Engineering of the DNA replication and repair machinery to develop binary mutators for rapid genome evolution of Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial workhorse for production of amino acids and chemicals. Although recently developed genome editing technologies have advanced the rational genetic engineering of C. glutamicum, continuous genome evolution based on genetic mutators is still unavailable. To address this issue, the DNA replication and repair machinery of C. glutamicum was targeted in this study. DnaQ, the homolog of ϵ subunit of DNA polymerase III responsible for proofreading in Escherichia coli, was proven irrelevant to DNA replication fidelity in C. glutamicum. However, the histidinol phosphatase (PHP) domain of DnaE1, the α subunit of DNA polymerase III, was characterized as the key proofreading element and certain variants with PHP mutations allowed elevated spontaneous mutagenesis. Repression of the NucS-mediated post-replicative mismatch repair pathway or overexpression of newly screened NucS variants also impaired the DNA replication fidelity. Simultaneous interference with the DNA replication and repair machinery generated a binary genetic mutator capable of increasing the mutation rate by up to 2352-fold. The mutators facilitated rapid evolutionary engineering of C. glutamicum to acquire stress tolerance and protein overproduction phenotypes. This study provides efficient tools for evolutionary engineering of C. glutamicum and could inspire the development of mutagenesis strategy for other microbial hosts.

Enhanced production of D-pantothenic acid in Corynebacterium glutamicum using an efficient CRISPR-Cpf1 genome editing method.

BACKGROUND: Corynebacterium glutamicum has industrial track records for producing a variety of valuable products such as amino acids. Although CRISPR-based genome editing technologies have undergone immense developments in recent years, the suicide-plasmid-based approaches are still predominant for C. glutamicum genome manipulation. It is crucial to develop a simple and efficient CRISPR genome editing method for C. glutamicum. RESULTS: In this study, we developed a RecombinAtion Prior to Induced Double-strand-break (RAPID) genome editing technology for C. glutamicum, as Cpf1 cleavage was found to disrupt RecET-mediated homologous recombination (HR) of the donor template into the genome. The RAPID toolbox enabled highly efficient gene deletion and insertion, and notably, a linear DNA template was sufficient for gene deletion. Due to the simplified procedure and iterative operation ability, this methodology could be widely applied in C. glutamicum genetic manipulations. As a proof of concept, a high-yield D-pantothenic acid (vitamin B5)-producing strain was constructed, which, to the best of our knowledge, achieved the highest reported titer of 18.62 g/L from glucose only. CONCLUSIONS: We developed a RecET-assisted CRISPR-Cpf1 genome editing technology for C. glutamicum that harnessed CRISPR-induced DSBs as a counterselection. This method is of great importance to C. glutamicum genome editing in terms of its practical applications, which also guides the development of CRISPR genome editing tools for other microorganisms.

Efficient fermentative production of L-theanine by Corynebacterium glutamicum.

L-Theanine is a unique non-protein amino acid found in tea plants that has been shown to possess numerous functional properties relevant to food science and human nutrition. L-Theanine has been commercially developed as a valuable additive for use in food and beverages, and its market is expected to expand substantially if the production cost can be lowered. Although the enzymatic approach holds considerable potential for use in L-theanine production, demand exists for developing more tractable methods (than those currently available) that can be implemented under mild conditions and will reduce operational procedures and cost. Here, we sought to engineer fermentative production of L-theanine in Corynebacterium glutamicum, an industrially safe host. For L-theanine synthesis, we used γ-glutamylmethylamide synthetase (GMAS), which catalyzes the ATP-dependent ligation of L-glutamate and ethylamine. First, distinct GMASs were expressed in C. glutamicum wild-type ATCC 13032 strain and GDK-9, an L-glutamate overproducing strain, to produce L-theanine upon ethylamine addition to the hosts. Second, the L-glutamate exporter in host cells was disrupted, which markedly increased the L-theanine titer in GDK-9 cells and almost eliminated the accumulation of L-glutamate in the culture medium. Third, a chromosomally gmasMm-integrated L-alanine producer was constructed and used, attempting to synthesize ethylamine endogenously by expressing plant-derived L-serine/L-alanine decarboxylases; however, these enzymes showed no L-alanine decarboxylase activity under our experimental conditions. The optimal engineered strain that we ultimately created produced ~ 42 g/L L-theanine, with a yield of 19.6%, in a 5-L fermentor. This is the first report of fermentative production of L-theanine achieved using ethylamine supplementation.

Metabolic Engineering of Corynebacterium glutamicum for the High-Level Production of l-Valine under Aerobic Conditions.

l-Valine, an essential amino acid, serves as a valuable compound in various industries. However, engineering strains with both high yield and purity are yet to be delivered for microbial l-valine production. We engineered a Corynebacterium glutamicum strain capable of highly efficient production of l-valine. We initially introduced an acetohydroxy acid synthase mutant from an industrial l-valine producer and optimized a cofactor-balanced pathway, followed by the activation of the nonphosphoenolpyruvate-dependent carbohydrate phosphotransferase system and the introduction of an exogenous Entner-Doudoroff pathway. Subsequently, we weakened anaplerotic pathways, and attenuated the tricarboxylic acid cycle via start codon substitution in icd, encoding isocitrate dehydrogenase. Finally, to balance bacterial growth and l-valine production, an l-valine biosensor-dependent genetic circuit was established to dynamically repress citrate synthase expression. The engineered strain Val19 produced 103 g/L of l-valine with a high yield of 0.35 g/g glucose and a productivity of 2.67 g/L/h. This represents the highest reported l-valine production in C. glutamicum via direct fermentation and exhibits potential for its industrial-scale production, leveraging the advantages of C. glutamicum over other microbes.

Directed evolution of linker helix as an efficient strategy for engineering LysR-type transcriptional regulators as whole-cell biosensors.

Whole-cell biosensors based on transcriptional regulators are powerful tools for rapid measurement, high-throughput screening, dynamic metabolic regulation, etc. To optimize the biosensing performance of transcriptional regulator, its effector-binding domain is commonly engineered. However, this strategy is encumbered by the limitation of diversifying such a large domain and the risk of affecting effector specificity. Molecular dynamics simulation of effector binding of LysG (an LysR-type transcriptional regulator, LTTR) suggests the crucial role of the short linker helix (LH) connecting effector- and DNA-binding domains in protein conformational change. Directed evolution of LH efficiently produced LysG variants with extended operational range and unaltered effector specificity. The whole-cell biosensor based on the best LysGE58V variant outperformed the wild-type LysG in enzyme high-throughput screening and dynamic regulation of l-lysine biosynthetic pathway. LH mutations are suggested to affect DNA binding and facilitate transcriptional activation upon effector binding. LH engineering was also successfully applied to optimize another LTTR BenM for biosensing. Since LTTRs represent the largest family of prokaryotic transcriptional regulators with highly conserved structures, LH engineering is an efficient and universal strategy for development and optimization of whole-cell biosensors.

Systems metabolic engineering of Escherichia coli for hyper-production of 5­aminolevulinic acid.

BACKGROUND: 5-Aminolevulinic acid (5-ALA) is a promising biostimulant, feed nutrient, and photodynamic drug with wide applications in modern agriculture and therapy. Although microbial production of 5-ALA has been improved realized by using metabolic engineering strategies during the past few years, there is still a gap between the present production level and the requirement of industrialization. RESULTS: In this study, pathway, protein, and cellular engineering strategies were systematically employed to construct an industrially competitive 5-ALA producing Escherichia coli. Pathways involved in precursor supply and product degradation were regulated by gene overexpression and synthetic sRNA-based repression to channel metabolic flux to 5-ALA biosynthesis. 5-ALA synthase was rationally engineered to release the inhibition of heme and improve the catalytic activity. 5-ALA transport and antioxidant defense systems were targeted to enhance cellular tolerance to intra- and extra-cellular 5-ALA. The final engineered strain produced 30.7 g/L of 5-ALA in bioreactors with a productivity of 1.02 g/L/h and a yield of 0.532 mol/mol glucose, represent a new record of 5-ALA bioproduction. CONCLUSIONS: An industrially competitive 5-ALA producing E. coli strain was constructed with the metabolic engineering strategies at multiple layers (protein, pathway, and cellular engineering), and the strategies here can be useful for developing industrial-strength strains for biomanufacturing.

Development of a growth-coupled selection platform for directed evolution of heme biosynthetic enzymes in Corynebacterium glutamicum.

Heme is an important tetrapyrrole compound, and has been widely applied in food and medicine industries. Although microbial production of heme has been developed with metabolic engineering strategies during the past 20 years, the production levels are relatively low due to the multistep enzymatic processes and complicated regulatory mechanisms of microbes. Previous studies mainly adopted the strategies of strengthening precursor supply and product transportation to engineer microbes for improving heme biosynthesis. Few studies focused on the engineering and screening of efficient enzymes involved in heme biosynthesis. Herein, a growth-coupled, high-throughput selection platform based on the detoxification of Zinc-protoporphyrin IX (an analogue of heme) was developed and applied to directed evolution of coproporphyrin ferrochelatase, catalyzing the insertion of metal ions into porphyrin ring to generate heme or other tetrapyrrole compounds. A mutant with 3.03-fold increase in k cat/K M was selected. Finally, growth-coupled directed evolution of another three key enzymes involved in heme biosynthesis was tested by using this selection platform. The growth-coupled selection platform developed here can be a simple and effective strategy for directed evolution of the enzymes involved in the biosynthesis of heme or other tetrapyrrole compounds.

Development of a Hyperosmotic Stress Inducible Gene Expression System by Engineering the MtrA/MtrB-Dependent NCgl1418 Promoter in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important workhorse for industrial production of diversiform bioproducts. Precise regulation of gene expression is crucial for metabolic balance and enhancing production of target molecules. Auto-inducible promoters, which can be activated without expensive inducers, are ideal regulatory tools for industrial-scale application. However, few auto-inducible promoters have been identified and applied in C. glutamicum. Here, a hyperosmotic stress inducible gene expression system was developed and used for metabolic engineering of C. glutamicum. The promoter of NCgl1418 (P NCgl1418 ) that was activated by the two-component signal transduction system MtrA/MtrB was found to exhibit a high inducibility under hyperosmotic stress conditions. A synthetic promoter library was then constructed by randomizing the flanking and space regions of P NCgl1418 , and mutant promoters exhibiting high strength were isolated via fluorescence activated cell sorting (FACS)-based high-throughput screening. The hyperosmotic stress inducible gene expression system was applied to regulate the expression of lysE encoding a lysine exporter and repress four genes involved in lysine biosynthesis (gltA, pck, pgi, and hom) by CRISPR interference, which increased the lysine titer by 64.7% (from 17.0 to 28.0 g/L) in bioreactors. The hyperosmotic stress inducible gene expression system developed here is a simple and effective tool for gene auto-regulation in C. glutamicum and holds promise for metabolic engineering of C. glutamicum to produce valuable chemicals and fuels.

An update of the suicide plasmid-mediated genome editing system in Corynebacterium glutamicum.

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 aroGfbr pheAfbr 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.

Betaine supplementation improved L-threonine fermentation of Escherichia coli THRD by upregulating zwf (glucose-6-phosphate dehydrogenase) expression

BACKGROUND: The supplementation of betaine, an osmoprotective compatible solute, in the cultivation media has been widely used to protect bacterial cells. To explore the effects of betaine addition on industrial fermentation, Escherichia coli THRD, an L-threonine producer, was used to examine the production of L-threonine with betaine supplementation and the underlying mechanism through which betaine functions was investigated. RESULTS: Betaine supplementation in the medium of E. coli THRD significantly improved L-threonine fermentation parameters. The transcription of zwf and corresponding enzyme activity of glucose-6-phosphate dehydrogenase were significantly promoted by betaine addition, which contributed to an enhanced expression of zwf that provided more nicotinamide adenine dinucleotide phosphate (NADPH) for L-threonine synthesis. In addition, as a result of the betaine addition, the betaine-stimulated expression of enhanced green fluorescent protein (eGFP) under the zwf promoter within a plasmid-based cassette proved to be a transcription-level response of zwf. Finally, the promoter of the phosphoenolpyruvate carboxylase gene ppc in THRD was replaced with that of zwf, while L-threonine fermentation of the new strain was promoted by betaine addition. Conclusions: We reveal a novel mode of betaine that facilitates the microbial production of useful compounds. Betaine supplementation upregulates the expression of zwf and increases the NADPH synthesis, which may be beneficial for the cell growth and thereby promote the production of L-threonine. This finding might be useful for the production of NADPH-dependent amino acids and derivatives in E. coli THRD or other E. coli strains.