Secretion of the cytoplasmic and high molecular weight ß-galactosidase of Paenibacillus wynnii with Bacillus subtilis.

BACKGROUND: The gram-positive bacterium Bacillus subtilis is widely used for industrial enzyme production. Its ability to secrete a wide range of enzymes into the extracellular medium especially facilitates downstream processing since cell disruption is avoided. Although various heterologous enzymes have been successfully secreted with B. subtilis, the secretion of cytoplasmic enzymes with high molecular weight is challenging. Only a few studies report on the secretion of cytoplasmic enzymes with a molecular weight > 100 kDa. RESULTS: In this study, the cytoplasmic and 120 kDa ß-galactosidase of Paenibacillus wynnii (ß-gal-Pw) was expressed and secreted with B. subtilis SCK6. Different strategies were focused on to identify the best secretion conditions. Tailormade codon-optimization of the ß-gal-Pw gene led to an increase in extracellular ß-gal-Pw production. Consequently, the optimized gene was used to test four signal peptides and two promoters in different combinations. Differences in extracellular ß-gal-Pw activity between the recombinant B. subtilis strains were observed with the successful secretion being highly dependent on the specific combination of promoter and signal peptide used. Interestingly, signal peptides of both the general secretory- and the twin-arginine translocation pathway mediated secretion. The highest extracellular activity of 55.2 ± 6 µkat/Lculture was reached when secretion was mediated by the PhoD signal peptide and expression was controlled by the PAprE promoter. Production of extracellular ß-gal-Pw was further enhanced 1.4-fold in a bioreactor cultivation to 77.5 ± 10 µkat/Lculture with secretion efficiencies of more than 80%. CONCLUSION: For the first time, the ß-gal-Pw was efficiently secreted with B. subtilis SCK6, demonstrating the potential of this strain for secretory production of cytoplasmic, high molecular weight enzymes.

Orotic acid production by Yarrowia lipolytica under conditions of limited pyrimidine.

Orotic acid (OA) is an intermediate of the pyrimidine biosynthesis with high industrial relevance due to its use as precursor for production of biochemical pyrimidines or its use as carrier molecule in drug formulations. It can be produced by fermentation of microorganisms with engineered pyrimidine metabolism. In this study, we surprisingly discovered the yeast Yarrowia lipolytica as a powerful producer of OA. The overproduction of OA in the Y. lipolytica strain PO1f was found to be caused by the deletion of the URA3 gene which prevents the irreversible decarboxylation of OA to uridine monophosphate. It was shown that the lack of orotidine-5'-phosphate decarboxylase was the reason for the accumulation of OA inside the cell since a rescue mutant of the URA3 deletion in Y. lipolytica PO1f completely prevented the OA secretion into the medium. In addition, pyrimidine limitation in the cell massively enhanced the OA accumulation followed by secretion due to intense overflow metabolism during bioreactor cultivations. Accordingly, supplementation of the medium with 200 mg/L uracil drastically decreased the OA overproduction by 91%. OA productivity was further enhanced in fed-batch cultivation with glucose and ammonium sulfate feed to a maximal yield of 9.62 ± 0.21 g/L. Y. lipolytica is one of three OA overproducing yeasts described in the literature so far, and in this study, the highest productivity was shown. This work demonstrates the potential of Y. lipolytica as a possible production organism for OA and provides a basis for further metabolic pathway engineering to optimize OA productivity.

Production and characterization of a new diamine oxidase from Yarrowia lipolytica.

A putative diamine oxidase (DAO) from Yarrowia lipolytica PO1f (DAO-1) was homologously recombinantly integrated into the genome of Y. lipolytica PO1f using the CRISPR-Cas9 system for the subsequent DAO production in a bioreactor. Thereby, it was proven that the DAO-1 produced was indeed a functional DAO. The cultivation yielded 2343 ± 98 nkat/Lculture with a specific DAO activity of 1301 ± 54.2 nkat/gprotein, which was a 93-fold increase of specific DAO activity compared to the native Y. lipolytica PO1f DAO-1 production. The DAO-1 showed a broad substrate selectivity with tyramine, histamine, putrescine and cadaverine being the most favored substrates. It was most active at 40 °C, pH 7.2 in Tris-HCl buffer (50 mM) (with histamine as substrate), which is comparable to human and porcine DAOs. The affinity of DAO-1 towards histamine was lower compared to mammalian DAOs (Km = 2.3 ± 0.2 mM). Nevertheless, DAO-1 degraded around 75% of the histamine used in a bioconversion experiment with a food-relevant concentration of 150 mg/L. With its broad selectivity for the most relevant biogenic amines in foods, DAO-1 from Y. lipolytica PO1f is an interesting enzyme for application in the food industry for the degradation of biogenic amines.

Molecular communication of the membrane insertase YidC with translocase SecYEG affects client proteins.

The membrane insertase YidC inserts newly synthesized proteins by its hydrophobic slide consisting of the two transmembrane (TM) segments TM3 and TM5. Mutations in this part of the protein affect the insertion of the client proteins. We show here that a quintuple mutation, termed YidC-5S, inhibits the insertion of the subunit a of the FoF1 ATP synthase but has no effect on the insertion of the Sec-independent M13 procoat protein and the C-tail protein SciP. Further investigations show that the interaction of YidC-5S with SecY is inhibited. The purified and fluorescently labeled YidC-5S did not approach SecYEG when both were co-reconstituted in proteoliposomes in contrast to the co-reconstituted YidC wild type. These results suggest that TM3 and TM5 are involved in the formation of a common YidC-SecYEG complex that is required for the insertion of Sec/YidC-dependent client proteins.

Polarity/charge as a determinant of translocase requirements for membrane protein insertion.

The YidC insertase of Escherichia coli inserts membrane proteins with small periplasmic loops (~20 residues). However, it has difficulty transporting loops that contain positively charged residues compared to negatively charged residues and, as a result, increasing the positive charge has an increased requirement for the Sec machinery as compared to negatively charged loops (Zhu et al., 2013; Soman et al., 2014). This suggested that the polarity and charge of the periplasmic regions of membrane proteins determine the YidC and Sec translocase requirements for insertion. Here we tested this polarity/charge hypothesis by showing that insertion of our model substrate protein procoat-Lep can become YidC/Sec dependent when the periplasmic loop was converted to highly polar even in the absence of any charged residues. Moreover, adding a number of hydrophobic amino acids to a highly polar loop can decrease the Sec-dependence of the otherwise strictly Sec-dependent membrane proteins. We also demonstrate that the length of the procoat-Lep loop is indeed a determinant for Sec-dependence by inserting alanine residues that do not markedly change the overall hydrophilicity of the periplasmic loop. Taken together, the results support the polarity/charge hypothesis as a determinant for the translocase requirement for procoat insertion.

Two Signal Recognition Particle Sequences Are Present in the Amino-Terminal Domain of the C-Tailed Protein SciP.

During their synthesis, the C-tailed membrane proteins expose the membrane-spanning segment late from the ribosome and consequently can insert into the membrane only posttranslationally. However, the C-tailed type 6 secretion system (T6SS) component SciP uses the bacterial signal recognition particle (SRP) system for membrane targeting, which operates cotranslationally. Analysis of possible sequence regions in the amino-terminal part of the protein revealed two candidates that were then tested for whether they function as SRP signal peptides. Both sequences were tested positive as synthetic peptides for binding to SRP. In addition, purified ribosomes with stalled nascent chains exposing either sequence were capable of binding to SRP and SRP-FtsY complexes with high affinity. Together, the data suggest that both peptides can serve as an SRP signal sequence promoting an early membrane targeting of SciP during its synthesis. Like observed for multispanning membrane proteins, the two cytoplasmic SRP signal sequences of SciP may also facilitate a retargeting event, making the targeting more efficient.IMPORTANCE C-tail proteins are anchored in the inner membrane with a transmembrane segment at the C terminus in an N-in/C-out topology. Due to this topology, membrane insertion occurs only posttranslationally. Nevertheless, the C-tail-anchored protein SciP is targeted cotranslationally by SRP. We report here that two amino-terminal hydrophobic stretches in SciP are individually recognized by SRP and target the nascent protein to FtsY. The presence of two signal sequences may enable a retargeting mechanism, as already observed for multispanning membrane proteins, to make the posttranslational insertion of SciP by YidC more efficient.

The SRP signal sequence of KdpD.

KdpD is a four-spanning membrane protein that has two large cytoplasmic domains at the amino- and at the carboxyterminus, respectively. During its biogenesis KdpD binds to the signal recognition particle (SRP) of Escherichia coli that consists of a 48-kDa protein Ffh and a 4.5S RNA. The protein is targeted to the inner membrane surface and is released after contacting the SRP receptor protein FtsY. The information within the KdpD protein that confers SRP interaction was found in the amino-terminal cytoplasmic domain of KdpD, particularly at residues 22-48. Within this sequence a Walker A motif is present at residues 30-38. To determine the actual sequence specificity to SRP, a collection of mutants was constructed. When the KdpD peptides (residues 22-48) were fused to sfGFP the targeting to the membrane was observed by fluorescence microscopy. Further, nascent chains of KdpD bound to ribosomes were purified and their binding to SRP was analysed by microscale thermophoresis. We found that the amino acid residues R22, K24 and K26 are important for SRP interaction, whereas the residues G30, G34 and G36, essential for a functional Walker A motif, can be replaced with alanines without affecting the affinity to SRP-FtsY and membrane targeting.

Each protomer of a dimeric YidC functions as a single membrane insertase.

The membrane insertase YidC catalyzes the entrance of newly synthesized proteins into the lipid bilayer. As an integral membrane protein itself, YidC can be found as a monomer, a dimer or also as a member of the holotranslocase SecYEGDF-YajC-YidC. To investigate whether the dimeric YidC is functional and whether two copies cooperate to insert a single substrate, we constructed a fusion protein where two copies of YidC are connected by a short linker peptide. The 120 kDa protein is stable and functional as it supports the membrane insertion of the M13 procoat protein, the C-tailed protein SciP and the fusion protein Pf3-Lep. Mutations that inhibit either protomer do not inactivate the insertase and rather keep it functional. When both protomers are defective, the substrate proteins accumulate in the cytoplasm. This suggests that the dimeric YidC operates as two insertases. Consistent with this, we show that the dimeric YidC can bind two substrate proteins simultaneously, suggesting that YidC indeed functions as a monomer.

Membrane Targeting and Insertion of the C-Tail Protein SciP.

C-tailed membrane proteins insert into the bilayer post-translationally because the hydrophobic anchor segment leaves the ribosome at the end of translation. Nevertheless, we find here evidence that the targeting of SciP to the membrane of Escherichia coli occurs co-translationally since signal elements in the N-terminal part of the SciP protein sequence are present. Two short hydrophobic sequences were identified that targeted a green fluorescent protein-SciP fusion protein to the membrane involving the signal recognition particle. After targeting, the membrane insertion of SciP is catalyzed by YidC independent of the SecYEG translocase. However, when the C-terminal tail of SciP was extended to 21 aa residues, we found that SecYEG becomes involved and makes its membrane insertion more efficient.

Combined B, T and NK Cell Deficiency Accelerates Atherosclerosis in BALB/c Mice.

This study focused on the unique properties of both the Ldlr knockout defect (closely mimicking the human situation) and the BALB/c (C) inbred mouse strain (Th-2 slanted immune response). We generated two immunodeficient strains with severe combined B- and T-cell immunodeficiency with or without a complete lack of natural killer cells to revisit the role of adaptive immune responses on atherogenesis. C-Ldlr-/- Rag1-/- mice, which show severe combined B- and T-cell immunodeficiency and C-Ldlr-/- Rag1-/- Il2rg-/- mice, which combine the T- and B-cell defect with a complete lack of natural killer cells and inactivation of multiple cytokine signalling pathways were fed an atherogenic Western type diet (WTD). Both B6-Ldlr-/- and C-Ldlr-/- immunocompetent mice were used as controls. Body weights and serum cholesterol levels of both immunodeficient strains were significantly increased compared to C-Ldlr-/- controls, except for cholesterol levels of C-Ldlr-/- Rag1-/- double mutants after 12 weeks on the WTD. Quantification of the aortic sinus plaque area revealed that both strains of immunodeficient mice developed significantly more atherosclerosis compared to C-Ldlr-/- controls after 24 weeks on the WTD. Increased atherosclerotic lesion development in C-Ldlr-/- Rag1-/- Il2rg-/- triple mutants was associated with significantly increased numbers of macrophages and significantly decreased numbers of smooth muscle cells compared to both C-Ldlr-/- wild type and C-Ldlr-/- Rag1-/- double mutants pointing to a plaque destabilizing effect of NK cell loss. Collectively, the present study reveals a previously unappreciated complexity with regard to the impact of lymphocytes on lipoprotein metabolism and the role of lymphocyte subsets in plaque composition.

The phosphopantetheinyl transferase KirP activates the ACP and PCP domains of the kirromycin NRPS/PKS of Streptomyces collinus Tü 365.

The main steps in the biosynthesis of complex secondary metabolites such as the antibiotic kirromycin are catalyzed by modular polyketide synthases (PKS) and/or nonribosomal peptide synthetases (NRPS). During antibiotic assembly, the biosynthetic intermediates are attached to carrier protein domains of these megaenzymes via a phosphopantetheinyl arm. This functional group of the carrier proteins is attached post-translationally by a phosphopantetheinyl transferase (PPTase). No experimental evidence exists about how such an activation of the carrier proteins of the kirromycin PKS/NRPS is accomplished. Here we report on the characterization of the PPTase KirP, which is encoded by a gene located in the kirromycin biosynthetic gene cluster. An inactivation of the kirP gene resulted in a 90% decrease in kirromycin production, indicating a substantial role for KirP in the biosynthesis of the antibiotic. In enzymatic assays, KirP was able to activate both acyl carrier protein and petidyl carrier domains of the kirromycin PKS/NRPS. In addition to coenzyme A (CoA), which is the natural substrate of KirP, the enzyme was able to transfer acyl-phosphopantetheinyl groups to the apo forms of the carrier proteins. Thus, KirP is very flexible in terms of both CoA substrate and carrier protein specificity. Our results indicate that KirP is the main PPTases that activates the carrier proteins in kirromycin biosynthesis.

Molecular analysis of the kirromycin biosynthetic gene cluster revealed beta-alanine as precursor of the pyridone moiety.

Kirromycin is a complex linear polyketide that acts as a protein biosynthesis inhibitor by binding to the bacterial elongation factor Tu. The kirromycin biosynthetic gene cluster was isolated from the producer, Streptomyces collinus Tü 365, and confirmed by targeted disruption of essential biosynthesis genes. Kirromycin is synthesized by a large hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) encoded by the genes kirAI-kirAVI. This complex involves some very unusual features, including the absence of internal acyltransferase (AT) domains in KirAI-KirAV, multiple split-ups of PKS modules on separate genes, and swapping in the domain organization. Interestingly, one PKS enzyme, KirAVI, contains internal AT domains. Based on in silico analysis, a route to pyridone formation involving PKS and NRPS steps was postulated. This hypothesis was experimentally proven by feeding studies with [U-13C3(15)N]beta-alanine and NMR and MS analyses of the isolated pure kirromycin.