Metabolic changes in a pyruvate kinase gene deletion mutant of Corynebacterium glutamicum ATCC 13032

To investigate primary effects of a pyruvate kinase (PYK) defect on glucose metabolism in Corynebacterium glutamicum, a pyk-deleted mutant was derived from wild-type C. glutamicum ATCC13032 using the double-crossover chromosome replacement technique. The mutant was then evaluated under glutamic acid-producing conditions induced by biotin limitation. The mutant showed an increased specific rate of glucose consumption, decreased growth, higher glutamic acid production, and aspartic acid formation during the glutamic acid production phase. A significant increase in phosphoenolpyruvate (PEP) carboxylase activity and a significant decrease in PEP carboxykinase activity occurred in the mutant, which suggested an enhanced overall flux of the anaplerotic pathway from PEP to oxaloacetic acid in the mutant. The enhanced anaplerotic flux may explain both the increased rate of glucose consumption and the higher productivity of glutamic acid in the mutant. Since the pyk-complemented strain had similar metabolic profiles to the wild-type strain, the observed changes represented intrinsic effects of pyk deletion on the physiology of C. glutamicum.     

Phenazine-1-carboxylic acid production in a chromosomally non-scar triple-deleted mutant Pseudomonas aeruginosa using statistical experimental designs to optimize yield

We constructed a non-scar triple-deleted mutant Pseudomonas aeruginosa to improve phenazine-1-carboxylic acid (PCA) yield and then optimized the culture conditions for PCA production. Using a non-scar deletion strategy, the 5′-untranslated region of the phz1 gene cluster and two genes, phzM and phzS, were knocked out of the P. aeruginosa strain M18 genome. The potential ability for high-yield PCA production in this triple-deleted mutant M18MSU1 was successfully realized by using statistical experimental designs. A 2^5–1 fractional factorial design was used to show that the three culture components of soybean meal, corn steep liquor and ethanol had the most significant effect on PCA production. Using a central composite design, the concentration of the three components was optimized. The maximum PCA production was predicted to be 4,725.1 mg/L. With the optimal medium containing soybean meal 74.25 g/L, corn steep liquor 13.01 g/L and ethanol 21.84 ml/L, a PCA production of 4,771.2 mg/L was obtained in the validation experiments, which was nearly twofold of that before optimization and tenfold of that in the wild-type strain. This non-scar triple-deleted mutant M18MSU1 may be a suitable strain for industrial production of this biologically synthesized fungicide due to its high PCA production, presumed safety, thermal adaptability and cost-effectiveness.     

Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae

The filamentous fungus Aspergillus oryzae is used as one of the most favored hosts for heterologous protein production due to its ability to secrete large amounts of proteins into the culture medium. We previously generated a hyper-producing mutant strain of A. oryzae, AUT1, which produced 3.2- and 2.6-fold higher levels of bovine chymosin (CHY) and human lysozyme (HLY), respectively, compared with the wild-type strain. However, further enhancement of heterologous protein production by multiple gene disruption is difficult because of the low gene-targeting efficiency in strain AUT1. Here, we disrupted the ligD gene, which is involved in nonhomologous recombination, and the pyrG gene to create uridine/uracil auxotrophy in strain AUT1, to generate a hyper-producing mutant applicable to pyrG marker recycling with highly efficient gene targeting. We generated single and double disruptants of the tripeptidyl peptidase gene AosedD and vacuolar sorting receptor gene Aovps10 in the hyper-producing mutant background, and found that all disruptants showed significant increases in heterologous protein production. Particularly, double disruption of the Aovps10 and AosedD genes increased the production levels of CHY and HLY by 1.6- and 2.1-fold, respectively, compared with the parental strain. Thus, we successfully generated a fungal host for further enhancing the heterologous protein production ability by combining mutational and molecular breeding techniques.     

Oxaloacetate hydrolase gene links the cytoplasmic route of oxalate formation to differentiation and virulence of entomopathogenic fungus Beauveria bassiana

Oxalic acid is used as a functional molecule by most filamentous fungi, and produced via cytoplasmic pathway and mitochondrial pathway. The cytoplasmic pathway of oxalate production from oxaloacetate is a one-step reaction catalyzed by oxaloacetate hydrolase. The entomopathogenic fungus Beauveria bassiana contains a unique oxaloacetate hydrolase gene (BbOAH). The role of cytoplasmic pathway of oxalate production in B. bassiana development and virulence was studied via construction of a targeted gene disruption mutant of BbOAH. Disruption of BbOAH resulted in a slight decrease (~ 30%) in oxalate production, but has no significant influence on fungal growth. The mutant strain displayed a significant delay at early stage of conidial development, and a significant defect in dimorphic transition. Additionally, bioassay using the greater waxmoth as host indicated a slight (~ 20%) decrease in mortality caused by the gene disruption strain. The phenotypic defects of the ΔBbOAH strain could be restored by ectopic integration of BbOAH. Our findings indicate that BbOAH gene connects the cytoplasmic route of oxalate production to fungal development and virulence, although the cytoplasmic route is not indispensable for oxalate production in B. bassiana.     

Efficient production of ethanol from crude glycerol by a Klebsiella pneumoniae mutant strain

A mutant strain of Klebsiella pneumoniae, termed GEM167, was obtained by γ irradiation, in which glycerol metabolism was dramatically affected on exposure to γ rays. Levels of metabolites of the glycerol reductive pathway, 1,3-propanediol (1,3-PD) and 3-hydroxypropionic acid (3-HP), were decreased in the GEM167 strain compared to a control strain, whereas the levels of metabolites derived from the oxidative pathway, 2,3-butanediol (2,3-BD), ethanol, lactate, and succinate, were increased. Notably, ethanol production from glycerol was greatly enhanced upon fermentation by the mutant strain, to a maximum production level of 21.5 g/l, with a productivity of 0.93 g/l/h. Ethanol production level was further improved to 25.0 g/l upon overexpression of Zymomonas mobilispdc and adhII genes encoding pyruvate decarboxylase (Pdc) and aldehyde dehydrogenase (Adh), respectively in the mutant strain GEM167.     

Effects of Disruption of Homocitrate Synthase Genes on Nostoc sp. Strain PCC 7120 Photobiological Hydrogen Production and Nitrogenase

In the case of nitrogenase-based photobiological hydrogen production systems of cyanobacteria, the inactivation of uptake hydrogenase (Hup) leads to significant increases in hydrogen production activity. However, the high-level-activity stage of the Hup mutants lasts only a few tens of hours under air, a circumstance which seems to be caused by sufficient amounts of combined nitrogen supplied by active nitrogenase. The catalytic FeMo cofactor of nitrogenase binds homocitrate, which is required for efficient nitrogen fixation. It was reported previously that the nitrogenase from the homocitrate synthase gene (nifV) disruption mutant of Klebsiella pneumoniae shows decreased nitrogen fixation activity and increased hydrogen production activity under N₂. The cyanobacterium Nostoc sp. strain PCC 7120 has two homocitrate synthase genes, nifV1 and nifV2, and with the ΔhupL variant of Nostoc sp. strain PCC 7120 as the parental strain, we have constructed two single mutants, the ΔhupL ΔnifV1 strain (with the hupL and nifV1 genes disrupted) and the ΔhupL ΔnifV2 strain, and a double mutant, the ΔhupL ΔnifV1 ΔnifV2 strain. Diazotrophic growth rates of the two nifV single mutants and the double mutant were decreased moderately and severely, respectively, compared with the rates of the parent ΔhupL strain. The hydrogen production activity of the ΔhupL ΔnifV1 mutant was sustained at higher levels than the activity of the parent ΔhupL strain after about 2 days of combined-nitrogen step down, and the activity in the culture of the former became higher than that in the culture of the latter. The presence of N₂ gas inhibited hydrogen production in the ΔhupL ΔnifV1 ΔnifV2 mutant less strongly than in the parent ΔhupL strain and the ΔhupL ΔnifV1 and ΔhupL ΔnifV2 mutants. The alteration of homocitrate synthase activity can be a useful strategy for improving sustained photobiological hydrogen production in cyanobacteria.     

Improving of lipid productivity of the biodiesel promising green microalga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> via low-energy ion implantation

High lipid content in microalgae is an essential parameter for adopting of microalgal biomass as a feedstock for biodiesel. Mutation is one approach to obtain desired algal strain with high lipid production. In this study, a mutant strain of Chlorella pyrenoidosa was isolated using 1.5?×?10¹⁵ ions cm^?2 s^?1 of N⁺ ion beam implantation technique, which has been widely used in mutagenesis of agricultural crops. N⁺ implantation slightly improved the growth of the mutant over the corresponding wild strain with significant increase in lipid content (32.4 % higher than the wild strain), which resulted in significant increase in lipid productivity by 35 %. In addition, ion implantation mutagenesis of C. pyrenoidosa resulted in 21.4 % decrease in total saturated fatty acids (SFAs) compared to the wild type, with a noticeable increase in polyunsaturated fatty acids (PUFAs). The increase in PUFAs was due mainly to stimulation of hexadecadienoic acid (C16:2) and octadecadienoic acid (C18:2) production. However, the SFA content of wild and mutant strains was 31.7 and 24.9 % of total fatty acids, respectively, highlighting the oxidative stability of biodiesel produced by both strains according to the European standards. Cultivation of C. pyrenoidosa mutant in selenite enrichment medium for five successive cultivation experiments showed insignificant changes in biomass productivity, lipid content, and lipid productivity alongside the study period, which confirms the genetic stability of the produced mutant. The present study confirmed the feasibility of generation of microalgae mutants with significant high lipid production using ion beam implantation.     

Isobutanol production as an alternative metabolic sink to rescue the growth deficiency of the glycogen mutant of Synechococcus elongatus PCC 7942

Glycogen synthesis initiated by glucose-1-phosphate adenylyltransferase (glgC) represents a major carbon storage route in cyanobacteria which could divert a significant portion of assimilated carbon. Significant growth retardation in cyanobacteria with glgC knocked out (ΔglgC) has been reported in high light conditions. Here, we knocked out the glgC gene and analyzed its effects on carbon distribution in an isobutanol-producing strain of Synechococcus elongatus PCC7942 and its parental wild-type strain. We showed that isobutanol production was able to partially rescue the growth of ΔglgC mutant where the growth rescue effect positively correlated with the rate of isobutanol production. Using NaH¹⁴CO₃ incorporation analysis, we observed a 28 % loss of total carbon fixation rate in the ΔglgC mutant compared to the wild-type. Upon expression of the isobutanol production pathway in ΔglgC mutant, the total carbon fixation rate was restored to the wild-type level. Furthermore, we showed that 52 % of the total carbon fixed was redirected into isobutanol biosynthesis in the ΔglgC mutant expressing enzymes for isobutanol production, which is 2.5 times higher than that of the wild-type expressing the same enzymes. These results suggest that biosynthesis of non-native product such as isobutanol can serve as a metabolic sink for replacing glycogen to rescue growth and restore carbon fixation rate. The rescue effect may further serve as a platform for cyanobacteria energy and carbon metabolism study.     

Screening,selection and development of Bacillus subtilis apr-IBL04 for hyper production of macromolecule alkaline protease

Bacillus subtilis microbe is commonly found in soil and produces proteases on nitrogen and carbon-containing sources and increases the fertility rate by degrading nitrogenous organic materials. The present study was aimed to develop hyper producing mutant strain of B. subtilis for the production of proteases, to improve the process variables by the response surface methodology (RSM) under central composite design (CCD) and the production of protease by the particular mutant strain in a liquid state fermentation media. The mutation of the strain was carried out using ethidium bromide. Pure B. subtilis strain was collected and screened for hyper-production of protease. The production of protease by mutant B. subtilis strain was optimized by varying temperature, inoculum size, pH and incubation time under liquid state fermentation. The CCD model were found to be reliable with r² of 0.999. The maximum enzyme activity of B. subtilis IBL-04 mutant with 3 mL/100 mL inoculum size, 72 h fermentation time, pH 8, and 45 °C temperature was developed with enzyme activity 631.09 U/mL, indicates 1–7-fold increase in enzyme activity than the parent strain having 82.32 U/mL activity. These characteristics render its potential use in industries for pharmaceutical and dairy formulation.     

Enhancement of Ethanol Fermentation in Saccharomyces cerevisiae Sake Yeast by Disrupting Mitophagy Function

Saccharomyces cerevisiae sake yeast strain Kyokai no. 7 has one of the highest fermentation rates among brewery yeasts used worldwide; therefore, it is assumed that it is not possible to enhance its fermentation rate. However, in this study, we found that fermentation by sake yeast can be enhanced by inhibiting mitophagy. We observed mitophagy in wild-type sake yeast during the brewing of Ginjo sake, but not when the mitophagy gene (ATG32) was disrupted. During sake brewing, the maximum rate of CO₂ production and final ethanol concentration generated by the atg32Δ laboratory yeast mutant were 7.50% and 2.12% higher than those of the parent strain, respectively. This mutant exhibited an improved fermentation profile when cultured under limiting nutrient concentrations such as those used during Ginjo sake brewing as well as in minimal synthetic medium. The mutant produced ethanol at a concentration that was 2.76% higher than the parent strain, which has significant implications for industrial bioethanol production. The ethanol yield of the atg32Δ mutant was increased, and its biomass yield was decreased relative to the parent sake yeast strain, indicating that the atg32Δ mutant has acquired a high fermentation capability at the cost of decreasing biomass. Because natural biomass resources often lack sufficient nutrient levels for optimal fermentation, mitophagy may serve as an important target for improving the fermentative capacity of brewery yeasts.     

Involvement of the Reserve Material Poly-β-Hydroxybutyrate in Azospirillum brasilense Stress Endurance and Root Colonization

When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-β-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.     

Properties of cytochrome c and methanol dehydrogenase produced by wild type and mutant strains of Methylomonas sp. in continuous culture

The cytochrome c production of the wild type strain and a mutant strain, YK 56, of Methylomonas sp. grown with excess methanol was higher than wild with limited methanol. The wild type strain grown under both conditions contained two soluble cytochromes c (c-I and c-II), though the mutant strain contained three (c-I, c-II, and c-III). The proportions of cytochromes c-II and c-III of the mutant strain damage changed according to the culture conditions.The methanol dehydrogenase of the wild type and mutant strains was purified and characterized. The enzymes were similar; they consisted of two subunits and their molecular weight was 120,000. The reactivity of cytochromes c with methanol dehydrogenase was investigated.     

Improvement of ethanol production of themophilic Clostridium sp. by Mutation

Three thermophilic Clostridium strains were isolated from soil as cellulose-fermenting bacteria wich produced ethanol, lactic acid, and acetic acid from cellulose at 60°C. To increase ethanol productivity, strains no. 187 was mutated with N-methyl-N′-nitro-N-nitrosguanidine. The resultant mutant, no 187-102-27, was superior to the original strain in ethanol production, and produced less lactic and acetic acid. The activities of some enzymes involved in the biosynthesis of the lactic and acetic acid of mutant no. 187-102-27 were lower than those of the original strain. These results are highly consistent with the acid production of the mutant strain being low.     

假单胞菌M18 relA 突变株的构建及其对吩嗪-1-羧酸合成的调控

假单胞菌M18是一株能同时合成吩嗪-1-羧酸(PCA)和藤黄绿菌素两种抗生素的植物根际分离细菌。RelA催化合成的效应分子ppGpp能介导细菌因营养饥饿引起的应激反应。以M18菌株染色体DNA为模板,PCR扩增获得relA基因,通过庆大霉素抗性片段插入失活与同源重组技术,构建假单胞菌M18的relA突变菌株M18RAG。在PPM培养基中进行PCA发酵分析,发现突变菌株M18RAG的PCA产量显著升高,约为野生型菌株的1.5-2倍。relA基因反式互补实验以及phzA′-′lacZ翻译融合测定结果,均进一步证明了RelA对PCA生物合成及其基因表达具有抑制作用。     

Synthetic Effect between Envelope Stress and Lack of Outer Membrane Vesicle Production in Escherichia coli

Outer membrane vesicles (OMVs) are composed of outer membrane and periplasmic components and are ubiquitously secreted by Gram-negative bacteria. OMVs can disseminate virulence factors for pathogenic bacteria as well as serve as an envelope stress response. From a transposon mutant screen for OMV phenotypes, it was discovered that an nlpA mutant of Escherichia coli produces fewer OMVs than the wild type, whereas a degP mutant produces higher levels of OMVs. NlpA is an inner-membrane-anchored lipoprotein that has a minor role in methionine import. DegP is a periplasmic chaperone/protease for misfolded envelope proteins that is critical when cells are heat shocked. To reveal how these proteins contribute to OMV production, the mutations were combined and the double mutant analyzed. The ΔnlpA ΔdegP strain displayed a high-temperature growth defect that corresponded to the production of fewer OMVs than produced by the ΔdegP strain. This phenotype also pertained to other undervesiculation mutations in a ΔdegP background. The hypovesiculation phenotype of ΔnlpA in the wild-type strain as well as in the degP deletion strain was found to be a stationary-phase phenomenon. The periplasm of the ΔnlpA ΔdegP strain was determined to contain significantly more protein in stationary phase than the wild type. Additionally, misfolded DegP substrate outer membrane porins were detected in ΔdegP mutant-derived OMVs. These data suggest that an accumulation of envelope proteins resulting from decreased vesiculation was toxic and contributed to the growth defect. We conclude that OMV production contributes to relieve the envelope of accumulated toxic proteins and that NlpA plays an important role in the production of vesicles in stationary phase.     

Applications of Gene Replacement Technology to Streptomyces clavuligerus Strain Development for Clavulanic Acid Production

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine -aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.     

Mapmi gene contributes to stress tolerance and virulence of the entomopathogenic fungus, Metarhizium acridum

Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate (Fru-6-P) and mannose 6-phosphate (Man-6-P), providing a link between glycolysis and the mannose metabolic pathway. In this study, we identified pmi gene (Mapmi) from the entomopathogenic fungus, Metarhizium acridum, and analyzed its functions using RNA interference (RNAi). Amending the growth medium with cell stress chemicals significantly reduced growth, conidial production and percent germination in Mapmi-RNAi mutant strain, compared to the wild-type strain. Growth of RNAi mutant was lower than the wild type strain with glucose or fructose as sole carbon source. RNAi mutant exhibited a normal growth phenotype with mannose at low concentrations, while trace or high concentration of mannose was more negatively impacted the growth of RNAi mutant than the wild type strain. Infection with Mapmi-RNAi mutant against Locusta migratoria manilensis (Meyen) led to a significantly reduced virulence compared to infection with the wild-type strain. These results suggest that Mapmi plays essential roles in stress tolerance and pathogenicity of M. acridum.     

Erythritol production with minimum By-product using Candida magnoliae mutant

In order to enhance erythritol production, mutants of Candida magnoliae DSM70638 were generated by ultraviolet and chemical mutagenesis. Erythritol productivity of samples was analyzed by TLC and HPLC with the refractive index detector. One of the mutants named mutant 12-2 gave a 2.4-fold increase in erythritol (20.32 g/L) and a 5.5-fold decrease in glycerol production compared to the wild strain. A sequence-based map of erythrose reductase gene in this mutant showed a replacement of the A³²¹ by G³²¹ that did not cause any amino acid exchange in protein structure. Therefore, the reason of higher erythritol production in C. magnoliae mutant 12-2 is probably the increase in expression of the open reading frame gene. This study revealed that a mutation or minor change in the sequence of genes involved in a production pathway can lead to a significant increase in protein translation.     

Identification and Isolation of Genes Involved in Poly(β-Hydroxybutyrate) Biosynthesis in Azospirillum brasilense and Characterization of a phbC Mutant

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly(β-hydroxybutyrate) (PHB) under suboptimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments, thus showing great potential for the improvement of bacterial inoculants for plants and soils. The three genes that are considered to be essential in the PHB biosynthetic pathway, phbA (β-ketothiolase), phbB (acetoacetyl coenzyme A reductase), and phbC (PHB synthase), were identified in Azospirillum brasilense strain Sp7, cloned, and sequenced. The phbA, -B, and -C genes were found to be linked together and located on the chromosome. An A. brasilense phbC mutant was obtained by insertion of a kanamycin resistance cassette within the phbC gene. No PHB production was detected in this mutant. The capability of the wild-type strain to endure starvation conditions was higher than that of the mutant strain. However, motility, cell aggregation, root adhesion, and exopolysaccharide (EPS) and capsular polysaccharide (CPS) production were higher in the phbC mutant strain than in the wild type.