Pta Gene Research Articles

Exploring the effect of lipid loading on acidogenic fermentation of food waste: Insights from microbiome, quorum sensing and carbon metabolic pathway

The high lipid loading in food waste (FW) is widely regarded as one of the main bottlenecks in the traditional anaerobic digestion, but the effect of lipid on FW acidogenic fermentation has been historically ignored. This study attempted to reveal the influence of lipid loading on volatile fatty acids (VFAs) production and the corresponding synthesis mechanisms during FW acidogenic fermentation. The VFAs production reached 27.10 ± 1.00 g COD/L when the lipid loading comprised 25 % of the FW dry weight, increasing by 36.88 % compared to the CK group. Lactobacillus, with a relative abundance of 59.43 %, was the predominant genus under lipid loading. In quorum sensing (QS) system, the relative abundance of luxS that is involved in the biosynthetic pathway of the Autoinducer-2 (AI-2) signal molecule increased from 0.066 % to 0.10 % in the presence of lipid. The relative abundance of Genetic Information Processing increased from 16.36 % to 24.25 % under lipid loading, with up-regulation of Translation and Replication and repair. Lipid loading not only promoted the conversion of acetyl-CoA to acetic acid, as evidenced by a 1.36-fold increase in pta gene abundance, but also concurrently suppressed the consumption of acetic acid, reflected in the 96.83 % and 77.16 % decreases in ALDH and aldB abundance, thus accumulating acetic acid. Carbon emission was also constrained under lipid loading, in which the conversion from formate to CO2 and passing Tricarboxylic acid (TCA) cycle were restricted over 60 %. Together, lipid loading in FW represents a potential strategy for enhancing VFAs production in FW acidogenic fermentation.

Minimizing acetate formation from overflow metabolism in Escherichia coli: comparison of genetic engineering strategies to improve robustness toward sugar gradients in large-scale fermentation processes.

Introduction: Escherichia coli, a well characterized workhorse in biotechnology, has been used to produce many recombinant proteins and metabolites, but have a major drawback in its tendency to revert to overflow metabolism. This phenomenon occurs when excess sugar triggers the production of mainly acetate under aerobic conditions, a detrimental by-product that reduces carbon efficiency, increases cell maintenance, and ultimately inhibits growth. Although this can be prevented by controlled feeding of the sugar carbon source to limit its availability, gradients in commercial-scale bioreactors can still induce it in otherwise carbon-limited cells. While the underlying mechanisms have been extensively studied, these have mostly used non-limited cultures. In contrast, industrial production typically employs carbon-limited processes, which results in a substantially different cell physiology. Objective: The objective of this study was to evaluate and compare the efficiency of different metabolic engineering strategies with the aim to reduce overflow metabolism and increase the robustness of an industrial 2'-O-fucosyllactose producing strain under industrially relevant conditions. Methods: Three distinct metabolic engineering strategies were compared: i) alterations to pathways leading to and from acetate, ii) increased flux towards the tricarboxylic acid (TCA) cycle, and iii) reduced glucose uptake rate. The engineered strains were evaluated for growth, acetate formation, and product yield under non-limiting batch conditions, carbon limited fed-batch conditions, and after a glucose pulse in fed-batch mode. Results and Discussion: The findings demonstrated that blockage of the major acetate production pathways by deletion of the pta and poxB genes or increased carbon flux into the TCA cycle by overexpression of the gltA and deletion of the iclR genes, were efficient ways to reduce acetate accumulation. Surprisingly, a reduced glucose uptake rate did not reduce acetate formation despite it having previously been shown as a very effective strategy. Interestingly, overexpression of gltA was the most efficient way to reduce acetate accumulation in non-limited cultures, whereas disruption of the poxB and pta genes was more effective for carbon-limited cultures exposed to a sudden glucose shock. Strains from both strategies showed increased tolerance towards a glucose pulse during carbon-limited growth indicating feasible ways to engineer industrial E. coli strains with enhanced robustness.

Inactivation of ackA and pta Genes Reduces GlpT Expression and Susceptibility to Fosfomycin in Escherichia coli.

Fosfomycin is used to treat a variety of bacterial infections, including urinary tract infections caused by Escherichia coli. In recent years, quinolone-resistant and extended-spectrum β-lactamase (ESBL)-producing bacteria have been increasing. Because fosfomycin is effective against many of these drug-resistant bacteria, the clinical importance of fosfomycin is increasing. Against this background, information on the mechanisms of resistance and the antimicrobial activity of this drug is desired to enhance the usefulness of fosfomycin therapy. In this study, we aimed to explore novel factors affecting the antimicrobial activity of fosfomycin. Here, we found that ackA and pta contribute to fosfomycin activity against E. coli. ackA and pta mutant E. coli had reduced fosfomycin uptake capacity and became less sensitive to this drug. In addition, ackA and pta mutants had decreased expression of glpT that encodes one of the fosfomycin transporters. Expression of glpT is enhanced by a nucleoid-associated protein, Fis. We found that mutations in ackA and pta also caused a decrease in fis expression. Thus, we interpret the decrease in glpT expression in ackA and pta defective strains to be due to a decrease in Fis levels in these mutants. Furthermore, ackA and pta are conserved in multidrug-resistant E. coli isolated from patients with pyelonephritis and enterohemorrhagic E. coli, and deletion of ackA and pta from these strains resulted in decreased susceptibility to fosfomycin. These results suggest that ackA and pta in E. coli contribute to fosfomycin activity and that mutation of these genes may pose a risk of reducing the effect of fosfomycin. IMPORTANCE The spread of drug-resistant bacteria is a major threat in the field of medicine. Although fosfomycin is an old type of antimicrobial agent, it has recently come back into the limelight because of its effectiveness against many drug-resistant bacteria, including quinolone-resistant and ESBL-producing bacteria. Since fosfomycin is taken up into the bacteria by GlpT and UhpT transporters, its antimicrobial activity fluctuates with changes in GlpT and UhpT function and expression. In this study, we found that inactivation of the ackA and pta genes responsible for the acetic acid metabolism system reduced GlpT expression and fosfomycin activity. In other words, this study shows a new genetic mutation that leads to fosfomycin resistance in bacteria. The results of this study will lead to further understanding of the mechanism of fosfomycin resistance and the creation of new ideas to enhance fosfomycin therapy.

Genomic and Biochemical Characterization of Bifidobacterium pseudocatenulatum JCLA3 Isolated from Human Intestine.

Bifidobacteria have been investigated due to their mutualistic microbe-host interaction with humans throughout their life. This work aims to make a biochemical and genomic characterization of Bifidobacterium pseudocatenulatum JCLA3. By multilocus analysis, the species of B. pseudocatenulatum JCLA3 was established as pseudocatenulatum. It contains one circular genome of 2,369,863 bp with G + C content of 56.6%, no plasmids, 1937 CDSs, 54 tRNAs, 16 rRNAs, 1 tmRNA, 1 CRISPR region, and 401 operons predicted, including a CRISPR-Cas operon; it encodes an extensive number of enzymes, which allows it to utilize different carbohydrates. The ack gene was found as part of an operon formed by xfp and pta genes. Two genes of ldh were found at different positions. Chromosomally encoded resistance to ampicillin and cephalothin, non-hemolytic activity, and moderate inhibition of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538 were demonstrated by B. pseudocatenulatum JCLA3; it can survive 100% in simulated saliva, can tolerate primary and secondary glyco- or tauro-conjugated bile salts but not in a mix of bile; the strain did not survive at pH 1.5-5. The cbh gene coding to choloylglycine hydrolase was identified in its genome, which could be related to the ability to deconjugate secondary bile salts. Intact cells showed twice as much antioxidant activity than debris. B. pseudocatenulatum JCLA3 showed 49% of adhesion to Caco-2 cells. The genome and biochemical analysis help to elucidate further possible biotechnological applications of B. pseudocatenulatum JCLA3.

Metabolic engineering of Aureobasidium melanogenum 9–1 for overproduction of liamocins by enhancing supply of acetyl-CoA and ATP

In this study, it was found that reducing consumption of acetyl-CoA in mitochondria, peroxisome and lipid biosynthesis could not obviously enhance liamocin biosynthesis by engineered strains of Aureobasidium melanogenm 9–1, but decreased cell growth of the mutants. On the contrary, expression of heterologous PTA gene for phosphotransacetylase in PK pathway and native ALD gene for acetaldehyde dehydrogenase and ACS gene encoding acetyl-CoA synthetase in the PDH bypass pathway reduced liamocin biosynthesis. However, expression the PK gene for phosphoketolase, the PDC gene encoding pyruvate decarboxylase and VHb gene coding for Vitreoscilla hemoglobin (VHb) in the glucose derepression mutants could greatly enhance liamocin production. The resulting strain V33 could produce 55.38 g/L of liamocin and 25.10 g/L of cell dry weight from 117.27 g/L of glucose within 168 h of 10-liter fermentation, leading to the yield of 0.47 g/g of glucose, the productivity of 0.33 g/L/h and rate of glucose utilization of 0.70 ± 0.01 g/L/h. This was a new and efficient strategy for overproduction of liamocin by A. melanogenm.

Molecular detection of some virulence genes of proteus mirabilis isolated from different samples in Babylon province /Iraq

A total of (130) alkaline urine sampls, as well as vaginal, wound, and burn sampls, were collected from patients with urinary tract infections of both sexes who visited Al-Hilla general Teaching Hospital in Al-Hilla governorate, Iraq, between September and December of 2021. Genotyping of P.mirabilis isolates was carried out at the molecular level to define genotypic features using a PCR approach targeting the key virulence factors genes. Three virulence genes (hpmA, rsbA, and pta) were screened from the total of 25 P.mirabilis positive samples. Twenty-five isolates of P.mirabilis were discovered. 17 (68%) isolates were obtained from patients with urinary tract infection, while 3 (12%) isolates were obtained from patients with vaginal infection, 3 (12%) isolates from patients with surgical wound infection, and 2 (8%) isolates were obtained from patients with burn infection. For the identification of the ureR gene, a specific PCR primer was utilized. The ureR gene was discovered in 25 (100%) isolates of P.mirabilis, including all UTI and vaginal infections, wound infections, and burn infections. A particular PCR primer was used to determine the presence of the hpmA gene. The hpmA gene was found in 24 P.mirabilis bacterial isolates (96%). PCR was used on genomic DNA from each P.mirabilis isolate to detect the rsbA gene using appropriate primers. PCR amplification of the rsbA gene revealed that only 20 (80%) of the 25 P.mirabilis isolates tested positive for this gene. Using specific PCR primers, the pta gene was also detected in P.mirabilis. Out of 25 isolates of P.mirabilis, the pta gene was found in 23 (92%) of P.mirabilis.

Validation of a real-time PCR method on pta gene for Clostridium tyrobutyricum quantification in milk

In the dairy industry, the late blowing defect in medium and long ripened cheeses causes important economic losses. This cheese defect, caused mainly by the anaerobic spore former Clostridium tyrobutyricum , is characterized by gas production resulting in holes formation in the paste, and in unpleasant aromas related to the butyrate production. Since longtime, the reference method used to detect the presence of total spore number in milk is the Most Probable Number (MPN), a semi-quantitative method which measures the bacterial growth and the gas production after heat treatment of the samples. This method often gives uncertain results, it requires long incubation periods and fails to differentiate between different species of spore-forming bacteria. A TaqMan real-time quantitative PCR targeting the phosphotransacetylase gene ( pta ) for C. tyrobutyricum was previously developed as an alternative technique to the traditional method, faster and more specific, and therefore of great interest to preventively determine the contamination of milk by C. tyrobutyricum . The aim of this work was the validation of this quantitative real-time PCR protocol for the C. tyrobutyricum detection in milk. Following the method validation, 120 samples of UHT and raw milk artificially contaminated with C. tyrobutyricum spores were analyzed in increasing concentrations, relating the results of the molecular method with those obtained by the MPN. Finally, 144 raw milk samples with a possible natural contamination were analyzed and demonstrated a positivity rate of 15.28% (CI95% 10.31–22.05%). • Validation of a real-time PCR for the C. tyrobutyricum detection in milk. • Method application on both spiked and naturally contaminated samples. • Rapid method to monitor the milk quality to avoid the late blowing defect.

The Physiological Responses of Escherichia coli Triggered by Phosphoribulokinase (PrkA) and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco).

Phosphoribulokinase (PrkA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been proposed to create a heterologous Rubisco-based engineered pathway in Escherichia coli for in situ CO2 recycling. While the feasibility of a Rubisco-based engineered pathway has been shown, heterologous expressions of PrkA and Rubisco also induced physiological responses in E. coli that may compete with CO2 recycling. In this study, the metabolic shifts caused by PrkA and Rubisco were investigated in recombinant strains where ppc and pta genes (encodes phosphoenolpyruvate carboxylase and phosphate acetyltransferase, respectively) were deleted from E. coli MZLF (E. coli BL21(DE3) Δzwf, ΔldhA, Δfrd). It has been shown that the demand for ATP created by the expression of PrkA significantly enhanced the glucose consumptions of E. coli CC (MZLF Δppc) and E. coli CA (MZLF Δppc, Δpta). The accompanying metabolic shift is suggested to be the mgsA route (the methylglyoxal pathway) which results in the lactate production for reaching the redox balance. The overexpression of Rubisco not only enhanced glucose consumption but also bacterial growth. Instead of the mgsA route, the overproduction of the reducing power was balanced by the ethanol production. It is suggested that Rubisco induces a high demand for acetyl-CoA which is subsequently used by the glyoxylate shunt. Therefore, Rubisco can enhance bacterial growth. This study suggests that responses induced by the expression of PrkA and Rubisco will reach a new energy balance profile inside the cell. The new profile results in a new distribution of the carbon flow and thus carbons cannot be majorly directed to the Rubisco-based engineered pathway.

Improvement of Streptococcus suis glutamate dehydrogenase expression in Escherichia coli through genetic modification of acetate synthesis pathway.

Escherichia coli generates acetate as an undesirable by-product that has several negative effects on protein expression, and the reduction of acetate accumulation by modifying genes of acetate synthesis pathway can improve the expression of recombinant proteins. In the present study, the effect of phosphotransacetylase (pta) or/and acetate kinase (ackA) deletion on glutamate dehydrogenase (GDH) expression was investigated. The results indicated that the disruptions of pta or/and ackA decreased the acetate accumulation and synthesis of per gram cell, and increased cell density, and GDH expression and synthesis of per gram cell. The pta gene was more important for acetate formation than the ackA gene. Using the strain with deletions of pta-ackA (SSGPA) for GDH expression, acetate accumulation (2·61gl-1 ) and acetate synthesis of per gram cell (0·229gg-1 ) were lowest, decreasing by 28·29 and 41·43% compared with those of the parental strain (SSG) respectively. The flux of acetate synthesis (6·6%) was decreased by 72·15% compared with that of SSG, and the highest cell density (11·38gl-1 ), GDH expression (2·78mgml-1 ), and GDH formation of per gram cell (0·2442mgmg-1 ) were obtained, which were 1·22-, 1·43- and 1·17-times higher than the parental strain respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: Acetate is the key undesirable by-product in Escherichia coli cultivation, and both biomass and production of desired products are increased by the reduction of acetate accumulation. In the present study, the strains with deletions of pta or/and ackA were constructed to reduce the acetate accumulation and improve the GDH expression, and the highest expression level of GDH was obtained using the strain with lesion in pta-ackA that was 1·17-times higher than that of the parental strain. The construction strategy of recombinant E. coli for decreasing the acetate excretion can be used for high expression level of other desired products.

Effect of photooxidative stress and mannitol on synthesis of OmpC-OmpF proteins of Escherichia coli in lake water

This study investigated the effects of photooxidative stress and mannitol on the synthesis of OmpC-OmpF porins of Escherichia coli in lake water. The synthesis of OmpF decreased independently of photooxidative stress, whereas the synthesis of OmpC decreased as a consequence of photooxidative stress in lake water. Thus, OmpC synthesis in E. coli was directly affected by photooxidative stress. Mutations in the envZ and pta genes had no effect on the control of OmpC and OmpF synthesis in E. coli under photooxidative stress in lake water. Mannitol is an antioxidant substance that provides protection from photooxidative stress. In this study, was found that ompC expression has a regulation mechanism during photooxidative stress. Mannitol was also found to have a relationship with EnvZ in the control of porin synthesis.

Impact of exogenous acetate on ethanol formation and gene transcription for key enzymes in Clostridium autoethanogenum grown on CO

Gas fermentation using C1 substrate (CO or CO2) is a novel and attractive technology for ethanol production. Clostridium autoethanogenum, a promising gas fermentation strain, can grow on 100 % CO and produce acetate and ethanol through the Wood-Ljungdahl pathway. Like other autotrophic acetogens, C. autoethanogenum produces ethanol primarily via the reduction of acetyl-CoA. In this study, we demonstrated that ethanol was also formed via acetate reduction. 13C-labeled acetate was employed to investigate the conversion of acetate in C. autoethanogenum during its autotrophic growth on CO. The addition of exogenous acetate can improve ethanol production in C. autoethanogenum, 13C abundance in ethanol increased significantly with the increase of exogenous 13C-labeled acetate, confirming that acetate can be transformed to ethanol. Moreover, transcriptional analysis of genes encoding phosphotransacetylase(pta), aldehyde:ferredoxin oxidoreductase(aor), carbon monoxide dehydrogenase(codh) and alcohol dehydrogenase(adh) in C. autoethanogenum revealed their differential expression under varied exogenous acetate levels. Among these genes, pta gene was down-regulated due to its involvement in acetate formation, whereas aor gene CAETHG_0102, codh gene CAETHG_3005, adh genes CAETHG_1841 and CAETHG_1813 were found to be highly up-regulated at higher levels of exogenous acetate, suggesting that they may play important roles in the conversion of acetate to ethanol.

Characterization of the phosphotransacetylase-acetate kinase pathway for ATP production in Porphyromonas gingivalis

ABSTRACTAcetyl phosphate (AcP) is generally produced from acetyl coenzyme A by phosphotransacetylase (Pta), and subsequent reaction with ADP, catalyzed by acetate kinase (Ack), produces ATP. The mechanism of ATP production in Porphyromonas gingivalis is poorly understood. The aim of this study was to explore the molecular basis of the Pta-Ack pathway in this microorganism. Pta and Ack from P. gingivalis ATCC 33277 were enzymatically and structurally characterized. Structural and mutational analyses suggest that Pta is a dimer with two substrate-binding sites in each subunit. Ack is also dimeric, with a catalytic cleft in each subunit, and structural analysis indicates a dramatic domain motion that opens and closes the cleft during catalysis. ATP formation by Ack proceeds via a sequential mechanism. Reverse transcription-PCR analysis demonstrated that the pta (PGN_1179) and ack (PGN_1178) genes, tandemly located in the genome, are cotranscribed as an operon. Inactivation of pta or ack in P. gingivalis by homologous recombination was successful only when the inactivated gene was expressed in trans. Therefore, both pta and ack genes are essential for this microorganism. Insights into the Pta-Ack pathway reported herein would be helpful to understand the energy acquisition in P. gingivalis.

Regulation of acetate metabolism and coordination with the TCA cycle via a processed small RNA

Bacterial regulatory small RNAs act as crucial regulators in central carbon metabolism by modulating translation initiation and degradation of target mRNAs in metabolic pathways. Here, we demonstrate that a noncoding small RNA, SdhX, is produced by RNase E-dependent processing from the 3'UTR of the sdhCDAB-sucABCD operon, encoding enzymes of the tricarboxylic acid (TCA) cycle. In Escherichia coli, SdhX negatively regulates ackA, which encodes an enzyme critical for degradation of the signaling molecule acetyl phosphate, while the downstream pta gene, encoding the enzyme critical for acetyl phosphate synthesis, is not significantly affected. This discoordinate regulation of pta and ackA increases the accumulation of acetyl phosphate when SdhX is expressed. Mutations in sdhX that abolish regulation of ackA lead to more acetate in the medium (more overflow metabolism), as well as a strong growth defect in the presence of acetate as sole carbon source, when the AckA-Pta pathway runs in reverse. SdhX overproduction confers resistance to hydroxyurea, via regulation of ackA SdhX abundance is tightly coupled to the transcription signals of TCA cycle genes but escapes all known posttranscriptional regulation. Therefore, SdhX expression directly correlates with transcriptional input to the TCA cycle, providing an effective mechanism for the cell to link the TCA cycle with acetate metabolism pathways.

Triple knockout of frdC gltA and pta genes enhanced pHA production in Escherichia coli

Polyhydroxyalkanoate (PHA) is a linear polyester produced through the fermentation of sugar or lipid. Biosynthesis of PHA comprises three enzymes known as acetyl-CoA acetyltransferase (phaA), acetoacetyl-CoA reductase (phaB) and PHA synthase (phaC). Comamonas sp. is one of the strains commonly used for PHA production. In order to develop higher PHA production from bacterial respond strategy, PHA biosynthesis operon of Comamonas sp. EB172 was introduced into Escherichia coli BW25113 through a pGEM-T vector. E. coli was chosen due to the complete genome information available and the absence of depolymerisation gene, phaZ. In this study, the deletion of several single genes, which are frdC, gltA, and pta, was found to be associated with PHA metabolism activity in E. coli BW25113. P1 transduction was performed to construct multiple genes knockout. The engineered strain, E. coli BW25113 frdCgltApta::kan/pGEM’-phaCABCo, yielded the highest PHA production at 64 wt.% with 1.4 fold higher than that of control strain of E. coli BW25113/pGEM’-phaCABCo. This strain is potential for industrial application for higher PHA production from E. coli.

Markerless genome editing in Clostridium beijerinckii using the CRISPR-Cpf1 system

CRISPR-Cpf1 is a type V CRISPR system that has recently been exploited for genome engineering purposes. Compared to the well-known Streptococcus pyogenes CRISPR-Cas9 system, the effector protein Cpf1 recognizes T-rich protospacer-adjacent motif (PAM) instead of G-rich PAM (used by CRISPR-Cas9), which could offer a substantial expansion of the existing genetic toolbox for genome editing. In this study, we report the implementation of the Acidaminococcus sp. Cpf1 (AsCpf1) for markerless genome engineering in Clostridium beijerinckii, a prominent species for biosolvent production through the well-known Acetone-Butanol-Ethanol (ABE) pathway. A lactose inducible promoter was used to control the expression of AsCpf1 to decrease its toxicity, while a constitutive small RNA promoter was employed to drive the expression of pre-crRNA. A One-Step-Assembly (OSA) approach was employed to construct the CRISPR-Cpf1-based vector in one single step, which simplified and streamlined the plasmid construction process. Using the customized CRISPR-Cpf1 system, we successfully deleted spo0A and pta genes in C. beijerinckii, with an editing efficiency of up to 100%. Altogether, our results demonstrated the easy programmability and high efficiency of the CRISPR-Cpf1 system for versatile genome engineering purposes. This study provides valuable guidance and essential references for repurposing the CRISPR-Cpf1 system for genome engineering in other microorganisms.

Impact of poxB, pta, and ackA genes on mineral-weathering of Enterobacter cloacae S71.

In this study, biotite weathering behaviors were compared between mineral-weathering bacteria Enterobacter cloacae S71, mutant strains created by the deletion of poxB, pta, and ackA genes involved in acetate formation, and their complemented strains. Compared to strain S71, a decrease in bacterial growth was observed during the early and middle stages for the mutant ΔpoxB and at the middle and later stages for the mutants Δpta and ΔackA. Dissolved Al and Fe concentrations were lower during the early stage for strain ΔpoxB, at the early or middle stage for strain Δpta, and at the middle and later stages and throughout the weathering process for strain ΔackA, compared to strain S71. Acetate production was depressed during the early stage for strain ΔpoxB, at the early and middle stages for strain Δpta, and throughout the weathering process for strain ΔackA. Overall, the ackA gene exhibited a larger impact on dissolved Fe and acetate concentrations than both the poxB and pta genes. Reduced bacterial growth and lower dissolved Al, Fe, and acetate concentrations recovered by the complemented strains. These results show that strain S71 promoted mineral weathering through the production of acetic acid with distinctive impacts by the genes involved in acetate.

The Effect of Cu-BPDCA-Ty on Antibacterial Activity and The Expression of mecA Gene in Clinical and Standard Strains of Methicillin-Resistant Staphylococcus aureus

Background: The antibiotic resistance of bacteria has increased in the last decade. The mecA gene plays an important role in the pathogenicity of Methicillin-Resistant Staphylococcus aureus (MRSA) by increasing antibiotics resistance. Recent studies have indicated that nanotechnology, as an antimicrobial agent, has had promising results. Objectives: The present study was conducted to determine the effect of Cu-BPDCA-Ty on antibacterial activity and mecA gene expression in clinical and standard strains of MRSA. Methods: The phenotypic tests were used to identify MRSA strain and confirmed with molecular detection of mecA gene. Synthesized Cu-BPDCA-Ty was confirmed with different techniques such as XRD and SEM analysis. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined by the micro broth dilution method. Real time PCR was used to investigate gene expression. Pta gene was considered as an endogenous control for normalization. Data were analyzed using one sample t test and paired t test in the SPSS software Version 22. Results: The findings indicated that the MIC and the MBC of Cu-BPDCA-Ty against the standard and clinical strains of MRSA were 0.5 mg/mL, 0.8 mg/mL, 0.46 ± 0.08 mg/mL, and 0.7 ± 0.1 mg/mL, respectively. Analysis of the real- time PCR indicated that all treated groups with Cu-BPDCA- Ty showed a significant decrease in the expression of the mecA gene compared to the control group (P < 0.05). Conclusions: Cu-BPDCA-Ty had an antibacterial effect on MRSA and induced downregulation of expression of the mecA gene.

Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum.

Essential gene functions remain largely underexplored in bacteria. Clostridium cellulolyticum is a promising candidate for consolidated bioprocessing; however, its genetic manipulation to reduce the formation of less-valuable acetate is technically challenging due to the essentiality of acetate-producing genes. Here we developed a Cas9 nickase-assisted chromosome-based RNA repression to stably manipulate essential genes in C. cellulolyticum. Our plasmid-based expression of antisense RNA (asRNA) molecules targeting the phosphotransacetylase (pta) gene successfully reduced the enzymatic activity by 35% in cellobiose-grown cells, metabolically decreased the acetate titer by 15 and 52% in wildtype transformants on cellulose and xylan, respectively. To control both acetate and lactate simultaneously, we transformed the repression plasmid into lactate production-deficient mutant and found the plasmid delivery reduced acetate titer by more than 33%, concomitant with negligible lactate formation. The strains with pta gene repression generally diverted more carbon into ethanol. However, further testing on chromosomal integrants that were created by double-crossover recombination exhibited only very weak repression because DNA integration dramatically lessened gene dosage. With the design of a tandem repetitive promoter-driven asRNA module and the use of a new Cas9 nickase genome editing tool, a chromosomal integrant (LM3P) was generated in a single step and successfully enhanced RNA repression, with a 27% decrease in acetate titer on cellulose in antibiotic-free medium. These results indicate the effectiveness of tandem promoter-driven RNA repression modules in promoting gene repression in chromosomal integrants. Our combinatorial method using a Cas9 nickase genome editing tool to integrate the gene repression module demonstrates easy-to-use and high-efficiency advantages, paving the way for stably manipulating genes, even essential ones, for functional characterization and microbial engineering.

Metabolic and stress responses of Acinetobacter oleivorans DR1 during long-chain alkane degradation.

Summary Acinetobacter oleivorans DR1 can utilize C12–C30 alkanes as a sole carbon source but not short‐chain alkanes (C6, C10). Two copies of each alkB‐, almA‐ and ladA‐type alkane hydroxylase (AH) are present in the genome of DR1 cells. Expression and mutational analyses of AHs showed that alkB1 and alkB2 are the major AH‐encoding genes under C12–C30, and the roles of other almA‐ and ladA genes are negligible. Our data suggested that AlkB1 is responsible for long‐chain alkane utilization (C24–C26), and AlkB2 is important for medium‐chain alkane (C12–C16) metabolism. Phylogenetic analyses revealed large incongruities between phylogenies of 16S rRNA and each AH gene, which implies that A. oleivorans DR1 has acquired multiple alkane hydroxylases through horizontal gene transfer. Transcriptomic and qRT‐PCR analyses suggested that genes participating in the synthesis of siderophore, trehalose and poly 3‐hydroxybutyrate (PHB) were expressed at much higher levels when cells used C30 than when used succinate as a carbon source. The following biochemical assays supported our gene expression analyses: (i) quantification of siderophore, (ii) measurement of trehalose and (iii) observation of PHB storage. Interestingly, highly induced both ackA gene encoding an acetate kinase A and pta gene encoding a phosphotransacetylase suggested unusual ATP synthesis during C30 alkane degradation, which was demonstrated by ATP measurement using the ΔackA mutant. Impaired growth of the ΔaceA mutant indicated that the glyoxylate shunt pathway is important when C30 alkane is utilized. Our data provide insight into long‐chain alkane degradation in soil microorganisms.

Metabolic engineering for isopropanol production by an engineered cyanobacterium, Synechococcus elongatus PCC 7942, under photosynthetic conditions

Cyanobacteria engineered for production of biofuels and biochemicals from carbon dioxide represent a promising area of research in relation to a sustainable economy. Previously, we have succeeded in producing isopropanol from cellular acetyl-CoA by means of Synechococcus elongatus PCC 7942 into which a synthetic metabolic pathway was introduced. The isopropanol production by this synthetic metabolic pathway requires acetate; therefore, the cells grown under photosynthetic conditions have to be transferred to a dark and anaerobic conditionsto produce acetate. In this study, we achieved acetate production under photosynthetic conditions by S.elongatus PCC 7942 into which we introduced the pta gene encoding phosphate acetyltransferase from Escherichia coli. The metabolic modification (via pta introduction) of the isopropanol-producing strain enabled production of isopropanol under photosynthetic conditions. During 14 days of production, the titer of isopropanol reached 0.55mM (33.1mg/l) with an intermediate product, acetone, at 0.21mM (12.2mg/l).