vip3A to acquire higher thermostability in a suitable host. Bt117 is a great host for Vip3A production due to protein production consistency, low protease activity in culture media, and large amounts of mostly full-length protein, but it produces Vip3A with lower thermostability (Vip3Aa35). The C-terminal region of Bt117 Vip3A was replaced with that of a Vip3A with higher thermostability (Vip3Aa64 from Bt294) to generate the recombinant Bt117-Vip3Aa64-C. Like the parental strain Bt117, this strain expressed mostly full-length protein and exhibited low protease activity and similar protein expression profiles in culture media but retained greater larvicidal activity upon 37°C storage like Bt294 Vip3Aa64. Importantly, every culture batch of Bt117-Vip3Aa64-C yielded over 200mg/l Vip3A, which is a notable improvement over the original Vip3Aa64-producing strain Bt294 where 45% of culture batches failed to produce Vip3A at the same level. Successfully, we combined the superior qualities of two Bt strains, Bt294, which produces thermostable Vip3A but at low and inconsistent levels, and Bt117, which produces Vip3A with low thermostability but at consistently high levels. Protein engineering of Vip3A in Bt117 ultimately yielded an improved strain producing a thermostable Vip3A with reliably high protein production.

Comparing the Soluble Form of Recombinant Human Insulin-like Growth Factor-1 (rhIGF-1) in Escherichia coli Using Thioredoxin as Fused and Co-expressed Protein.

Insulin-like growth factor-1 (IGF-1) is a single-chain polypeptide with various physiological functions. Escherichia coli is one of the most desirable hosts for recombinant protein production, especially for human proteins whose post-translation modifications are not essential for their bioactivity, such as hIGF-1. In this study, bacterial thioredoxin (Trx) was studied as a fused and non-fused protein to convert the insoluble form of recombinant human IGF-1 (rhIGF-1) to its soluble form in E. coli. The rhIGF-1 was expressed in the E. coli Origami strain in the form of fused-Trx. It was co-expressed with Trx and then purified and quantified. In the next step, the biological activity of rhIGF-1 was evaluated by alkaline phosphatase (ALP) activity assay in human adipose-derived stem cells (hASCs) regarding the differentiation enhancement effect of IGF-1 through the osteogenic process. Results showed that Trx in both the fused and non-fused forms had a positive effect on the production of the soluble form of rhIGF-1. A significant increase in ALP activity in hASCs after rhIGF-1 treatment was observed, confirming protein bioactivity. It was strongly suggested that the overproduction of Trx could increase the solubility of co-expressed recombinant proteins by changing the redox state in E. coli cells.

Enhancing extracellular membrane vesicle productivity of Shewanella vesiculosa HM13, a prospective host for vesiculation-mediated protein secretion, by weakening outer membrane-peptidoglycan linkage

Shewanella vesiculosa HM13, a psychrotrophic gram-negative bacterium isolated from the intestinal contents of horse mackerel, produces abundant extracellular membrane vesicles (EMVs) by budding the outer membrane. The EMVs of this bacterium carry a single major cargo protein, P49, of unknown function, which may be useful as a carrier for the secretory production of heterologous proteins as cargoes of EMVs. In this study, to increase the utility of S.vesiculosa HM13 as a host for EMV-mediated protein production, we improved its EMV productivity by weakening the linkage between the outer membrane and underlying peptidoglycan layer. In gram-negative bacteria, the outer membrane is connected to peptidoglycans predominantly through Braun's lipoprotein (Lpp), and the formation of this linkage is catalyzed by an l,d-transpeptidase (Ldt). We constructed gene-disrupted mutants of Lpp and Ldt and assessed their EMV productivity. The EMVs of the lpp- and ldt-disrupted mutants grown at 18°C were evaluated using nanoparticle tracking analysis, and their morphologies were observed using transmission electron microscopy. As a result, an approximately 2.5-fold increase in EMV production was achieved, whereas the morphology of the EMVs of these mutants remained almost identical to that of the parent strain. In accordance with the increase in EMV production, the mutants secreted approximately 2-fold higher amounts of P49 than the parent strain into the culture broth as the EMV cargo. These findings will contribute to the development of an EMV-based secretory production system for heterologous proteins using S.vesiculosa HM13 as a host.

Sodium acetate increases the productivity of HEK293 cells expressing the ECD-Her1 protein in batch cultures: experimental results and metabolic flux analysis.

Human Embryonic Kidney cells (HEK293) are a popular host for recombinant protein expression and production in the biotechnological industry. This has driven within both, the scientific and the engineering communities, the search for strategies to increase their protein productivity. The present work is inserted into this search exploring the impact of adding sodium acetate (NaAc) into a batch culture of HEK293 cells. We monitored, as a function of time, the cell density, many external metabolites, and the supernatant concentration of the heterologous extra-cellular domain ECD-Her1 protein, a protein used to produce a candidate prostate cancer vaccine. We observed that by adding different concentrations of NaAc (0, 4, 6 and 8mM), the production of ECD-Her1 protein increases consistently with increasing concentration, whereas the carrying capacity of the medium decreases. To understand these results we exploited a combination of experimental and computational techniques. Metabolic Flux Analysis (MFA) was used to infer intracellular metabolic fluxes from the concentration of external metabolites. Moreover, we measured independently the extracellular acidification rate and oxygen consumption rate of the cells. Both approaches support the idea that the addition of NaAc to the culture has a significant impact on the metabolism of the HEK293 cells and that, if properly tuned, enhances the productivity of the heterologous ECD-Her1 protein.

Exploring the secretome of Corynebacterium glutamicum ATCC 13032.

The demand for alternative sources of food proteins is increasing due to the limitations and challenges associated with conventional food production. Advances in biotechnology have enabled the production of proteins using microorganisms, thus prompting the exploration of attractive microbial hosts capable of producing functional proteins in high titers. Corynebacterium glutamicum is widely used in industry for the production of amino acids and has many advantages as a host organism for recombinant protein production. However, its performance in this area is limited by low yields of target proteins and high levels of native protein secretion. Despite representing a challenge for heterologous protein production, the C. glutamicum secretome has not been fully characterized. In this study, state-of-the-art mass spectrometry-based proteomics was used to identify and analyze the proteins secreted by C. glutamicum. Both the wild-type strain and a strain that produced and secreted a recombinant β-lactoglobulin protein were analyzed. A total of 427 proteins were identified in the culture supernatants, with 148 predicted to possess a secretion signal peptide. MS-based proteomics on the secretome enabled a comprehensive characterization and quantification (based on abundance) of the secreted proteins through label-free quantification (LFQ). The top 12 most abundant proteins accounted for almost 80% of the secretome. These are uncharacterized proteins of unknown function, resuscitation promoting factors, protein PS1, Porin B, ABC-type transporter protein and hypothetical membrane protein. The data can be leveraged for protein production by, e.g., utilizing the signal peptides of the most abundant proteins to improve secretion of heterologous proteins. In addition, secretory stress can potentially be alleviated by inactivating non-essential secreted proteins. Here we provide targets by identifying the most abundant, secreted proteins of which majority are of unknown function. The data from this study can thus provide valuable insight for researchers looking to improve protein secretion and optimize C. glutamicum as a host for secretory protein production.

Synergic kinetic and physiological control to improve the efficiency of Komagataella phaffii recombinant protein production bioprocesses.

The yeast Komagataella phaffii (Pichia pastoris) is currently considered a versatile and highly efficient host for recombinant protein production (RPP). Interestingly, the regulated application of specific stress factors as part of bioprocess engineering strategies has proven potential for increasing the production of recombinant products. This study aims to evaluate the impact of controlled oxygen-limiting conditions on the performance of K. phaffii bioprocesses for RPP in combination with the specific growth rate (μ) in fed-batch cultivations. In this work, Candida rugosa lipase 1 (Crl1) production, regulated by the constitutive GAP promoter, growing at different nominal μ (0.030, 0.065, 0.100 and 0.120 h-1 ) under both normoxic and hypoxic conditions in carbon-limiting fed-batch cultures is analysed. Hypoxic fermentations were controlled at a target respiratory quotient (RQ) of 1.4, with excellent performance, using an innovative automated control based on the stirring rate as the manipulated variable developed during this study. The results conclude that oxygen limitation positively affects bioprocess efficiency under all growing conditions compared. The shift from respiratory to respiro-fermentative metabolism increases bioprocess productivity by up to twofold for the specific growth rates evaluated. Moreover, the specific product generation rate (qp ) increases linearly with μ, regardless of oxygen availability. Furthermore, this hypoxic boosting effect was also observed in the production of Candida antarctica lipase B (CalB) and pro-Rhizopus oryzae lipase (proRol), thus proving the synergic effect of kinetic and physiological stress control. Finally, the Crl1 production scale-up was conducted successfully, confirming the strategy's scalability and the robustness of the results obtained at the bench-scale level.

Antigen surface display in two novel whole genome sequenced food grade strains, Lactiplantibacillus pentosus KW1 and KW2

BackgroundUtilization of commensal bacteria for delivery of medicinal proteins, such as vaccine antigens, is an emerging strategy. Here, we describe two novel food-grade strains of lactic acid bacteria, Lactiplantibacillus pentosus KW1 and KW2, as well as newly developed tools for using this relatively unexplored but promising bacterial species for production and surface-display of heterologous proteins.ResultsWhole genome sequencing was performed to investigate genomic features of both strains and to identify native proteins enabling surface display of heterologous proteins. Basic characterization of the strains revealed the optimum growth temperatures for both strains to be 35–37 °C, with peak heterologous protein production at 33 °C (KW1) and 37 °C (KW2). Negative staining revealed that only KW1 produces closely bound exopolysaccharides. Production of heterologous proteins with the inducible pSIP-expression system enabled high expression in both strains. Exposure to KW1 and KW2 skewed macrophages toward the antigen presenting state, indicating potential adjuvant properties. To develop these strains as delivery vehicles, expression of the mycobacterial H56 antigen was fused to four different strain-specific surface-anchoring sequences.ConclusionAll experiments that enabled comparison of heterologous protein production revealed KW1 to be the better recombinant protein production host. Use of the pSIP expression system enabled successful construction of L. pentosus strains for production and surface display of an antigen, underpinning the potential of these strains as novel delivery vehicles.

Agrobacterium tumefaciens-mediated transformation for the genetic modification of the biotechnologically relevant fungus Aspergillus vadensis through synthetic biology

In the last years, many research efforts have been applied for the development of filamentous fungi as hosts for heterologous protein production. Aspergillus vadensis CBS 113365, a close relative of the industrial workhorse Aspergillus niger, has been suggested as a more suitable cell factory as it does not acidify the culture medium and produces very low levels of secreted proteases. Therefore, efficient methods and tools that allow the genetic manipulation and exploitation of this biotechnologically relevant fungus are needed. To date, only protoplast-mediated transformation and classical cloning strategies have been implemented for A. vadensis genetic modification, which decreases the exploitation capacity of this fungus at the industrial level. In this study, we have adapted and implemented an Agrobacterium tumefaciens-mediated transformation protocol for A. vadensis for the first time, and applied the FungalBraid system to genetically modify this species by means of synthetic biology. As proof of concept, we have successfully complemented and fluorescently labelled a uridine auxotrophic A. vadensis pyrA- strain and generated A. vadensis mutants carrying the Penicillium expansum-based expression cassette for the heterologous production of the antifungal protein PeAfpA from P. expansum. Even though we have yet to find the conditions that trigger PeAfpA production in this species, the implementation of the ATMT method reported here, along with the application of the FungalBraid system, will greatly aid in this task and will facilitate the exploitation of A. vadensis as a fungal workhorse for protein production for multiple biotechnological applications.

Effect of biotin starvation on gene expression in <i>Komagataella phaffii</i> cells

Methylotrophic yeast Komagataella phaffii are widely used in biotechnology as a host for recombinant protein production. Due to the practical significance of this yeast, it is extremely important to properly select the cultivation conditions and optimize the media composition. In this study the effect of biotin starvation on K. phaffii gene expression was investigated at transcriptomic level. It was demonstrated, that K. phaffii cell response to biotin deficiency strongly depends on the carbon source in the medium. In media containing glycerol, biotin deficiency led to activation of genes involved in biotin metabolism, glyoxylate cycle and synthesis of acetyl-CoA in cytoplasm, as well as repression of genes, involved in lipo- and gluconeogenesis. In methanol containing media, biotin deficiency primarily led to repression of genes, involved in protein synthesis, and activation of cell response to oxidative stress.

Genome-wide identification and in-silico analysis of papain-family cysteine protease encoding genes in Tetrahymena thermophila

Tetrahymena thermophila is a promising host for recombinant protein production, but its utilization in biotechnology is mostly limited due to the presence of intracellular and extracellular papain-family cysteine proteases (PFCPs). In this study, we employed bioinformatics approaches to investigate the T. thermophila PFCP genes and their encoded proteases (TtPFCPs), the most prominent protease family in the genome. Results from the multiple sequence alignment, protein modeling, and conserved motif analyses revealed that all TtPFCPs showed considerably high homology with mammalian cysteine cathepsins and contained conserved amino acid motifs. The total of 121 TtPFCP-encoding genes, 14 of which were classified as non-peptidase homologs, were found. Remaining 107 true TtPFCPs were divided into four distinct subgroups depending on their homology with mammalian lysosomal cathepsins: cathepsin L-like (TtCATLs), cathepsin B-like (TtCATBs), cathepsin C-like (TtCATCs), and cathepsin X-like (TtCATXs) PFCPs. The majority of true TtPFCPs (96 out of the total) were in TtCATL-like peptidase subgroup. Both phylogenetic and chromosomal localization analyses of TtPFCPs supported the hypothesis that TtPFCPs likely evolved through tandem gene duplication events and predominantly accumulated on micronuclear chromosome 5. Additionally, more than half of the identified TtPFCP genes are expressed in considerably low quantities compared to the rest of the TtPFCP genes, which are expressed at a higher level. However, their expression patterns fluctuate based on the stage of the life cycle. In conclusion, this study provides the first comprehensive in-silico analysis of TtPFCP genes and encoded proteases. The results would help designing an effective strategy for protease knockout mutant cell lines to discover biological function and to improve the recombinant protein production in T. thermophila.

AOX1 promoter-driven expression of yeast-enhanced green fluorescent protein in Pichia pastoris

Green fluorescent protein (GFP) is a widely employed reporter for studying various cellular functions in genetic engineering. Methylotrophic yeast; Pichia pastoris is an excellent host for heterologous protein production. Codon optimized yeast-enhanced green fluorescent protein (yEGFP) has been reported to have higher expression as a yeast reporter which can be used to study many aspects of P. pastoris. The present investigation was focused on studying the yEGFP intracellular expression pattern in the P. pastoris GS115 strain. yEGFP is cloned into the pPICZ A vector under the control of an AOX1 (Alcohol Oxidase) promoter that was induced by adding methanol to the growth medium. However, methanol concentration and time of induction could affect the optimum expression of yEGFP. Hence, those conditions were tested for manifesting optimum yEGFP expression by monitoring the fluorescence intensities in minimal methanol histidine media supplemented with 0.5%, 1%, 2%, and 2.5% methanol. The pH of the media and the composition of the media (minimal or complex) also affect recombinant protein production. The effect of pH and the media composition was investigated in buffered media at pH 6 and unbuffered media of minimal methanol histidine media and methanol complex media. The results showed that the yEGFP expression increased up to the 5th day of induction and then decreased on the 6th day, 2% methanol showed the highest expression, and the pH and the media composition did not have any effect on the yEGFP expression. In conclusion, the optimum yEGFP expression can be obtained in 2% methanol on the 5th day of induction irrespective of minimal or complex media used.

Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains.

Komagataella phaffii (Pichia pastoris) is a widely known microbial host for recombinant protein production and an emerging model organism in fundamental research. The development of new materials and techniques on this yeast improves heterologous protein synthesis. One of the most prominent ways to enhance protein production efficiency is to select K. phaffii strains with multiple expression cassettes and generate MutS strains using various vectors. In this study, we demonstrate approaches to expand the applications of pPICZ series vectors. Procedures based on PCR amplification and in vivo cloning allow rapid exchange of selectable markers. The combination of PCR amplification with split-marker-mediated transformation allows the development of K. phaffii MutS strains with two expression cassettes using pPICZ vectors. Both PCR-based approaches were applied to efficiently produce interleukin-2 mimetic Neo-2/15 in K. phaffii. The described techniques provide alternative ways to generate and improve K. phaffii strains without the need for obtaining new specific vectors or additional cloning of expression cassettes.

A CRISPR/Cas9-based multicopy integration system for protein production in Aspergillus niger.

The filamentous fungus Aspergillus niger is well known for its high protein secretion capacity and a preferred host for homologous and heterologous protein production. To improve the protein production capacity of A. niger even further, a set of dedicated protein production strains was made containing up to 10 glucoamylase landing sites (GLSs) at predetermined sites in the genome. These GLSs replace genes encoding enzymes abundantly present or encoding unwanted functions. Each GLS contains the promotor and terminator region of the glucoamylase gene (glaA), one of the highest expressed genes in A. niger. Integrating multiple gene copies, often realized by random integration, is known to boost protein production yields. In our approach the GLSs allow for rapid targeted gene replacement using CRISPR/Cas9-mediated genome editing. By introducing the same or different unique DNA sequences (dubbed KORE sequences) in each GLS and designing Cas9-compatible single guide RNAs, one is able to select at which GLS integration of a target gene occurs. In this way a set of identical strains with different copy numbers of the gene of interest can be easily and rapidly made to compare protein production levels. As an illustration of its potential, we successfully used the expression platform to generate multicopy A. niger strains producing the Penicillium expansum PatE::6xHis protein catalysing the final step in patulin biosynthesis. The A. niger strain expressing 10 copies of the patE::6xHis expression cassette produced about70 μg·mL-1 PatE protein in the culture medium with a purity just under 90%.

Recombinant Cas9 protein production in an endotoxin-free system and evaluation with editing the BCL11A gene in human cells

Many therapeutic proteins are expressed in Escherichia coli bacteria for the low cost and high yield obtained. However, these gram-negative bacteria also generate undesirable endotoxin byproducts such as lipopolysaccharides (LPS). These endotoxins can induce a human immune response and cause severe inflammation. To mitigate this problem, we have employed the ClearColi BL21 (DE3) endotoxin-free cells as an expression host for Cas9 protein production. Cas9 is an endonuclease enzyme that plays a key role in the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated protein 9 (CRISPR/Cas9) genome editing technique. This technology is very promising for use in diagnostics as well as treatment of diseases, especially for genetic diseases such as thalassemia. The potential uses for this technology thus generate a considerable interest for Cas9 utilization as a therapeutic protein in clinical treatment. Therefore, special care in protein production should be a major concern. Accordingly, we expressed the Cas9 protein in endotoxin-free bacterial cells achieving 99% purity with activity comparable to commercially available Cas9. Our protocol therefore yields a cost-effective product suitable for invitro experiments with stem cells.

Heterologous Expression of Plantaricin 423 and Mundticin ST4SA in Saccharomyces cerevisiae

Antimicrobial peptides or bacteriocins are excellent candidates for alternative antimicrobials, but high manufacturing costs limit their applications. Recombinant gene expression offers the potential to produce these peptides more cost-effectively at a larger scale. Saccharomyces cerevisiae is a popular host for recombinant protein production, but with limited success reported on antimicrobial peptides. Individual recombinant S. cerevisiae strains were constructed to secrete two class IIa bacteriocins, plantaricin 423 (PlaX) and mundticin ST4SA (MunX). The native and codon-optimised variants of the plaA and munST4SA genes were cloned into episomal expression vectors containing either the S. cerevisiae alpha mating factor (MFα1) or the Trichoderma reesei xylanase 2 (XYNSEC) secretion signal sequences. The recombinant peptides retained their activity and stability, with the MFα1 secretion signal superior to the XYNSEC secretion signal for both bacteriocins. An eight-fold increase in activity against Listeria monocytogenes was observed for MunX after codon optimisation, but not for PlaX-producing strains. After HPLC-purification, the codon-optimised genes yielded 20.9 mg/L of MunX and 18.4 mg/L of PlaX, which displayed minimum inhibitory concentrations (MICs) of 108.52 nM and 1.18 µM, respectively, against L. monocytogenes. The yields represent a marked improvement relative to an Escherichia coli expression system previously reported for PlaX and MunX. The results demonstrated that S. cerevisiae is a promising host for recombinant bacteriocin production that requires a simple purification process, but the efficacy is sensitive to codon usage and secretion signals.

Developing a metabolic model‐based fed‐batch feeding strategy for Pichia pastoris fermentation through fine‐tuning of the methanol utilization pathway

Pichia pastoris is a commonly used microbial host for recombinant protein production. It is mostly cultivated in fed-batch mode, in which the environment of the cell is continuously changing. Hence, it is vital to understand the influence of feeding strategy parameters on the intracellular reaction network to fine-tune bioreactor performance. This study used dynamic flux balance analysis (DFBA) integrated with transcriptomics data to simulate the recombinant P. pastoris (Muts ) growth during the induction phase for three fed-batch strategies, conducted at constant specific growth rates (μ-stat). The induction phase was split into equal time intervals, and the correlated reactions with protein yield were identified in the three fed-batch strategies using the Pearson correlation coefficient. Subsequently, principal component analysis (PCA) was applied to cluster induction phase time intervals and identify the role of correlated reactions on metabolic differentiation of time intervals. It was found that increasing fluxes through the methanol dissimilation pathway increased protein yield. By adding a methanol assimilation pathway inhibitor (HgCl2 ) to the shake flask medium growing on glycerol: methanol mixture (10%: 90%, v/v), the protein titre increased by 60%. As per DFBA, the higher the methanol to biomass flux ratio (Rmeoh/Δx ), the higher the protein yield. Finally, a novel feeding strategy was developed to increase the amount of Rmeoh/Δx compared to the three feeding strategies. The concentration of recombinant human growth hormone (rhGH), used as the model protein, increased by 16% compared to the optimal culture result obtained previously (800 mg L-1 to 928 mg L-1 ), while production yield improved by 85% (24.8 mg gDCW -1 to 46 mg gDCW -1 ).