The ybcF Gene of Escherichia coli Encodes a Local Orphan Enzyme, Catabolic Carbamate Kinase.

Escherichia coli can use allantoin as its sole nitrogen source under anaerobic conditions. The ureidoglycolate produced by double release of ammonia from allantoin can flow into either the glyoxylate shunt or further catabolic transcarbamoylation. Although the former pathway is well studied, the genes of the latter (catabolic) pathway are not known. In the catabolic pathway, ureidoglycolate is finally converted to carbamoyl phosphate (CP) and oxamate, and then CP is dephosphorylated to carbamate by a catabolic carbamate kinase (CK), whereby ATP is formed. We identified the ybcF gene in a gene cluster containing fdrA-ylbE-ylbF-ybcF that is located downstream of the allDCE-operon. Reverse transcription PCR of total mRNA confirmed that the genes fdrA, ylbE, ylbF, and ybcF are co-transcribed. Deletion of ybcF caused only a slight increase in metabolic flow into the glyoxylate pathway, probably because CP was used to de novo synthesize pyrimidine and arginine. The activity of the catabolic CK was analyzed using purified YbcF protein. The Vmax is 1.82 U/mg YbcF for CP and 1.94 U/mg YbcF for ADP, and the KM value is 0.47 mM for CP and 0.43 mM for ADP. With these results, it was experimentally revealed that the ybcF gene of E. coli encodes catabolic CK, which completes anaerobic allantoin degradation through substrate-level phosphorylation. Therefore, we suggest renaming the ybcF gene as allK.

Functional Analysis of Feedback Inhibition-Insensitive Variants of N-Acetyl Glutamate Kinase Found in Sake Yeast Mutants with Ornithine Overproduction.

In the yeast Saccharomyces cerevisiae, N-acetyl glutamate kinase (NAGK), which catalyzes the phosphorylation of N-acetyl glutamate to form N-acetyl glutamyl-5-phosphate, is one of the rate-limiting enzymes in the ornithine and arginine biosynthetic pathways. NAGK activity is strictly regulated via feedback inhibition by the end product, arginine. We previously reported that the Thr340Ile variant of NAGK was insensitive to arginine feedback inhibition and that the interaction between Lys336 and Thr340 in NAGK may be important for arginine recognition. In the present study, we demonstrated that amino acid changes of Thr340 to Ala, Leu, Arg, Glu, Ile, and Asn removed arginine feedback inhibition, although the Thr340Ser variant was subject to the feedback inhibition. Therefore, these results indicate that the arginine-binding cavity formed via the interaction between the carbonyl group in the main chain of Lys336 and the hydroxyl group in the side chain of the residue at position 340 is critical for arginine recognition of NAGK. In addition, we newly identified two mutations in the ARG5,6 gene encoding the Cys119Tyr or Val267Ala variant of NAGK of sake yeast mutants with intracellular ornithine accumulation. Although it is unlikely that Cys119 and Val267 are directly involved in arginine recognition, we found here that two variants of NAGK were insensitive to arginine feedback inhibition and contributed to high-level production of ornithine. Structural analysis of NAGK suggests that these two amino acid substitutions influence the sensitivity to Arg feedback inhibition through alterations in local conformation around each residue. IMPORTANCE Ornithine has a number of physiological benefits in humans. Thus, an Orn-rich alcoholic beverage is expected to relieve feelings of fatigue after drinking. In the yeast Saccharomyces cerevisiae, N-acetyl glutamate kinase (NAGK) encoded by the ARG5,6 gene catalyzes the second step in ornithine and arginine biosynthesis, and its activity is subjected to feedback inhibition by arginine. Here, we revealed a role of key residues in the formation of the arginine-binding cavity which is critical for arginine recognition of NAGK. In addition, we analyzed novel arginine feedback inhibition-insensitive variants of NAGK in sake yeast mutants with ornithine overproduction and proposed that the amino acid substitutions in the NAGK variants destabilize the arginine-binding cavity, leading to the lower sensitivity to arginine feedback inhibition of NAGK activity. These findings provide new insight into the allosteric regulation of NAGK activity and will help to construct superior industrial yeast strains for high-level production of ornithine.

CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an L-proline exporter for L-proline hyperproduction.

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.

Split NanoLuc technology allows quantitation of interactions between PII protein and its receptors with unprecedented sensitivity and reveals transient interactions.

PII proteins constitute a widespread signal transduction superfamily in the prokaryotic world. The canonical PII signal proteins sense metabolic state of the cells by binding the metabolite molecules ATP, ADP and 2-oxoglutarate. Depending on bound effector molecule, PII proteins interact with and modulate the activity of multiple target proteins. To investigate the complexity of interactions of PII with target proteins, analytical methods that do not disrupt the native cellular context are required. To this purpose, split luciferase proteins have been used to develop a novel complementation reporter called NanoLuc Binary Technology (NanoBiT). The luciferase NanoLuc is divided in two subunits: a 18 kDa polypeptide termed "Large BiT" and a 1.3 kDa peptide termed "Small BiT", which only weakly associate. When fused to proteins of interest, they reconstitute an active luciferase when the proteins of interest interact. Therefore, we set out to develop a new NanoBiT sensor based on the interaction of PII protein from Synechocystis sp. PCC6803 with PII-interacting protein X (PipX) and N-acetyl-L-glutamate kinase (NAGK). The novel NanoBiT sensor showed unprecedented sensitivity, which made it possible to detect even weak and transient interactions between PII variants and their interacting partners, thereby shedding new light in PII signalling processes.

Enzymology of the pathway for ATP production by arginine breakdown.

In cells, the breakdown of arginine to ornithine and ammonium ion plus carbon dioxide is coupled to the generation of metabolic energy in the form of ATP. The arginine breakdown pathway is minimally composed of arginine deiminase, ornithine transcarbamoylase, carbamate kinase, and an arginine/ornithine antiporter; ammonia and carbon dioxide most likely diffuse passively across the membrane. The genes for the enzymes and transporter have been cloned and expressed, and the proteins have been purified from Lactococcus lactis IL1403 and incorporated into lipid vesicles for sustained production of ATP. Here, we study the kinetic parameters and biochemical properties of the individual enzymes and the antiporter, and we determine how the physicochemical conditions, effector composition, and effector concentration affect the enzymes. We report the KM and VMAX values for catalysis and the native oligomeric state of all proteins, and we measured the effect of pathway intermediates, pH, temperature, freeze-thaw cycles, and salts on the activity of the cytosolic enzymes. We also present data on the protein-to-lipid ratio and lipid composition dependence of the antiporter.

More than just a ticket canceller: the mitochondrial processing peptidase tailors complex precursor proteins at internal cleavage sites.

Most mitochondrial proteins are synthesized as precursors that carry N-terminal presequences. After they are imported into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase (MPP). Using the mitochondrial tandem protein Arg5,6 as a model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into mitochondria and subsequently separated into two distinct enzymes. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor at its N-terminus and at an internal site. The peculiar organization of Arg5,6 is conserved across fungi and reflects the polycistronic arginine operon in prokaryotes. MPP cleavage sites are also present in other mitochondrial fusion proteins from fungi, plants, and animals. Hence, besides its role as a "ticket canceller" for removal of presequences, MPP exhibits a second conserved activity as an internal processing peptidase for complex mitochondrial precursor proteins.

High-level production of ornithine by expression of the feedback inhibition-insensitive N-acetyl glutamate kinase in the sake yeast Saccharomyces cerevisiae.

We previously reported that intracellular proline (Pro) confers tolerance to ethanol on the yeast Saccharomyces cerevisiae. In this study, to improve the ethanol productivity of sake, a traditional Japanese alcoholic beverage, we successfully isolated several Pro-accumulating mutants derived from diploid sake yeast of S. cerevisiae by a conventional mutagenesis. Interestingly, one of them (strain A902-4) produced more than 10-fold greater amounts of ornithine (Orn) and Pro compared to the parent strain (K901). Orn is a non-proteinogenic amino acid and a precursor of both arginine (Arg) and Pro. It has some physiological functions, such as amelioration of negative states such as lassitude and improvement of sleep quality. We also identified a homo-allelic mutation in the ARG5,6 gene encoding the Thr340Ile variant N-acetylglutamate kinase (NAGK) in strain A902-4. The NAGK activity of the Thr340Ile variant was extremely insensitive to feedback inhibition by Arg, leading to intracellular Orn accumulation. This is the first report of the removal of feedback inhibition of NAGK activity in the industrial yeast, leading to high levels of intracellular Orn. Moreover, sake and sake cake brewed with strain A902-4 contained 4-5 times more Orn than those brewed with strain K901. The approach described here could be a practical method for the development of industrial yeast strains with overproduction of Orn.

Effects of a novel variant of the yeast γ-glutamyl kinase Pro1 on its enzymatic activity and sake brewing.

Sake is a traditional Japanese alcoholic beverage brewed with the yeast Saccharomyces cerevisiae. Sake taste is affected by sugars, organic acids, and amino acids. We previously isolated mutants resistant to the proline analogue azetidine-2-carboxylate derived from a diploid sake yeast strain. Some of the mutants produced a greater amount of proline in the brewed sake. One of them (strain K-9-AZC) carried a novel mutation in the PRO1 gene encoding the Gln79His variant of the γ-glutamyl kinase Pro1, a key enzyme in proline biosynthesis in S. cerevisiae. This mutation resulted in extreme desensitization to feedback inhibition by proline, leading to proline overproduction. Interestingly, sake brewed with K-9-AZC contained 3.7-fold more proline, but only 25% less succinate than sake brewed with the parent strain. Metabolome analysis suggests that the decrease in succinate was attributable to a lower level of 2-oxoglutarate, which is converted into glutamate. The approach here could be a practical method for breeding of yeast strains involved in the diversity of sake taste.

Staphylococcus aureus Fatty Acid Kinase FakA Modulates Pathogenesis during Skin Infection via Proteases.

Staphylococcus aureus fatty acid kinase FakA is necessary for the incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that the inactivation of fakA leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin in vitro, and that the ΔfakA mutant causes larger lesions than the wild type (WT) during murine skin infection. As expected, necrosis is Hla dependent in the presence or absence of FakA, as both hla and hla ΔfakA mutants are unable to cause necrosis of the skin. At day 4 postinfection, while the ΔfakA mutant maintains larger and more necrotic abscesses, bacterial numbers are similar to those of the WT, indicating the enhanced tissue damage of mice infected with the ΔfakA mutant is not due to an increase in bacterial burden. At this early stage of infection, skin infected with the ΔfakA mutant has decreased levels of proinflammatory cytokines, such as interleukin-17A (IL-17A) and IL-1α, compared to those of WT-infected skin. At a later stage of infection (day 7), abscess resolution and bacterial clearance are hindered in ΔfakA mutant-infected mice. The paradoxical findings of decreased Hla in vitro but increased necrosis in vivo led us to investigate the role of the proteases regulated by FakA. Utilizing Δaur and ΔsspAB mutants in both the WT and fakA mutant backgrounds, we found that the absence of these proteases in a fakA mutant reduced dermonecrosis to levels similar to those of the WT strain. These studies suggest that the overproduction of proteases is one factor contributing to the enhanced pathogenesis of the ΔfakA mutant during skin infection.

Development of a Proline-Based Selection System for Reliable Genetic Engineering in Chinese Hamster Ovary Cells.

Chinese hamster ovary (CHO) cells are the superior host cell culture models used for the bioproduction of therapeutic proteins. One of the prerequisites for bioproduction using CHO cell lines is the need to generate stable CHO cell lines with optimal expression output. Antibiotic selection is commonly employed to isolate and select CHO cell lines with stable expression, despite its potential negative impact on cellular metabolism and expression level. Herein, we present a novel proline-based selection system for the isolation of stable CHO cell lines. The system exploits a dysfunctional proline metabolism pathway in CHO cells by using a pyrroline-5-carboxylate synthase gene as a selection marker, enabling selection to be made using proline-free media. The selection system was demonstrated by expressing green fluorescent protein (GFP) and a monoclonal antibody. When GFP was expressed, more than 90% of stable transfectants were enriched within 2 weeks of the selection period. When a monoclonal antibody was expressed, we achieved comparable titers (3.35 ± 0.47 µg/mL) with G418 and Zeocin-based selections (1.65 ± 0.46 and 2.25 ± 0.07 µg/mL, respectively). We further developed a proline-based coselection by using S. cerevisiae PRO1 and PRO2 genes as markers, which enables the generation of 99.5% double-transgenic cells. The proline-based selection expands available selection tools and provides an alternative to antibiotic-based selections in CHO cell line development.

Characterization of a butyrate kinase from Desulfovibrio vulgaris str. Hildenborough.

Short and branched chain fatty acid kinases participate in both bacterial anabolic and catabolic processes, including fermentation, through the reversible, ATP-dependent synthesis of acyl phosphates. This study reports biochemical properties of a predicted butyrate kinase from Desulfovibrio vulgaris str. Hildenborough (DvBuk) expressed heterologously and purified from Escherichia coli. Gel filtration chromatography indicates purified DvBuk is active as a dimer. The optimum temperature and pH for DvBuk activity is 44°C and 7.5, respectively. The enzyme displays enhanced thermal stability in the presence of substrates as observed for similar enzymes. Measurement of kcat and KM for various substrates reveals DvBuk exhibits the highest catalytic efficiencies for butyrate, valerate and isobutyrate. In particular, these measurements reveal this enzyme's apparent high affinity for C4 fatty acids relative to other butyrate kinases. These results have implications on structure and function relationships within the ASKHA superfamily of phosphotransferases, particularly regarding the acyl binding pocket, as well as potential physiological roles for this enzyme in Desulfovibrio vulgaris str. Hildenborough.

Lactobacillus rhamnosus GG supplementation modulates the gut microbiota to promote butyrate production, protecting against deoxynivalenol exposure in nude mice.

Deoxynivalenol (DON) is the most common mycotoxin in grains, and DON exposure causes gastrointestinal inflammation and systemic immunosuppression. The immunosuppression caused by DON has raised serious concerns about whether it is safe to use probiotics in immunocompromised hosts. Gut microbiota remodeling by Lactobacillus is a potential effective strategy to prevent DON exposure. The athymic nude mice were chose as the model of immunocompromised animals. We tested the effect of the probiotic Lactobacillus rhamnosus GG (LGG) or Lactobacillus acidophilus (LA) supplementation on host protection against DON exposure and the underlying mechanisms in nude mice. DON exposure induced endoplasmic reticulum (ER) stress and impaired intestinal barrier function and microbiota, which were relieved by LGG supplementation but not LA supplementation. LGG supplementation significantly enhanced the intestinal barrier function, increased the body weight and the survival rate in nude mice that exposed to DON for two weeks. Furthermore, LGG supplementation modulated the gut microbiota by increasing the abundance of Bacteroidetes and the levels of the butyrate-producing genes But and Buk to promote butyrate production. Butyrate inhibited the IRE1α/XBP1 signaling pathway to reduce DON-induced intestine injury. In conclusion, LGG supplementation modulated the gut microbiota to promote butyrate production, protecting against DON exposure in nude mice. Both LGG and butyrate show promise for use in protecting against DON exposure.

PII Signal Transduction Protein GlnK Alleviates Feedback Inhibition of N-Acetyl-l-Glutamate Kinase by l-Arginine in Corynebacterium glutamicum.

PII signal transduction proteins are ubiquitous and highly conserved in bacteria, archaea, and plants and play key roles in controlling nitrogen metabolism. However, research on biological functions and regulatory targets of PII proteins remains limited. Here, we illustrated experimentally that the PII protein Corynebacterium glutamicum GlnK (CgGlnK) increased l-arginine yield when glnK was overexpressed in Corynebacterium glutamicum Data showed that CgGlnK regulated l-arginine biosynthesis by upregulating the expression of genes of the l-arginine metabolic pathway and interacting with N-acetyl-l-glutamate kinase (CgNAGK), the rate-limiting enzyme in l-arginine biosynthesis. Further assays indicated that CgGlnK contributed to alleviation of the feedback inhibition of CgNAGK caused by l-arginine. In silico analysis of the binding interface of CgGlnK-CgNAGK suggested that the B and T loops of CgGlnK mainly interacted with C and N domains of CgNAGK. Moreover, F11, R47, and K85 of CgGlnK were identified as crucial binding sites that interact with CgNAGK via hydrophobic interaction and H bonds, and these interactions probably had a positive effect on maintaining the stability of the complex. Collectively, this study reveals PII-NAGK interaction in nonphotosynthetic microorganisms and further provides insights into the regulatory mechanism of PII on amino acid biosynthesis in corynebacteria.IMPORTANCE Corynebacteria are safe industrial producers of diverse amino acids, including l-glutamic acid and l-arginine. In this study, we showed that PII protein GlnK played an important role in l-glutamic acid and l-arginine biosynthesis in C. glutamicum Through clarifying the molecular mechanism of CgGlnK in l-arginine biosynthesis, the novel interaction between CgGlnK and CgNAGK was revealed. The alleviation of l-arginine inhibition of CgNAGK reached approximately 48.21% by CgGlnK addition, and the semi-inhibition constant of CgNAGK increased 1.4-fold. Furthermore, overexpression of glnK in a high-yield l-arginine-producing strain and fermentation of the recombinant strain in a 5-liter bioreactor led to a remarkably increased production of l-arginine, 49.978 g/liter, which was about 22.61% higher than that of the initial strain. In conclusion, this study provides a new strategy for modifying amino acid biosynthesis in C. glutamicum.

Nitrogen storage regulation by PII protein: lessons learned from taxonomic outliers.

The paper 'Interaction of N-acetyl-l-glutamate kinase with the PII signal transducer in the non-photosynthetic alga Polytomella parva: Co-evolution towards a hetero-oligomeric enzyme' by Selim et al. highlights how the study of a true taxonomic oddity, the heterotrophic unicellular alga P. parva, has been instrumental in uncovering the large potential for adaptive variation in the signaling complex of PII with the enzyme N-acetylglutamate kinase (NAGK). This complex modifies the regulatory properties of NAGK, allowing nitrogen stockpiling as arginine. In P. parva, a stable PII-NAGK complex is formed which lacks regulation by canonical PII effectors but which exhibits novel adaptive responses to nitrogen abundance mediated by glutamine, a neo-effector of PII proteins of photosynthetic eukaryotes.

Interaction of N-acetyl-l-glutamate kinase with the PII signal transducer in the non-photosynthetic alga Polytomella parva: Co-evolution towards a hetero-oligomeric enzyme.

During evolution, several algae and plants became heterotrophic and lost photosynthesis; however, in most cases, a nonphotosynthetic plastid was maintained. Among these organisms, the colourless alga Polytomella parva is a special case, as its plastid is devoid of any DNA, but is maintained for specific metabolic tasks carried out by nuclear encoded enzymes. This makes P. parva attractive to study molecular events underlying the transition from autotrophic to heterotrophic lifestyle. Here we characterize metabolic adaptation strategies of P. parva in comparison to the closely related photosynthetic alga Chlamydomonas reinhardtii with a focus on the role of plastid-localized PII signalling protein. Polytomella parva accumulates significantly higher amounts of most TCA cycle intermediates as well as glutamate, aspartate and arginine, the latter being specific for the colourless plastid. Correlating with the altered metabolite status, the carbon/nitrogen sensory PII signalling protein and its regulatory target N-acetyl-l-glutamate-kinase (NAGK; the controlling enzyme of arginine biosynthesis) show unique features: They have co-evolved into a stable hetero-oligomeric complex, irrespective of effector molecules. The PII signalling protein, so far known as a transiently interacting signalling protein, appears as a permanent subunit of the enzyme NAGK. NAGK requires PII to properly sense the feedback inhibitor arginine, and moreover, PII tunes arginine-inhibition in response to glutamine. No other PII effector molecules interfere, indicating that the PII-NAGK system in P. parva has lost the ability to estimate the cellular energy and carbon status but has specialized to provide an entirely glutamine-dependent arginine feedback control, highlighting the evolutionary plasticity of PII signalling system.

A synthetic metabolic network for physicochemical homeostasis.

One of the grand challenges in chemistry is the construction of functional out-of-equilibrium networks, which are typical of living cells. Building such a system from molecular components requires control over the formation and degradation of the interacting chemicals and homeostasis of the internal physical-chemical conditions. The provision and consumption of ATP lies at the heart of this challenge. Here we report the in vitro construction of a pathway in vesicles for sustained ATP production that is maintained away from equilibrium by control of energy dissipation. We maintain a constant level of ATP with varying load on the system. The pathway enables us to control the transmembrane fluxes of osmolytes and to demonstrate basic physicochemical homeostasis. Our work demonstrates metabolic energy conservation and cell volume regulatory mechanisms in a cell-like system at a level of complexity minimally needed for life.

Effects of arginine on Polytomella parva growth, PII protein levels and lipid body formation.

MAIN CONCLUSION: L-Arginine supports growth and resulted in increased PII signaling protein levels and lipid droplet accumulation in the colorless green alga Polytomella parva. Polytomella parva, a model system for nonphotosynthetic green algae, utilizes ammonium and several carbon sources, including ethanol and acetate. We previously reported that P. parva accumulates high amounts of arginine with the key enzyme of the ornithine/arginine biosynthesis pathway, N-acetyl-L-glutamate kinase, exhibiting high activity. Here we demonstrate that L-arginine can be used by this alga as a nitrogen source. Externally supplied arginine directly influenced the levels of PII signaling protein and formation of triacylglycerol (TAG)-filled lipid bodies (LBs). Our results suggest that the nitrogen source, but not nitrogen starvation, may be critical for the accumulation of LBs in a PII-independent manner in P. parva.

Quinoline Derivatives Kill Mycobacterium tuberculosis by Activating Glutamate Kinase.

There is a great need for identification and development of new anti-tuberculosis drugs with novel targets. Recent drug-discovery efforts typically focus on identifying inhibitors but not activators that perturb metabolic enzymes' functions as a means to kill Mycobacterium tuberculosis (Mtb). Here, we describe a class of quinoline compounds, Z0933/Z0930, which kill Mtb by acting as activators of glutamate kinase (GK), a previously untargeted enzyme catalyzing the first step of proline biosynthesis. We further show that Z0933/Z0930 augment proline production and induce Mtb killing via proline-derived redox imbalance and production of reactive oxygen species. This work highlights the effectiveness of gain-of-function probes against Mtb and provides a framework for the discovery of next-generation allosteric activators of GK.

Phosphorylation Mechanism of N-Acetyl-l-glutamate Kinase, a QM/MM Study.

In microorganisms and plants, N-acetyl-l-glutamate kinase (NAGK) catalyzes the second step in l-arginine synthesis, the phosphorylation of N-Acetyl-l-glutamate (NAG) to give N-acetyl-l-glutamate-5-phosphate. NAGK is only present in microorganisms and plants but absent in mammals, which makes it an attractive target for antimicrobial or biocidal development. Understanding the substrate binding mode and reaction mechanism of NAGK is crucial for targeting the kinase to develop potential therapies. Here, the substrate binding mode was studied by comparing the conformational change of NAGK in the presence and in the absence of the NAG substrate based on molecular dynamics simulations. We revealed that with substrate binding, the catalytic site of the kinase involving three loops in NAGK exhibits a closed conformation, which is predominantly controlled by an interaction between Arg98 and the α-COO- of NAG. Lys41 is found to guide phosphate transfer through the interactions with the ß-,γ-, and γ-phosphate oxygen atoms of adenosine 5'-triphosphate surrounded by two highly conserved glycine residues (Gly44 and Gly76), while Arg98 helps to position the NAG substrate in the catalytic site, which facilitates the phosphate transfer. Furthermore, we elucidated phosphate-transfer reaction mechanism using hybrid density functional theory-based quantum mechanics/molecular mechanics calculations (B97D/AMBER99) and found that the catalysis follows a dissociative mechanism.

Regulation of ATP levels in Escherichia coli using CRISPR interference for enhanced pinocembrin production.

BACKGROUND: Microbial biosynthesis of natural products holds promise for preclinical studies and treating diseases. For instance, pinocembrin is a natural flavonoid with important pharmacologic characteristics and is widely used in preclinical studies. However, high yield of natural products production is often limited by the intracellular cofactor level, including adenosine triphosphate (ATP). To address this challenge, tailored modification of ATP concentration in Escherichia coli was applied in efficient pinocembrin production. RESULTS: In the present study, a clustered regularly interspaced short palindromic repeats (CRISPR) interference system was performed for screening several ATP-related candidate genes, where metK and proB showed its potential to improve ATP level and increased pinocembrin production. Subsequently, the repression efficiency of metK and proB were optimized to achieve the appropriate levels of ATP and enhancing the pinocembrin production, which allowed the pinocembrin titer increased to 102.02 mg/L. Coupled with the malonyl-CoA engineering and optimization of culture and induction condition, a final pinocembrin titer of 165.31 mg/L was achieved, which is 10.2-fold higher than control strains. CONCLUSIONS: Our results introduce a strategy to approach the efficient biosynthesis of pinocembrin via ATP level strengthen using CRISPR interference. Furthermore coupled with the malonyl-CoA engineering and induction condition have been optimized for pinocembrin production. The results and engineering strategies demonstrated here would hold promise for the ATP level improvement of other flavonoids by CRISPRi system, thereby facilitating other flavonoids production.