Improving glucose oxidase catalysis in Aspergillus niger via Vitreoscilla hemoglobin fusion protein.

Oxygen is crucial for converting glucose to gluconic acid catalyzed by glucose oxidase (Gox). However, industrial gluconic acid production faces oxygen supply limitations. To enhance Gox efficiency, Vitreoscilla hemoglobin (VHb) has been considered as an efficient oxygen transfer carrier. This study identified GoxA, a specific isoform of Gox in the industrial gluconic acid-producing strain of Aspergillus niger. Various forms of VHb expression in A. niger were tested to improve GoxA's catalytic efficiency. Surprisingly, the expression of free VHb, both intracellularly and extracellularly, did not promote gluconic acid production during shake flask fermentation. Then, five fusion proteins were constructed by linking Gox and VHb using various methods. Among these, VHb-GS1-GoxA, where VHb's C-terminus connected to GoxA's N-terminus via the flexible linker GS1, demonstrated a significantly higher Kcat/Km value (96% higher) than GoxA. Unfortunately, the expression of VHb-GS1-GoxA in A. niger was limited, resulting in a low gluconic acid production of 3.0 g/L. To overcome the low expression problem, single- and dual-strain systems were designed with tools of SpyCatcher/SpyTag and SnoopCatcher/SnoopTag. In these systems, Gox and VHb were separately expressed and then self-assembled into complex proteins. Impressively, the single-strain system outperformed the GoxA overexpression strain S1971, resulting in 23% and 9% higher gluconic acid production under 0.6 vvm and 1.2 vvm aeration conditions in the bioreactor fermentation, respectively. The successful construction of Gox and VHb fusion or complex proteins, as proposed in this study, presents promising approaches to enhance Gox catalytic efficiency and lower aerodynamic costs in gluconic acid production. KEY POINTS: • Overexpressing free VHb in A. niger did not improve the catalytic efficiency of Gox • The VHb-GS1-GoxA showed an increased Kcat/Km value by 96% than GoxA • The single-strain system worked better in the gluconic acid bioreactor fermentation.

Engineering a Phosphoketolase Pathway to Supplement Cytosolic Acetyl-CoA in Aspergillus niger Enables a Significant Increase in Citric Acid Production.

Citric acid is widely used in the food, chemical and pharmaceutical industries. Aspergillus niger is the workhorse used for citric acid production in industry. A canonical citrate biosynthesis that occurred in mitochondria was well established; however, some research suggested that the cytosolic citrate biosynthesis pathway may play a role in this chemical production. Here, the roles of cytosolic phosphoketolase (PK), acetate kinase (ACK) and acetyl-CoA synthetase (ACS) in citrate biosynthesis were investigated by gene deletion and complementation in A. niger. The results indicated that PK, ACK and ACS were important for cytosolic acetyl-CoA accumulation and had significant effects on citric acid biosynthesis. Subsequently, the functions of variant PKs and phosphotransacetylase (PTA) were evaluated, and their efficiencies were determined. Finally, an efficient PK-PTA pathway was reconstructed in A. niger S469 with Ca-PK from Clostridium acetobutylicum and Ts-PTA from Thermoanaerobacterium saccharolyticum. The resultant strain showed an increase of 96.4% and 88% in the citrate titer and yield, respectively, compared with the parent strain in the bioreactor fermentation. These findings indicate that the cytosolic citrate biosynthesis pathway is important for citric acid biosynthesis, and increasing the cytosolic acetyl-CoA level can significantly enhance citric acid production.

Effective production of kojic acid in engineered Aspergillus niger.

BACKGROUND: Kojic acid (KA) is a widely used compound in the cosmetic, medical, and food industries, and is typically produced by Aspergillus oryzae. To meet increasing market demand, it is important to optimize KA production through seeking alternatives that are more economic than current A. oryzae-based methods. RESULTS: In this study, we achieved the first successful heterologous production of KA in Aspergillus niger, an industrially important fungus that does not naturally produce KA, through the expression of the kojA gene from A. oryzae. Using the resulting KA-producing A. niger strain as a platform, we identified four genes (nrkA, nrkB, nrkC, and nrkD) that negatively regulate KA production. Knocking down nrkA or deleting any of the other three genes resulted in a significant increase in KA production in shaking flask cultivation. The highest KA titer (25.71 g/L) was achieved in a pH controlled batch bioreactor using the kojA overexpression strain with a deletion of nrkC, which showed a 26.7% improvement compared to the KA titer (20.29 g/L) that was achieved in shaking flask cultivation. CONCLUSION: Our study demonstrates the potential of using A. niger as a platform for studying KA biosynthesis and regulation, and for the cost-effective production of KA in industrial strain development.

Identification and genetic characterization of mitochondrial citrate transporters in Aspergillus niger.

Aspergillus niger is a major cell factory for citric acid production, and the process of citrate export from mitochondria to cytoplasm is predicted to be one of rate-limiting steps in citric acid accumulation. Currently, the mitochondrial citrate transporters (Ctps) in A. niger are not fully characterized. Here, six putative Ctp encoding genes (ctpA to ctpF) were identified based on their homology with a mitochondrial citrate transporter ScCtp1 from Saccharomyces cerevisiae. Disruption of individual ctpA to ctpF caused varying degrees of decline in citric acid accumulation at different fermentation stages, whereas a mutant strain S1696 with disruption of all six ctps showed complete loss of citiric acid production. S1696 also exhibited delayed growth, reduced conidia formation, and decreased pigmentogenesis. Exogenous addition of citrate partially restored the conidia formation and pigmentogenesis in S1696 mutant. Reintroduction of individual ctps (ctpA to ctpF) into S1696 at the amyA locus showed that ctpA, ctpB, and ctpD restored the citric acid titers to 88.5, 93.8, and 94.6% of the parent strain, respectively. Additionally, the formation of conidia and pigment production was partially restored after reintroduction of ctpA, ctpB, or ctpD. Overexpression of respective ctpA, ctpB, and ctpD in the parent strain resulted in increases in citric acid accumulation by 32.8, 19.3, and 24.2%, respectively. These results demonstrate that CtpA, CtpB, and CtpD play important roles in citric acid transport across the mitochondrial membrane and function in a redundant manner. Enhancement of citric acid transport process can serve as a target for boosting citric acid accumulation in A. niger.

[Engineering the precursor supply pathway in Streptomyces gilvosporeus for overproduction of natamycin].

Natamycin is a safe and efficient antimycotics which is widely used in food and medicine industry. The polyene macrolide compound, produced by several bacterial species of the genus Streptomyces, is synthesized by type Ⅰ polyketide synthases using acetyl-CoA, malonyl-CoA, and methylmalonyl-CoA as substrates. In this study, four pathways potentially responsible for the supply of the three precursors were evaluated to identify the effective precursor supply pathway which can support the overproduction of natamycin in Streptomyces gilvosporeus, a natamycin-producing wild-type strain. The results showed that over-expressing acetyl-CoA synthetase and methylmalonyl-CoA mutase increased the yield of natamycin by 44.19% and 20.51%, respectively, compared with the wild type strain under shake flask fermentation. Moreover, the yield of natamycin was increased by 66.29% compared with the wild-type strain by co-overexpression of acetyl-CoA synthetase and methylmalonyl-CoA mutase. The above findings will facilitate natamycin strain improvement as well as development of strains for producing other polyketide compounds.

Is hyaluronic acid production transcriptionally regulated? A transcriptional repressor gene deletion study in Streptococcus zooepidemicus.

Hyaluronic acid (HA) is a multiple-function biopolymer that is widely used in food, cosmetic, and biomedical fields. In group C streptococci, the major workhorse of HA production in industry, the HA biosynthetic pathway has been proposed, while how HA synthesis is regulated is unclear. In this study, we identified twenty-five putative transcriptional repressors in S. zooepidemicus and studied whether they regulate HA synthesis or not. The individual gene deletion strain was firstly constructed, and the phenotypic changes of the corresponding deletion strains in stress tolerance and HA production were detected. The hrcA deletion strain is more sensitive to high temperature, and the rex deletion strain is more resistant to the oxidative stress. Three transcriptional repressor deletions resulted significantly decreased transcriptional levels of hasA, among which the scrR deletion strain shows most dramatical decrease in HA production. The regulatory mechanism of how ScrR affects the production of HA was further explored by transcriptional expression analysis of scrA and scrB, two direct target genes of ScrR regulon. Our results indicates that the deficiency of ScrR results in the unbalanced expression of scrA and scrB, which might also partly account for the decreasing production of HA. In agreement with the speculation, overexpression of scrB in ΔscrR genetic background results in 80% improvement in HA production. Taken together, the systemic genetic study of transcriptional repressors expands our understanding for the physiological regulation process of S. zooepidemicus and should help in the development of high-performance industrial strains for the efficient production of HA. KEY POINTS: • Twenty-two transcriptional repressor genes in S. zooepidemicus were deleted individually, and the phenotypes of corresponding mutants on a variety of conditions were characterized. • HrcA deficiency showed inferior cell tolerance to high temperature, and Rex deficiency showed superior cell tolerance to reactive oxygen stress, and four repressors deficiency showed inferior hyaluronic acid synthesis, among which the transcriptional levels of hasA of three mutants decreased significantly. • Optimizing sucrose metabolic flux can enhance hyaluronic acid synthesis significantly.

Characterization of FtsH Essentiality in Streptococcus mutans via Genetic Suppression.

FtsH belongs to the AAA+ ATP-dependent family of proteases, which participate in diverse cellular processes and are ubiquitous among bacteria, chloroplasts, and mitochondria. FtsH is poorly characterized in most organisms, especially compared to other major housekeeping proteases. In the current study, we examined the source of FtsH essentiality in the human oral microbiome species Streptococcus mutans, one of the primary etiological agents of dental caries. By creating a conditionally lethal ftsH mutant, we were able to identify a secondary suppressor missense mutation in the vicR gene, encoding the response regulator of the essential VicRK two-component system (TCS). Transcriptomic analysis of the vicR (G195R) mutant revealed significantly reduced expression of 46 genes, many of which were located within the genomic island Tnsmu2, which harbors the mutanobactin biosynthetic gene cluster. In agreement with the transcriptomic data, deletion of the mutanobactin biosynthetic gene cluster suppressed ftsH essentiality in S. mutans. We also explored the role of FtsH in S. mutans physiology and demonstrated its critical role in stress tolerance, especially acid stress. The presented results reveal the first insights within S. mutans for the pleiotropic regulatory function of this poorly understood global regulator.

Identification of Streptococcus mutans genes involved in fluoride resistance by screening of a transposon mutant library.

Fluoride has been used as an effective anticaries agent for more than 70 years, which might result in the emergence of fluoride-resistant strains. However, the fluoride resistance mechanism and the cariogenic properties of fluoride-resistant mutant for cariogenic bacterial species Streptococcus mutans remain largely unknown. We describe here the construction and characterization of a mariner-based transposon system designed to be used in S. mutans, which is also potentially applicable to other streptococci. To identify genetic determinants of fluoride resistance in S. mutans, we constructed a library of S. mutans transposon insertion mutants and screened this library to identify mutants exhibiting fluoride resistance phenotype. Two mutants were found to carry transposon insertion in two different genetic loci (smu.396 and smu.1291c), respectively. Our subsequent genetic study indicates the fluoride-resistant phenotype for the mutant with the insertion in smu.1291c is resulting from the constitutive overexpression of downstream operon smu.1290c-89c, which is consistent with the previous reports. We also demonstrate for the first time that the deletion of smu.396 is responsible for the fluoride-resistant phenotype and that the combining of smu1290c-89c overexpression and smu.396 deletion in one strain could attribute an additive effect on the fluoride resistance. In addition, our results suggest that the biological fitness of those fluoride-resistant mutants is reduced compared to that of wild-type strain. Overall, our identification and characterization of genetic determinants responsible for fluoride resistance in S. mutans expand our understanding of the fluoride resistance mechanism and the biological consequence of the fluoride resistance strains.

Identification and engineering a C4-dicarboxylate transporter for improvement of malic acid production in Aspergillus niger.

Modification of C4-dicarboxylate transport processes is an important strategy for the development of efficient malic acid producing cell factory in Aspergillus niger. However, there is a lack of identification and functional research of malic acid transport proteins, which seriously hinders the construction of high-yield malic acid metabolic engineering strains. A C4-dicarboxylate transport protein (DCT) DCT1 is identified as major malic acid transport protein and exhibits significant elevation in malic acid production when overexpressed. DCT1 is found by homology searches and domain analyses with SpMAE1 from Schizosaccharomyces pombe as the template. Phylogenetic and domain analyses show that DCTs belong to voltage-dependent slow-anion channel transporter (SLAC1) family and are members of Tellurite-resistance/Dicarboxylate Transporter (TDT) Family. DCT1 disruption dramatically decreases malic acid titer by about 85.6% and 96.2% at 3 days and 5 days compared with the parent strain, respectively. Meanwhile, the citric acid titers increase by 36.4% and 13.7% at 3 days and 5 days upon DCT1 deficiency. These results suggest that DCT1 is the major malic acid transporter in A. niger. Overexpression of dct1 with its native promoter significantly improves malic acid production yielding up to 13.86 g/L and 30.79 g/L at 3 days and 5 days, respectively, which is 36.8% and 22.8% higher than those in the parent strain. However, the citric acid has no significant change during the 5-day fermentation. These results demonstrate the importance of C4-dicarboxylate transporters for the efficient production of malic acid. Furthermore, enhancement of malic acid transport process is a feasible approach of efficient malic acid production in this citric acid producing A. niger strain. KEY POINTS: • A dicarboxylate transporter DCT1 is identified as a major malic acid transporter. • DCT1 deficiency results in significant decrease of malic acid. • DCT1 overexpression leads to increased titers of malic acid. • Enhancement of malic acid transport is vital for malic acid production in A. niger.

Regulatory control of the Streptococcus mutans HdrRM LytTR Regulatory System functions via a membrane sequestration mechanism.

Bacteria sense and respond to environmental changes via several broad categories of sensory signal transduction systems. Recently, we described the key features of a previously unrecognized, but widely conserved class of prokaryotic sensory system that we refer to as the LytTR Regulatory System (LRS). Our previous studies suggest that most, if not all, prokaryotic LRS membrane proteins serve as inhibitors of their cognate transcription regulators, but the inhibitory mechanisms employed have thus far remained a mystery. Using the Streptococcus mutans HdrRM LRS as a model, we demonstrate how the LRS membrane protein HdrM inhibits its cognate transcription regulator HdrR by tightly sequestering HdrR in a membrane-localized heteromeric HdrR/M complex. Membrane sequestration of HdrR prevents the positive feedback autoregulatory function of HdrR, thereby maintaining a low basal expression of the hdrRM operon. However, this mechanism can be antagonized by ectopically expressing a competitive inhibitor mutant form of HdrR that lacks its DNA binding ability while still retaining its HdrM interaction. Our results indicate that sequestration of HdrR is likely to be the only mechanism required to inhibit its transcription regulator function, suggesting that endogenous activation of the HdrRM LRS is probably achieved through a modulation of the HdrR/M interaction.

Reductase of Mutanobactin Synthetase Triggers Sequential C-C Macrocyclization, C-S Bond Formation, and C-C Bond Cleavage.

Mutanobactins (MUBs) and their congeners that contain a macrocycle and/or a thiazepane ring are lipopeptides from Streptococcus mutans, a major causative agent of dental caries. Here we show that the C-terminal reductase domain of MubD releases the lipohexapeptide intermediates in an aldehyde form, which enables a spontaneous C-C macrocyclization. In the presence of a thiol group, the macrocyclized MUBs can further undergo spontaneous C-S bond formation and C-C bond cleavage to generate diverse MUB congeners.

Development of a Cre-loxP-based genetic system in Aspergillus niger ATCC1015 and its application to construction of efficient organic acid-producing cell factories.

The filamentous fungus Aspergillus niger is widely used in the biotechnology industry for the production of chemicals and enzymes. Engineering of this valuable organism to improve its productivity is currently hampered by the lack of efficient genetic tools. Here, a Cre-loxP-based system for gene editing in A. niger was developed and its application in construction of A. niger cell factories to produce various organic acids was explored. Two established inducible systems, the xylanase A gene promoter Pxln and Tet-on system, were examined for driving cre expression and thus selection marker hyh deletion. Under inducing conditions, the efficiency of loxP site-specific recombination in the strain with cre driven by Pxln is about 2%, while cre driven by Tet-on system is about 34% which was used as the platform strain for further genetic engineering. As a proof of application of this system, strains containing different copies of oxaloacetate acetylhydrolase-encoding gene (oahA) were constructed, and the resultant strain S428 showed as high as 3.1-fold increase in oxalic acid production. Furthermore, an efficient malate-producing strain was generated through four-step genetic manipulation (oahA deletion, pyc, mdh3 and C4-dicarboxylate transporter gene c4t318 insertion). The resultant strain S575 achieved a titer 120.38 g/L malic acid with the flask culture, and a titer 201.24 g/L malic acid in fed-batch fermentation. These results demonstrated that this modified Cre-loxP system is a powerful tool for genetic engineering in A. niger, which has the potential to be genetically modified as a viable aciduric platform strain to produce high levels of various organic acids.

An anaerobic bacterium host system for heterologous expression of natural product biosynthetic gene clusters.

Anaerobic bacteria represent an overlooked rich source of biological and chemical diversity. Due to the challenge of cultivation and genetic intractability, assessing the capability of their biosynthetic gene clusters (BGCs) for secondary metabolite production requires an efficient heterologous expression system. However, this kind of host system is still unavailable. Here, we use the facultative anaerobe Streptococcus mutans UA159 as a heterologous host for the expression of BGCs from anaerobic bacteria. A natural competence based large DNA fragment cloning (NabLC) technique was developed, which can move DNA fragments up to 40-kb directly and integrate a 73.7-kb BGC to the genome of S. mutans UA159 via three rounds of NabLC cloning. Using this system, we identify an anti-infiltration compound, mutanocyclin, from undefined BGCs from human oral bacteria. We anticipate this host system will be useful for heterologous expression of BGCs from anaerobic bacteria.

Identification of a Quorum Sensing System Regulating Capsule Polysaccharide Production and Biofilm Formation in Streptococcus zooepidemicus.

Streptococcus zooepidemicus is an important opportunistic pathogen of several species including humans. This organism is also well-known as the main producing strain in industrial production of hyaluronic acid (HA), which is the component of its capsule polysaccharide. How its virulence and capsule polysaccharide production is regulated remains poorly understood. Intercellular chemical signaling among bacteria provides communities of microbes the opportunity to coordinate gene expression to facilitate group behavior, such as pathogenicity, capsule polysaccharide production, etc. Yet no conserved cell-to-cell signaling system has been elucidated in S. zooepidemicus. Encoded within the genome of S. zooepidemicus is one Rgg regulator encoding gene (rgg) with low similarity to both rgg2 and rgg3 from Streptococcus pyogenes. A small ORF (named as shp) encoding a novel short hydrophobic peptide (SHP) was found in the vicinity of rgg. We found that the active form of pheromone is short and hydrophobic (LLLLKLA), corresponding to the C terminal 7 amino acids of the pre-peptide Shp, which shows divergent sequence to all peptide pheromones reported in streptococci. In response to active SHP, Rgg functions as a transcriptional activator to induce the expression of shp, forming a positive feedback circuit. Bacteria social behaviors, such as capsule polysaccharide production and biofilm formation, were significantly affected when the rgg-shp pathway was inactivated. These data provide the first demonstration that Rgg/Shp signaling pathway comprises an active quorum sensing system in S. zooepidemicus.

LytTR Regulatory Systems: A potential new class of prokaryotic sensory system.

The most commonly studied prokaryotic sensory signal transduction systems include the one-component systems, phosphosignaling systems, extracytoplasmic function (ECF) sigma factor systems, and the various types of second messenger systems. Recently, we described the regulatory role of two separate sensory systems in Streptococcus mutans that jointly control bacteriocin gene expression, natural competence development, as well as a cell death pathway, yet they do not function via any of the currently recognized signal transduction paradigms. These systems, which we refer to as LytTR Regulatory Systems (LRS), minimally consist of two proteins, a transcription regulator from the LytTR Family and a transmembrane protein inhibitor of this transcription regulator. Here, we provide evidence suggesting that LRS are a unique uncharacterized class of prokaryotic sensory system. LRS exist in a basal inactive state. However, when LRS membrane inhibitor proteins are inactivated, an autoregulatory positive feedback loop is triggered due to LRS regulator protein interactions with direct repeat sequences located just upstream of the -35 sequences of LRS operon promoters. Uncharacterized LRS operons are widely encoded by a vast array of Gram positive and Gram negative bacteria as well as some archaea. These operons also contain unique direct repeat sequences immediately upstream of their operon promoters indicating that positive feedback autoregulation is a globally conserved feature of LRS. Despite the surprisingly widespread occurrence of LRS operons, the only characterized examples are those of S. mutans. Therefore, the current study provides a useful roadmap to investigate LRS function in the numerous other LRS-encoding organisms.

d-Sedoheptulose-7-phosphate is a common precursor for the heptoses of septacidin and hygromycin B.

Seven-carbon-chain-containing sugars exist in several groups of important bacterial natural products. Septacidin represents a group of l-heptopyranoses containing nucleoside antibiotics with antitumor, antifungal, and pain-relief activities. Hygromycin B, an aminoglycoside anthelmintic agent used in swine and poultry farming, represents a group of d-heptopyranoses-containing antibiotics. To date, very little is known about the biosynthesis of these compounds. Here we sequenced the genome of the septacidin producer and identified the septacidin gene cluster by heterologous expression. After determining the boundaries of the septacidin gene cluster, we studied septacidin biosynthesis by in vivo and in vitro experiments and discovered that SepB, SepL, and SepC can convert d-sedoheptulose-7-phosphate (S-7-P) to ADP-l-glycero-ß-d-manno-heptose, exemplifying the involvement of ADP-sugar in microbial natural product biosynthesis. Interestingly, septacidin, a secondary metabolite from a gram-positive bacterium, shares the same ADP-heptose biosynthesis pathway with the gram-negative bacterium LPS. In addition, two acyltransferase-encoding genes sepD and sepH, were proposed to be involved in septacidin side-chain formation according to the intermediates accumulated in their mutants. In hygromycin B biosynthesis, an isomerase HygP can recognize S-7-P and convert it to ADP-d-glycero-ß-d-altro-heptose together with GmhA and HldE, two enzymes from the Escherichia coli LPS heptose biosynthetic pathway, suggesting that the d-heptopyranose moiety of hygromycin B is also derived from S-7-P. Unlike the other S-7-P isomerases, HygP catalyzes consecutive isomerizations and controls the stereochemistry of both C2 and C3 positions.

[Secondary metabolites from Streptococcus mutans and their ecological roles in dental biofilm].

Dental biofilms are composed of hundreds of bacterial species, among which Streptococcus mutans is widely recognized as the major pathogen of dental caries. The cariogenic potential of S. mutans is related to its ability to form a robust biofilm on the tooth surface and its acidogenic and acid-tolerant properties. Co-evolution of S. mutans with the host has resulted in the diversity of secondary metabolism of S. mutans in strain level. A variety of secondary metabolites, including 10 bacteriocins (mutacins) and one hybrid Polyketide/Non-Ribosomal Peptide type compound, have been characterized. Studies on these secondary metabolites indicate that they play a significant role either in interspecies or in inter-kingdom interactions in the dental biofilm. As more S. mutans strains are isolated and sequenced, additional secondary metabolites with novel functions will be discovered. The study of secondary metabolites in S. mutans is anticipated to be helpful for oral disease treatment and prevention by providing new strategies.

Neomycin biosynthesis is regulated positively by AfsA-g and NeoR in Streptomyces fradiae CGMCC 4.7387.

Neomycins are a group of aminoglycoside antibiotics with both clinical and agricultural applications. To elucidate the regulatory mechanism of neomycin biosynthesis, we completed draft genome sequencing of a neomycin producer Streptomyces fradiae CGMCC 4.7387 from marine sediments, and the neomycin biosynthesis gene cluster was identified. Inactivation of the afsA-g gene encoding a γ-butyrolactone (GBL) synthase in S. fradiae CGMCC 4.7387 resulted in a significant decrease of neomycin production. Quantitative RT-PCR analysis revealed that the transcriptional level of neoR and the aphA-neoGH operon were reduced in the afsA-g::aac(3)IV mutant. Interestingly, a conserved binding site of AdpA, a key activator in the GBL regulatory cascade, was discovered upstream of neoR, a putative regulatory gene encoding a protein with an ATPase domain and a tetratricopeptide repeat domain. When neoR was inactivated, the neomycin production was reduced about 40% in comparison with the WT strain. Quantitative RT-PCR analysis revealed that the transcriptional levels of genes in the aphA-neoGH operon were reduced clearly in the neoR::aac(3)IV mutant. Finally, the titers of neomycin were improved considerably by overexpression of afsA-g and neoR in S. fradiae CGMCC 4.7387.

An external substrate-free blue/white screening system in Escherichia coli.

Since the lacZα-based blue/white screening system was introduced to molecular biology, several different visual reporter systems were developed and used for various purposes in Escherichia coli. A common limit to the existent visual reporter systems is that an extracellular chromogenic substrate has to be added for the visible pigment production. In this study, we developed a new blue/white screening system based on a non-ribosomal peptide synthetase encoded by idgS from Streptomyces and a phosphopantetheinyl transferase encoded by sfp from Bacillus. When IdgS is activated from an apo-form to a holo-form via a posttranslational modification catalyzed by Sfp, it can synthesize a blue pigment indigoidine using L-glutamine, the amino acid abundant in cells, as a substrate. The new blue/white screening system contains a recipient E. coli strain with an optimized idgS gene cassette and a cloning vector harboring an sfp gene with an in-frame insertion of a multiple cloning site close to its N-terminal. We demonstrated that the IdgS/Sfp-based blue/white screening system is a powerful alternative to the lacZα-based screening system, which does not require any external substrate addition.

Identification of New Degrons in Streptococcus mutans Reveals a Novel Strategy for Engineering Targeted, Controllable Proteolysis.

Recently, controllable, targeted proteolysis has emerged as one of the most promising new strategies to study essential genes and otherwise toxic mutations. One of the principal limitations preventing the wider adoption of this approach is due to the lack of easily identifiable species-specific degrons that can be used to trigger the degradation of target proteins. Here, we report new advancements in the targeted proteolysis concept by creating the first prokaryotic N-terminal targeted proteolysis system. We demonstrate how proteins from the LexA-like protein superfamily can be exploited as species-specific reservoirs of N- and/or C-degrons, which are easily identifiable due to their proximity to strictly conserved residues found among LexA-like proteins. Using the LexA-like regulator HdiR of Streptococcus mutans, we identified two separate N-degrons derived from HdiR that confer highly efficient constitutive proteolysis upon target proteins when added as N-terminal peptide tags. Both degrons mediate degradation via AAA+ family housekeeping proteases with one degron primarily targeting FtsH and the other targeting the ClpP-dependent proteases. To modulate degron activity, our approach incorporates a hybrid N-terminal protein tag consisting of the ubiquitin-like protein NEDD8 fused to an HdiR degron. The NEDD8 fusion inhibits degron function until the NEDD8-specific endopeptidase NEDP1 is heterologously expressed to expose the N-degron. By fusing the NEDD8-degron tag onto GFP, luciferase, and the pleiotropic regulator RNase J2, we demonstrate that the N-terminal proteolysis approach exhibits far superior performance compared to the classic transcriptional depletion approach and is similarly applicable for the study of highly toxic mutations.