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Bacteriocins are antimicrobial peptides applied in food preservation and are interesting candidates as alternatives to conventional antibiotics or as microbiome modulators. Recently, we established Corynebacterium glutamicum as a suitable production host for various bacteriocins including garvicin Q (GarQ). Here, we establish secretion of GarQ by C. glutamicum via the Sec translocon achieving GarQ titers of about 7 mg L–1 in initial fermentations. At neutral pH, the cationic peptide is efficiently adsorbed to the negatively charged envelope of producer bacteria limiting availability of the bacteriocin in culture supernatants. A combination of CaCl2 and Tween 80 efficiently reduces GarQ adsorption to C. glutamicum. Moreover, cultivation in minimal medium supplemented with CaCl2 and Tween 80 improves GarQ production by C. glutamicum to about 15 mg L–1 but Tween 80 resulted in reduced GarQ activity at later timepoints. Using a reporter strain and proteomic analyses, we identified HtrA, a protease associated with secretion stress, as another potential factor limiting GarQ production. Transferring production to HtrA-deficient C. glutamicum K9 improves GarQ titers to close to 40 mg L–1. Applying conditions of low aeration prevented loss in activity at later timepoints and improved GarQ titers to about 100 mg L–1. This is about 50-fold higher than previously shown with a C. glutamicum strain employing the native GarQ transporter GarCD for secretion and in the range of levels observed with the native producer Lactococcus petauri B1726. Additionally, we tested several synthetic variants of GarQ and were able to show that exchange of the methionine in position 5 to a phenylalanine (GarQM5F) results in markedly increased activity against Lactococcus lactis and Listeria monocytogenes. In summary, our findings shed light on several aspects of recombinant GarQ production that may also be of relevance for production with natural producers and other bacteriocins.

The establishment of sustainable processes for the production of commodity chemicals is one of today’s central challenges for biotechnological industries. The chemo-autotrophic fixation of CO2 and the subsequent production of acetate by acetogenic bacteria via anaerobic gas fermentation represents a promising platform for the ecologically sustainable production of high-value biocommodities via sequential fermentation processes. In this study, the applicability of acetate-containing cell-free spent medium of the gas-fermenting acetogenic bacterium A. woodii WP1 as the feeder strain for growth and the recombinant production of P. aeruginosa PAO1 mono-rhamnolipids in the well-established nonpathogenic producer strain P. putida KT2440 were investigated. Additionally, the potential possibility of a simplified production process without the necessary separation of feeder strain cells was elucidated via the cultivation of P. putida in cell-containing A. woodii culture broth. For these cultures, the content of both strains was investigated by examining the relative quantification of strain-exclusive genes via qPCR. The recombinant production of mono-rhamnolipids was successfully achieved with maximum titers of approximately 360–400 mg/L for both cell-free and cell-containing A. woodii spent medium. The reported processes therefore represent a successful proof of principle for gas fermentation-derived acetate as a potential sustainable carbon source for future recombinant rhamnolipid production processes by P. putida KT2440.

Abstract Bacteriocins are ribosomally synthesized antimicrobial peptides, that either kill target bacteria or inhibit their growth. Bacteriocins are used in food preservation and are of increasing interest as potential alternatives to conventional antibiotics. In the present study, we show that Lactococcus petauri B1726, a strain isolated from fermented balsam pear, produces a heat-stable and protease-sensitive compound. Following genome sequencing, a gene cluster for production of a class IId bacteriocin was identified consisting of garQ (encoding for the bacteriocin garvicin Q), garI (for a putative immunity protein), garC, and garD (putative transporter proteins). Growth conditions were optimized for increased bacteriocin activity in supernatants of L. petauri B1726 and purification and mass spectrometry identified the compound as garvicin Q. Further experiments suggest that garvicin Q adsorbs to biomass of various susceptible and insusceptible bacteria and support the hypothesis that garvicin Q requires a mannose-family phosphotransferase system (PTSMan) as receptor to kill target bacteria by disruption of membrane integrity. Heterologous expression of a synthetic garQICD operon was established in Corynebacterium glutamicum demonstrating that genes garQICD are responsible for biosynthesis and secretion of garvicin Q. Moreover, production of garvicin Q by the recombinant C. glutamicum strain was improved by using a defined medium yet product levels were still considerably lower than with the natural L. petauri B1726 producer strain. Collectively, our data identifies the genetic basis for production of the bacteriocin garvicin Q by L. petauri B1726 and provides insights into the receptor and mode of action of garvicin Q. Moreover, we successfully performed first attempts towards biotechnological production of this interesting bacteriocin using natural and heterologous hosts.

Abstract Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B • Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control • Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany

ABSTRACT Genome analysis of Corynebacterium lactis revealed a bacteriocin gene cluster encoding a putative bacteriocin of the linaridin family of ribosomally synthesized and posttranslationally modified peptides (RiPPs). The locus harbors typical linaridin modification enzymes but lacks genes for a decarboxylase and methyltransferase, which is unusual for type B linaridins. Supernatants of Corynebacterium lactis RW3-42 showed antimicrobial activity against Corynebacterium glutamicum. Deletion of the precursor gene crdA clearly linked the antimicrobial activity of the producer strain to the identified gene cluster. Following purification, we observed potent activity of the peptide against Actinobacteria, mainly other members of the genus Corynebacterium, including the pathogenic species Corynebacterium striatum and Corynebacterium amycolatum. Also, low activity against some Firmicutes was observed, but there was no activity against Gram-negative species. The peptide is resilient towards heat but sensitive to proteolytic degradation by trypsin and proteinase K. Analysis by mass spectrometry indicates that corynaridin is processed by cleaving off the leader sequence at a conserved motif and posttranslationally modified by dehydration of all threonine and serin residues, resulting in a monoisotopic mass of 3,961.19 Da. Notably, time-kill kinetics and experiments using live biosensors to monitor membrane integrity suggest bactericidal activity that does not involve formation of pores in the cytoplasmic membrane. As Corynebacterium species are ubiquitous in nature and include important commensals and pathogens of mammalian organisms, secretion of bacteriocins by species of this genus could be a hitherto neglected trait with high relevance for intra- and interspecies competition and infection. IMPORTANCE Bacteriocins are antimicrobial peptides produced by bacteria to fend off competitors in ecological niches and are considered to be important factors influencing the composition of microbial communities. However, bacteriocin production by bacteria of the genus Corynebacterium has been a hitherto neglected trait, although its species are ubiquitous in nature and make up large parts of the microbiome of humans and animals. In this study, we describe and characterize a novel linaridin family bacteriocin from Corynebacterium lactis and show its narrow-spectrum activity, mainly against other actinobacteria. Moreover, we were able to extend the limited knowledge on linaridin bioactivity in general and for the first time describe the bactericidal activity of such a bacteriocin. Interestingly, the peptide, which was named corynaridin, appears bactericidal, but without formation of pores in the bacterial membrane.

Abstract Background The bacteriocin nisin is naturally produced by Lactococcus lactis as an inactive prepeptide that is modified posttranslationally resulting in five (methyl-)lanthionine rings characteristic for class Ia bacteriocins. Export and proteolytic cleavage of the leader peptide results in release of active nisin. By targeting the universal peptidoglycan precursor lipid II, nisin has a broad target spectrum including important human pathogens such as Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains. Industrial nisin production is currently performed using natural producer strains resulting in rather low product purity and limiting its application to preservation of dairy food products. Results We established heterologous nisin production using the biotechnological workhorse organism Corynebacterium glutamicum in a two-step process. We demonstrate successful biosynthesis and export of fully modified prenisin and its activation to mature nisin by a purified, soluble variant of the nisin protease NisP (sNisP) produced in Escherichia coli. Active nisin was detected by a L. lactis sensor strain with strictly nisin-dependent expression of the fluorescent protein mCherry. Following activation by sNisP, supernatants of the recombinant C. glutamicum producer strain cultivated in standard batch fermentations contained at least 1.25 mg/l active nisin. Conclusions We demonstrate successful implementation of a two-step process for recombinant production of active nisin with C. glutamicum. This extends the spectrum of bioactive compounds that may be produced using C. glutamicum to a bacteriocin harboring complex posttranslational modifications. Our results provide a basis for further studies to optimize product yields, transfer production to sustainable substrates and purification of pharmaceutical grade nisin.

Today production of (bulk) chemicals and fuels almost exclusively relies on petroleum-based sources, which are connected to greenhouse gas release, fueling climate change. This increases the urgence to develop alternative bio-based technologies and processes. Gaseous and liquid C1 compounds are available at low cost and often occur as waste streams. Acetogenic bacteria can directly use C1 compounds like CO, CO2, formate or methanol anaerobically, converting them into acetate and ethanol for higher-value biotechnological products. However, these microorganisms possess strict energetic limitations, which in turn pose limitations to their potential for biotechnological applications. Moreover, efficient genetic tools for strain improvement are often missing. However, focusing on the metabolic abilities acetogens provide, they can prodigiously ease these technological disadvantages. Producing acetate and ethanol from C1 compounds can fuel via bio-based intermediates conversion into more energy-demanding, higher-value products, by deploying aerobic organisms that are able to grow with acetate/ethanol as carbon and energy source. Promising new approaches have become available combining these two fermentation steps in sequential approaches, either as separate fermentations or as integrated two-stage fermentation processes. This review aims at introducing, comparing, and evaluating the published approaches of sequential C1 fermentations, delivering a list of promising organisms for the individual fermentation steps and giving an overview of the existing broad spectrum of products based on acetate and ethanol. Understanding of these pioneering approaches allows collecting ideas for new products and may open avenues toward making full use of the technological potential of these concepts for establishment of a sustainable biotechnology.

Cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger discovered in Bacillus subtilis and involved in potassium homeostasis, cell wall maintenance and/or DNA stress response. As the role of c-di-AMP has been mostly studied in Firmicutes, we sought to increase the understanding of its role in Actinobacteria, namely in Corynebacterium glutamicum. This organism is a well-known industrial production host and a model organism for pathogens, such as C. diphtheriae or Mycobacterium tuberculosis. Here, we identify and analyze the minimal set of two C. glutamicum enzymes, the diadenylate cyclase DisA and the phosphodiesterase PdeA, responsible for c-di-AMP metabolism. DisA synthesizes c-di-AMP from two molecules of ATP, whereas PdeA degrades c-di-AMP, as well as the linear degradation intermediate phosphoadenylyl-(3′→5′)-adenosine (pApA) to two molecules of AMP. Here, we show that a ydaO/kimA-type c-di-AMP-dependent riboswitch controls the expression of the strictly regulated cell wall peptidase gene nlpC in C. glutamicum. In contrast to previously described members of the ydaO/kimA-type riboswitches, our results suggest that the C. glutamicum nlpC riboswitch likely affects the translation instead of the transcription of its downstream gene. Although strongly regulated by different mechanisms, we show that the absence of nlpC, the first known regulatory target of c-di-AMP in C. glutamicum, is not detrimental for this organism under the tested conditions.

(1) Background: Hyaluronic acid (HA) is a polyanionic mucopolysaccharide extensively used in biomedical and cosmetic industries due to its unique rheological properties. Recombinant HA production using other microbial platforms has received increasing interest to avoid potential toxin contamination associated with its production by streptococcal fermentation. In this study, the Gram-negative strains Escherichia coli (pLysY/Iq), E. coli Rosetta2, E. coli Rosetta (DE3) pLysS, E. coli Rosetta2 (DE3), E. coli Rosetta gammiB(DE3)pLysS, and the Gram-positive Bacillus megaterium (MS941) were investigated as new platforms for the heterologous production of HA. (2) Results: The HA biosynthesis gene hasA, cloned from Streptococcus equi subsp. zoopedemicus, was ligated into plasmid pMM1522 (MoBiTec), resulting in pMM1522 hasA, which was introduced into E. coli Rosetta-2(DE3) and B. megaterium (MS941). The initial HA titer by the two hosts in the LB medium was 5 mg/L and 50 mg/L, respectively. Streptococcal hasABC and hasABCDE genes were ligated into plasmid pPT7 (MoBiTec) and different E. coli host strains were then transformed with the resulting plasmids pPT7hasABC and pPT7hasABCDE. For E. coli Rosetta-gamiB(DE3)pLysS transformed with pPT7hasABC, HA production was 500 ± 11.4 mg/L in terrific broth (TB) medium. Productivity was slightly higher (585 ± 2.9 mg/L) when the same host was transformed with pPT7 carrying the entire HA operon. We also transformed B. megaterium (MS941) protoplasts carrying T7-RNAP with pPT7hasABC and pPT7hasABCDE. In comparison, the former plasmid resulted in HA titers of 2116.7 ± 44 and 1988.3 ± 19.6 mg/L in LB media supplemented with 5% sucrose and A5 medium + MOPSO, respectively; the latter plasmid boosted the titer final concentration further to reach 2476.7 ± 14.5 mg/L and 2350 ± 28.8 mg/L in the two media, respectively. The molecular mass of representative HA samples ranged from 105 − 106 Daltons (Da), and the polydispersity index (PDI) was E. coli Rosetta-gamiB(DE3)pLysS, while no HA capsules were produced by B. megaterium. (3) Conclusions: Our results suggested that Gram-positive bacteria are probably superior host strains for recombinant HA production over their Gram-negative counters. The titers and the molecular weight (MW) of HA produced by B. megaterium were significantly higher than those obtained by different E. coli host strains used in this study.

The compatible solute mannosylglycerate (MG) has exceptional properties in terms of protein stabilization and protection under salt, heat, and freeze-drying stresses as well as against protein aggregation. Due to these characteristics, MG possesses large potential for clinical and biotechnological applications. To achieve efficient MG production, Corynebacterium glutamicum was equipped with a bifunctional MG synthase (encoded by mgsD and catalyzing the condensation of 3-phosphoglycerate and GDP-mannose to MG) from Dehalococcoides mccartyi. The resulting strain C. glutamicum (pEKEx3 mgsD) intracellularly accumulated about 111 mM MG (60 ± 9 mg gCDW−1) with 2% glucose as a carbon source. To enable efficient mannose metabolization, the native manA gene, encoding mannose 6-phosphate isomerase, was overexpressed. Combined overexpression of manA and mgsD from two plasmids in C. glutamicum resulted in intracellular MG accumulation of up to ca. 329 mM [corresponding to 177 mg g cell dry weight (CDW)−1] with glucose, 314 mM (168 mg gCDW−1) with glucose plus mannose, and 328 mM (176 mg gCDW−1) with mannose as carbon source(s), respectively. The product was successfully extracted from cells by using a cold water shock, resulting in up to 5.5 mM MG (1.48 g L−1) in supernatants. The two-plasmid system was improved by integrating the mgsD gene into the manA-bearing plasmid and the resulting strain showed comparable production but faster growth. Repeated cycles of growth/production and extraction of MG in a bacterial milking-like experiment showed that cells could be recycled, which led to a cumulative MG production of 19.9 mM (5.34 g L−1). The results show that the newly constructed C. glutamicum strain produces MG from glucose and mannose and that a cold water shock enables extraction of MG from the cytosol into the medium.

Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) is an acetylated amino sugar nucleotide that naturally serves as precursor in bacterial cell wall synthesis and is involved in prokaryotic and eukaryotic glycosylation reactions. UDP-GlcNAc finds application in various fields including the production of oligosaccharides and glycoproteins with therapeutic benefits. At present, nucleotide sugars are produced either chemically or in vitro by enzyme cascades. However, chemical synthesis is complex and non-economical, and in vitro synthesis requires costly substrates and often purified enzymes. A promising alternative is the microbial production of nucleotide sugars from cheap substrates. In this study, we aimed to engineer the non-pathogenic, Gram-positive soil bacterium Corynebacterium glutamicum as a host for UDP-GlcNAc production. The native glmS, glmU, and glmM genes and glmM of Escherichia coli, encoding the enzymes for UDP-GlcNAc synthesis from fructose-6-phosphate, were over-expressed in different combinations and from different plasmids in C. glutamicum GRS43, which lacks the glucosamine-6-phosphate deaminase gene (nagB) for glucosamine degradation. Over-expression of glmS, glmU and glmM, encoding glucosamine-6-phosphate synthase, the bifunctional glucosamine-1-phosphate acetyltransferase/N-acetyl glucosamine-1-phosphate uridyltransferase and phosphoglucosamine mutase, respectively, was confirmed using activity assays or immunoblot analysis. While the reference strain C. glutamicum GlcNCg1 with an empty plasmid in the exponential growth phase contained intracellularly only about 0.25 mM UDP-GlcNAc, the best engineered strain GlcNCg4 accumulated about 14 mM UDP-GlcNAc. The extracellular UDP-GlcNAc concentrations in the exponential growth phase did not exceed 2 mg/L. In the stationary phase, about 60 mg UDP-GlcNAc/L was observed extracellularly with strain GlcNCg4, indicating the potential of C. glutamicum to produce and to release the activated sugar into the culture medium. To our knowledge, the observed UDP-GlcNAc levels are the highest obtained with microbial hosts, emphasizing the potential of C. glutamicum as a suitable platform for activated sugar production.

Post-translationally modified, (methyl-)lanthionine-containing peptides are produced by several Gram-positive bacteria. These so-called lantibiotics have potent activity against various bacterial pathogens including multidrug-resistant strains and are thus discussed as alternatives to antibiotics. Several naturally occurring mechanisms of resistance against lantibiotics have been described for bacteria, including cell envelope modifications, ABC-transporters, lipoproteins and peptidases. Corynebacterium species are widespread in nature and comprise important pathogens, commensals as well as environmentally and biotechnologically relevant species. Yet, little is known about lantibiotic biosynthesis and resistance in this genus. Here, we present a comprehensive in silico prediction of lantibiotic resistance traits in this important group of Gram-positive bacteria. Our analyses suggest that enzymes for cell envelope modification, peptidases as well as ABC-transporters involved in peptide resistance are widely distributed in the genus. Based on our predictions, we analyzed the susceptibility of six Corynebacterium species to nisin and found that those without dedicated resistance traits are more susceptible and unable to adapt to higher concentrations. In addition, we were able to identify lantibiotic resistance operons encoding for peptidases, ABC-transporters and two-component systems with an unusual predicted structure that are conserved in the genus Corynebacterium. Heterologous expression shows that these operons indeed confer resistance to the lantibiotic nisin.

Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide to bicarbonate and maintains the balance of CO2/HCO3- in the intracellular environment, specifically for carboxylation/decarboxylation reactions. In Corynebacterium glutamicum, two putative genes, namely the bca (cg2954) and gca (cg0155) genes, coding for β-type and γ-type carbonic anhydrase, respectively, have been identified. We here analyze the transcriptional organization of these genes. The transcriptional start site (TSS) of the bca gene was shown to be the first nucleotide "A" of its putative translational start codon (ATG) and thus, bca codes for a leaderless transcript. The TSS of the gca gene was identified as an "A" residue located at position -20 relative to the first nucleotide of the annotated translational start codon of the cg0154 gene, which is located immediately upstream of gca. Comparative expression analysis revealed carbon source-dependent regulation of the bca gene, with 1.5- to 2-fold lower promoter activity in cells grown on acetate as compared to glucose as sole carbon source. Based on higher expression of bca in a mutant deficient of the regulator of acetate metabolism RamA as compared to the wild-type of C. glutamicum and based on the binding of His-tagged RamA protein to the bca promoter region, we here present evidence that RamA negatively regulates expression of bca in C. glutamicum. Functional characterization of a gca deletion mutant of C. glutamicum revealed the same growth characteristics of C. glutamicum ∆gca as that of wild-type C. glutamicum and no effect on expression of the bca gene.