Sesquiterpene Cyclase BcBOT2 Promotes the Unprecedented Wagner-Meerwein Rearrangement of the Methoxy Group.

Presilphiperfolan-8ß-ol synthase (BcBOT2), a substrate-promiscuous sesquiterpene cyclase (STC) of fungal origin, is capable of converting two new farnesyl pyrophosphate (FPP) derivatives modified at C7 of farnesyl pyrophosphate (FPP) bearing either a hydroxymethyl group or a methoxymethyl group. These substrates were chosen based on a computationally generated model. Biotransformations yielded five new oxygenated terpenoids. Remarkably, the formation of one of these tricyclic products can only be explained by a cationically induced migration of the methoxy group, presumably via a Meerwein-salt intermediate, unprecedented in synthetic chemistry and biosynthesis. The results show the great principle and general potential of terpene cyclases for mechanistic studies of unusual cation chemistry and for the creation of new terpene skeletons.

Digitalization concepts in academic bioprocess development.

Digitalization with integrated devices, digital and physical assistants, automation, and simulation is setting a new direction for laboratory work. Even with complex research workflows, high staff turnover, and a limited budget some laboratories have already shown that digitalization is indeed possible. However, academic bioprocess laboratories often struggle to follow the trend of digitalization. Due to their diverse research circumstances, high variety of team composition, goals, and limitations the concepts are substantially different. Here, we will provide an overview on different aspects of digitalization and describe how academic laboratories successfully digitalized their working environment. The key aspect is the collaboration and communication between IT-experts and scientific staff. The developed digital infrastructure is only useful if it supports the laboratory worker and does not complicate their work. Thereby, laboratory researchers have to collaborate closely with IT-experts in order for a well-developed and maintainable digitalization concept that fits their individual needs and level of complexity. This review may serve as a starting point or a collection of ideas for the transformation toward a digitalized laboratory.

Endospore production of Bacillus spp. for industrial use.

The increased occurrence of antibiotic resistance and the harmful use of pesticides are a major problem of modern times. A ban on the use of antibiotics as growth promoters in animal breeding has put a focus on the probiotics market. Probiotic food supplements are versatile and show promising results in animal and human nutrition. Chemical pesticides can be substituted by biopesticides, which are very effective against various pests in plants due to increased research. What these fields have in common is the use of spore-forming bacteria. The endospore-forming Bacillus spp. belonging to this group offer an effective solution to the aforementioned problems. Therefore, the biotechnological production of sufficient qualities of such endospores has become an innovative and financially viable field of research. In this review, the production of different Bacillus spp. endospores will be reviewed. For this purpose, the media compositions, cultivation conditions and bioprocess optimization methods of the last 20 years are presented and reflected.

Bioprocess development for endospore production by Bacillus coagulans using an optimized chemically defined medium.

Bacillus coagulans is a promising probiotic, because it combines probiotic properties of Lactobacillus and the ability of Bacillus to form endospores. Due to this hybrid relationship, cultivation of this organism is challenging. As the probiotics market continues to grow, there is a new focus on the production of these microorganisms. In this work, a strain-specific bioprocess for B. coagulans was developed to support growth on one hand and ensure sporulation on the other hand. This circumstance is not trivial, since these two metabolic states are contrary. The developed bioprocess uses a modified chemically defined medium which was further investigated in a one-factor-at-a-time assay after adaptation. A transfer from the shake flask to the bioreactor was successfully demonstrated in the scope of this work. The investigated process parameters included temperature, agitation and pH-control. Especially the pH-control improved the sporulation in the bioreactor when compared to shake flasks. The bioprocess resulted in a sporulation efficiency of 80%-90%. This corresponds to a sevenfold increase in sporulation efficiency due to a transfer to the bioreactor with pH-control. Additionally, a design of experiment (DoE) was conducted to test the robustness of the bioprocess. This experiment validated the beforementioned sporulation efficiency for the developed bioprocess. Afterwards the bioprocess was then scaled up from a 1 L scale to a 10 L bioreactor scale. A comparable sporulation efficiency of 80% as in the small scale was achieved. The developed bioprocess facilitates the upscaling and application to an industrial scale, and can thus help meet the increasing market for probiotics.

Investigation and evaluation of a 3D-printed optical modified cultivation vessel for improved scattered light measurement of biotechnologically relevant organisms.

In the field of bioprocess development miniaturization, parallelization and flexibility play a key role reducing costs and time. To precisely meet these requirements, additive manufacturing (3D-printing) is an ideal technology. 3D-printing enables rapid prototyping and cost-effective fabrication of individually designed devices with complex geometries on demand. For successful bioprocess development, monitoring of process-relevant parameters, such as pH, dissolved oxygen (DO), and biomass, is crucial. Online monitoring is preferred as offline sampling is time-consuming and leads to loss of information. In this study, 3D-printed cultivation vessels with optical prisms are evaluated for the use in upstream processes of different industrially relevant microorganisms and cell lines. It was shown, that the 3D-printed optically modified well (OMW) is of benefit for a wide range of biotechnologically relevant microorganisms and even for mammalian suspension cells. Evaluation tests with Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and Chinese hamster ovary (CHO) cells were performed, providing highly reproducible results. Growth behavior of OMW cultures was comparable to behavior of shake flask (SF) cultivations and the signal to noise ratio in online biomass measurement was shown to be reduced up to 95.8% by using the OMW. Especially the cultivation phases with low turbidity respective optical densities below 1.0 rel.AU could be monitored accurately for the first time. Furthermore, it was demonstrated that the 3D-printed optics are transferable to different well geometries and sizes, enabling efficient biomass monitoring for individual requirements with tailor-made 3D-printed cultivation vessels in small scale.

Mobile SARS­CoV­2 screening facilities for rapid deployment and university-based diagnostic laboratory.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created a public crisis. Many medical and public institutions and businesses went into isolation in response to the pandemic. Because SARS-CoV-2 can spread irrespective of a patient's course of disease, these institutions' continued operation or reopening based on the assessment and control of virus spread can be supported by targeted population screening. For this purpose, virus testing in the form of polymerase chain reaction (PCR) analysis and antibody detection in blood can be central. Mobile SARS-CoV-2 screening facilities with a built-in biosafety level (BSL)-2 laboratory were set up to allow the testing offer to be brought close to the subject group's workplace. University staff members, their expertise, and already available equipment were used to implement and operate the screening facilities and a certified diagnostic laboratory. This operation also included specimen collection, transport, PCR and antibody analysis, and informing subjects as well as public health departments. Screening facilities were established at different locations such as educational institutions, nursing homes, and companies providing critical supply chains for health care. Less than 4 weeks after the first imposed lockdown in Germany, a first mobile testing station was established featuring a build-in laboratory with two similar stations commencing operation until June 2020. During the 15-month project period, approximately 33,000 PCR tests and close to 7000 antibody detection tests were collected and analyzed. The presented approach describes the required procedures that enabled the screening facilities and laboratories to collect and process several hundred specimens each day under difficult conditions. This report can assist others in establishing similar setups for pandemic scenarios.

Sensors and Techniques for On-Line Determination of Cell Viability in Bioprocess Monitoring.

In recent years, the bioprocessing industry has experienced significant growth and is increasingly emerging as an important economic sector. Here, efficient process management and constant control of cellular growth are essential. Good product quality and yield can only be guaranteed with high cell density and high viability. Whereas the on-line measurement of physical and chemical process parameters has been common practice for many years, the on-line determination of viability remains a challenge and few commercial on-line measurement methods have been developed to date for determining viability in industrial bioprocesses. Thus, numerous studies have recently been conducted to develop sensors for on-line viability estimation, especially in the field of optical spectroscopic sensors, which will be the focus of this review. Spectroscopic sensors are versatile, on-line and mostly non-invasive. Especially in combination with bioinformatic data analysis, they offer great potential for industrial application. Known as soft sensors, they usually enable simultaneous estimation of multiple biological variables besides viability to be obtained from the same set of measurement data. However, the majority of the presented sensors are still in the research stage, and only a few are already commercially available.

Comparison of two antibody screening systems for SARS-CoV-2 antibody detection in recovered and vaccinated subjects - test performance and possible indicators for immunity.

BACKGROUND: Detection of seroconversion after SARS-CoV-2-infection or vaccination is relevant to discover subclinical cases and recognize patients with a possible immunity. OBJECTIVES: Test performance, effects of age, time-point of seroconversion and immune status regarding neutralizing antibodies (NAbs) and T-cell-reactivity were investigated. STUDY DESIGN: Two antibody assays (Viramed-Test for S/N-specific IgG, Roche-Test for N-specific IgA, -M, -G) were evaluated with classified samples. In total, 381 subjects aged 6-99 years, who had either recovered from the disease or had been vaccinated, were screened for SARS-CoV-2-specific antibodies. This screening was part of an open observational study with working adults. Additionally, children and adults were analyzed in a longitudinal COVID-19 study in schools. For immunity evaluation, virus neutralization tests and ELISpot tests were performed in a subgroup of subjects. RESULTS: Viramed revealed a slightly lower test performance than Roche, but test quality was equally well in samples from very young or very old donors. The time-point of seroconversion after the respective immunization detected by the two tests was not significantly different. N-specific antibodies, detected with Roche, highly correlated with NAbs in recovered subjects, whereas a positive Viramed-Test result was paralleled by a positive ELISpot result. CONCLUSION: Viramed-Test was not as sensitive as Roche-Test, but highly specific and beneficial to distinguish between recovered and vaccinated status. For both tests correlations with humoral and cellular immunity were found. Of note, the expected early detection of IgA and IgM by the Roche-Test did not prove to be an advantage over IgG testing by Viramed.

Study on the development and integration of 3D-printed optics in small-scale productions of single-use cultivation vessels.

Integrating optical sensors and 3D-printed optics into single-use (SU) cultivation vessels for customized, tailor-made equipment could be a next big step in the bioreactor and screening platform development enabling online bioprocess monitoring. Many different parameters such as pH, oxygen, carbon dioxide and optical density (OD) can be monitored more easily using online measuring instruments compared to offline sampling. Space-saving integrated sensors in combination with adapted optics such as prisms open up vastly new possibilities to precisely guide light through SU vessels. This study examines how optical prisms can be 3D-printed with a 3D-inkjet printer, modified and then evaluated in a custom made optical bench. The prisms are coated or bonded with thin cover glasses. For the examination of reflectance performance and conformity prisms are compared on the basis of measured characteristics of a conventional glass prism. In addition, the most efficient and reproducible prism geometry and modification technique is applied to a customized 3D-printed cultivation vessel. The vessel is evaluated on a commercial sensor-platform, a shake flask reader (SFR) vario, to investigate its sensing-characteristics while monitoring scattered light with the turbidity standard formazine and a cell suspension of Saccharomyces cerevisiae as model organism. It is demonstrated that 3D-printed prisms can be used in combination with commercial scattered light sensor-platforms to determine OD of a microbial culture and that a 3D-printed unibody design with integrated optics in a cultivation vessel is feasible. In the range of OD600 0-1.16 rel.AU a linear correlation between sensor amplitude and offline determined OD can be achieved. Thus, enabling for the first time a measurement of low cell densities with the SFR vario platform. Moreover, sensitivity is also at least three times higher compared to the commonly used method.

Facilitated endospore detection for Bacillus spp. through automated algorithm-based image processing.

Bacillus spp. endospores are important dormant cell forms and are distributed widely in environmental samples. While these endospores can have important industrial value (e.g. use in animal feed as probiotics), they can also be pathogenic for humans and animals, emphasizing the need for effective endospore detection. Standard spore detection by colony forming units (CFU) is time-consuming, elaborate and prone to error. Manual spore detection by spore count in cell counting chambers via phase-contrast microscopy is less time-consuming. However, it requires a trained person to conduct. Thus, the development of a facilitated spore detection tool is necessary. This work presents two alternative quantification methods: first, a colorimetric assay for detecting the biomarker dipicolinic acid (DPA) adapted to modern needs and applied for Bacillus spp. and second, a model-based automated spore detection algorithm for spore count in phase-contrast microscopic pictures. This automated spore count tool advances manual spore detection in cell counting chambers, and does not require human overview after sample preparation. In conclusion, this developed model detected various Bacillus spp. endospores with a correctness of 85-89%, and allows an automation and time-saving of Bacillus endospore detection. In the laboratory routine, endospore detection and counting was achieved within 5-10 min, compared to up to 48 h with conventional methods. The DPA-assay on the other hand enabled very accurate spore detection by simple colorimetric measurement and can thus be applied as a reference method.

3D-Printed microfluidic device for protein purification in batch chromatography.

Modern 3D printers enable not only rapid prototyping, but also high-precision printing-microfluidic devices with channel diameters of just a few micrometres can now be readily assembled using this technology. Such devices offer a myriad of benefits (including miniaturization) that significantly reduce sample and buffer volumes and lead to lower process costs. Although such microfluidic devices are already widely used in the field of biotechnology, there is a lack of research regarding the potential of miniaturization by 3D-printed devices in lab-scale chromatography. In this study, the efficacy of a 3D-printed microfluidic device which provides a substantially lower dead-volume compared to established chromatography systems is demonstrated for batch purification applications. Furthermore, this device enables straightforward integration of various components (such as microfluidic valves and chromatographic units) in an unprecedentedly flexible fashion. Initial proof-of-concept experiments demonstrate successful gradient elution with bovine serum albumin (BSA), and the purification of a pharmaceutically relevant IgG monoclonal antibody (mAb).

A versatile ceramic capillary membrane reactor system for continuous enzyme-catalyzed hydrolysis.

As an alternative to classical batch processes, enzyme-catalyzed hydrolysis can also be carried out continuously. To facilitate this, a continuous ceramic capillary membrane reactor system (CCCMRS) was developed which can be operated with various proteolytic enzymes immobilized on the porous ceramic capillary membranes. This system has several advantages over common batch processes regarding stability, reproducibility and controllability and can easily be adapted to optimal reaction conditions and individual preferences. Two exemplary applications utilizing the CCCMRS were carried out and investigated in long-term stability studies. In the first application the continuous enzymatic cleavage of human IgG into the antibody fragments Fab and Fc by immobilized papain was performed. A total volume of 22 mL of 1 mg mL-1 IgG-solution was enzymatically cleaved over a period of 33.3 h. The antibody cleavage products could be detected in an SEC-HPLC over the whole process time thus indicating long-term stability of the continuous hydrolysis process. The second application investigated the continuous digestion of pea and almond protein isolates by immobilized Alcalase resulting in the generation of a large variety of different peptides. This peptide fingerprint remains constant over a long period of time enabling fractionation and thus making the peptides accessible for further bioactivity studies in sufficient quantities. The constant peptide fingerprint could be shown in the RP-HPLC analysis for all 30 samples with a total volume of 29.7 mL collected over a period of 45 h.

Implementing a digital infrastructure for the lab using a central laboratory server and the SiLA2 communication standard.

In this report, a fully integrated solution for laboratory digitization is presented. The approach presents a flexible and complete integration method for the digitally assisted workflow. The worker in the laboratory performs procedures in direct interaction with the digitized infrastructure that guides through the process and aids while performing tasks. The digital transformation of the laboratory starts with standardized integration of both new and "smart" lab devices, as well as legacy devices through a hardware gateway module. The open source Standardization in Lab Automation 2 standard is used for device communication. A central lab server channels all device communication and keeps a database record of every measurement, task and result generated or used in the lab. It acts as a central entry point for process management. This backbone enables a process control system to guide the worker through the lab process and provide additional assistance, like results of automated calculations or safety information. The description of the infrastructure and architecture is followed by a practical example on how to implement a digitized workflow. This approach is highly useful for - but not limited to - the biotechnological laboratory and has the potential to increase productivity in both industry and research for example by enabling automated documentation.

Digitalization and Bioprocessing: Promises and Challenges.

The production of pharmaceuticals, industrial chemicals, and food ingredients from biotechnological processes is a vast and rapidly growing industry. While advances in synthetic biology and metabolic engineering have made it possible to produce thousands of new molecules from cells, few of these molecules have reached the market. The traditional methods of strain and bioprocess development that transform laboratory results to industrial processes are slow and use computers and networks only for data acquisition and storage. Digitalization, machine learning (ML), and artificial intelligence (AI) methods are transforming many fields - how can they be applied to bioprocessing to overcome current bottlenecks? What are the challenges, especially for regulatory issues, in the production of biopharmaceuticals? This chapter begins with a discussion of the current challenges for strain and bioprocess development and then considers how digitalization can be used to approach these tasks in completely new ways. Finally, regulatory considerations are addressed, with the goal of incorporating these issues from the outset as new digitalization methods are created.

Accelerated production of α2,8- and α2,9-linked polysialic acid in recombinant Escherichia coli using high cell density cultivation.

Polysialic acid (polySia) are α2,8- and/or α2,9-linked homopolymers with interesting properties for meningococcal vaccine development or the cure of human neurodegenerative disorders. With the goal to avoid large scale production of pathogenic bacteria, we compare in the current study the efficacy of conventional polySia production to recombinant approaches using the engineered laboratory safety strain E. coli BL21. High cell density cultivation (HCDC) experiments were performed in two different bioreactor systems. Increased cell densities of up to 11.3 (±0.4) g/L and polySia concentrations of up to 774 (±18) mg/L were reached in E. coli K1. However, cultivation of engineered E. coli BL21 strains delivered comparable cell densities but a maximum of only 133 mg/L polySia. Using established downstream procedures, host cell DNA and proteins were removed. All recombinant polySia products showed an identical degree of polymerization >90. Polymers with different glycosidic linkages could be successfully differentiated by nuclear magnetic resonance spectroscopy.

Whole-Cell Production of Patchouli Oil Sesquiterpenes in Escherichia coli: Metabolic Engineering and Fermentation Optimization in Solid-Liquid Phase Partitioning Cultivation.

Patchouli oil is a major ingredient in perfumery, granting a dark-woody scent due to its main constituent (-)-patchoulol. The growing demand for patchouli oil has raised interest in the development of a biotechnological process to assure a reliable supply. Herein, we report the production of patchouli oil sesquiterpenes by metabolically engineered Escherichia coli strains, using solid-liquid phase partitioning cultivation. The (-)-patchoulol production was possible using the endogenous methylerythritol phosphate pathway and overexpressing a (-)-patchoulol synthase isoform from Pogostemon cablin but at low titers. To improve the (-)-patchoulol production, the exogenous mevalonate pathway was overexpressed in the multi-plasmid PTS + Mev strain, which increased the (-)-patchoulol titer 5-fold. Fermentation was improved further by evaluating several defined media, and optimizing the pH and temperature of culture broth, enhancing the (-)-patchoulol titer 3-fold. To augment the (-)-patchoulol recovery from fermentation, the solid-liquid phase partitioning cultivation was analyzed by screening polymeric adsorbers, where the Diaion HP20 adsorber demonstrated the highest (-)-patchoulol recovery from all tests. Fermentation was scaled-up to fed-batch bioreactors, reaching a (-)-patchoulol titer of 40.2 mg L-1 and productivity of 20.1 mg L-1 d-1. The terpene profile and aroma produced from the PTS + Mev strain were similar to the patchouli oil, comprising (-)-patchoulol as the main product, and α-bulnesene, trans-ß-caryophyllene, ß-patchoulene, and guaia-5,11-diene as side products. This investigation represents the first study of (-)-patchoulol production in E. coli by solid-liquid phase partitioning cultivation, which provides new insights for the development of sustainable bioprocesses for the microbial production of fragrant terpenes.

Bringing IoT to the Lab: SiLA2 and Open-Source-Powered Gateway Module for Integrating Legacy Devices into the Digital Laboratory.

In this article a gateway module to integrate legacy laboratory devices into the network of the digital laboratory in the 21st century is introduced. The device is based on ready to buy consumer hardware that is easy to get and inexpensive. Depending on the specific requirements of the desired application (bare embedded computer, RS232 serial port connector, IP65 certified casing and connectors) the needed investment ranges from about 95 € up to 200 €. The embedded computer runs an open source Linux operating system and can in principle be used to run any kind of software needed for communicating with the laboratory device. Here the open source SiLA2 standard is used for presenting the device's functions in the network. As an example the digital integration of a magnetic stirrer is shown and can be used as a template for other applications. A method for easy remote integration of the device to ensure an easy and consistent workflow in development, testing and usage is also presented. This incorporates a method for remote installation of SiLA2 servers on the box as well as a web frontend for administration, debugging and management of those.

Optimization of factors influencing enzyme activity and product selectivity and the role of proton transfer in the catalytic mechanism of patchoulol synthase.

The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (-)-patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH-value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2-(N-morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl2 at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism.

Membrane Adsorber for the Fast Purification of a Monoclonal Antibody Using Protein A Chromatography.

Monoclonal antibodies are conquering the biopharmaceutical market because they can be used to treat a variety of diseases. Therefore, it is very important to establish robust and optimized processes for their production. In this article, the first step of chromatography (Protein A chromatography) in monoclonal antibody purification was optimized with a focus on the critical elution step. Therefore, different buffers (citrate, glycine, acetate) were tested for chromatographic performance and product quality. Membrane chromatography was evaluated because it promises high throughputs and short cycle times. The membrane adsorber Sartobind® Protein A 2 mL was used to accelerate the purification procedure and was further used to perform a continuous chromatographic run with a four-membrane adsorber-periodic counter-current chromatography (4MA-PCCC) system. It was found that citrate buffer at pH 3.5 and 0.15 M NaCl enabled the highest recovery of >95% and lowest total aggregate content of 0.26%. In the continuous process, the capacity utilization of the membrane adsorber was increased by 20%.

Improved Production and In Situ Recovery of Sesquiterpene (+)-Zizaene from Metabolically-Engineered E. coli.

The sesquiterpene (+)-zizaene is the direct precursor of khusimol, the main fragrant compound of the vetiver essential oil from Chrysopogon zizanioides and used in nearly 20% of men's fine perfumery. The biotechnological production of such fragrant sesquiterpenes is a promising alternative towards sustainability; nevertheless, product recovery from fermentation is one of the main constraints. In an effort to improve the (+)-zizaene recovery from a metabolically-engineered Escherichia coli, we developed an integrated bioprocess by coupling fermentation and (+)-zizaene recovery using adsorber extractants. Initially, (+)-zizaene volatilization was confirmed from cultivations with no extractants but application of liquid-liquid phase partitioning cultivation (LLPPC) improved (+)-zizaene recovery nearly 4-fold. Furthermore, solid-liquid phase partitioning cultivation (SLPPC) was evaluated by screening polymeric adsorbers, where Diaion HP20 reached the highest recovery. Bioprocess was scaled up to 2 L bioreactors and in situ recovery configurations integrated to fermentation were evaluated. External recovery configuration was performed with an expanded bed adsorption column and improved (+)-zizaene titers 2.5-fold higher than LLPPC. Moreover, internal recovery configuration (IRC) further enhanced the (+)-zizaene titers 2.2-fold, whereas adsorption velocity was determined as critical parameter for recovery efficiency. Consequently, IRC improved the (+)-zizaene titer 8.4-fold and productivity 3-fold from our last report, achieving a (+)-zizaene titer of 211.13 mg L-1 and productivity of 3.2 mg L-1 h-1. This study provides further knowledge for integration of terpene bioprocesses by in situ product recovery, which could be applied for many terpene studies towards the industrialization of fragrant molecules.