Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus

The transglutaminase secreted by Streptoverticillium mobaraense is a useful enzyme in the food industry. A fragment of transglutaminase was secreted by Corynebacterium glutamicum when it was coupled on a plasmid to the promoter and signal peptide of a cell surface protein from C. glutamicum. We analyzed the signal peptide and the pro-domain of the transglutaminase gene and found that the signal peptide consists of 31 amino acid residues and the pro-domain consists of 45 residues. When the pro-domain of the transglutaminase was used, the pro-transglutaminase was secreted efficiently by C. glutamicum but had no enzymatic activity. However, when the plasmid carrying the S. mobaraense transglutaminase also encoded SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus, the peptide bond to the C side of 41-Ser of the pro-transglutaminase was hydrolyzed, and the pro-transglutaminase was converted to an active form. Our findings suggest that C. glutamicum has potential as a host for industrial-scale protein production.     

Production of Native-Type Streptoverticillium mobaraense Transglutaminase in Corynebacterium glutamicum

We previously observed secretion of active-form transglutaminase in Corynebacterium glutamicum by coexpressing the subtilisin-like protease SAM-P45 from Streptomyces albogriseolus to process the prodomain. However, the N-terminal amino acid sequence of the transglutaminase differed from that of the native Streptoverticillium mobaraense enzyme. In the present work we have used site-directed mutagenesis to generate an optimal SAM-P45 cleavage site in the C-terminal region of the prodomain. As a result, native-type transglutaminase was secreted.     

Production of <Emphasis Type="Italic">Chryseobacterium proteolyticum</Emphasis> protein-glutaminase using the twin-arginine translocation pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The protein glutaminase (PG) secreted by the Gram-negative bacterium Chryseobacterium proteolyticum can deamidate glutaminyl residues in several substrate proteins, including insoluble wheat glutens. This enzyme therefore has potential application in the food industry. We assessed the possibility to produce PG containing a pro-domain in Corynebacterium glutamicum which we have successfully used for production of several kinds of proteins at industrial-scale. When it was targeted to the general protein secretion pathway (Sec) via its own signal sequence, the protein glutaminase was not secreted in this strain. In contrast, we showed that pro-PG could be efficiently produced using the recently discovered twin-arginine translocation (Tat) pathway when the typical Sec-dependent signal peptide was replaced by a Tat-dependent signal sequence from various bacteria. The accumulation of pro-PG in C. glutamicum ATCC13869 reached 183 mg/l, and the pro-PG was converted to an active form as the native one by SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus. The successful secretion of PG via this approach confirms that the Tat pathway of C. glutamicum is an efficient alternative for the industrial-scale production of proteins that are not efficiently secreted by other systems.     

TatABC Overexpression Improves Corynebacterium glutamicum Tat-Dependent Protein Secretion

The twin-arginine translocation (Tat) pathway in Corynebacterium glutamicum has been described previously. The minimal functional Tat system in C. glutamicum required TatA and TatC but did not require TatB, although this component was required for maximal efficiency of Tat-dependent secretion. We previously demonstrated that Chryseobacterium proteolyticum pro-protein glutaminase (pro-PG) and Streptomyces mobaraensis pro-transglutaminase (pro-TG) could be secreted via the Tat pathway in C. glutamicum. Here we report that the amounts of pro-PG secreted were more than threefold larger when TatC or TatAC was overexpressed, and there was a further threefold increase when TatABC was overexpressed. These results show that the amount of TatC protein is the first bottleneck and the amount of TatB protein is the second bottleneck in Tat-dependent protein secretion in C. glutamicum. In addition, the amount of pro-TG that accumulated via the Tat pathway when TatABC was overexpressed with the TorA signal peptide in C. glutamicum was larger than the amount that accumulated via the Sec pathway. We concluded that TatABC overexpression improves Tat-dependent pro-PG and pro-TG secretion in C. glutamicum.     

Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria

We previously reported on the secretion of Streptomyces mobaraensis transglutaminase by Corynebacterium glutamicum ATCC13869 (formerly classified as Brevibacterium lactofermentum). In the present work, we investigated whether any other coryneform bacteria showed higher productivity than C. glutamicum ATCC13869. We found that most coryneform species secreted pro-transglutaminase efficiently. Moreover, we confirmed that Corynebacterium ammoniagenes ATCC6872 produced about 2.5 g/l pro-transglutaminase over a 71-h period in a jar fermentor. Our findings suggest that some other coryneform bacteria, especially C. ammoniagenes ATCC6872, are potential hosts for industrial scale protein production.     

Influence of SigB inactivation on Corynebacterium glutamicum protein secretion

The non-essential Corynebacterium glutamicum sigma factor, sigB, modulates global gene expression during the transition from exponential growth to the stationary phase. Utilizing a signal peptide derived from C. glutamicum R CgR_0949, a sigB disruption mutant able to secrete 3- to 5-fold more green fluorescence protein (GFP) and α-amylase than the wild type strain was isolated. The signal peptide selectively enabled the mutant to produce greater amounts of both proteins, which were in turn secreted in culture medium in greater quantities than previously acknowledged. A peak GFP productivity of 2.8 g/l was attained, representing the highest GFP productivity reported in C. glutamicum to date. CgR_0949 signal sequence length (30 residues), type (Tat) or the target protein identity (GFP or α-amylase) had no measurable effect on the magnitude of the protein accumulation and consequent secretion. It therefore follows that actual experimentation remains the fastest way to identify suitable signal sequences in C. glutamicum. More secretion studies may reveal even greater secretion productivity by C. glutamicum and consequently present an attractive avenue to further enhance the utility of C. glutamicum as an industrial workhorse.     

Functional Analysis of the Twin-Arginine Translocation Pathway in Corynebacterium glutamicum ATCC 13869

Compared to those of other gram-positive bacteria, the genetic structure of the Corynebacterium glutamicum Tat system is unique in that it contains the tatE gene in addition to tatA, tatB, and tatC. The tatE homologue has been detected only in the genomes of gram-negative enterobacteria. To assess the function of the C. glutamicum Tat pathway, we cloned the tatA, tatB, tatC, and tatE genes from C. glutamicum ATCC 13869 and constructed mutants carrying deletions of each tat gene or of both the tatA and tatE genes. Using green fluorescent protein (GFP) fused with the twin-arginine signal peptide of the Escherichia coli TorA protein, we demonstrated that the minimal functional Tat system required TatA and TatC. TatA and TatE provide overlapping function. Unlike the TatB proteins from gram-negative bacteria, C. glutamicum TatB was dispensable for Tat function, although it was required for maximal efficiency of secretion. The signal peptide sequence of the isomaltodextranase (IMD) of Arthrobacter globiformis contains a twin-arginine motif. We showed that both IMD and GFP fused with the signal peptide of IMD were secreted via the C. glutamicum Tat pathway. These observations indicate that IMD is a bona fide Tat substrate and imply great potential of the C. glutamicum Tat system for industrial production of heterologous folded proteins.     

Variations in the sequences of BMP2 imply different mechanisms for the evolution of morphological diversity in vertebrates

Bone morphogenetic protein 2 (BMP2) plays an important role in skeletogenesis, osteoblastic differentiation and limb patterning. Its protein coding region consists of the signal peptide, the pro-domain (that regulates post-translational control of synthesis) and the mature domain (that carries out gene function). This gene has been considered previously to be conserved. By re-analyzing the coding region of BMP2 in 31 species of vertebrates, we found that the mature domain region is indeed conserved in mammals, but not among non-mammalian taxa. Moreover, compared to the mature domain, the signal peptide and pro-domain have experienced dramatic variation in all vertebrates. Six amino acid sites in the pro-domain were identified to be under diversifying Darwinian selection in mammals. These results indicate that the signal peptide and pro-domain of BMP2 may be involved in skeletal poly-morphology during mammal evolution and the mature domain may also contribute to this function in non-mammals. This supports the hypothesis that morphological variations in mammals result mainly from a change in post-translational control of synthesis, whereas in non-mammals they result mainly from gene functional change.     

A novel 7-β-(4-carboxybutanamido)-cephalosporanic acid acylase isolated from Pseudomonas strain C427 and its high-level production in Escherichia coli

We cloned the gene for 7-β-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA) acylase from Pseudomonas strain C427. The DNA sequence revealed an open reading frame of 2154 bp coding for 718 amino acid residues. The deduced amino acid sequence consists of 4 structural domains: (i) a signal peptide (positions 1–27), (ii) a small subunit of the acylase (positions 28–190), designated as α, (iii) a spacer peptide (positions 191–198), (iv) a large subunit (positions 199–718), designated as β. Plasmids were constructed to direct the synthesis of the acylase in Escherichia coli and the following results were obtained. The active acylase consists of two subunits which are processed from a single precursor protein, removing the spacer peptide during processing. A proportion of active acylase is secreted into the periplasm and the remainder is retained in the cytoplasm. The amount of precursor protein accumulated in the cytoplasm is greatly reduced when plasmids for the acylase lacking the signal sequence are expressed. Therefore, processing is independent of the translocation of the gene product through the cytoplasmic membrane, in contrast to the situation for penicillin G acylase. A high level of active enzyme production was achieved with a plasmid coding for an acylase in which the amino terminal sequence (positions 1–32) of native acylase is replaced by MFPTT.     

Role of the C-terminal pro-domain of aqualysin I in the maturation and translocation of the precursor across the cytoplasmic membrane in Escherichia coli

Aqualysin I, which is a subtilisin-type, extracellular protease secreted by Thermus aquaticus YT-1, is synthesized as a unique precursor bearing pro-domains at both N- and C-terminus of the mature protease domain as well as an N-terminal signal peptide. To investigate the function of the C-terminal pro-domain in maturation and export pathway of the precursor in E. coli cells, aqualysin I variants were constructed in which deletion mutants of the C-terminal pro-domain lacking its own signal peptide were inserted into pIN-III-ompA3. When E. coli harboring wild type and mutant plasmids were induced by 0.2 mM IPTG, active aqualysin I was produced by heat treatment at 65 °C. Aqualysin I precursors with deletions of more than 5 amino acid residues at the C-terminal end of pro-domain were much more rapidly processed than that of wild type, indicating that the C-terminal pro-domain functions as a inhibitor for processing of aqualysin I precursor. With the wild type, most of aqualysin I was present in membrane fraction (probably the outer membrane), whereas for the truncated mutants, it remained in the cytoplasm, indicating that for deletion mutants, their precursors expressed in cells were not translocated across the cytoplasmic membrane, despite the existence of an N-terminal signal peptide.     

Efficient markerless gene replacement in Corynebacterium glutamicum using a new temperature-sensitive plasmid

Random chemical mutation of a Corynebacterium glutamicum-Escherichia coli shuttle vector derived from plasmid pCGR2 was done using hydroxylamine. It brought about amino acid substitutions G109D and E180K within the replicase superfamily domain of the plasmid's RepA protein and rendered the plasmid highly unstable, especially at higher incubation temperatures. Colony formation of C. glutamicum was consequently completely inhibited at 37 °C but not at 25 °C. G109 is a semi-conserved residue mutation which resulted in major temperature sensitivity. E180 on the other hand is not conserved even among RepA proteins of closely related C. glutamicum pCG1 family plasmids and its independent mutation caused relatively moderate plasmid instability. Nonetheless, simultaneous mutation of both residues was required to achieve temperature-sensitive colony formation. This new pCGR2-derived temperature-sensitive plasmid enabled highly efficient chromosomal integration in a variety of C. glutamicum wild-type strains, proving its usefulness in gene disruption studies. Based on this, an efficient markerless gene replacement system was demonstrated using a selection system incorporating the temperature-sensitive replicon and Bacillus subtilis sacB selection marker, a system hitherto not used in this bacterium. Single-crossover integrants were accurately selected by temperature-dependent manner and 93% of the colonies obtained by the subsequent sucrose selection were successful double-crossover recombinants.     

Phosphate Starvation-Inducible Gene ushA Encodes a 5′ Nucleotidase Required for Growth of Corynebacterium glutamicum on Media with Nucleotides as the Phosphorus Source

Phosphorus is an essential component of macromolecules, like DNA, and central metabolic intermediates, such as sugar phosphates, and bacteria possess enzymes and control mechanisms that provide an optimal supply of phosphorus from the environment. UDP-sugar hydrolases and 5′ nucleotidases may play roles in signal transduction, as they do in mammals, in nucleotide salvage, as demonstrated for UshA of Escherichia coli, or in phosphorus metabolism. The Corynebacterium glutamicum gene ushA was found to encode a secreted enzyme which is active as a 5′ nucleotidase and a UDP-sugar hydrolase. This enzyme was synthesized and secreted into the medium when C. glutamicum was starved for inorganic phosphate. UshA was required for growth of C. glutamicum on AMP and UDP-glucose as sole sources of phosphorus. Thus, in contrast to UshA from E. coli, C. glutamicum UshA is an important component of the phosphate starvation response of this species and is necessary to access nucleotides and related compounds as sources of phosphorus.     

Identification of new secreted proteins and secretion of heterologous amylase by <Emphasis Type="Italic">C. glutamicum</Emphasis>

In this study, secreted Corynebacterium glutamicum proteins were investigated by two-dimensional gel electrophoresis. Around 100 spots observed in the pH range 4.5–5.5 had molecular masses that varied from 10 to 50 kDa. Upon N-terminal amino acid sequence analysis by Edman degradation, two of them were hits to two hypothetical proteins encoded by cgR_1176 and cgR_2070 on C. glutamicum R genome, respectively. Active-form α-amylase derived from Geobacillus stearothermophilus was successfully secreted by using the predicted cgR_1176 and cgR_2070 signal sequences, indicating that these hypothetical proteins were secreted proteins. Analysis using a disruption mutant of the twin-arginine translocation (Tat) export pathway machinery of C. glutamicum suggested that one is Tat pathway dependent secretion while the other is independent of the pathway. Our results demonstrate that C. glutamicum can secrete exoproteins by using its own signal sequences, indicating its potential as a host for protein productions.     

The promoter-proximal region of the Bacillus licheniformis penicillinase gene: Nucleotide sequence and predicted leader peptide sequence

Penicillinase (β-lactamase) is a major species of secreted protein produced by Bacillus licheniformis 749. From the pTB2 recombinant plasmid containing the cloned entire penicillinase (penP) gene, we have isolated and sequenced a 446-bp HpaII fragment carrying the beginning of penP. The 3′-end coding region of 216-bp on this DNA fragment codes for the first 72 amino acids of the prepenicillinase protein. The deduced structure of the leader peptide consists of a 34 amino acid signal sequence with a hydrophilic N-terminal region and a central hydrophobic core.     

Functional analysis of arabinofuranosidases and a xylanase of <Emphasis Type="Italic">Corynebacterium alkanolyticum</Emphasis> for arabinoxylan utilization in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Xylooligosaccharides (XOSs) and arabinoxylooligosaccharides (AXOSs) are major oligosaccharides derived from arabinoxylan. In our previous report, Corynebacterium glutamicum was engineered to utilize XOSs by introducing Corynebacterium alkanolyticum xyloside transporter and β-xylosidase. However, this strain was unable to consume AXOSs due to the absence of α-l-arabinofuranosidase activity. In this study, to confer AXOS utilization ability on C. glutamicum, two putative arabinofuranosidase genes (abf51A and abf51B) were isolated from C. alkanolyticum by the combination of degenerate PCR and genome walking methods. Recombinant Abf51A and Abf51B heterologously expressed in Escherichia coli showed arabinofuranosidase activities toward 4-nitrophenyl-α-l-arabinofuranoside with k _cat values of 150 and 63, respectively, with optimum at pH 6.0 to 6.5. However, Abf51A showed only a slight activity toward AXOSs and was more susceptible to product inhibition by arabinose and xylose than Abf51B. Introduction of abf51B gene into the C. glutamicum XOS-utilizing strain enabled it to utilize AXOSs as well as XOSs. The xylI gene encoding a putative xylanase was found upstream of the C. alkanolyticum xyloside transporter genes. A signal peptide was predicted at the N-terminus of the xylI-encoding polypeptide, which indicated XylI was a secreted protein. Recombinant mature XylI protein heterologously expressed in E. coli showed a xylanase activity toward xylans from various plant sources with optimum at pH 6.5, and C. glutamicum recombinant strain expressing native XylI released xylose, xylobiose, xylotriose, and arabino-xylobiose from arabinoxylan. Finally, introduction of the xylI gene into the C. glutamicum AXOS-utilizing strain enabled it to directly utilize arabinoxylan.     

Heterologous Leaky Production of Transglutaminase in Lactococcus lactis Significantly Enhances the Growth Performance of the Host

This study describes a novel strategy to improve the growth performance of Lactococcus lactis by heterologous production of food-grade transglutaminase. The mtg gene from Streptoverticillium mobaraense that encodes the transglutaminase mature protein was cloned into a nisin-inducible expression vector and transformed into L. lactis subsp. cremoris NZ9000. The leaky expression of the mtg gene from the nisA promoter resulted in ammonia formation and carbon flux redistribution at the pyruvate branch. As a consequence, medium acidification was lessened and energy utilization was improved. This led to significantly higher biomass production under aerobic conditions and particularly under non-pH-controlled conditions (up to a 12-fold increase). The results presented here provide a novel way to enhance the growth yield of L. lactis, which is an important step for the purposes of producing proteins of commercial interest using L. lactis as a host.