Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii.

BACKGROUND: Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii. RESULTS: In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system. CONCLUSIONS: In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.

Online monitoring of protein refolding in inclusion body processing using intrinsic fluorescence.

Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of recombinant protein production despite the need for additional processing steps, i.e., isolation, solubilization and refolding. Industrial process development of protein refolding is a labor-intensive task based largely on empirical approaches rather than knowledge-driven strategies. A prerequisite for knowledge-driven process development is a reliable monitoring strategy. This work explores the potential of intrinsic tryptophan and tyrosine fluorescence for real-time and in situ monitoring of protein refolding. In contrast to commonly established process analytical technology (PAT), this technique showed high sensitivity with reproducible measurements for protein concentrations down to 0.01 g L - 1 . The change of protein conformation during refolding is reflected as a shift in the position of the maxima of the tryptophan and tyrosine fluorescence spectra as well as change in the signal intensity. The shift in the peak position, expressed as average emission wavelength of a spectrum, was correlated to the amount of folding intermediates whereas the intensity integral correlates to the extent of aggregation. These correlations were implemented as an observation function into a mechanistic model. The versatility and transferability of the technique were demonstrated on the refolding of three different proteins with varying structural complexity. The technique was also successfully applied to detect the effect of additives and process mode on the refolding process efficiency. Thus, the methodology presented poses a generic and reliable PAT tool enabling real-time process monitoring of protein refolding.

Poly-ß-hydroxybutyrate production by Synechocystis MT_a24 in a raceway pond using urban wastewater.

Poly-ß-hydroxybutyrate (PHB) is a potential source of biodegradable plastics that are environmentally friendly due to their complete degradation to water and carbon dioxide. This study aimed to investigate PHB production in the cyanobacterium Synechocystis sp. PCC6714 MT_a24 in an outdoor bioreactor using urban wastewater as a sole nutrient source. The culture was grown in a thin-layer raceway pond with a working volume of 100 L, reaching a biomass density of up to 3.5 g L-1 of cell dry weight (CDW). The maximum PHB content was found under nutrient-limiting conditions in the late stationary phase, reaching 23.7 ± 2.2% PHB per CDW. These data are one of the highest reported for photosynthetic production of PHB by cyanobacteria, moreover using urban wastewater in pilot-scale cultivation which multiplies the potential of sustainable cultivation approaches. Contamination by grazers (Poterioochromonas malhamensis) was managed by culturing Synechocystis in a highly alkaline environment (pH about 10.5) which did not significantly affect the culture growth. Furthermore, the strain MT_a24 showed significant wastewater nutrient remediation removing about 72% of nitrogen and 67% of phosphorus. These trials demonstrate that the photosynthetic production of PHB by Synechocystis sp. PCC6714 MT_a24 in the outdoor thin-layer bioreactor using urban wastewater and ambient carbon dioxide. It shows a promising approach for the cost-effective and sustainable production of biodegradable carbon-negative plastics. KEY POINTS: • High PHB production by cyanobacteria in outdoor raceway pond • Urban wastewater used as a sole source of nutrients for phototrophic growth • Potential for cost-effective and sustainable production of biodegradable plastics.

Application of Quantum Cascade Laser-Infrared Spectroscopy and Chemometrics for In-Line Discrimination of Coeluting Proteins from Preparative Size Exclusion Chromatography.

An external-cavity quantum cascade laser (EC-QCL)-based flow-through mid-infrared (IR) spectrometer was placed in line with a preparative size exclusion chromatography system to demonstrate real-time analysis of protein elutions with strongly overlapping chromatographic peaks. Two different case studies involving three and four model proteins were performed under typical lab-scale purification conditions. The large optical path length (25 µm), high signal-to-noise ratios, and wide spectral coverage (1350 to 1750 cm-1) of the QCL-IR spectrometer allow for robust spectra acquisition across both the amide I and II bands. Chemometric analysis by self-modeling mixture analysis and multivariate curve resolution enabled accurate quantitation and structural fingerprinting across the protein elution transient. The acquired concentration profiles were found to be in excellent agreement with the off-line high-performance liquid chromatography reference analytics performed on the collected effluent fractions. These results demonstrate that QCL-IR detectors can be used effectively for in-line, real-time analysis of protein elutions, providing critical quality attribute data that are typically only accessible through time-consuming and resource-intensive off-line methods.

Coelastrella terrestris for Adonixanthin Production: Physiological Characterization and Evaluation of Secondary Carotenoid Productivity.

A novel strain of Coelastrella terrestris (Chlorophyta) was collected from red mucilage in a glacier foreland in Iceland. Its morphology showed characteristic single, ellipsoidal cells with apical wart-like wall thickenings. Physiological characterization revealed the presence of the rare keto-carotenoid adonixanthin, as well as high levels of unsaturated fatty acids of up to 85%. Initial screening experiments with different carbon sources for accelerated mixotrophic biomass growth were done. Consequently, a scale up to 1.25 L stirred photobioreactor cultivations yielded a maximum of 1.96 mg·L-1 adonixanthin in free and esterified forms. It could be shown that supplementing acetate to the medium increased the volumetric productivity after entering the nitrogen limitation phase compared to autotrophic control cultures. This study describes a promising way of biotechnological adonixanthin production using Coelastrella terrestris.

Reducing phenotypic instabilities of a microbial population during continuous cultivation based on cell switching dynamics.

Predicting the fate of individual cells among a microbial population (i.e., growth and gene expression) remains a challenge, especially when this population is exposed to very dynamic environmental conditions, such as those encountered during continuous cultivation. Indeed, the dynamic nature of a continuous cultivation process implies the potential diversification of the microbial population resulting in genotypic and phenotypic heterogeneity. The present work focused on the induction of the arabinose operon in Escherichia coli as a model system to study this diversification process in continuous cultivations. As a preliminary step, the green fluorescent protein (GFP) level triggered by an arabinose-inducible ParaBAD promoter was tracked by flow cytometry in chemostat cultivations with glucose-arabinose co-feeding. For a wide range of glucose-arabinose co-feeding concentrations in the chemostats, the simultaneous occurrence of GFP positive and negative subpopulation was observed. In the second set of experiments, continuous cultivation was performed by adding glucose continuously and arabinose based on the capability of individual cells to switch from low GFP to high GFP expression states, performed with a technology setup called segregostat. In the segregostat cultivation mode, on-line flow cytometry analysis was used for adjusting the arabinose/glucose transitions based on the phenotypic switching profiles of the microbial population. This strategy allowed finding an appropriate arabinose pulsing frequency, leading to prolonged maintenance of the induction level with a limited increase in the phenotypic diversity for more than 60 generations. The results suggest that the steady forcing of individual cells into a given phenotypic trajectory may not be the best strategy for controlling cell populations. Instead, allowing individual cells to switch periodically around a predefined threshold seems to be a more robust strategy leading to oscillations, but within a predictable cell population behavior range.

Microbial technologies for biotherapeutics production: Key tools for advanced biopharmaceutical process development and control.

Current trends in the biopharmaceutical market such as the diversification of therapies as well as the increasing time-to-market pressure will trigger the rethinking of bioprocess development and production approaches. Thereby, the importance of development time and manufacturing costs will increase, especially for microbial production. In the present review, we investigate three technological approaches which, to our opinion, will play a key role in the future of biopharmaceutical production. The first cornerstone of process development is the generation and effective utilization of platform knowledge. Building processes on well understood microbial and technological platforms allows to accelerate early-stage bioprocess development and to better condense this knowledge into multi-purpose technologies and applicable mathematical models. Second, the application of verified scale down systems and in silico models for process design and characterization will reduce the required number of large scale batches before dossier submission. Third, the broader availability of mathematical process models and the improvement of process analytical technologies will increase the applicability and acceptance of advanced control and process automation in the manufacturing scale. This will reduce process failure rates and subsequently cost of goods. Along these three aspects we give an overview of recently developed key tools and their potential integration into bioprocess development strategies.

Production of Active Recombinant Hyaluronidase Inclusion Bodies from Apis mellifera in E. coli Bl21(DE3) and characterization by FT-IR Spectroscopy.

The bacterium E. coli is one of the most important hosts for recombinant protein production. The benefits are high growth rates, inexpensive media, and high protein titers. However, complex proteins with high molecular weight and many disulfide bonds are expressed as inclusion bodies (IBs). In the last decade, the overall perception of these IBs being not functional proteins changed, as enzyme activity was found within IBs. Several applications for direct use of IBs are already reported in literature. While fluorescent proteins or protein tags are used for determination of IB activity to date, direct measurements of IB protein activity are scacre. The expression of recombinant hyaluronidase from Apis mellifera in E. coli BL21(DE3) was analyzed using a face centered design of experiment approach. Hyaluronidase is a hard to express protein and imposes a high metabolic burden to the host. Conditions giving a high specific IB titer were found at 25 °C at low specific substrate uptake rates and induction times of 2 to 4 h. The protein activity of hyaluronidase IBs was verified using (Fourier transform) FT-IR spectroscopy. Degradation of the substrate hyaluronan occurred at increased rates with higher IB concentrations. Active recombinant hyaluronidase IBs can be immediately used for direct degradation of hyaluronan without further down streaming steps. FT-IR spectroscopy was introduced as a method for tracking IB activity and showed differences in degradation behavior of hyaluronan dependent on the applied active IB concentration.

Perspectives of inclusion bodies for bio-based products: curse or blessing?

The bacterium Escherichia coli is a major host for recombinant protein production of non-glycosylated products. Depending on the expression strategy, the recombinant protein can be located intracellularly, which often leads to protein aggregates inside of the cytoplasm, forming so the called inclusion bodies (IBs). When compared to other protein expression strategies, inclusion body formation allows high product titers and also the possibility of expressing proteins being toxic for the host. In the past years, the comprehension of inclusion bodies being only inactive protein aggregates changed, and the new term of non-classical inclusion bodies emerged. These inclusion bodies are believed to contain a reasonable amount of active protein within their structure. However, subsequent downstream processing, such as homogenisation of cells, centrifugation or solubilisation of IBs, is prone to variable process performance and is often known to result in low extraction yields. It is hypothesised that variations in IB quality attributes are responsible for those effects and that such attributes can be controlled by upstream process conditions. In this review, we address the impact of process design (process parameters) in the upstream on defined inclusion body quality attributes. The following topics are therefore addressed: (i) an overview of the range of inclusion body applications (including emerging technologies); (ii) analytical methods to determine quality attributes; and (iii) screws in process engineering to achieve the desired quality attributes for different inclusion body-based applications. Process parameters in the upstream can be used to trigger different quality attributes including protein activity, but are not exploited to a satisfying content yet. Design by quality approaches in the upstream are already considered for a multitude of existing processes. Further intensifying this approach may pave the industrial application for new IB-based products and improves IB processing, as discussed within this review.

Custom made inclusion bodies: impact of classical process parameters and physiological parameters on inclusion body quality attributes.

BACKGROUND: The bacterium E. coli is a major host for recombinant protein production of non-glycosylated products. Depending on the expression strategy, the recombinant protein can be located intracellularly. In many cases the formation of inclusion bodies (IBs), protein aggregates inside of the cytoplasm of the cell, is favored in order to achieve high productivities and to cope with toxic products. However, subsequent downstream processing, including homogenization of the cells, centrifugation or solubilization of the IBs, is prone to variable process performance or can be characterized by low extraction yields as published elsewhere. It is hypothesized that variations in IB quality attributes (QA) are responsible for those effects and that such attributes can be controlled by upstream process conditions. This contribution is aimed at analyzing how standard process parameters, such as pH and temperature (T) as well as different controlled levels of physiological parameters, such as specific substrate uptake rates, can vary IB quality attributes. RESULTS: Classical process parameters like pH and T influence the expression of analyzed IB. The effect on the three QAs titer, size and purity could be successfully revealed. The developed data driven model showed that low temperatures and low pH are favorable for the expression of the two tested industrially relevant proteins. Based on this knowledge, physiological control using specific substrate feeding rate (of glucose) qs,Glu is altered and the impact is tested for one protein. CONCLUSIONS: Time dependent monitoring of IB QA-titer, purity, IB bead size-showed a dependence on classical process parameters pH and temperature. These findings are confirmed using a second industrially relevant strain. Optimized process conditions for pH and temperature were used to determine dependence on the physiological parameters, the specific substrate uptake rate (qs,Glu). Higher qs,Glu were shown to have a strong influence on the analyzed IB QAs and drastically increase the titer and purity in early time stages. We therefore present a novel approach to modulate-time dependently-quality attributes in upstream processing to enable robust downstream processing.

Bioconversion of Furanic Compounds by Chlorella vulgaris-Unveiling Biotechnological Potentials.

Lignocellulosic biomass is abundant on Earth, and there are multiple acidic pretreatment options to separate the cellulose, hemicellulose, and lignin fraction. By doing so, the fermentation inhibitors 5-Hydroxymethylfurfural (HMF) and furfural (FF) are produced in varying concentrations depending on the hydrolyzed substrate. In this study, the impact of these furanic compounds on Chlorella vulgaris growth and photosynthetic activity was analyzed. Both compounds led to a prolonged lag phase in Chlorella vulgaris growth. While the photosynthetic yield Y(II) was not significantly influenced in cultivations containing HMF, FF significantly reduced Y(II). The conversion of 5-Hydroxymethylfurfural and furfural to 5-Hydroxymethyl-2-Furoic Acid and 2-Furoic Acid was observed. In total, 100% of HMF and FF was converted in photoautotrophic and mixotrophic Chlorella vulgaris cultivations. The results demonstrate that Chlorella vulgaris is, as of now, the first known microalgal species converting furanic compounds.

Corrigendum: State-of-the-art and novel approaches to mild solubilization of inclusion bodies.

[This corrects the article DOI: 10.3389/fbioe.2023.1249196.].

Inclusion Bodies: Status Quo and Perspectives.

Multiple E. coli cultivations, producing recombinant proteins, lead to the formation of inclusion bodies (IBs). IBs historically were considered as nondesired by-products, due to their time- and cost-intensive purification. Nowadays, many obstacles in IB processing can be overcome. As a consequence, several industrial processes with E. coli favor IB formation over soluble production options due to the high space time yields obtained. Within this chapter, we discuss the state-of-the art biopharmaceutical IB process, review its challenges, highlight the recent developments and perspectives, and also propose alternative solutions, compared to the state-of-the art processing.

Fundamental insights in early-stage inclusion body formation.

Early-stage inclusion body formation is still mysterious. Literature is ambiguous about the existence of rod-shaped protein aggregates, a potential sponge-like inclusion body scaffold as well as the number of inclusion bodies per Escherichia coli cell. In this study, we verified the existence of rod-shaped inclusion bodies, confirmed their porous morphology, the presence of multiple protein aggregates per cell and modelled inclusion body formation as function of the number of generations.

State-of-the-art and novel approaches to mild solubilization of inclusion bodies.

Throughout the twenty-first century, the view on inclusion bodies (IBs) has shifted from undesired by-products towards a targeted production strategy for recombinant proteins. Inclusion bodies can easily be separated from the crude extract after cell lysis and contain the product in high purity. However, additional solubilization and refolding steps are required in the processing of IBs to recover the native protein. These unit operations remain a highly empirical field of research in which processes are developed on a case-by-case basis using elaborate screening strategies. It has been shown that a reduction in denaturant concentration during protein solubilization can increase the subsequent refolding yield due to the preservation of correctly folded protein structures. Therefore, many novel solubilization techniques have been developed in the pursuit of mild solubilization conditions that avoid total protein denaturation. In this respect, ionic liquids have been investigated as promising agents, being able to solubilize amyloid-like aggregates and stabilize correctly folded protein structures at the same time. This review briefly summarizes the state-of-the-art of mild solubilization of IBs and highlights some challenges that prevent these novel techniques from being yet adopted in industry. We suggest mechanistic models based on the thermodynamics of protein unfolding with the aid of molecular dynamics simulations as a possible approach to solve these challenges in the future.

Evaluation of reference genes for transcript analyses in Komagataella phaffii (Pichia pastoris).

BACKGROUND: The yeast Komagataella phaffii (Pichia pastoris) is routinely used for heterologous protein expression and is suggested as a model organism for yeast. Despite its importance and application potential, no reference gene for transcript analysis via RT-qPCR assays has been evaluated to date. In this study, we searched publicly available RNASeq data for stably expressed genes to find potential reference genes for relative transcript analysis by RT-qPCR in K. phaffii. To evaluate the applicability of these genes, we used a diverse set of samples from three different strains and a broad range of cultivation conditions. The transcript levels of 9 genes were measured and compared using commonly applied bioinformatic tools. RESULTS: We could demonstrate that the often-used reference gene ACT1 is not very stably expressed and could identify two genes with outstandingly low transcript level fluctuations. Consequently, we suggest the two genes, RSC1, and TAF10 to be simultaneously used as reference genes in transcript analyses by RT-qPCR in K. phaffii in future RT-qPCR assays. CONCLUSION: The usage of ACT1 as a reference gene in RT-qPCR analysis might lead to distorted results due to the instability of its transcript levels. In this study, we evaluated the transcript levels of several genes and found RSC1 and TAF10 to be extremely stable. Using these genes holds the promise for reliable RT-qPCR results.

A Guideline to Set Up Cascaded Continuous Cultivation with E. coli Bl21 (DE3).

Continuous processing allows to maximize space-time yields and is implemented in many industrial branches. However, in manufacturing of value added compounds produced with microbial hosts, continuous processing is not state-of-the-art yet. This is because fluctuating productivity causes unwanted process deviations. Cascaded continuous bioprocessing, unlike conventional continuous process modes, was found to result in stable productivity. This manuscript serves as a guideline how to set up a cascaded continuous cultivation with Escherichia coli BL21 DE(3).

At-Line Reversed Phase Liquid Chromatography for In-Process Monitoring of Inclusion Body Solubilization.

Refolding is known as the bottleneck in inclusion body (IB) downstream processing in the pharmaceutical industry: high dilutions leading to large operating volumes, slow refolding kinetics and low refolding yields are only a few of the problems that impede industrial application. Solubilization prior to refolding is often carried out empirically and the effects of the solubilizate on the subsequent refolding step are rarely investigated. The results obtained in this study, however, indicate that the quality of the IB solubilizate has a severe effect on subsequent refolding. As the solubilizate contains chaotropic reagents in high molarities, it is commonly analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE, however, suffers from a long analysis time, making at-line analytical implementation difficult. In this study, we established an at-line reversed phase liquid chromatography method to investigate the time-dependent quality of the solubilizate. To verify the necessity of at-line solubilization monitoring, we varied the essential solubilization conditions for horseradish peroxidase IBs. The solubilization time was found to have a major influence on subsequent refolding, underlining the high need for an at-line analysis of solubilization. Furthermore, we used the developed reversed phase liquid chromatography method for an in-process control (IPC). In conclusion, the presented reversed phase liquid chromatography method allows a proper control of IB solubilization applicable for tailored refolding.

Cascaded processing enables continuous upstream processing with E. coli BL21(DE3).

In many industrial sectors continuous processing is already the golden standard to maximize productivity. However, when working with living cells, subpopulation formation causes instabilities in long-term cultivations. In cascaded continuous cultivation, biomass formation and recombinant protein expression can be spatially separated. This cultivation mode was found to facilitate stable protein expression using microbial hosts, however mechanistic knowledge of this cultivation strategy is scarce. In this contribution we present a method workflow to reduce workload and accelerate the establishment of stable continuous processes with E. coli BL21(DE3) exclusively based on bioengineering methods.

Repetitive Fed-Batch: A Promising Process Mode for Biomanufacturing With E. coli.

Recombinant protein production with Escherichia coli is usually carried out in fed-batch mode in industry. As set-up and cleaning of equipment are time- and cost-intensive, it would be economically and environmentally favorable to reduce the number of these procedures. Switching from fed-batch to continuous biomanufacturing with microbials is not yet applied as these cultivations still suffer from time-dependent variations in productivity. Repetitive fed-batch process technology facilitates critical equipment usage, reduces the environmental fingerprint and potentially increases the overall space-time yield. Surprisingly, studies on repetitive fed-batch processes for recombinant protein production can be found for yeasts only. Knowledge on repetitive fed-batch cultivation technology for recombinant protein production in E. coli is not available until now. In this study, a mixed feed approach, enabling repetitive fed-batch technology for recombinant protein production in E. coli, was developed. Effects of the cultivation mode on the space-time yield for a single-cycle fed-batch, a two-cycle repetitive fed-batch, a three-cycle repetitive fed batch and a chemostat cultivation were investigated. For that purpose, we used two different E. coli strains, expressing a model protein in the cytoplasm or in the periplasm, respectively. Our results demonstrate that a repetitive fed-batch for E. coli leads to a higher space-time yield compared to a single-cycle fed-batch and can potentially outperform continuous biomanufacturing. For the first time, we were able to show that repetitive fed-batch technology is highly suitable for recombinant protein production in E. coli using our mixed feeding approach, as it potentially (i) improves product throughput by using critical equipment to its full capacity and (ii) allows implementation of a more economic process by reducing cleaning and set-up times.