AutoBioTech─A Versatile Biofoundry for Automated Strain Engineering.

The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of Escherichia coli with plasmids assembled via modular cloning. A CRISPR/Cas9 toolbox compatible with existing modular cloning frameworks allows automated and flexible genome editing of E. coli. In addition, novel workflows have been established for the fully automated transformation of the Gram-positive model organism Corynebacterium glutamicum by conjugation and electroporation, with the latter proving to be the more robust technique. Overall, the AutoBioTech platform excels at versatility due to the modularity of workflows and seamless transitions between modules. This will accelerate strain engineering of Gram-negative and Gram-positive bacteria.

A type III polyketide synthase cluster in the phylum Planctomycetota is involved in alkylresorcinol biosynthesis.

Members of the bacterial phylum Planctomycetota have recently emerged as promising and for the most part untapped sources of novel bioactive compounds. The characterization of more than 100 novel species in the last decade stimulated recent bioprospection studies that start to unveil the chemical repertoire of the phylum. In this study, we performed systematic bioinformatic analyses based on the genomes of all 131 described members of the current phylum focusing on the identification of type III polyketide synthase (PKS) genes. Type III PKSs are versatile enzymes involved in the biosynthesis of a wide array of structurally diverse natural products with potent biological activities. We identified 96 putative type III PKS genes of which 58 are encoded in an operon with genes encoding a putative oxidoreductase and a methyltransferase. Sequence similarities on protein level and the genetic organization of the operon point towards a functional link to the structurally related hierridins recently discovered in picocyanobacteria. The heterologous expression of planctomycetal type III PKS genes from strains belonging to different families in an engineered Corynebacterium glutamicum strain led to the biosynthesis of pentadecyl- and heptadecylresorcinols. Phenotypic assays performed with the heterologous producer strains and a constructed type III PKS gene deletion mutant suggest that the natural function of the identified compounds differs from that confirmed in other bacterial alkylresorcinol producers. KEY POINTS: • Planctomycetal type III polyketide synthases synthesize long-chain alkylresorcinols. • Phylogenetic analyses suggest an ecological link to picocyanobacterial hierridins. • Engineered C. glutamicum is suitable for an expression of planctomycete-derived genes.

Beyond rational-biosensor-guided isolation of 100 independently evolved bacterial strain variants and comparative analysis of their genomes.

BACKGROUND: In contrast to modern rational metabolic engineering, classical strain development strongly relies on random mutagenesis and screening for the desired production phenotype. Nowadays, with the availability of biosensor-based FACS screening strategies, these random approaches are coming back into fashion. In this study, we employ this technology in combination with comparative genome analyses to identify novel mutations contributing to product formation in the genome of a Corynebacterium glutamicum L-histidine producer. Since all known genetic targets contributing to L-histidine production have been already rationally engineered in this strain, identification of novel beneficial mutations can be regarded as challenging, as they might not be intuitively linkable to L-histidine biosynthesis. RESULTS: In order to identify 100 improved strain variants that had each arisen independently, we performed > 600 chemical mutagenesis experiments, > 200 biosensor-based FACS screenings, isolated > 50,000 variants with increased fluorescence, and characterized > 4500 variants with regard to biomass formation and L-histidine production. Based on comparative genome analyses of these 100 variants accumulating 10-80% more L-histidine, we discovered several beneficial mutations. Combination of selected genetic modifications allowed for the construction of a strain variant characterized by a doubled L-histidine titer (29 mM) and product yield (0.13 C-mol C-mol-1) in comparison to the starting variant. CONCLUSIONS: This study may serve as a blueprint for the identification of novel beneficial mutations in microbial producers in a more systematic manner. This way, also previously unexplored genes or genes with previously unknown contribution to the respective production phenotype can be identified. We believe that this technology has a great potential to push industrial production strains towards maximum performance.

Membrane manipulation by free fatty acids improves microbial plant polyphenol synthesis.

Microbial synthesis of nutraceutically and pharmaceutically interesting plant polyphenols represents a more environmentally friendly alternative to chemical synthesis or plant extraction. However, most polyphenols are cytotoxic for microorganisms as they are believed to negatively affect cell integrity and transport processes. To increase the production performance of engineered cell factories, strategies have to be developed to mitigate these detrimental effects. Here, we examine the accumulation of the stilbenoid resveratrol in the cell membrane and cell wall during its production using Corynebacterium glutamicum and uncover the membrane rigidifying effect of this stilbenoid experimentally and with molecular dynamics simulations. A screen of free fatty acid supplements identifies palmitelaidic acid and linoleic acid as suitable additives to attenuate resveratrol's cytotoxic effects resulting in a three-fold higher product titer. This cost-effective approach to counteract membrane-damaging effects of product accumulation is transferable to the microbial production of other polyphenols and may represent an engineering target for other membrane-active bioproducts.

Engineering and application of a biosensor with focused ligand specificity.

Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an L-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate L-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.

CRISPR/Cas12a Mediated Genome Editing To Introduce Amino Acid Substitutions into the Mechanosensitive Channel MscCG of Corynebacterium glutamicum.

Against the background of a growing demand for the implementation of environmentally friendly production processes, microorganisms are engineered for the large-scale biosynthesis of chemicals, fuels, or food and feed additives from sustainable resources. Since strain development is expensive and time-consuming, continuous improvement of molecular tools for the genetic modification of the microbial production hosts is absolutely vital. Recently, the CRISPR/Cas12a technology for the engineering of Corynebacterium glutamicum as an important platform organism for industrial amino acid production has been introduced. Here, this system was advanced by designing an easy-to-construct crRNA delivery vector using simple oligonucleotides. In combination with a C. glutamicum strain engineered for the chromosomal expression of the ß-galactosidase-encoding lacZ gene, this new plasmid was used to investigate CRISPR/Cas12a targeting and editing at various positions relative to the PAM site. Finally, we used this system to perform codon saturation mutagenesis at critical positions in the mechanosensitive channel MscCG to identify new gain-of-function mutations for increased l-glutamate export. The mutations obtained can be explained by particular demands of the channel on its immediate lipid environment to allow l-glutamate efflux.

AftD functions as an α1 → 5 arabinofuranosyltransferase involved in the biosynthesis of the mycobacterial cell wall core.

Arabinogalactan (AG) is an essential structural macromolecule present in the cell wall of Mycobacterium tuberculosis, serving to connect peptidoglycan with the outer mycolic acid layer. The D-arabinan segment is a highly branched component of AG and is assembled in a step-wise fashion by a variety of arabinofuranosyltransferases (AraT). We have previously used Corynebacterium glutamicum as a model organism to study these complex processes which are otherwise essential in mycobacteria. In order to further our understanding of the molecular basis of AG assembly, we investigated the role of a fourth AraT, now termed AftD by generating single (ΔaftD) and double deletion (ΔaftB ΔaftD) mutants of C. glutamicum. We demonstrate that AftD functions as an α(1 → 5) AraT and reveal the point at which it exerts its activity in the AG biosynthetic pathway.

The myo-inositol/proton symporter IolT1 contributes to d-xylose uptake in Corynebacterium glutamicum.

Corynebacterium glutamicum has been engineered to utilize d-xylose as sole carbon and energy source. Recently, a C. glutamicum strain has been optimized for growth on defined medium containing d-xylose by laboratory evolution, but the mutation(s) attributing to the improved-growth phenotype could not be reliably identified. This study shows that loss of the transcriptional repressor IolR is responsible for the increased growth performance on defined d-xylose medium in one of the isolated mutants. Underlying reason is derepression of the gene for the glucose/myo-inositol permease IolT1 in the absence of IolR, which could be shown to also contribute to d-xylose uptake in C. glutamicum. IolR-regulation of iolT1 could be successfully repealed by rational engineering of an IolR-binding site in the iolT1-promoter. This minimally engineered C. glutamicum strain bearing only two nucleotide substitutions mimics the IolR loss-of-function phenotype and allows for a high growth rate on d-xylose-containing media (µmax = 0.24 ±â€¯0.01 h-1).

Mutations in MurE, the essential UDP-N-acetylmuramoylalanyl-D-glutamate 2,6-diaminopimelate ligase of Corynebacterium glutamicum: effect on L-lysine formation and analysis of systemic consequences.

OBJECTIVES: To explore systemic effects of mutations in the UDP-N-acetylmuramoylalanyl-D-glutamate 2,6-diaminopimelate ligase (MurE) of Corynebacterium glutamicum, that leads to extracellular L-lysine accumulation by this bacterium. RESULTS: The analysis of a mutant cohort of C. glutamicum strains carrying all possible 20 amino acids at position 81 of MurE revealed unexpected effects on cellular properties. With increasing L-lysine accumulation the growth rate of the producing strain is reduced. A dynamic flux balance analysis including the flux over MurE fully supports this finding and suggests that further reductions at this flux control point would enhance L-lysine accumulation even further. The strain carrying the best MurE variant MurE-G81K produces 37 mM L-lysine with a yield of 0.17 g/g (L-lysine·HCl/glucose·H2O), bearing no other genetic modification. Interestingly, among the strains with high L-lysine titers, strain variants occur which, despite possessing the desired amino acid substitutions in MurE, have regained close to normal growth and correspondingly lower L-lysine accumulation. Genome analyses of such variants revealed the transposition of mobile genetic elements which apparently annulled the favorable consequences of the MurE mutations on L-lysine formation. CONCLUSION: MurE is an attractive target to achieve high L-lysine accumulation, and product formation is inversely related to the specific growth rate. Moreover, single point mutations leading to elevated L-lysine titers may cause systemic effects on different levels comprising also major genome modifications. The latter caused by the activity of mobile genetic elements, most likely due to the stress conditions being characteristic for microbial metabolite producers.

Formation of xylitol and xylitol-5-phosphate and its impact on growth of d-xylose-utilizing Corynebacterium glutamicum strains.

Wild-type Corynebacterium glutamicum has no endogenous metabolic activity for utilizing the lignocellulosic pentose d-xylose for cell growth. Therefore, two different engineering approaches have been pursued resulting in platform strains harbouring a functional version of either the Isomerase (ISO) or the Weimberg (WMB) pathway for d-xylose assimilation. In a previous study we found for C. glutamicum WMB by-product formation of xylitol during growth on d-xylose and speculated that the observed lower growth rates are due to the growth inhibiting effect of this compound. Based on a detailed phenotyping of the ISO, WMB and the wild-type strain of C. glutamicum, we here show that this organism has a natural capability to synthesize xylitol from d-xylose under aerobic cultivation conditions. We furthermore observed the intracellular accumulation of xylitol-5-phosphate as a result of the intracellular phosphorylation of xylitol, which was particularly pronounced in the C. glutamicum ISO strain. Interestingly, low amounts of supplemented xylitol strongly inhibit growth of this strain on d-xylose, d-glucose and d-arabitol. These findings demonstrate that xylitol is a suitable substrate of the endogenous xylulokinase (XK, encoded by xylB) and its overexpression in the ISO strain leads to a significant phosphorylation of xylitol in C. glutamicum. Therefore, in order to circumvent cytotoxicity by xylitol-5-phosphate, the WMB pathway represents an interesting alternative route for engineering C. glutamicum towards efficient d-xylose utilization.

Identification of the phd gene cluster responsible for phenylpropanoid utilization in Corynebacterium glutamicum.

Phenylpropanoids as abundant, lignin-derived compounds represent sustainable feedstocks for biotechnological production processes. We found that the biotechnologically important soil bacterium Corynebacterium glutamicum is able to grow on phenylpropanoids such as p-coumaric acid, ferulic acid, caffeic acid, and 3-(4-hydroxyphenyl)propionic acid as sole carbon and energy sources. Global gene expression analyses identified a gene cluster (cg0340-cg0341 and cg0344-cg0347), which showed increased transcription levels in response to phenylpropanoids. The gene cg0340 (designated phdT) encodes for a putative transporter protein, whereas cg0341 and cg0344-cg0347 (phdA-E) encode enzymes involved in the ß-oxidation of phenylpropanoids. The phd gene cluster is transcriptionally controlled by a MarR-type repressor encoded by cg0343 (phdR). Cultivation experiments conducted with C. glutamicum strains carrying single-gene deletions showed that loss of phdA, phdB, phdC, or phdE abolished growth of C. glutamicum with all phenylpropanoid substrates tested. The deletion of phdD (encoding for putative acyl-CoA dehydrogenase) additionally abolished growth with the α,ß-saturated phenylpropanoid 3-(4-hydroxyphenyl)propionic acid. However, the observed growth defect of all constructed single-gene deletion strains could be abolished through plasmid-borne expression of the respective genes. These results and the intracellular accumulation of pathway intermediates determined via LC-ESI-MS/MS in single-gene deletion mutants showed that the phd gene cluster encodes for a CoA-dependent, ß-oxidative deacetylation pathway, which is essential for the utilization of phenylpropanoids in C. glutamicum.

Anaerobic growth of Corynebacterium glutamicum via mixed-acid fermentation.

Corynebacterium glutamicum, a model organism in microbial biotechnology, is known to metabolize glucose under oxygen-deprived conditions to l-lactate, succinate, and acetate without significant growth. This property is exploited for efficient production of lactate and succinate. Our detailed analysis revealed that marginal growth takes place under anaerobic conditions with glucose, fructose, sucrose, or ribose as a carbon and energy source but not with gluconate, pyruvate, lactate, propionate, or acetate. Supplementation of glucose minimal medium with tryptone strongly enhanced growth up to a final optical density at 600 nm (OD600) of 12, whereas tryptone alone did not allow growth. Amino acids with a high ATP demand for biosynthesis and amino acids of the glutamate family were particularly important for growth stimulation, indicating ATP limitation and a restricted carbon flux into the oxidative tricarboxylic acid cycle toward 2-oxoglutarate. Anaerobic cultivation in a bioreactor with constant nitrogen flushing disclosed that CO2 is required to achieve maximal growth and that the pH tolerance is reduced compared to that under aerobic conditions, reflecting a decreased capability for pH homeostasis. Continued growth under anaerobic conditions indicated the absence of an oxygen-requiring reaction that is essential for biomass formation. The results provide an improved understanding of the physiology of C. glutamicum under anaerobic conditions.

The contest for precursors: channelling L-isoleucine synthesis in Corynebacterium glutamicum without byproduct formation.

L-Isoleucine is an essential amino acid, which is required as a pharma product and feed additive. Its synthesis shares initial steps with that of L-lysine and L-threonine, and four enzymes of L-isoleucine synthesis have an enlarged substrate specificity involved also in L-valine and L-leucine synthesis. As a consequence, constructing a strain specifically overproducing L-isoleucine without byproduct formation is a challenge. Here, we analyze for consequences of plasmid-encoded genes in Corynebacterium glutamicum MH20-22B on L-isoleucine formation, but still obtain substantial accumulation of byproducts. In a different approach, we introduce point mutations into the genome of MH20-22B to remove the feedback control of homoserine dehydrogenase, hom, and threonine dehydratase, ilvA, and we assay sets of genomic promoter mutations to increase hom and ilvA expression as well as to reduce dapA expression, the latter gene encoding the dihydrodipicolinate synthase. The promoter mutations are mirrored in the resulting differential protein levels determined by a targeted LC-MS/MS approach for the three key enzymes. The best combination of genomic mutations was found in strain K2P55, where 53 mM L-isoleucine could be obtained. Whereas in fed-batch fermentations with the plasmid-based strain, 94 mM L-isoleucine with L-lysine as byproduct was formed; with the plasmid-less strain K2P55, 109 mM L-isoleucine accumulated with no substantial byproduct formation. The specific molar yield with the latter strain was 0.188 mol L-isoleucine (mol glucose)(-1) which characterizes it as one of the best L-isoleucine producers available and which does not contain plasmids.

Acyl-CoA sensing by FasR to adjust fatty acid synthesis in Corynebacterium glutamicum.

Corynebacterium glutamicum, like Mycobacterium tuberculosis, is a member of the Corynebacteriales, which have linear fatty acids and as branched fatty acids the mycolic acids. We identified accD1 and fasA as key genes of fatty acid synthesis, encoding the ß-subunit of the acetyl-CoA carboxylase and a type-I fatty acid synthase, respectively, and observed their repression during growth on minimal medium with acetate. We also identified the transcriptional regulator FasR and its binding sites in the 5' upstream regions of accD1 and fasA. In the present work we establish by co-isolation and gel-mobility shifts oleoyl-CoA and palmitoyl-CoA as effectors of FasR, and show by DNA microarray analysis that in presence of exogeneous fatty acids accD1 and fasA are repressed. These results are evidence that acyl-CoA derivatives derived from extracellular fatty acids interact with FasR to repress the genes of fatty acid synthesis. This model also explains the observed repression of accD1 and fasA during growth on acetate, where apparently the known high intracellular acetyl-CoA concentration during growth on this substrate requires reduced accD1 and fasA expression for fine control of de novo fatty acid synthesis. Consequently, this mechanism ensures that membrane lipid homeostasis is maintained when specific nutrients are available resulting in increased acetyl-CoA concentration, as is the case with acetate, or when fatty acids are directly available from the extracellular environment. However, the genes specific to mycolic acid synthesis, which are in part shared with linear fatty acid synthesis, are not controlled by FasR, which is in agreement with the fact that they can not be supplied from the extracellular environment but that their synthesis fully depends on a constant supply of linear fatty acid chains.

Engineering of Corynebacterium glutamicum for minimized carbon loss during utilization of D-xylose containing substrates.

Biomass-derived d-xylose represents an economically interesting substrate for the sustainable microbial production of value-added compounds. The industrially important platform organism Corynebacterium glutamicum has already been engineered to grow on this pentose as sole carbon and energy source. However, all currently described C. glutamicum strains utilize d-xylose via the commonly known isomerase pathway that leads to a significant carbon loss in the form of CO2, in particular, when aiming for the synthesis of α-ketoglutarate and its derivatives (e.g. l-glutamate). Driven by the motivation to engineer a more carbon-efficient C. glutamicum strain, we functionally integrated the Weimberg pathway from Caulobacter crescentus in C. glutamicum. This five-step pathway, encoded by the xylXABCD-operon, enabled a recombinant C. glutamicum strain to utilize d-xylose in d-xylose/d-glucose mixtures. Interestingly, this strain exhibited a tri-phasic growth behavior and transiently accumulated d-xylonate during d-xylose utilization in the second growth phase. However, this intermediate of the implemented oxidative pathway was re-consumed in the third growth phase leading to more biomass formation. Furthermore, C. glutamicum pEKEx3-xylXABCDCc was also able to grow on d-xylose as sole carbon and energy source with a maximum growth rate of µmax=0.07±0.01h(-1). These results render C. glutamicum pEKEx3-xylXABCDCc a promising starting point for the engineering of efficient production strains, exhibiting only minimal carbon loss on d-xylose containing substrates.

Benzothiazinones mediate killing of Corynebacterineae by blocking decaprenyl phosphate recycling involved in cell wall biosynthesis.

Benzothiazinones (BTZs) are a new class of sulfur containing heterocyclic compounds that target DprE1, an oxidoreductase involved in the epimerization of decaprenyl-phosphoribose (DPR) to decaprenyl-phosphoarabinose (DPA) in the Corynebacterineae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis. As a result, BTZ inhibition leads to inhibition of cell wall arabinan biosynthesis. Previous studies have demonstrated the essentiality of dprE1. In contrast, Cg-UbiA a ribosyltransferase, which catalyzes the first step of DPR biosynthesis prior to DprE1, when genetically disrupted, produced a viable mutant, suggesting that although BTZ biochemically targets DprE1, killing also occurs through chemical synthetic lethality, presumably through the lack of decaprenyl phosphate recycling. To test this hypothesis, a derivative of BTZ, BTZ043, was examined in detail against C. glutamicum and C. glutamicum::ubiA. The wild type strain was sensitive to BTZ043; however, C. glutamicum::ubiA was found to be resistant, despite possessing a functional DprE1. When the gene encoding C. glutamicum Z-decaprenyl-diphosphate synthase (NCgl2203) was overexpressed in wild type C. glutamicum, resistance to BTZ043 was further increased. This data demonstrates that in the presence of BTZ, the bacilli accumulate DPR and fail to recycle decaprenyl phosphate, which results in the depletion of decaprenyl phosphate and ultimately leads to cell death.

Differential arabinan capping of lipoarabinomannan modulates innate immune responses and impacts T helper cell differentiation.

Toll-like receptors (TLRs) recognize pathogens by interacting with pathogen-associated molecular patterns, such as the phosphatidylinositol-based lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM). Such structures are present in several pathogens, including Mycobacterium tuberculosis, being important for the initiation of immune responses. It is well established that the interaction of LM and LAM with TLR2 is a process dependent on the structure of the ligands. However, the implications of structural variations on TLR2 ligands for the development of T helper (Th) cell responses or in the context of in vivo responses are less studied. Herein, we used Corynebacterium glutamicum as a source of lipoglycan intermediates for host interaction studies. In this study, we have deleted a putative glycosyltransferase, NCgl2096, from C. glutamicum and found that it encodes for a novel α(1→2)arabinofuranosyltransferase, AftE. Biochemical analysis of the lipoglycans obtained in the presence (wild type) or absence of NCgl2096 showed that AftE is involved in the biosynthesis of singular arabinans of LAM. In its absence, the resulting molecule is a hypermannosylated (hLM) form of LAM. Both LAM and hLM were recognized by dendritic cells, mainly via TLR2, and triggered the production of several cytokines. hLM was a stronger stimulus for in vitro cytokine production and, as a result, a more potent inducer of Th17 responses. In vivo data confirmed hLM as a stronger inducer of cytokine responses and suggested the involvement of pattern recognition receptors other than TLR2 as sensors for lipoglycans.

A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level.

We present a novel method for visualizing intracellular metabolite concentrations within single cells of Escherichia coli and Corynebacterium glutamicum that expedites the screening process of producers. It is based on transcription factors and we used it to isolate new L-lysine producing mutants of C. glutamicum from a large library of mutagenized cells using fluorescence-activated cell sorting (FACS). This high-throughput method fills the gap between existing high-throughput methods for mutant generation and genome analysis. The technology has diverse applications in the analysis of producer populations and screening of mutant libraries that carry mutations in plasmids or genomes.

Deletion of manC in Corynebacterium glutamicum results in a phospho-myo-inositol mannoside- and lipoglycan-deficient mutant.

Mannose is an important constituent of the immunomodulatory glycoconjugates of the mycobacterial cell wall: lipoarabinomannan (LAM), lipomannan (LM) and the related phospho-myo-inositol mannosides (PIMs). In Mycobacterium tuberculosis and the related bacillus Corynebacterium glutamicum, mannose is either imported from the medium or derived from glycolysis, and is subsequently converted into the nucleotide-based sugar donor guanosine diphosphomannose (GDP-mannose). This can be utilized by the glycosyltranferases of the GT-A/B superfamily or converted to the lipid-based donor polyprenyl monophosphomannose, and used as a substrate by the transmembrane glycosyltransferases of the GT-C superfamily. To investigate GDP-mannose biosynthesis in detail, the gene encoding a putative ManC in C. glutamicum was deleted. Deletion of manC resulted in a slow-growing mutant, with reduced but not totally abrogated guanosine diphosphomannose pyrophosphorylase activity. However, a comprehensive cell wall analysis revealed that C. glutamicumΔmanC is deficient in PIMs and LM/LAM. Closer inspection suggests that promiscuous ManC activity is contributed by additional putative nucleotidyltransferases, PmmB, WbbL1, GalU and GlmU, and a hypothetical protein, NCgl0715. Furthermore, complementation analyses of C. glutamicumΔmanC with Rv3264c suggested that it is a true homologue of ManC in M. tuberculosis, and the essentiality of PIMs in M. tuberculosis makes it an attractive drug target.

MmpL genes are associated with mycolic acid metabolism in mycobacteria and corynebacteria.

Mycolic acids are vital components of the cell wall of the tubercle bacillus Mycobacterium tuberculosis and are required for viability and virulence. While mycolic acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. We investigated the role of large membrane proteins encoded by mmpL genes in mycolic acid transport in mycobacteria and the related corynebacteria. MmpL3 was found to be essential in mycobacteria and conditional depletion of MmpL3 in Mycobacterium smegmatis resulted in loss of cell wall mycolylation, and of the cell wall-associated glycolipid, trehalose dimycolate. In parallel, an accumulation of trehalose monomycolate (TMM) was observed, suggesting that mycolic acids were transported as TMM. In contrast to mycobacteria, we found redundancy in the role of two mmpL genes, in Corynebacterium glutamicum; a complete loss of trehalose-associated and cell wall bound corynomycolates was observed in an NCgl0228-NCgl2769 double mutant, but not in individual single mutants. Our studies highlight the role of mmpL genes in mycolic acid metabolism and identify potential new targets for anti-TB drug development.