Analysis of protein secretion in Bacillus subtilis by combining a secretion stress biosensor strain with an in vivo split GFP assay.

BACKGROUND: Bacillus subtilis is one of the workhorses in industrial biotechnology and well known for its secretion potential. Efficient secretion of recombinant proteins still requires extensive optimization campaigns and screening with activity-based methods. However, not every protein can be detected by activity-based screening. We therefore developed a combined online monitoring system, consisting of an in vivo split GFP assay for activity-independent target detection and an mCherry-based secretion stress biosensor. The split GFP assay is based on the fusion of a target protein to the eleventh ß-sheet of sfGFP, which can complement a truncated sfGFP that lacks this ß-sheet named GFP1-10. The secretion stress biosensor makes use of the CssRS two component quality control system, which upregulates expression of mCherry in the htrA locus thereby allowing a fluorescence readout of secretion stress. RESULTS: The biosensor strain B. subtilis PAL5 was successfully constructed by exchanging the protease encoding gene htrA with mCherry via CRISPR/Cas9. The Fusarium solani pisi cutinase Cut fused to the GFP11 tag (Cut11) was used as a model enzyme to determine the stress response upon secretion mediated by signal peptides SPPel, SPEpr and SPBsn obtained from naturally secreted proteins of B. subtilis. An in vivo split GFP assay was developed, where purified GFP1-10 is added to the culture broth. By combining both methods, an activity-independent high-throughput method was created, that allowed optimization of Cut11 secretion. Using the split GFP-based detection assay, we demonstrated a good correlation between the amount of secreted cutinase and the enzymatic activity. Additionally, we screened a signal peptide library and identified new signal peptide variants that led to improved secretion while maintaining low stress levels. CONCLUSION: Our results demonstrate that the combination of a split GFP-based detection assay for secreted proteins with a secretion stress biosensor strain enables both, online detection of extracellular target proteins and identification of bottlenecks during protein secretion in B. subtilis. In general, the system described here will also enable to monitor the secretion stress response provoked by using inducible promoters governing the expression of different enzymes.

[Development, Evaluation and Implementation of Digital Health Interventions (Part 1) - Discussion Paper of the Digital Health Working Group of the German Network for Health Services Research (DNVF)]. / Digitale Gesundheitsinterventionen entwickeln, evaluieren und implementieren (Teil I) ­ Diskussionspapier der Arbeitsgruppe Digital Health des Deutschen Netzwerk Versorgungsforschung (DNVF).

The development and application of digital interventions in health-related topics are gaining momentum in health service research. Digital interventions are often complex and need to be evaluated and implemented in complex settings. Due to their characteristics, this poses methodological challenges for health services research that have to be identified and addressed. Hence, the Working Group on Digital Health of the German Network for Health Services Research (DNVF) has prepared a discussion paper. This paper discusses methodological, practical and theoretical challenges associated with the development and evaluation of digital interventions from the perspective of health services research. Possible solutions are suggested and future research needs to address these methodological challenges are identified.

[Developing, Evaluating and Implementing Digital Health Interventions Part II - Discussion Paper of the Digital Health Working Group of the German Network for Health Services Research (DNVF)]. / Digitale Gesundheitsinterventionen entwickeln, evaluieren und implementieren Teil II ­ Diskussionspapier der Arbeitsgruppe Digital Health des Deutschen Netzwerk Versorgungsforschung (DNVF).

The methodological challenges of evaluating digital interventions (DI) for health services research are omnipresent. The Digital Health Working Group of the German Network for Health Services Research (DNVF) presented and discussed these challenges in a two-part discussion paper. The first part addressed challenges in definition, development and evaluation of DI. In this paper, which represents the second part, the definition of outcomes, reporting of results, synthesis of evidence, and implementation are addressed as methodological challenges of DI. Potential solutions are presented and the need to address these challenges in future research are discussed.

Accelerated strain construction and characterization of C. glutamicum protein secretion by laboratory automation.

Secretion of bacterial proteins into the culture medium simplifies downstream processing by avoiding cell disruption for target protein purification. However, a suitable signal peptide for efficient secretion needs to be identified, and currently, there are no tools available to predict optimal combinations of signal peptides and target proteins. The selection of such a combination is influenced by several factors, including protein biosynthesis efficiency and cultivation conditions, which both can have a significant impact on secretion performance. As a result, a large number of combinations must be tested. Therefore, we have developed automated workflows allowing for targeted strain construction and secretion screening using two platforms. Key advantages of this experimental setup include lowered hands-on time and increased throughput. In this study, the automated workflows were established for the heterologous production of Fusarium solani f. sp. pisi cutinase in Corynebacterium glutamicum. The target protein was monitored in culture supernatants via enzymatic activity and split GFP assay. Varying spacer lengths between the Shine-Dalgarno sequence and the start codon of Bacillus subtilis signal peptides were tested. Consistent with previous work on the secretory cutinase production in B. subtilis, a ribosome binding site with extended spacer length to up to 12 nt, which likely slows down translation initiation, does not necessarily lead to poorer cutinase secretion by C. glutamicum. The best performing signal peptides for cutinase secretion with a standard spacer length were identified in a signal peptide screening. Additional insights into the secretion process were gained by monitoring secretion stress using the C. glutamicum K9 biosensor strain. KEY POINTS: • Automated workflows for strain construction and screening of protein secretion • Comparison of spacer, signal peptide, and host combinations for cutinase secretion • Signal peptide screening for secretion by C. glutamicum using the split GFP assay.

Biosensor-Based Optimization of Cutinase Secretion by Corynebacterium glutamicum.

The industrial microbe Corynebacterium glutamicum is gaining substantial importance as a platform host for recombinant protein secretion. We recently developed a fluorescence-based (eYFP) C. glutamicum reporter strain for the quantification of Sec-dependent protein secretion by monitoring the secretion-related stress response and now demonstrate its applicability in optimizing the secretion of the heterologous enzyme cutinase from Fusarium solani pisi. To drive secretion, either the poor-performing PelSP or the potent NprESP Sec signal peptide from Bacillus subtilis was used. To enable easy detection and quantification of the secreted cutinase we implemented the split green fluorescent protein (GFP) assay, which relies on the GFP11-tag fused to the C-terminus of the cutinase, which can complement a truncated GFP thereby reconstituting its fluorescence. The reporter strain was transformed with different mutant libraries created by error-prone PCR, which covered the region of the signal peptide and the N-terminus of the cutinase. Fluorescence-activated cell sorting (FACS) was performed to isolate cells that show increased fluorescence in response to increased protein secretion stress. Five PelSP variants were identified that showed a 4- to 6-fold increase in the amount and activity of the secreted cutinase (up to 4,100 U/L), whereas two improved NprESP variants were identified that showed a ∼35% increase in secretion, achieving ∼5,500 U/L. Most of the isolated variants carried mutations in the h-region of the signal peptide that increased its overall hydrophobicity. Using site-directed mutagenesis it was shown that the combined mutations F11I and P16S within the hydrophobic core of the PelSP are sufficient to boost cutinase secretion in batch cultivations to the same level as achieved by the NprESP. Screening of a PelSP mutant library in addition resulted in the identification of a cutinase variant with an increased specific activity, which was attributed to the mutation A85V located within the substrate-binding region. Taken together the biosensor-based optimization approach resulted in a substantial improvement of cutinase secretion by C. glutamicum, and therefore represents a valuable tool that can be applied to any secretory protein of interest.

[Interdisciplinary care path and potential IT support for people with rare diseases in Germany]. / Interdisziplinärer Versorgungspfad und mögliche IT-Unterstützung für Menschen mit Seltenen Erkrankungen in Deutschland.

INTRODUCTION: Due to the high variability and, at the same time, rare occurrence of rare diseases, the diagnosis of these patients (approx. 4 million people in Germany) can turn into an odyssey. The large time interval between the appearance of symptoms and the final diagnosis of the rare disease leads to a delay in the appropriate treatment. The often long period of uncertainty about the cause of symptoms as well as non-specific or even wrong therapies can have negative effects on both the course of disease and the patients' quality of life. For a better understanding of the current care situation and IT landscape, the interdisciplinary care pathway for people with rare diseases will be modelled and the possible uses of IT applications identified that have the potential to improve diagnosis, treatment and therapy of rare diseases. METHODS: In order to achieve these goals, an initial care pathway was modelled on the basis of process descriptions which are commonly used in the literature, discussed in detail, and agreed upon in a first workshop with six experts from outpatient and inpatient care as well as employees of Centers for Rare Diseases. In a second workshop, ten experts analyzed the resulting care pathway with regard to the possible use of IT applications, and the identification was agreed upon. The experts included those involved in the process, in particular physicians, patients / patient representatives, health care researchers, and experts in hospital IT, IT security, and data protection. RESULTS: The two workshops resulted in process models including the specification of possible uses for IT applications. The most important steps in the care pathway for people with rare diseases in Germany include: neonatal screening, seeking medical advice, outpatient care by general practitioners, outpatient care by specialists, care by specialist outpatient clinic, care by clinic, care by a Center for Rare Diseases: case review and case conference and treatment and therapy. The discussion of the possible uses of IT applications resulted in a focus on registers (e. g. with regard to experts, treatment and therapy options) as well as on digital tools, such as "digital findings and findings platform" and "digital referral with referral tracking". DISCUSSION: Our results show that the care pathway is very heterogeneous and complex. Thus, the sub-processes show different variants with many branches and repetitions. They also illustrate that the care for people with rare diseases requires a high level of interdisciplinary collaboration; diagnosis as well as treatment and therapy often take place across sectors and in cooperation between different medical health care institutions and professions. When analyzing the current IT landscape, it becomes clear that IT applications can be used at many process steps in the care for people with rare diseases and have a high potential. Therefore, they must be used to inform decisions about the adequate diagnosis and treatment as well as communication about the clinical pictures and the patient's case between practitioners and medical care sectors. CONCLUSION: The interdisciplinary collaboration highlights the need for cooperation between the various parties involved in the process, which requires the identification and implementation of interfaces between the stakeholders and their systems. However, it is not enough to include the view of the processes; the data perspective is also required. Creating interoperability also enables the use of IT applications. The basis for this is the results obtained.

The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis.

BACKGROUND: Bacillus subtilis is one of the most important microorganisms for recombinant protein production. It possesses the GRAS (generally recognized as safe) status and a potent protein secretion capacity. Secretory protein production greatly facilitates downstream processing and thus significantly reduces costs. However, not all heterologous proteins are secreted and intracellular production poses difficulties for quantification. To tackle this problem, we have established a so-called intracellular split GFP (iSplit GFP) assay in B. subtilis as a tool for the in vivo protein detection during expression in batch cultures and at a single-cell level. For the iSplit GFP assay, the eleventh ß-sheet of sfGFP is fused to a target protein and can complement a detector protein consisting of the respective truncated sfGFP (GFP1-10) to form fluorescent holo-GFP. RESULTS: As proof of concept, the GFP11-tag was fused C-terminally to the E. coli ß-glucuronidase GUS, resulting in fusion protein GUS11. Variable GUS and GUS11 production levels in B. subtilis were achieved by varying the ribosome binding site via spacers of increasing lengths (4-12 nucleotides) for the GUS-encoding gene. Differences in intracellular enzyme accumulation were determined by measuring the GUS11 enzymatic activity and subsequently by adding the detector protein to respective cell extracts. Moreover, the detector protein was co-produced with the GUS11 using a two-plasmid system, which enabled the in vivo detection and online monitoring of glucuronidase production. Using this system in combination with flow cytometry and microfluidics, we were able to monitor protein production at a single-cell level thus yielding information about intracellular protein distribution and culture heterogeneity. CONCLUSION: Our results demonstrate that the iSplit GFP assay is suitable for the detection, quantification and online monitoring of recombinant protein production in B. subtilis during cultivation as well as for analyzing production heterogeneity and intracellular localization at a single-cell level.

Use of Patient-Reported Outcome Measures and Patient-Reported Experience Measures Within Evaluation Studies of Telemedicine Applications: Systematic Review.

BACKGROUND: With the rise of digital health technologies and telemedicine, the need for evidence-based evaluation is growing. Patient-reported outcome measures (PROMs) and patient-reported experience measures (PREMs) are recommended as an essential part of the evaluation of telemedicine. For the first time, a systematic review has been conducted to investigate the use of PROMs and PREMs in the evaluation studies of telemedicine covering all application types and medical purposes. OBJECTIVE: This study investigates the following research questions: in which scenarios are PROMs and PREMs collected for evaluation purposes, which PROM and PREM outcome domains have been covered and how often, which outcome measurement instruments have been used and how often, does the selection and quantity of PROMs and PREMs differ between study types and application types, and has the use of PROMs and PREMs changed over time. METHODS: We conducted a systematic literature search of the MEDLINE and Embase databases and included studies published from inception until April 2, 2020. We included studies evaluating telemedicine with patients as the main users; these studies reported PROMs and PREMs within randomized controlled trials, controlled trials, noncontrolled trials, and feasibility trials in English and German. RESULTS: Of the identified 2671 studies, 303 (11.34%) were included; of the 303 studies, 67 (22.1%) were feasibility studies, 70 (23.1%) were noncontrolled trials, 20 (6.6%) were controlled trials, and 146 (48.2%) were randomized controlled trials. Health-related quality of life (n=310; mean 1.02, SD 1.05), emotional function (n=244; mean 0.81, SD 1.18), and adherence (n=103; mean 0.34, SD 0.53) were the most frequently assessed outcome domains. Self-developed PROMs were used in 21.4% (65/303) of the studies, and self-developed PREMs were used in 22.3% (68/303). PROMs (n=884) were assessed more frequently than PREMs (n=234). As the evidence level of the studies increased, the number of PROMs also increased (τ=-0.45), and the number of PREMs decreased (τ=0.35). Since 2000, not only has the number of studies using PROMs and PREMs increased, but the level of evidence and the number of outcome measurement instruments used have also increased, with the number of PREMs permanently remaining at a lower level. CONCLUSIONS: There have been increasingly more studies, particularly high-evidence studies, which use PROMs and PREMs to evaluate telemedicine. PROMs have been used more frequently than PREMs. With the increasing maturity stage of telemedicine applications and higher evidence level, the use of PROMs increased in line with the recommendations of evaluation guidelines. Health-related quality of life and emotional function were measured in almost all the studies. Simultaneously, health literacy as a precondition for using the application adequately, alongside proper training and guidance, has rarely been reported. Further efforts should be pursued to standardize PROM and PREM collection in evaluation studies of telemedicine.

Effect of Photocaged Isopropyl ß-d-1-thiogalactopyranoside Solubility on the Light Responsiveness of LacI-controlled Expression Systems in Different Bacteria.

Photolabile protecting groups play a significant role in controlling biological functions and cellular processes in living cells and tissues, as light offers high spatiotemporal control, is non-invasive as well as easily tuneable. In the recent past, photo-responsive inducer molecules such as 6-nitropiperonyl-caged IPTG (NP-cIPTG) have been used as optochemical tools for Lac repressor-controlled microbial expression systems. To further expand the applicability of the versatile optochemical on-switch, we have investigated whether the modulation of cIPTG water solubility can improve the light responsiveness of appropriate expression systems in bacteria. To this end, we developed two new cIPTG derivatives with different hydrophobicity and demonstrated both an easy applicability for the light-mediated control of gene expression and a simple transferability of this optochemical toolbox to the biotechnologically relevant bacteria Pseudomonas putida and Bacillus subtilis. Notably, the more water-soluble cIPTG derivative proved to be particularly suitable for light-mediated gene expression in these alternative expression hosts.

[Evaluation of a future scenario concerning the use of big data applications to improve the care of people with rare diseases]. / Evaluation eines Zukunftsszenarios zur Nutzung von Big-Data-Anwendungen für die Verbesserung der Versorgung von Menschen mit seltenen Erkrankungen.

INTRODUCTION: In Germany there are about 4 million people living with a rare disease. Studies have shown that big data applications can improve diagnosis of and research on rare diseases more effectively. However, no concrete comprehensive concept for the use of big data in the care of people with rare diseases has so far been established in Germany. As part of the project "BIDA-SE", which is funded by the German Ministry of Health, a first scenario has been designed to show how big data applications can be usefully incorporated into the care of people with rare diseases. METHODS: The aim of the present study was to evaluate this scenario with regard to acceptance, (clinical) benefits, economic aspects, and limitations and barriers to its implementation. To evaluate the scenario, an online survey was conducted in Germany in October/November 2019 amongst a total of N = 9 physicians, N = 69 patients with rare diseases/patient representatives, N = 14 IT experts and N = 21 health care researchers. The online questionnaire consisted of both standardized, validated questions taken from already tested survey instruments and additional questions which were constructed on the basis of a preceding literature analysis. The evaluation of the survey was primarily descriptive, with a calculation of frequencies, mean values and standard deviations. RESULTS: The results of the evaluation show that the scenario has been accepted by a majority of all groups surveyed (physicians, patients/patient representatives, IT experts and health care researchers). From the point of view of physicians, patients/patient representatives and health care researchers, the scenario has the potential to accelerate the diagnosis and initiation of therapy and to improve cross-sectoral treatment. From the physician's and health care researcher's perspective, investments in the application presented in the scenario would be profitable. Financing the scenario would, however, require adjusting the reimbursement situation. The limitations and barriers identified by all groups for a medium-term implementation of the scenario can be grouped into seven thematic areas where action is needed: (1) financing and investment, (2) data protection and data security, (3) standards/data sources/data quality, (4) acceptance of technology, (5) integration into the daily work routine, (6) knowledge about availability as well as (7) habits and preferences/physician's role. DISCUSSION: With the present study, a first interdisciplinary, practical scenario using big data applications was evaluated with regard to acceptance, benefits and limitations/barriers. The scenario is widely accepted among the majority of all surveyed target groups and is considered (clinically) useful, although legal, organisational and technical barriers still need to be overcome for its medium-term implementation. The evaluation results contribute to the derivation of recommendations for action to ensure the medium-term implementation of the scenario and to channel access to the Centres for Rare Diseases in the future. CONCLUSION: Many activities have been initiated at a national level to improve the health care situation of people with rare diseases. The scenario developed in the "BIDA-SE" project complements these research activities and illustrates how big data applications can be usefully implemented into practice to improve the diagnosis and therapy of people with rare diseases in a sustainable way.

Parkinson Network Eastern Saxony (PANOS): Reaching Consensus for a Regional Intersectoral Integrated Care Concept for Patients with Parkinson's Disease in the Region of Eastern Saxony, Germany.

As integrated care is recognized as crucial to meet the challenges of chronic conditions such as Parkinson's disease (PD), integrated care networks have emerged internationally and throughout Germany. One of these networks is the Parkinson Network Eastern Saxony (PANOS). PANOS aims to deliver timely and equal care to PD patients with a collaborative intersectoral structured care pathway. Additional components encompass personalized case management, an electronic health record, and communicative and educative measures. To reach an intersectoral consensus of the future collaboration in PANOS, a structured consensus process was conducted in three sequential workshops. Community-based physicians, PD specialists, therapists, scientists and representatives of regulatory authorities and statutory health insurances were asked to rate core pathway-elements and supporting technological, personal and communicative measures. For the majority of core elements/planned measures, a consensus was reached, defined as an agreement by >75% of participants. Additionally, six representatives from all partners involved in the network-design independently assessed PANOS based on the Development Model for Integrated Care (DMIC), a validated model addressing the comprehensiveness and maturity of integrated care concepts. The results show that PANOS is currently in an early maturation state but has the potential to comprehensively represent the DMIC if all planned activities are implemented successfully. Despite the favorable high level of consensus regarding the PANOS concept and despite its potential to become a balanced integrated care concept according to the DMIC, its full implementation remains a considerable challenge.

The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis.

BACKGROUND: Bacillus subtilis is widely used for the industrial production of recombinant proteins, mainly due to its high secretion capacity, but higher production yields can be achieved only if bottlenecks are removed. To this end, a crucial process is translation initiation which takes place at the ribosome binding site enclosing the Shine Dalgarno sequence, the start codon of the target gene and a short spacer sequence in between. Here, we have studied the effects of varying spacer sequence lengths in vivo on the production yield of different intra- and extracellular proteins. RESULTS: The shuttle vector pBSMul1 containing the strong constitutive promoter PHpaII and the optimal Shine Dalgarno sequence TAAGGAGG was used as a template to construct a series of vectors with spacer lengths varying from 4 to 12 adenosines. For the intracellular proteins GFPmut3 and ß-glucuronidase, an increase of spacer lengths from 4 to 7-9 nucleotides resulted in a gradual increase of product yields up to 27-fold reaching a plateau for even longer spacers. The production of secreted proteins was tested with cutinase Cut and swollenin EXLX1 which were N-terminally fused to one of the Sec-dependent signal peptides SPPel, SPEpr or SPBsn. Again, longer spacer sequences resulted in up to tenfold increased yields of extracellular proteins. Fusions with signal peptides SPPel or SPBsn revealed the highest production yields with spacers of 7-10nt length. Remarkably, fusions with SPEpr resulted in a twofold lower production yield with 6 or 7nt spacers reaching a maximum with 10-12nt spacers. This pattern was observed for both secreted proteins fused to SPEpr indicating a dominant role also of the nucleotide sequence encoding the respective signal peptide for translation initiation. This conclusion was corroborated by RT qPCR revealing only slightly different amounts of transcript. Also, the effect of a putative alternative translation initiation site could be ruled out. CONCLUSION: Our results confirm the importance of the 5' end sequence of a target gene for translation initiation. Optimizing production yields thus may require screenings for optimal spacer sequence lengths. In case of secreted proteins, the 5' sequence encoding the signal peptide for Sec-depended secretion should also be considered.

Fast automated online xylanase activity assay using HPAEC-PAD.

In contrast to biochemical reactions, which are often carried out under automatic control and maintained overnight, the automation of chemical analysis is usually neglected. Samples are either analyzed in a rudimentary fashion using in situ techniques, or aliquots are withdrawn and stored to facilitate more precise offline measurements, which can result in sampling and storage errors. Therefore, in this study, we implemented automated reaction control, sampling, and analysis. As an example, the activities of xylanases on xylotetraose and soluble xylan were examined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The reaction was performed in HPLC vials inside a temperature-controlled Dionex™ AS-AP autosampler. It was started automatically when the autosampler pipetted substrate and enzyme solution into the reaction vial. Afterwards, samples from the reaction vial were injected repeatedly for 60 min onto a CarboPac™ PA100 column for analysis. Due to the rapidity of the reaction, the analytical method and the gradient elution of 200 mM sodium hydroxide solution and 100 mM sodium hydroxide with 500 mM sodium acetate were adapted to allow for an overall separation time of 13 min and a detection limit of 0.35-1.83 mg/L (depending on the xylooligomer). This analytical method was applied to measure the soluble short-chain products (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and longer xylooligomers) that arise during enzymatic hydrolysis. Based on that, the activities of three endoxylanases (EX) were determined as 294 U/mg for EX from Aspergillus niger, 1.69 U/mg for EX from Bacillus stearothermophilus, and 0.36 U/mg for EX from Bacillus subtilis. Graphical abstract Xylanase activity assay automation.

Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis.

BACKGROUND: Bacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production. Identified variants with beneficial effects on production were sequenced and analyzed regarding B. subtilis growth behavior, extracellular lipase activity and amount as well as changes in lipase transcript levels. RESULTS: In total, 26 LipA variants were identified showing an up to twofold increase in either amount or activity of extracellular lipase. These variants harbor single amino acid or codon substitutions that did not substantially affect B. subtilis growth. Subsequent exemplary combination of beneficial single amino acid substitutions revealed an additive effect solely at the level of extracellular lipase amount; however, lipase amount and activity could not be increased simultaneously. CONCLUSIONS: Single amino acid and codon substitutions can affect LipA secretion and production by B. subtilis. Several codon-related effects were observed that either enhance lipA transcription or promote a more efficient folding of LipA. Single amino acid substitutions could improve LipA production by increasing its secretion or stability in the culture supernatant. Our findings indicate that optimization of the expression system is not sufficient for efficient protein production in B. subtilis. The sequence of the target protein should also be considered as an optimization target for successful protein production. Our results further suggest that variants with improved properties might be identified much faster and easier if mutagenesis is prioritized towards elements that contribute to enzymatic activity or structural integrity.

Activity-independent screening of secreted proteins using split GFP.

The large-scale industrial production of proteins requires efficient secretion, as provided, for instance, by the Sec system of Gram-positive bacteria. Protein engineering approaches to optimize secretion often involve the screening of large libraries, e.g. comprising a target protein fused to many different signal peptides. Respective high-throughput screening methods are usually based on photometric or fluorimetric assays enabling fast and simple determination of enzymatic activities. Here, we report on an alternative method for quantification of secreted proteins based on the split GFP assay. We analyzed the secretion by Bacillus subtilis of a homologous lipase and a heterologous cutinase by determination of GFP fluorescence and enzyme activity assays. Furthermore, we identified from a signal peptide library a variant of the biotechnologically relevant B. subtilis protein swollenin EXLX1 with up to 5-fold increased secretion. Our results demonstrate that the split GFP assay can be used to monitor secretion of enzymatic and non-enzymatic proteins in B. subtilis in a high-throughput manner.

Novel Tools for the Functional Expression of Metagenomic DNA.

Functional expression of genes from metagenomic libraries is limited by various factors including inefficient transcription and/or translation of target genes as well as improper folding and assembly of the corresponding proteins caused by the lack of appropriate chaperones and cofactors. It is now well accepted that the use of different expression hosts of distinct phylogeny and physiology can dramatically increase the rate of success. In the following chapter, we therefore describe tools and protocols allowing for the comparative heterologous expression of genes in five bacterial expression hosts, namely Escherichia coli, Pseudomonas putida, Bacillus subtilis, Burkholderia glumae, and Rhodobacter capsulatus. Different broad-host-range shuttle vectors are described that allow activity-based screening of metagenomic DNA in these bacteria. Furthermore, we describe the newly developed transfer-and-expression system TREX which comprises genetic elements essential to allow for expression of large clusters of functionally coupled genes in different microbial species.

Mutations improving production and secretion of extracellular lipase by Burkholderia glumae PG1.

Burkholderia glumae is a Gram-negative phytopathogenic bacterium known as the causative agent of rice panicle blight. Strain B. glumae PG1 is used for the production of a biotechnologically relevant lipase, which is secreted into the culture supernatant via a type II secretion pathway. We have comparatively analyzed the genome sequences of B. glumae PG1 wild type and a lipase overproducing strain obtained by classical strain mutagenesis. Among a total number of 72 single nucleotide polymorphisms (SNPs) identified in the genome of the production strain, two were localized in front of the lipAB operon and were analyzed in detail. Both mutations contribute to a 100-fold overproduction of extracellular lipase in B. glumae PG1 by affecting transcription of the lipAB operon and efficiency of lipase secretion. We analyzed each of the two SNPs separately and observed a stronger influence of the promoter mutation than of the signal peptide modification but also a cumulative effect of both mutations. Furthermore, fusion of the mutated LipA signal peptide resulted in a 2-fold increase in secretion of the heterologous reporter alkaline phosphatase from Escherichia coli.

Genome-wide RNA sequencing analysis of quorum sensing-controlled regulons in the plant-associated Burkholderia glumae PG1 strain.

Burkholderia glumae PG1 is a soil-associated motile plant-pathogenic bacterium possessing a cell density-dependent regulation system called quorum sensing (QS). Its genome contains three genes, here designated bgaI1 to bgaI3, encoding distinct autoinducer-1 (AI-1) synthases, which are capable of synthesizing QS signaling molecules. Here, we report on the construction of B. glumae PG1 ΔbgaI1, ΔbgaI2, and ΔbgaI3 mutants, their phenotypic characterization, and genome-wide transcriptome analysis using RNA sequencing (RNA-seq) technology. Knockout of each of these bgaI genes resulted in strongly decreased motility, reduced extracellular lipase activity, a reduced ability to cause plant tissue maceration, and decreased pathogenicity. RNA-seq analysis of all three B. glumae PG1 AI-1 synthase mutants performed in the transition from exponential to stationary growth phase revealed differential expression of a significant number of predicted genes. In comparison with the levels of gene expression by wild-type strain B. glumae PG1, 481 genes were differentially expressed in the ΔbgaI1 mutant, 213 were differentially expressed in the ΔbgaI2 mutant, and 367 were differentially expressed in the ΔbgaI3 mutant. Interestingly, only a minor set of 78 genes was coregulated in all three mutants. The majority of the QS-regulated genes were linked to metabolic activities, and the most pronounced regulation was observed for genes involved in rhamnolipid and Flp pilus biosynthesis and the type VI secretion system and genes linked to a clustered regularly interspaced short palindromic repeat (CRISPR)-cas gene cluster.