Heterologous production of long-chain rhamnolipids from Burkholderia glumae in Pseudomonas putida-a step forward to tailor-made rhamnolipids.

Rhamnolipids are biosurfactants consisting of rhamnose (Rha) molecules linked through a ß-glycosidic bond to 3-hydroxyfatty acids with various chain lengths, and they have an enormous potential for various industrial applications. The best known native rhamnolipid producer is the human pathogen Pseudomonas aeruginosa, which produces short-chain rhamnolipids mainly consisting of a Rha-Rha-C10-C10 congener. Bacteria from the genus Burkholderia are also able to produce rhamnolipids, which are characterized by their long-chain 3-hydroxyfatty acids with a predominant Rha-Rha-C14-C14 congener. These long-chain rhamnolipids offer different physicochemical properties compared to their counterparts from P. aeruginosa making them very interesting to establish novel potential applications. However, widespread applications of rhamnolipids are still hampered by the pathogenicity of producer strains and-even more important-by the complexity of regulatory networks controlling rhamnolipid production, e.g., the so-called quorum sensing system. To overcome encountered challenges of the wild type, the responsible genes for rhamnolipid biosynthesis in Burkholderia glumae were heterologously expressed in the non-pathogenic Pseudomonas putida KT2440. Our results show that long-chain rhamnolipids from Burkholderia spec. can be produced in P. putida. Surprisingly, the heterologous expression of the genes rhlA and rhlB encoding an acyl- and a rhamnosyltransferase, respectively, resulted in the synthesis of two different mono-rhamnolipid species containing one or two 3-hydroxyfatty acid chains in equal amounts. Furthermore, mixed biosynthetic rhlAB operons with combined genes from different organisms were created to determine whether RhlA or RhlB is responsible to define the fatty acid chain lengths in rhamnolipids.

Novel insights into biosynthesis and uptake of rhamnolipids and their precursors.

The human pathogenic bacterium Pseudomonas aeruginosa produces rhamnolipids, glycolipids with functions for bacterial motility, biofilm formation, and uptake of hydrophobic substrates. Rhamnolipids represent a chemically heterogeneous group of secondary metabolites composed of one or two rhamnose molecules linked to one or mostly two 3-hydroxyfatty acids of various chain lengths. The biosynthetic pathway involves rhamnosyltransferase I encoded by the rhlAB operon, which synthesizes 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) followed by their coupling to one rhamnose moiety. The resulting mono-rhamnolipids are converted to di-rhamnolipids in a third reaction catalyzed by the rhamnosyltransferase II RhlC. However, the mechanism behind the biosynthesis of rhamnolipids containing only a single fatty acid is still unknown. To understand the role of proteins involved in rhamnolipid biosynthesis the heterologous expression of rhl-genes in non-pathogenic Pseudomonas putida KT2440 strains was used in this study to circumvent the complex quorum sensing regulation in P. aeruginosa. Our results reveal that RhlA and RhlB are independently involved in rhamnolipid biosynthesis and not in the form of a RhlAB heterodimer complex as it has been previously postulated. Furthermore, we demonstrate that mono-rhamnolipids provided extracellularly as well as HAAs as their precursors are generally taken up into the cell and are subsequently converted to di-rhamnolipids by P. putida and the native host P. aeruginosa. Finally, our results throw light on the biosynthesis of rhamnolipids containing one fatty acid, which occurs by hydrolyzation of typical rhamnolipids containing two fatty acids, valuable for the production of designer rhamnolipids with desired physicochemical properties.

Work of Breathing in Fixed and Pressure Relief Continuous Positive Airway Pressure (C-Flex™): A post hoc Analysis.

BACKGROUND: Expiratory pressure relief continuous positive airway pressure (pressure relief CPAP; C-Flex™) causes increases in inspiratory duty cycle and shortening of expiratory time. It has been suggested that these changes are caused by an increase in work of breathing. OBJECTIVES: We studied the effects of C-Flex on work of breathing and intrinsic positive end-expiratory pressure as compared to fixed CPAP. METHODS: Work of breathing was analyzed in 24 patients with obstructive sleep apnea during treatment with fixed CPAP and C-Flex with 3 different pressure relief settings in a randomized order during rapid-eye-movement (REM) and non-REM sleep. Work of breathing was assessed on a breath-by-breath basis using a piezoelectric esophageal pressure catheter and a pneumotachograph for measuring airflow. RESULTS: We found there was no increase in inspiratory work of breathing observed using C-Flex compared to fixed CPAP. Instead, we found a linear decrease in inspiratory work of breathing with increasing pressure relief, with a mean difference of 1.22 J/min between CPAP and maximum pressure release (C-Flex 3; 90% of the value with nasal CPAP); however, the decrease was not statistically significant. The decrease in inspiratory work of breathing associated with C-Flex has a significant inverse correlation with BMI. CONCLUSIONS: The C-Flex technology does not change work of breathing but shows a tendency towards a reduction of inspiratory work of breathing in patients with a lower BMI using higher C-Flex. The effect is probably caused by diminishing airway resistance generated by the positive end-expiratory pressure. Our findings may lead to additional fields of application of the C-Flex technology, such as chronic obstructive pulmonary disease or muscular dystrophy.

Exploring the protein stability landscape: Bacillus subtilis lipase A as a model for detergent tolerance.

A systematic study was conducted with Bacillus subtilis lipase A (BSLA) to determine the effect of every single amino acid substitution on detergent tolerance. BSLA is a minimal α/ß-hydrolase of 181 amino acids with a known crystal structure. It can be expressed in Escherichia coli and is biochemically well characterized. Site saturation mutagenesis resulted in a library of 3439 variants, each with a single amino acid exchange as confirmed by DNA sequencing. The library was tested against four detergents, namely SDS, CTAB, Tween 80, and sulfobetaine. Surface remodeling emerged as an effective engineering strategy to increase tolerance towards detergents. Amino acid residues that significantly affect the tolerance for each of the four detergents were identified. In summary, this systematic analysis provides an experimental dataset to help derive novel protein engineering strategies as well as to direct modeling efforts.

Uml2 is a novel CalB-type lipase of Ustilago maydis with phospholipase A activity.

CalB of Pseudozyma aphidis (formerly named Candida antarctica) is one of the most widely applied enzymes in industrial biocatalysis. Here, we describe a protein with 66 % sequence identity to CalB, designated Ustilago maydis lipase 2 (Uml2), which was identified as the product of gene um01422 of the corn smut fungus U. maydis. Sequence analysis of Uml2 revealed the presence of a typical lipase catalytic triad, Ser-His-Asp with Ser125 located in a Thr-Xaa-Ser-Xaa-Gly pentapeptide. Deletion of the uml2 gene in U. maydis diminished the ability of cells to hydrolyse fatty acids from tributyrin or Tween 20/80 substrates, thus demonstrating that Uml2 functions as a lipase that may contribute to nutrition of this fungal pathogen. Uml2 was heterologously produced in Pichia pastoris and recombinant N-glycosylated Uml2 protein was purified from the culture medium. Purified Uml2 released short- and long-chain fatty acids from p-nitrophenyl esters and Tween 20/80 substrates. Furthermore, phosphatidylcholine substrates containing long-chain saturated or unsaturated fatty acids were effectively hydrolysed. Both esterase and phospholipase A activity of Uml2 depended on the Ser125 catalytic residue. These results indicate that Uml2, in contrast to CalB, exhibits not only esterase and lipase activity but also phospholipase A activity. Thus, by genome mining, we identified a novel CalB-like lipase with different substrate specificities.

Identification of amino acids involved in the hydrolytic activity of lipase LipBL from Marinobacter lipolyticus.

The lipolytic enzyme family VIII currently includes only seven members but represents a group of lipolytic enzymes with interesting properties. Recently, we identified a gene encoding the family VIII lipase LipBL from the halophilic bacterium Marinobacter lipolyticus. This enzyme, like most lipolytic enzymes from family VIII, possesses two possible nucleophilic serines located in an S-X-X-K ß-lactamase motif and a G-X-S-X-G lipase motif. The serine in the S-X-X-K motif is a catalytic residue, but the role of serine within the common lipase consensus sequence G-X-S-X-G has not yet been systematically studied. Here, the previously reported time-intensive procedure for purification of recombinant LipBL was replaced by one-step metal-affinity chromatography purification in the presence of ATP. Heterologous co-expression of His(6)-tagged LipBL with the cytoplasmic molecular chaperones GroEL/GroES was necessary to obtain catalytically active LipBL. Site-directed mutagenesis performed to map the active site of LipBL revealed that mutation of serine and lysine in the ß-lactamase motif (S(72)-M-T-K(75)) to alanine abolished the enzyme activity of LipBL, in contrast to mutation of the serine in the lipase consensus motif (S321A). Furthermore, mutagenesis was performed to understand the role of the G-X-S-X-G motif and other amino acids that are conserved among family VIII esterases. We describe how mutations in the conserved G-X-S-X-G motif altered the biochemical properties and substrate specificity of LipBL. Molecular modelling results indicate the location of the G-X-S(321)-X-G motif in a loop close to the catalytic centre of LipBL, presumably representing a substrate-binding site of LipBL.

The lipase LipA (PA2862) but not LipC (PA4813) from Pseudomonas aeruginosa influences regulation of pyoverdine production and expression of the sigma factor PvdS.

A key element in iron-dependent regulation of iron metabolism and virulence-related functions for Pseudomonas aeruginosa is the sigma factor PvdS. PvdS expression itself is also influenced by iron-independent stimuli. We show that pyoverdine production and pvdS expression depend on one of the two lipases of P. aeruginosa.

Glycosylation is required for outer membrane localization of the lectin LecB in Pseudomonas aeruginosa.

The fucose-/mannose-specific lectin LecB from Pseudomonas aeruginosa is transported to the outer membrane; however, the mechanism used is not known so far. Here, we report that LecB is present in the periplasm of P. aeruginosa in two variants of different sizes. Both were functional and could be purified by their affinity to mannose. The difference in size was shown by a specific enzyme assay to be a result of N glycosylation, and inactivation of the glycosylation sites was shown by site-directed mutagenesis. Furthermore, we demonstrate that this glycosylation is required for the transport of LecB.

Autotransporters with GDSL passenger domains: molecular physiology and biotechnological applications.

Autotransporters are large proteins produced and secreted by Gram-negative bacteria. They consist of an N-terminal passenger domain, which typically harbours enzymatic activity and exerts a virulence function, and a C-terminal membrane anchor domain. Somehow, the membrane domain facilitates the transport of the passenger domain into the extracellular space. Several autotransporters possess hydrolase passenger domains that belong to the GDSL family of lipolytic enzymes. GDSL autotransporters represent a functionally distinct family and are characterized by several features of their passenger domains; these include 1) the absence of a conserved right-handed parallel ß-helix, 2) lipolytic activity, and thus the capability to hydrolyse membranes, and 3) covalent attachment to the respective C-terminal ß-domain, with the hydrolase domain exposed to the exterior. The esterase EstA of Pseudomonas aeruginosa is a typical enzyme of this type. Its physiological role was studied, its potential biotechnological application has been demonstrated, and its crystal structure was solved recently. Furthermore, it is capable of displaying different classes of enzymes in a range of Gram-negative bacteria including Escherichia coli, and FACS-based high-throughput screening for enantioselective esterases could be achieved using EstA.

Growth independent rhamnolipid production from glucose using the non-pathogenic Pseudomonas putida KT2440.

BACKGROUND: Rhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen Pseudomonas aeruginosa during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular P. aeruginosa. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly. RESULTS: Here, the metabolic engineering of a rhamnolipid producing Pseudomonas putida KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, P. putida KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli. P. putida KT2440 expressing the rhlAB-genes from P. aeruginosa PAO1 produces mono-rhamnolipids of P. aeruginosa PAO1 type (mainly C(10):C(10)). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of P. aeruginosa PAO1 at yields of about 0.15 g/g(glucose) corresponding to 32% of the theoretical optimum. What's more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation. CONCLUSIONS: A functional alternative to the pathogenic rhamnolipid producer P. aeruginosa was constructed and characterized. P. putida KT24C1 pVLT31_rhlAB featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer P. aeruginosa.

The Pseudomonas aeruginosa patatin-like protein PlpD is the archetype of a novel Type V secretion system.

We discovered a novel secreted protein by Pseudomonas aeruginosa, PlpD, as a member of the bacterial lipolytic enzyme family of patatin-like proteins (PLPs). PlpD is synthesized as a single molecule consisting of a secreted domain fused to a transporter domain. The N-terminus of PlpD includes a classical signal peptide followed by the four PLP conserved blocks that account for its lipase activity. The C-terminus consists of a POTRA (polypeptide transport-associated) motif preceding a putative 16-stranded beta-barrel similar to those of TpsB transporters of Type Vb secretion system. We showed that the C-terminus remains inserted into the outer membrane while the patatin moiety is secreted. The association between a TpsB component and a passenger protein is a unique hybrid organization that we propose to classify as Type Vd. More than 200 PlpD orthologues exist among pathogenic and environmental bacteria, which suggests that bacteria secrete numerous PLPs using this newly defined mechanism.

Extracellular enzymes affect biofilm formation of mucoid Pseudomonas aeruginosa.

Pseudomonas aeruginosa secretes a variety of hydrolases, many of which contribute to virulence or are thought to play a role in the nutrition of the bacterium. As most studies concerning extracellular enzymes have been performed on planktonic cultures of non-mucoid P. aeruginosa strains, knowledge of the potential role of these enzymes in biofilm formation in mucoid (alginate-producing) P. aeruginosa remains limited. Here we show that mucoid P. aeruginosa produces extracellular hydrolases during biofilm growth. Overexpression of the extracellular lipases LipA and LipC, the esterase EstA and the proteolytic elastase LasB from plasmids revealed that some of these hydrolases affected the composition and physicochemical properties of the extracellular polymeric substances (EPS). While no influence of LipA was observed, the overexpression of estA and lasB led to increased concentrations of extracellular rhamnolipids with enhanced levels of mono-rhamnolipids, elevated amounts of total carbohydrates and decreased alginate concentrations, resulting in increased EPS hydrophobicity and viscosity. Moreover, we observed an influence of the enzymes on cellular motility. Overexpression of estA resulted in a loss of twitching motility, although it enhanced the ability to swim and swarm. The lasB-overexpression strain showed an overall enhanced motility compared with the parent strain. Moreover, the EstA- and LasB-overproduction strains completely lost the ability to form 3D biofilms, whereas the overproduction of LipC increased cell aggregation and the heterogeneity of the biofilms formed. Overall, these findings indicate that directly or indirectly, the secreted enzymes EstA, LasB and LipC can influence the formation and architecture of mucoid P. aeruginosa biofilms as a result of changes in EPS composition and properties, as well as the motility of the cells.

Probing enzyme promiscuity of SGNH hydrolases.

Several hydrolases of the SGNH superfamily, including the lipase SrLip from Streptomyces rimosus (Q93MW7), the acyl-CoA thioesterase I TesA from Pseudomonas aeruginosa (Q9HZY8) and the two lipolytic enzymes EstA (from P. aeruginosa, O33407) and EstP (from Pseudomonas putida, Q88QS0), were examined for promiscuity. These enzymes were tested against four chemically different classes of a total of 34 substrates known to be hydrolysed by esterases, thioesterases, lipases, phospholipases, Tweenases and proteases. Furthermore, they were also analysed with respect to their amino acid sequences and structural homology, and their phylogenetic relationship was determined. The Pseudomonas esterases EstA and EstP each have an N-terminal domain with catalytic activity together with a C-terminal autotransporter domain, and so the hybrid enzymes EstA(N)-EstP(C) and EstP(N)-EstA(C) were constructed by swapping the corresponding N- and C-terminal domains, and their hydrolytic activities were compared. Interestingly, substrate specificity and kinetic measurements indicated a significant influence of the autotransporter domains on the catalytic activities of these enzymes in solution. TesA, EstA and EstP were shown to function as esterases with different affinities and catalytic efficacies towards p-nitrophenyl butyrate. Of all the enzymes tested, only SrLip revealed lipase, phospholipase, esterase, thioesterase and Tweenase activities.

The Distribution of Alpha-1 Antitrypsin Genotypes Between Patients with COPD/Emphysema, Asthma and Bronchiectasis.

Purpose: Alpha-1-antitrypsin deficiency (AATD) is a rare hereditary condition characterized by low circulating levels of alpha-1antitrypsin (AAT). While the association between AATD and COPD/emphysema is undisputed, the association between AATD and asthma or bronchiectasis is still a matter of debate. Aims and Objectives: Our study aimed to investigate the distribution of AAT genotypes between patients with COPD/emphysema, asthma and bronchiectasis. To back up the diagnostic labels, we described symptoms associated with the diagnosis. Methods: Between September 2003 and March 2020, 29,465 testing kits (AlphaKit®) were analyzed in the AAT laboratory, University of Marburg, Germany. The diagnosis of AATD has been made based on the measurements of AAT serum levels, followed by genotyping, phenotyping or whole gene sequencing depending on the availability and/or the need for more detailed interpretation of the results. The respiratory symptoms were recorded as well. Results: Regarding the distribution of the wild type allele M and the most frequent mutations S (E264V) and Z (E342K), no significant differences could be found between COPD/emphysema [Pi*MM (58.24%); Pi*SZ (2.49%); Pi*ZZ (9.12%)] and bronchiectasis [Pi*MM (59.30%) Pi*SZ (2.81%); Pi*ZZ (7.02%)]. When COPD/emphysema and bronchiectasis were recorded in the same patient, the rate of Pi* ZZ (14.78%) mutations was even higher. Asthma patients exhibited significantly less deficient genotypes [Pi*MM (54.81%); Pi*SZ (2%); Pi*ZZ (2.77%)] than two other groups. Associated respiratory symptoms confirmed the diagnosis. Conclusion: COPD/emphysema and bronchiectasis, but not asthma patients, exhibit higher frequency of AATD genotypes. Our data suggest that AATD testing should be offered to patients with COPD/emphysema and bronchiectasis.

Mutations towards enantioselectivity adversely affect secretion of Pseudomonas aeruginosa lipase.

Lipases are important biocatalysts used as detergent additives to manufacture biodiesel, and in particular, for the production of enantiopure compounds such as alcohols, amines and carboxylic acids. Extensive efforts were conducted trying to optimize lipase properties and lipase LipA of Pseudomonas aeruginosa comprises the best-studied example in terms of optimizing enantioselectivity by application of numerous directed evolution methods. Its enantioselectivity in the asymmetric hydrolysis of the model substrate 2-methyldecanoic acid p-nitrophenyl ester was increased from E=1.1 for the wild-type enzyme to E=51 for the best (S)-enantioselective variant which carried six amino acid exchanges. We have observed that overexpression of this variant in the homologous host resulted in only marginal yields of enzyme in the bacterial culture supernatant, suggesting that the enantioselective LipA variant was secreted with only low efficiency. Hence, we have analysed the secretion of this lipase variant and compared it to variants carrying either the respective single mutations or some combinations. We report here the identification of two amino acid substitutions located on the protein surface, which significantly impair lipase secretion.

Lectin-mediated reversible immobilization of human cells into a glycosylated macroporous protein hydrogel as a cell culture matrix.

3D cell culture is a helpful approach to study cell-cell interaction in a native-like environment, but is often limited due the challenge of retrieving cells from the material. In this study, we present the use of recombinant lectin B, a sugar-binding protein with four binding cavities, to enable reversible cell integration into a macroporous protein hydrogel matrix. By functionalizing hydrogel precursors with saccharose, lectin B can both bind to sugar moieties on the cellular surface as well as to the modified hydrogel network. Confocal microscopy and flow cytometry analysis revealed cells to be integrated into the network and to adhere and proliferate. Furthermore, the specificity and reversibility was investigated by using a recombinantly produced yellow fluorescent - lectin B fusion protein and a variety of sugars with diverging affinities for lectin B at different concentrations and elution times. Cells could be eluted within minutes by addition of L-fucose to the cell-loaded hydrogels to make cells available for further analysis.

A membrane-bound esterase PA2949 from Pseudomonas aeruginosa is expressed and purified from Escherichia coli.

Pseudomonas aeruginosa strain 1001 produces an esterase (EstA) that can hydrolyse the racemic methyl ester of ß-acetylthioisobutyrate to produce the (D)-enantiomer, which serves as a precursor of captopril, a drug used for treatment of hypertension. We show here that PA2949 from P. aeruginosa PA01, a homologue of EstA, can efficiently be expressed in an enzymatically active form in E. coli. The enzyme is membrane-associated as demonstrated by cell fractionation studies. PA2949 was purified to homogeneity after solubilisation with the nonionic detergent, Triton X-100, and was shown to possess a conserved esterase catalytic triad consisting of Ser137-His258-Asp286. Our results should allow the development of an expression and purification strategy to produce this biotechnologically relevant esterase in a pure form with a high yield.

Metagenomic discovery of novel enzymes and biosurfactants in a slaughterhouse biofilm microbial community.

DNA derived from environmental samples is a rich source of novel bioactive molecules. The choice of the habitat to be sampled predefines the properties of the biomolecules to be discovered due to the physiological adaptation of the microbial community to the prevailing environmental conditions. We have constructed a metagenomic library in Escherichia coli DH10b with environmental DNA (eDNA) isolated from the microbial community of a slaughterhouse drain biofilm consisting mainly of species from the family Flavobacteriaceae. By functional screening of this library we have identified several lipases, proteases and two clones (SA343 and SA354) with biosurfactant and hemolytic activities. Sequence analysis of the respective eDNA fragments and subsequent structure homology modelling identified genes encoding putative N-acyl amino acid synthases with a unique two-domain organisation. The produced biosurfactants were identified by NMR spectroscopy as N-acyltyrosines with N-myristoyltyrosine as the predominant species. Critical micelle concentration and reduction of surface tension were similar to those of chemically synthesised N-myristoyltyrosine. Furthermore, we showed that the newly isolated N-acyltyrosines exhibit antibiotic activity against various bacteria. This is the first report describing the successful application of functional high-throughput screening assays for the identification of biosurfactant producing clones within a metagenomic library.

A generic system for the Escherichia coli cell-surface display of lipolytic enzymes.

EstA is an outer membrane-anchored esterase from Pseudomonas aeruginosa. An inactive EstA variant was used as an anchoring motif for the Escherichia coli cell-surface display of lipolytic enzymes. Flow cytometry analysis and measurement of lipase activity revealed that Bacillus subtilis lipase LipA, Fusarium solani pisi cutinase and one of the largest lipases presently known, namely Serratia marcescens lipase were all efficiently exported by the EstA autotransporter and also retained their lipolytic activities upon cell surface exposition. EstA provides a useful tool for surface display of lipases including variant libraries generated by directed evolution thereby enabling the identification of novel enzymes with interesting biological and biotechnological ramifications.

Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts.

Enzyme libraries displayed on the surface of microbial cells or microbeads can be screened with fluorogenic substrates that provide a physical linkage of the reaction product to the corresponding enzyme. Libraries exceeding 10(9) different variants can be quantitatively analysed and screened by flow cytometry at a rate of 30 000 cells/second. The promise of screening methods based on fluorescence-activated cell sorting for directed enzyme evolution is being realized and significantly improved enzymes have been reported recently.