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The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria , a large bacterial phylum that includes the pathogen Mycobacterium tuberculosis , lack the canonical FtsZ-membrane anchors and Z-ring regulators described for E. coli . Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to interact with the conserved C-terminal tail of FtsZ. We show an essential interdependence of FtsZ and SepF for formation of a functional Z-ring in C. glutamicum . The crystal structure of the SepF–FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and suggests a reversible oligomerization interface. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, indicating that SepF has multiple roles at the cell division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function. The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Here, Sogues et al. study the interaction between FtsZ and SepF in Corynebacterium glutamicum , showing an essential interdependence of these proteins for formation of a functional Z-ring.

Cysteine, a common amino acid used in food, cosmetic, and pharmaceutical industries, has a growth inhibitory effect. This growth inhibition by cysteine poses a problem, as the production of cysteine using microbial cells results in decreased cell growth and cysteine productivity. The underlying mechanism of growth inhibition by cysteine is unclear. This study aims to understand the mechanism of growth inhibition by cysteine in Corynebacterium glutamicum . To do this, cysteine-resistant mutants of C. glutamicum were isolated based on adaptive laboratory evolution (ALE) and their characteristics were analyzed. Genome resequencing revealed that mutations in the open reading frame of the ilvN gene encoding the regulatory small subunit of acetohydroxyacid synthase (AHAS), which is involved in branched-chain amino acid biosynthesis, were found in ALE cell populations and the isolated cysteine-resistant mutants. The ilvN mutations which are responsible for increased valine production resulted in improved cell growth in the presence of cysteine. Moreover, the addition of valine to the culture medium mitigated growth inhibition by cysteine, whereas the addition of leucine and isoleucine showed a slight mitigation. Additionally, the activity of AHAS from C. glutamicum was inhibited by cysteine, whereas AHAS from the strains carrying ilvN mutations exhibited resistance to cysteine. These results indicate that growth inhibition by cysteine is caused by perturbations in the biosynthesis of branched-chain amino acids, particularly valine in C. glutamicum . Furthermore, the cysteine-resistant mutants obtained by ALE demonstrated enhanced cysteine production as production hosts, suggesting that cysteine resistance is a useful phenotype for cysteine production in C. glutamicum . Key points • Cysteine-resistant mutants of C. glutamicum obtained by ALE were analyzed. • Perturbation of valine biosynthesis by cysteine results in growth inhibition in C. glutamicum. • Cysteine resistance is a useful phenotype for cysteine production by C. glutamicum.

Summary Corynebacterium glutamicum is an important industrial microorganism, but the availability of tools for its genetic modification has lagged compared to other model microorganisms such as Escherichia coli. Despite great progress in CRISPR‐based technologies, the most feasible genome editing method in C. glutamicum is suicide plasmid‐mediated, the editing efficiency of which is low due to high false‐positive rates of sacB counter selection, and the requirement for tedious two‐round selection and verification of rare double‐cross‐over events. In this study, an rpsL mutant conferring streptomycin resistance was harnessed for counter selection, significantly increasing the positive selection rate. More importantly, with the aid of high selection efficiencies through the use of antibiotics, namely kanamycin and streptomycin, the two‐step verification strategy can be simplified to just one‐step verification of the final edited strain. As proof of concept, a 2.5‐kb DNA fragment comprising aroGfbrpheAfbr expressing cassettes was integrated into the genome of C. glutamicum, with an efficiency of 20% out of the theoretical 50%. The resulting strain produced 110 mg l−1 l‐tyrosine in shake‐flask fermentation. This updated suicide plasmid‐mediated genome editing system will greatly facilitate genetic manipulations including single nucleotide mutation, gene deletion and gene insertion in C. glutamicum and can be easily applied to other microbes. Mutated rpsL gene encoding small ribosomal protein S12P enables Corynebacterium glutamicum resistance to streptomycin. The second‐crossover strains can be high‐efficiently selected by harnessing rpsL counter‐selection in the suicide plasmid‐mediated genome editing method for C. glutamicum. Taking advantage of the biotics, kanamycin and streptomycin selection, the procedure can be simplified to one‐step verification of edited strains.

Development of hyperproducing strains is important for biomanufacturing of biochemicals and biofuels but requires extensive efforts to engineer cellular metabolism and discover functional components. Herein, we optimize and use the CRISPR-assisted editing and CRISPRi screening methods to convert a wild-type Corynebacterium glutamicum to a hyperproducer of l -proline, an amino acid with medicine, feed, and food applications. To facilitate l -proline production, feedback-deregulated variants of key biosynthetic enzyme γ-glutamyl kinase are screened using CRISPR-assisted single-stranded DNA recombineering. To increase the carbon flux towards l -proline biosynthesis, flux-control genes predicted by in silico analysis are fine-tuned using tailored promoter libraries. Finally, an arrayed CRISPRi library targeting all 397 transporters is constructed to discover an l -proline exporter Cgl2622. The final plasmid-, antibiotic-, and inducer-free strain produces l -proline at the level of 142.4 g/L, 2.90 g/L/h, and 0.31 g/g. The CRISPR-assisted strain development strategy can be used for engineering industrial-strength strains for efficient biomanufacturing. Corynebacterium glutamicum is a major workhorse in industrial biomanufacturing of amino acids. Here, the authors employ CRISPR-assisted rational flux-tuning and CRISPRi screening of a L-proline exporter to covert a wild-type C. glutamicum to a hyperproducer of L-proline.

Malonyl-CoA is an important central metabolite for the production of diverse valuable chemicals including natural products, but its intracellular availability is often limited due to the competition with essential cellular metabolism. Several malonyl-CoA biosensors have been developed for high-throughput screening of targets increasing the malonyl-CoA pool. However, they are limited for use only in Escherichia coli and Saccharomyces cerevisiae and require multiple signal transduction steps. Here we report development of a colorimetric malonyl-CoA biosensor applicable in three industrially important bacteria: E. coli, Pseudomonas putida, and Corynebacterium glutamicum. RppA, a type III polyketide synthase producing red-colored flaviolin, was repurposed as a malonyl-CoA biosensor in E. coli. Strains with enhanced malonyl-CoA accumulation were identifiable by the colorimetric screening of cells showing increased red color. Other type III polyketide synthases could also be repurposed as malonyl-CoA biosensors. For target screening, a 1,858 synthetic small regulatory RNA library was constructed and applied to find 14 knockdown gene targets that generally enhanced malonyl-CoA level in E. coli. These knockdown targets were applied to produce two polyketide (6-methylsalicylic acid and aloesone) and two phenylpropanoid (resveratrol and naringenin) compounds. Knocking down these genes alone or in combination, and also in multiple different E. coli strains for two polyketide cases, allowed rapid development of engineered strains capable of enhanced production of 6-methylsalicylic acid, aloesone, resveratrol, and naringenin to 440.3, 30.9, 51.8, and 103.8 mg/L, respectively. The malonyl-CoA biosensor developed here is a simple tool generally applicable to metabolic engineering of microorganisms to achieve enhanced production of malonyl-CoA–derived chemicals.

Methanol, a one-carbon compound, can be utilized by a variety of bacteria and other organisms as carbon and energy source and is regarded as a promising substrate for biotechnological production. In this study, a strain of non-methylotrophic Corynebacterium glutamicum, which was able to produce the polyamide building block cadaverine as non-native product, was engineered for co-utilization of methanol. Expression of the gene encoding NAD⁺-dependent methanol dehydrogenase (Mdh) from the natural methylotroph Bacillus methanolicus increased methanol oxidation. Deletion of the endogenous aldehyde dehydrogenase genes ald and fadH prevented methanol oxidation to carbon dioxide and formaldehyde detoxification via the linear formaldehyde dissimilation pathway. Heterologous expression of genes for the key enzymes hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase of the ribulose monophosphate (RuMP) pathway in this strain restored growth in the presence of methanol or formaldehyde, which suggested efficient formaldehyde detoxification involving RuMP key enzymes. While growth with methanol as sole carbon source was not observed, the fate of ¹³C-methanol added as co-substrate to sugars was followed and the isotopologue distribution indicated incorporation into central metabolites and in vivo activity of the RuMP pathway. In addition, ¹³C-label from methanol was traced to the secreted product cadaverine. Thus, this synthetic biology approach led to a C. glutamicum strain that converted the non-natural carbon substrate methanol at least partially to the non-native product cadaverine.

Methyl anthranilate (MANT) is a widely used compound to give grape scent and flavor, but is currently produced by petroleum-based processes. Here, we report the direct fermentative production of MANT from glucose by metabolically engineered Escherichia coli and Corynebacterium glutamicum strains harboring a synthetic plantderived metabolic pathway. Optimizing the key enzyme anthranilic acid (ANT) methyltransferase1 (AAMT1) expression, increasing the direct precursor ANT supply, and enhancing the intracellular availability and salvage of the cofactor S-adenosyl-L-methionine required by AAMT1, results in improved MANT production in both engineered microorganisms. Furthermore, in situ two-phase extractive fermentation using tributyrin as an extractant is developed to overcome MANT toxicity. Fed-batch cultures of the final engineered E. coli and C. glutamicum strains in two-phase cultivation mode led to the production of 4.47 and 5.74 g/L MANT, respectively, in minimal media containing glucose. The metabolic engineering strategies developed here will be useful for the production of volatile aromatic esters including MANT.

Hyaluronan is widely used in cosmetics and pharmaceutics. Development of robust and safe cell factories and cultivation approaches to efficiently produce hyaluronan is of many interests. Here, we describe the metabolic engineering of Corynebacterium glutamicum and application of a fermentation strategy to manufacture hyaluronan with different molecular weights. C. glutamicum is engineered by combinatorial overexpression of type I hyaluronan synthase, enzymes of intermediate metabolic pathways and attenuation of extracellular polysaccharide biosynthesis. The engineered strain produces 34.2 g L −1 hyaluronan in fed-batch cultures. We find secreted hyaluronan encapsulates C. glutamicum , changes its cell morphology and inhibits metabolism. Disruption of the encapsulation with leech hyaluronidase restores metabolism and leads to hyper hyaluronan productions of 74.1 g L −1 . Meanwhile, the molecular weight of hyaluronan is also highly tunable. These results demonstrate combinatorial optimization of cell factories and the extracellular environment is efficacious and likely applicable for the production of other biopolymers. Bioproduction of hyaluronan needs increases in yield and greater diversity of the molecular weights. Here, the author increases hyaluronan production and diversifies the molecular weights through engineering the hyaluronan biosynthesis pathway and disruption of Corynebacterium glutamicum encapsulation caused by secreted hyaluronan.

The Corynebacterineae suborder includes a number of major bacterial pathogens. These organisms grow by polar extension unlike most well-studied model bacteria, which grow by inserting wall material at dispersed sites along their length. Members of the Corynebacterineae suborder of bacteria, including major pathogens such as Mycobacterium tuberculosis , grow via the insertion of new cell wall peptidoglycan (PG) material at their poles. This mode of elongation differs from that used by Escherichia coli and other more well-studied model organisms that grow by inserting new PG at dispersed sites along their cell body. Dispersed cell elongation is known to strictly require the SEDS-type PG synthase called RodA, whereas the other major class of PG synthases called class A penicillin-binding proteins (aPBPs) are not required for this mode of growth. Instead, they are thought to be important for maintaining the integrity of the PG matrix in organisms growing by dispersed elongation. In contrast, based on prior genetic studies in M. tuberculosis and related members of the Corynebacterineae suborder, the aPBPs are widely believed to be essential for polar growth, with RodA being dispensable. However, polar growth has not been directly assessed in mycobacterial or corynebacterial mutants lacking aPBP-type PG synthases. We therefore investigated the relative roles of aPBPs and RodA in polar growth using Corynebacterium glutamicum as a model member of Corynebacterineae . Notably, we discovered that the aPBPs are dispensable for polar growth and that this growth mode can be mediated by either an aPBP-type or a SEDS-type enzyme functioning as the sole elongation PG synthase. Thus, our results reveal that the mechanism of polar elongation is fundamentally flexible and, unlike dispersed elongation, can be effectively mediated in C. glutamicum by either a SEDS-bPBP or an aPBP-type synthase. IMPORTANCE The Corynebacterineae suborder includes a number of major bacterial pathogens. These organisms grow by polar extension unlike most well-studied model bacteria, which grow by inserting wall material at dispersed sites along their length. A better understanding of polar growth promises to uncover new avenues for targeting mycobacterial and corynebacterial infections. Here, we investigated the roles of the different classes of cell wall synthases for polar growth using Corynebacterium glutamicum as a model. We discovered that the polar growth mechanism is surprisingly flexible in this organism and, unlike dispersed synthesis, can function using either of the two known types of cell wall synthase enzymes.

The introduction of the key non-oxidative glycolytic (NOG) pathway enzyme, phosphoketolases (PKTs), into heterologous hosts can improve the yield of a variety of acetyl CoA-derived products of interest. However, the low specific activity of existing PKTs compared with that of 6-phosphofructokinase (PFK), the key EMP pathway enzyme, largely limits their potential applications. To improve PKT activity, previous attempts have focused on increasing intracellular PKT concentration via the use of strong promoters. Herein, we report the establishment of a growth-coupled evolution strategy for the enrichment and selection of PKT mutants with improved specific activity in Corynebacterium glutamicum hosts with defective PFK. Five mutants from 9 Bifidobacterium adolescentis -source PKT (BA-PKT) mutant libraries were obtained. Site-directed mutagenesis analysis revealed 11 mutant sites which contributed to improved BA-PKT specific activity. Further structural analysis revealed that the mutant sites were located far away from the enzyme active site, which makes them almost unpredictable using a rational design approach. Mutant site recombination led to the construction of a novel mutant, PKT T2A/I6T/H260Y , with V max 29.77 ± 1.58 U/mg and K cat / K m 0.32 ± 0.01 s −1 /mM, which corresponds to 73.27 ± 3.25% and 80.16 ± 3.38% improvements, respectively, compared with the wildtype ( Vmax ; 17.17 ± 0.59 U/mg, K cat / K m ; 0.17 ± 0.01 s −1 /mM). Expression of PKT T2A/I6T/H260 in C. glutamicum Z188 resulted in 16.67 ± 2.24% and 18.19 ± 0.53% improvement in l -glutamate titer and yield, respectively, compared with the wildtype BA-PKT. Our findings provide an efficient approach for improving the activity of PKTs. Furthermore, the novel mutants could serve as useful tools in improving the yield of l -glutamate and other acetyl CoA-associated products.