Tuning the antimicrobial activity of microbial glycolipid biosurfactants through chemical modification.

Sophorolipids, glycolipid biosurfactants derived from microorganisms such as Starmerella bombicola, possess distinctive surface-active and bioactive properties, holding potential applications in cosmetics, pharmaceuticals and bioremediation. However, the limited structural variability in wild-type sophorolipids restricts their properties and applications. To address this, metabolic engineering efforts have allowed to create a portfolio of molecules. In this study, we went one step further by chemically modifying microbially produced sophorosides, produced by an engineered S. bombicola. Twenty-four new sophoroside derivatives were synthesized, including sophoroside amines with varying alkyl chain lengths (ethyl to octadecyl) on the nitrogen atom and their corresponding quaternary ammonium salts. Additionally, six different microbially produced glycolipid biosurfactants were hydrogenated to achieve fully saturated lipid tails. These derivatives, along with microbially produced glycolipids and three benchmark biosurfactants (di-rhamnolipids, alkyl polyglucosides, cocamidopropyl betaine), were assessed for antimicrobial activity against bacteria (Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa) and yeast (Candida albicans). Results indicated that microbially produced glycolipids, such as bola sophorosides, acidic sophorolipids and acidic glucolipids exhibit selective antimicrobial activity against the test organisms. Conversely, lactonic sophorolipids, sophoroside amines and quaternary ammonium salts display a broad antimicrobial activity. N-octyl, N-dodecyl and N-octadecyl derivatives exhibit the lowest minimal inhibitory concentrations, ranging from 0.014 to 20.0 mg mL-1. This study demonstrates the potential synergy of thoughtful biotechnology and targeted chemistry to precisely tailor glycolipid biosurfactants to meet specific requirements across applications.

An exploratory in silico comparison of open-source codon harmonization tools.

BACKGROUND: Not changing the native constitution of genes prior to their expression by a heterologous host can affect the amount of proteins synthesized as well as their folding, hampering their activity and even cell viability. Over the past decades, several strategies have been developed to optimize the translation of heterologous genes by accommodating the difference in codon usage between species. While there have been a handful of studies assessing various codon optimization strategies, to the best of our knowledge, no research has been performed towards the evaluation and comparison of codon harmonization algorithms. To highlight their importance and encourage meaningful discussion, we compared different open-source codon harmonization tools pertaining to their in silico performance, and we investigated the influence of different gene-specific factors. RESULTS: In total, 27 genes were harmonized with four tools toward two different heterologous hosts. The difference in %MinMax values between the harmonized and the original sequences was calculated (ΔMinMax), and statistical analysis of the obtained results was carried out. It became clear that not all tools perform similarly, and the choice of tool should depend on the intended application. Almost all biological factors under investigation (GC content, RNA secondary structures and choice of heterologous host) had a significant influence on the harmonization results and thus must be taken into account. These findings were substantiated using a validation dataset consisting of 8 strategically chosen genes. CONCLUSIONS: Due to the size of the dataset, no complex models could be developed. However, this initial study showcases significant differences between the results of various codon harmonization tools. Although more elaborate investigation is needed, it is clear that biological factors such as GC content, RNA secondary structures and heterologous hosts must be taken into account when selecting the codon harmonization tool.

Engineering Comamonas testosteroni for the production of 2-pyrone-4,6-dicarboxylic acid as a promising building block.

BACKGROUND: Plastics are an indispensable part of our daily life. However, mismanagement at their end-of-life results in severe environmental consequences. The microbial conversion of these polymers into new value-added products offers a promising alternative. In this study, we engineered the soil-bacterium Comamonas testosteroni KF-1, a natural degrader of terephthalic acid, for the conversion of the latter to the high-value product 2-pyrone-4,6-dicarboxylic acid. RESULTS: In order to convert terephthalic acid to 2-pyrone-4,6-dicarboxylic acid, we deleted the native PDC hydrolase and observed only a limited amount of product formation. To test whether this was the result of an inhibition of terephthalic acid uptake by the carbon source for growth (i.e. glycolic acid), the consumption of both carbon sources was monitored in the wild-type strain. Both carbon sources were consumed at the same time, indicating that catabolite repression was not the case. Next, we investigated if the activity of pathway enzymes remained the same in the wild-type and mutant strain. Here again, no statistical differences could be observed. Finally, we hypothesized that the presence of a pmdK variant in the degradation operon could be responsible for the observed phenotype and created a double deletion mutant strain. This newly created strain accumulated PDC to a larger extent and again consumed both carbon sources. The double deletion strain was then used in a bioreactor experiment, leading to the accumulation of 6.5 g/L of product in 24 h with an overall productivity of 0.27 g/L/h. CONCLUSIONS: This study shows the production of the chemical building block 2-pyrone-4,6-dicarboxylic acid from terephthalic acid through an engineered C. testosteroni KF-1 strain. It was observed that both a deletion of the native PDC hydrolase as well as a pmdK variant is needed to achieve high conversion yields. A product titer of 6.5 g/L in 24 h with an overall productivity of 0.27 g/L/h was achieved.

MariClus: Your One-Stop Platform for Information on Marine Natural Products, Their Gene Clusters and Producing Organisms.

BACKGROUND: The marine environment hosts the vast majority of living species and marine microbes that produce natural products with great potential in providing lead compounds for drug development. With over 70% of Earth's surface covered in water and the high interaction rate associated with liquid environments, this has resulted in many marine natural product discoveries. Our improved understanding of the biosynthesis of these molecules, encoded by gene clusters, along with increased genomic information will aid us in uncovering even more novel compounds. RESULTS: We introduce MariClus (https://www.mariclus.com), an online user-friendly platform for mining and visualizing marine gene clusters. The first version contains information on clusters and the predicted molecules for over 500 marine-related prokaryotes. The user-friendly interface allows scientists to easily search by species, cluster type or molecule and visualize the information in table format or graphical representation. CONCLUSIONS: This new online portal simplifies the exploration and comparison of gene clusters in marine species for scientists and assists in characterizing the bioactive molecules they produce. MariClus integrates data from public sources, like GenBank, MIBiG and PubChem, with genome mining results from antiSMASH. This allows users to access and analyze various aspects of marine natural product biosynthesis and diversity.

Comprehensive metabolomics reveals correlation between sophorolipid biosynthesis and autophagy.

Sophorolipids are biobased and biodegradable glycolipid surface-active agents contributing to the shift from petroleum to biobased surfactants, associated with clear environmental benefits. However, their production cost is currently too high to allow commercialisation. Therefore, a continuous sophorolipid production process was evaluated, i.e., a retentostat with an external filtration unit. Despite an initial increase in volumetric productivity, productivity eventually declined to almost 0 g L-1 h-1. Following comprehensive metabolomics on supernatant obtained from a standardised retentostat, we hypothesised exhaustion of the N-starvation-induced autophagy as the main mechanism responsible for the decline in bolaform sophorolipid productivity. Thirty-six metabolites that correlate with RNA/protein autophagy and high sophorolipid productivity were putatively identified. In conclusion, our results unveil a plausible cause of this bola sophorolipid productivity decline in an industrially relevant bioreactor set-up, which may thus impact majorly on future yeast biosurfactant regulation studies and the finetuning of bola sophorolipid production processes.

Industrial side streams as sustainable substrates for microbial production of poly(3-hydroxybutyrate) (PHB).

Poly(3-hydroxybutyrate) (PHB) is a microbially produced biopolymer that is emerging as a propitious alternative to petroleum-based plastics owing to its biodegradable and biocompatible properties. However, to date, the relatively high costs related to the PHB production process are hampering its widespread commercialization. Since feedstock costs add up to half of the total production costs, ample research has been focusing on the use of inexpensive industrial side streams as carbon sources. While various industrial side streams such as second-generation carbohydrates, lignocellulose, lipids, and glycerol have been extensively investigated in liquid fermentation processes, also gaseous sources, including carbon dioxide, carbon monoxide, and methane, are gaining attention as substrates for gas fermentation. In addition, recent studies have investigated two-stage processes to convert waste gases into PHB via organic acids or alcohols. In this review, a variety of different industrial side streams are discussed as more sustainable and economical carbon sources for microbial PHB production. In particular, a comprehensive overview of recent developments and remaining challenges in fermentation strategies using these feedstocks is provided, considering technical, environmental, and economic aspects to shed light on their industrial feasibility. As such, this review aims to contribute to the global shift towards a zero-waste bio-economy and more sustainable materials.

From bumblebee to bioeconomy: Recent developments and perspectives for sophorolipid biosynthesis.

Sophorolipids are biobased compounds produced by the genera Starmerella and Pseudohyphozyma that gain exponential interest from academic and industrial stakeholders due to their mild and environmental friendly characteristics. Currently, industrially relevant sophorolipid volumetric productivities are reached up to 3.7 g∙L-1∙h-1 and sophorolipids are used in the personal care and cleaning industry at small scale. Moreover, applications in crop protection, food, biohydrometallurgy and medical fields are being extensively researched. The research and development of sophorolipids is at a crucial stage. Therefore, this work presents an overview of the state-of-the-art on sophorolipid research and their applications, while providing a critical assessment of scientific techniques and standardisation in reporting. In this review, the genuine sophorolipid producing organisms and the natural role of sophorolipids are discussed. Subsequently, an evaluation is made of innovations in production processes and the relevance of in-situ product recovery for process performance is discussed. Furthermore, a critical assessment of application research and its future perspectives are portrayed with a focus on the self-assembly of sophorolipid molecules. Following, genetic engineering strategies that affect the sophorolipid physiochemical properties are summarised. Finally, the impact of sophorolipids on the bioeconomy are uncovered, along with relevant future perspectives.

A multi-omics study to boost continuous bolaform sophorolipid production.

Biodegradable and biobased surface active agents are renewable and environmentally friendly alternatives to petroleum derived or oleochemical surfactants. However, they are accompanied by relatively high production costs. In this study, the aim was to reduce the production costs for an innovative type of microbial biosurfactant: bolaform sophorolipids, produced by the yeast Starmerella bombicola ΔsbleΔat. A novel continuous retentostat set-up was performed whereby continuous broth microfiltration retained the biomass in the bioreactor while performing an in situ product separation of bolaform sophorolipids. Although a mean volumetric productivity of 0.56 g L-1 h-1 was achieved, it was not possible to maintain this productivity, which collapsed to almost 0 g L-1 h-1. Therefore, two process adaptations were evaluated, a sequential batch strategy and a phosphate limitation alleviation strategy. The sequential batch set-up restored the mean volumetric productivity to 0.66 g L-1 h-1 for an additional 132 h but was again followed by a productivity decline. A similar result was obtained with the phosphate limitation alleviation strategy where a mean volumetric productivity of 0.54 g L-1 h-1 was reached, but a productivity decline was also observed. Whole genome variant analysis uncovered no evidence for genomic variations for up to 1306 h of retentostat cultivation. Untargeted metabolomics analysis identified 8-hydroxyguanosine, a biomarker for oxidative RNA damage, as a key metabolite correlating with high bolaform sophorolipid productivity. This study showcases the application of a retentostat to increase bolaform sophorolipid productivity and lays the basis of a multi-omics platform for in depth investigation of microbial biosurfactant production with S. bombicola.

Advanced PHB fermentation strategies with CO2-derived organic acids.

Over the past decade, formic acid and acetic acid have gained increasing attention as alternative feedstocks for poly-3-hydroxybutyrate (PHB) production as these potentially CO2-derived molecules are naturally assimilated by Cupriavidus necator. Both organic acids were individually evaluated in fed-batch fermentations at bioreactor scale. Acetic acid was revealed as the most promising carbon source yielding 42.3 g L-1 PHB, whereas no significant amount of PHB was produced from formic acid. Hence, acetic acid was further used as the substrate during process intensification. Key performance characteristics, including process stability, PHB titer, and productivity were optimized by introducing NH4-acetate as the nitrogen source, extending the growth phase, and implementing a repeated fed-batch procedure, respectively. These advanced fermentation strategies resulted in the establishment of a stable fermentation process reaching 58.5 g L-1 PHB, while doubling the productivity to 0.93 g L-1 h-1 PHB.

Elucidation of the Natural Function of Sophorolipids Produced by Starmerella bombicola.

The yeast Starmerella bombicola distinguishes itself from other yeasts by its potential of producing copious amounts of the secondary metabolites sophorolipids (SLs): these are glycolipid biosurfactants composed out of a(n) (acetylated) sophorose moiety and a lipid tail. Although SLs are the subject of numerous research papers and have been commercialized, e.g., in eco-friendly cleaning solutions, the natural function of SLs still remains elusive. This research article investigates several hypotheses for why S. bombicola invests that much energy in the production of SLs, and we conclude that the main natural function of SLs in S. bombicola is niche protection: (1) the extracellular storage of an energy-rich, yet metabolically less accessible carbon source that can be utilized by S. bombicola upon conditions of starvation with (2) antimicrobial properties. In this way, S. bombicola creates a dual advantage in competition with other microorganisms. Additionally, SLs can expedite growth on rapeseed oil, composed of triacylglycerols which are hydrophobic substrates present in the yeasts' environment, for a non-SL producing strain (Δcyp52M1). It was also found that-at least under lab conditions-SLs do not provide protection against high osmotic pressure prevalent in sugar-rich environments such as honey or nectar present in the natural habitat of S. bombicola.

Novel Alkaloids from Marine Actinobacteria: Discovery and Characterization.

The marine environment is an excellent resource for natural products with therapeutic potential. Its microbial inhabitants, often associated with other marine organisms, are specialized in the synthesis of bioactive secondary metabolites. Similar to their terrestrial counterparts, marine Actinobacteria are a prevalent source of these natural products. Here, we discuss 77 newly discovered alkaloids produced by such marine Actinobacteria between 2017 and mid-2021, as well as the strategies employed in their elucidation. While 12 different classes of alkaloids were unraveled, indoles, diketopiperazines, glutarimides, indolizidines, and pyrroles were most dominant. Discoveries were mainly based on experimental approaches where microbial extracts were analyzed in relation to novel compounds. Although such experimental procedures have proven useful in the past, the methodologies need adaptations to limit the chance of compound rediscovery. On the other hand, genome mining provides a different angle for natural product discovery. While the technology is still relatively young compared to experimental screening, significant improvement has been made in recent years. Together with synthetic biology tools, both genome mining and extract screening provide excellent opportunities for continued drug discovery from marine Actinobacteria.

Comprehensive study on Escherichia coli genomic expression: Does position really matter?

As a biorefinery platform host, Escherichia coli has been used extensively to produce metabolites of commercial interest. Integration of foreign DNA onto the bacterial genome allows for stable expression overcoming the need for plasmid expression and its associated instability. Despite the development of numerous tools and genome editing technologies, the question of where to incorporate a synthetic pathway remains unanswered. To address this issue, we studied the genomic expression in E. coli and linked it not only to 26 rationally selected genomic locations, but also to the gene direction in relation to the DNA replication fork, to the carbon and nitrogen source, to DNA folding and supercoiling, and to metabolic burden. To enable these experiments, we have designed a fluorescent expression cassette to eliminate specific local effects on gene expression. Overall it can be concluded that although the expression range obtained by changing the genomic location of a pathway is small compared to the range typically seen in promoter-RBS libraries, the effect of culture medium, environmental stress and metabolic burden can be substantial. The characterization of multiple effects on genomic expression, and the associated libraries of well-characterized strains, will only stimulate and improve the creation of stable production hosts fit for industrial settings.

Alkaloids from Marine Fungi: Promising Antimicrobials.

Resistance of pathogenic microorganisms against antimicrobials is a major threat to contemporary human society. It necessitates a perpetual influx of novel antimicrobial compounds. More specifically, Gram- pathogens emerged as the most exigent danger. In our continuing quest to search for novel antimicrobial molecules, alkaloids from marine fungi show great promise. However, current reports of such newly discovered alkaloids are often limited to cytotoxicity studies and, moreover, neglect to discuss the enigma of their biosynthesis. Yet, the latter is often a prerequisite to make them available through sufficiently efficient processes. This review aims to summarize novel alkaloids with promising antimicrobial properties discovered in the past five years and produced by marine fungi. Several discovery strategies are summarized, and knowledge gaps in biochemical production routes are identified. Finally, links between the structure of the newly discovered molecules and their activity are proposed. Since 2015, a total of 35 new antimicrobial alkaloids from marine fungi were identified, of which 22 showed an antibacterial activity against Gram- microorganisms. Eight of them can be classified as narrow-spectrum Gram- antibiotics. Despite this promising ratio of novel alkaloids active against Gram- microorganisms, the number of newly discovered antimicrobial alkaloids is low, due to the narrow spectrum of discovery protocols that are used and the fact that antimicrobial properties of newly discovered alkaloids are barely characterized. Alternatives are proposed in this review. In conclusion, this review summarizes novel findings on antimicrobial alkaloids from marine fungi, shows their potential as promising therapeutic candidates, and hints on how to further improve this potential.

5' untranslated regions: the next regulatory sequence in yeast synthetic biology.

When developing industrial biotechnology processes, Saccharomyces cerevisiae (baker's yeast or brewer's yeast) is a popular choice as a microbial host. Many tools have been developed in the fields of synthetic biology and metabolic engineering to introduce heterologous pathways and tune their expression in yeast. Such tools mainly focus on controlling transcription, whereas post-transcriptional regulation is often overlooked. Herein we discuss regulatory elements found in the 5' untranslated region (UTR) and their influence on protein synthesis. We provide not only an overall picture, but also a set of design rules on how to engineer a 5' UTR. The reader is also referred to currently available models that allow gene expression to be tuned predictably using different 5' UTRs.

Production of long-chain hydroxy fatty acids by Starmerella bombicola.

To decrease our dependency for the diminishing source of fossils resources, bio-based alternatives are being explored for the synthesis of commodity and high-value molecules. One example in this ecological initiative is the microbial production of the biosurfactant sophorolipids by the yeast Starmerella bombicola. Sophorolipids are surface-active molecules mainly used as household and laundry detergents. Because S. bombicola is able to produce high titers of sophorolipids, the yeast is also used to increase the portfolio of lipophilic compounds through strain engineering. Here, the one-step microbial production of hydroxy fatty acids by S. bombicola was accomplished by the selective blockage of three catabolic pathways through metabolic engineering. Successful production of 17.39 g/l (ω-1) linked hydroxy fatty acids was obtained by the successive blockage of the sophorolipid biosynthesis, the ß-oxidation and the ω-oxidation pathways. Minor contamination of dicarboxylic acids and fatty aldehydes were successfully removed using flash chromatography. This way, S. bombicola was further expanded into a flexible production platform of economical relevant compounds in the chemical, food and cosmetic industries.

Hidden antibiotics: Where to uncover?

The struggle of humans versus pathogens is a never ending battle. Since the discovery of antibiotics humans have tipped the scales in their favour, but today bacteria are nullifying this advantage by developing resistance mechanisms against these molecules. The plethora of different antibiotics active against pathogens is shrinking while the discovery of new molecules is arduous. Especially the development of drugs active against Gram- pathogens continues slowly. New strategies to discover novel, potent antibiotics are hence needed. Adopting the optimistic view of technological singularity, innovative and disruptive approaches are required and hence proposed to lift the current conundrum. In this review, questions are answered on where and how to look for new natural product hit molecules with antibacterial activity, on how the field of synthetic biology can aid the contemporary pharmaceutical challenge and whether we are ready to make the transition towards other approaches, such as narrow-spectrum antibiotics and phage therapy.

Synthesis of bolaform biosurfactants by an engineered Starmerella bombicola yeast.

Bola-amphiphilic surfactants are molecules with fascinating properties. Their unique configuration consisting of a long hydrophobic spacer connecting two hydrophilic entities renders the molecule more water soluble than the average surfactant, but still allows formation of supramolecular structures. These properties make them extremely suitable for applications in in nanotechnology, electronics, and gene and drug delivery. In general, these compounds are obtained by chemical synthesis. We report here an efficient microbial production process for the fully green synthesis of bolaform surfactants. A sophorolipid-producing Starmerella bombicola yeast strain was disabled in its sophorolipid acetyltransferase and lactone esterase, which should logically result in synthesis of non-acetylated acidic sophorolipids; molecules with the classic amphiphilic structure. Surprisingly, also bolaform glycolipids were obtained, with an additional sophorose linked to the free carboxyl end of the acidic sophorolipids as confirmed by MS and NMR analysis. The obtained titers of 27.7 g/L total product are comparable to wild type values, and the novel molecules account for at least 74% of this. Bola-amphiphile biosynthesis proved to be attributed to the promiscuous activity of both UDP-glucosyltransferases UGTA1 and UGTB1 from the core sophorolipid pathway, displaying activity toward non-acetylated intermediates. The absence of acetyl groups seems to trigger formation of bolaform compounds starting from acidic sophorolipids. Hence, wild type S. bombicola produces these compounds only at marginal amounts in general not reaching detection limits. We created a strain knocked-out in its sophorolipid acetyltransferase and lactone esterase able to produce these novel compounds in economical relevant amounts, opening doors for the application of biological-derived bolaform structures. Biotechnol. Bioeng. 2016;113: 2644-2651. © 2016 Wiley Periodicals, Inc.

High yield 1,3-propanediol production by rational engineering of the 3-hydroxypropionaldehyde bottleneck in Citrobacter werkmanii.

BACKGROUND: Imbalance in cofactors causing the accumulation of intermediates in biosynthesis pathways is a frequently occurring problem in metabolic engineering when optimizing a production pathway in a microorganism. In our previous study, a single knock-out Citrobacter werkmanii ∆dhaD was constructed for improved 1,3-propanediol (PDO) production. Instead of an enhanced PDO concentration on this strain, the gene knock-out led to the accumulation of the toxic intermediate 3-hydroxypropionaldehyde (3-HPA). The hypothesis was emerged that the accumulation of this toxic intermediate, 3-HPA, is due to a cofactor imbalance, i.e. to the limited supply of reducing equivalents (NADH). Here, this bottleneck is alleviated by rationally engineering cell metabolism to balance the cofactor supply. RESULTS: By eliminating non-essential NADH consuming enzymes (such as lactate dehydrogenase coded by ldhA, and ethanol dehydrogenase coded by adhE) or by increasing NADH producing enzymes, the accumulation of 3-HPA is minimized. Combining the above modifications in C. werkmanii ∆dhaD resulted in the strain C. werkmanii ∆dhaD∆ldhA∆adhE::ChlFRT which provided the maximum theoretical yield of 1.00 ± 0.03 mol PDO/mol glycerol when grown on glucose/glycerol (0.33 molar ratio) on flask scale under anaerobic conditions. On bioreactor scale, the yield decreased to 0.73 ± 0.01 mol PDO/mol glycerol although no 3-HPA could be measured, which indicates the existence of a sink of glycerol by a putative glycerol dehydrogenase, channeling glycerol to the central metabolism. CONCLUSIONS: In this study, a multiple knock-out was created in Citrobacter species for the first time. As a result, the concentration of the toxic intermediate 3-HPA was reduced to below the detection limit and the maximal theoretical PDO yield on glycerol was reached.

1,3-propanediol production with Citrobacter werkmanii DSM17579: effect of a dhaD knock-out.

BACKGROUND: 1,3-propanediol (PDO) is a substantially industrial metabolite used in the polymer industry. Although several natural PDO production hosts exist, e.g. Klebsiella sp., Citrobacter sp. and Clostridium sp., the PDO yield on glycerol is insufficient for an economically viable bio-process. Enhancing this yield via strain improvement can be achieved by disconnecting the production and growth pathways. In the case of PDO formation, this approach results in a microorganism metabolizing glycerol strictly for PDO production, while catabolizing a co-substrate for growth and maintenance. We applied this strategy to improve the PDO production with Citrobacter werkmanii DSM17579. RESULTS: Genetic tools were developed and used to create Citrobacter werkmanii DSM17579 ∆dhaD in which dhaD, encoding for glycerol dehydrogenase, was deleted. Since this strain was unable to grow on glycerol anaerobically, both pathways were disconnected. The knock-out strain was perturbed with 13 different co-substrates for growth and maintenance. Glucose was the most promising, although a competition between NADH-consuming enzymes and 1,3-propanediol dehydrogenase emerged. CONCLUSION: Due to the deletion of dhaD in Citrobacter werkmanii DSM17579, the PDO production and growth pathway were split. As a consequence, the PDO yield on glycerol was improved 1,5 times, strengthening the idea that Citrobacter werkmanii DSM17579 could become an industrially interesting host for PDO production.

Unraveling the dha cluster in Citrobacter werkmanii: comparative genomic analysis of bacterial 1,3-propanediol biosynthesis clusters.

In natural 1,3-propanediol (PDO) producing microorganisms such as Klebsiella pneumoniae, Citrobacter freundii and Clostridium sp., the genes coding for PDO producing enzymes are grouped in a dha cluster. This article describes the dha cluster of a novel candidate for PDO production, Citrobacter werkmanii DSM17579 and compares the cluster to the currently known PDO clusters of Enterobacteriaceae and Clostridiaceae. Moreover, we attribute a putative function to two previously unannotated ORFs, OrfW and OrfY, both in C. freundii and in C. werkmanii: both proteins might form a complex and support the glycerol dehydratase by converting cob(I)alamin to the glycerol dehydratase cofactor coenzyme B12. Unraveling this biosynthesis cluster revealed high homology between the deduced amino acid sequence of the open reading frames of C. werkmanii DSM17579 and those of C. freundii DSM30040 and K. pneumoniae MGH78578, i.e., 96 and 87.5 % identity, respectively. On the other hand, major differences between the clusters have also been discovered. For example, only one dihydroxyacetone kinase (DHAK) is present in the dha cluster of C. werkmanii DSM17579, while two DHAK enzymes are present in the cluster of K. pneumoniae MGH78578 and Clostridium butyricum VPI1718.