Construction and characterization of ack deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid and hydrogen production

Clostridium tyrobutyricum produces butyrate, acetate, H(2), and CO(2) as its main fermentation products from glucose and xylose. To improve butyric acid and hydrogen production, integrational mutagenesis was used to create a metabolically engineered mutant with inactivated ack gene, encoding acetate kinase (AK) associated with the acetate formation pathway. A non-replicative plasmid containing the acetate kinase gene (ack) fragment was constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome should inactivate the target ack gene and produce ack-deleted mutant, PAK-Em. Enzyme activity assays showed that the AK activity in PAK-Em decreased by approximately 50%; meanwhile, phosphotransacetylase (PTA) and hydrogenase activities each increased by approximately 40%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the expression of protein with approximately 32 kDa molecular mass was reduced significantly in the mutant. Compared to the wild type, the mutant grew more slowly at pH 6.0 and 37 degrees C, with a lower specific growth rate of 0.14 h(-1) (vs 0.21 h(-1) for the wild type), likely due to the partially impaired PTA-AK pathway. However, the mutant produced 23.5% more butyrate (0.42 vs 0.34 g/g glucose) at a higher final concentration of 41.7 g/L (vs 19.98 g/L) as a result of its higher butyrate tolerance as indicated in the growth kinetics study using various intial concentrations of butyrate in the media. The mutant also produced 50% more hydrogen (0.024 g/g) from glucose than the wild type. Immobilized-cell fermentation of PAK-Em in a fibrous-bed bioreactor (FBB) further increased the final butyric acid concentration (50.1 g/L) and the butyrate yield (0.45 g/g glucose). Furthermore, in the FBB fermentation at pH 5.0 with xylose as the substrate, only butyric acid was produced by the mutant, whereas the wild type produced large amounts of acetate (0.43 g/g xylose) and lactate (0.61 g/g xylose) and little butyrate (0.05 g/g xylose), indicating a dramatic metabolic pathway shift caused by the ack deletion in the mutant.     

Construction and characterization of pta gene-deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid fermentation

Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium, producing butyrate and acetate as its main fermentation products. In order to decrease acetate and increase butyrate production, integrational mutagenesis was used to disrupt the gene associated with the acetate formation pathway in C. tyrobutyricum. A nonreplicative integrational plasmid containing the phosphotransacetylase gene (pta) fragment cloned from C. tyrobutyricum by using degenerate primers and an erythromycin resistance cassette were constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome inactivated the target pta gene and produced the pta-deleted mutant (PTA-Em), which was confirmed by Southern hybridization. SDS-PAGE and two-dimensional protein electrophoresis results indicated that protein expression was changed in the mutant. Enzyme activity assays using the cell lysate showed that the activities of PTA and acetate kinase (AK) in the mutant were reduced by more than 60% for PTA and 80% for AK. The mutant grew more slowly in batch fermentation with glucose as the substrate but produced 15% more butyrate and 14% less acetate as compared to the wild-type strain. Its butyrate productivity was approximately 2-fold higher than the wild-type strain. Moreover, the mutant showed much higher tolerance to butyrate inhibition, and the final butyrate concentration was improved by 68%. However, inactivation of pta gene did not completely eliminate acetate production in the fermentation, suggesting the existence of other enzymes (or pathways) also leading to acetate formation. This is the first-reported genetic engineering study demonstrating the feasibility of using a gene-inactivation technique to manipulate the acetic acid formation pathway in C. tyrobutyricum in order to improve butyric acid production from glucose.     

Disruption of the acetate kinase (<Emphasis Type="Italic">ack</Emphasis>) gene of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> results in delayed acetate production

In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack (-) strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack (-) and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (-50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia.     

Enhanced propionic acid fermentation by Propionibacterium acidipropionici mutant obtained by adaptation in a fibrous-bed bioreactor

Fed-batch fermentations of glucose by P. acidipropionici ATCC 4875 in free-cell suspension culture and immobilized in a fibrous-bed bioreactor (FBB) were studied. The latter produced a much higher propionic acid concentration (71.8 +/- 0.8 g/L vs. 52.2 +/- 1.1 g/L), indicating enhanced tolerance to propionic acid inhibition by cells adapted in the FBB. Compared to the free-cell fermentation, the FBB culture produced 20-59% more propionate (0.40-0.65 +/- 0.02 g/g vs. 0.41 +/- 0.02 g/g), 17% less acetate (0.10 +/- 0.01 g/g vs. 0.12 +/- 0.02 g/g), and 50% less succinate (0.09 +/- 0.02 g/g vs. 0.18 +/- 0.03 g/g) from glucose. The higher propionate production in the FBB was attributed to mutations in two key enzymes, oxaloacetate transcarboxylase and propionyl CoA: succinyl CoA transferase, leading to the production of propionic acid from pyruvate. Both showed higher specific activity and lower sensitivity to propionic acid inhibition in the mutant than in the wild type. In contrast, the activity of PEP carboxylase, which converts PEP directly to oxaloacetate and leads to the production of succinate from glucose, was generally lower in the mutant than in the wild type. For phosphotransacetylase and acetate kinase in the acetate formation pathway, however, there was no significant difference between the mutant and the wild type. In addition, the mutant had a striking change in its morphology. With a threefold increase in its length and approximately 24% decrease in its diameter, the mutant cell had an approximately 10% higher specific surface area that should have made the mutant more efficient in transporting substrates and metabolites across the cell membrane. A slightly lower membrane-bound ATPase activity found in the mutant also indicated that the mutant might have a more efficient proton pump to allow it to better tolerate propionic acid. In addition, the mutant had more longer-chain saturated fatty acids (C17:0) and less unsaturated fatty acids (C18:1), both of which could decrease membrane fluidity and might have contributed to the increased propionate tolerance. The enhanced propionic acid production from glucose by P. acidipropionici was thus attributed to both a high viable cell density maintained in the reactor and favorable mutations resulted from adaptation by cell immobilization in the FBB.     

Transposon-induced norfloxacin-sensitive mutants of Bacteroides thetaiotaomicron

To elucidate the mechanism of norfloxacin (a fluoroquinolone) resistance of Bacteroides thetaiotaomicron, a member of the B. fragilis group, we isolated transposon-induced mutants sensitive to this agent using Tn4351. Four norfloxacin-sensitive mutants showed reduced levels of resistance, at least, to ethidium bromide. Cloning and sequencing of three chromosomal fragments adjacent to Tn4351 from the mutants revealed that two partial open reading frames (orfs) were disrupted by a transposon. Amino acid sequences of partial orf products had strong homologies to those of Escherichia coli RecB and B. ovatus transketolase. Two mutants carried a recB homolog inserted by Tn4351 together with R751 (cointegration) and by itself (simple transposition) at the amino- and carboxyl-terminal portions, respectively. Since mutations in recB produce E. coli cells sensitive to DNA-damaging treatments by quinolones, it is concluded that decreases of the minimum inhibitory concentrations (MICs) of the agents for B. thetaiotaomicron resulted from disruption of the recB homolog. Another mutant carried a transketolase gene inserted by Tn4351. There is no reasonable explanation why disruption of the transketolase gene caused a decrease of the MIC of norfloxacin for this organism, although Streptococcus pneumoniae RecP related to DNA recombination was reported to be transketolase.     

The adenosine transporter of Toxoplasma gondii. Identification by insertional mutagenesis, cloning, and recombinant expression

Purine transport into the protozoan parasite Toxoplasma gondii plays an indispensable nutritional function for this pathogen. To facilitate genetic and biochemical characterization of the adenosine transporter of the parasite, T. gondii tachyzoites were transfected with an insertional mutagenesis vector, and clonal mutants were selected for resistance to the cytotoxic adenosine analog adenine arabinoside (Ara-A). Whereas some Ara-A-resistant clones exhibited disruption of the adenosine kinase (AK) locus, others displayed normal AK activity, suggesting that a second locus had been tagged by the insertional mutagenesis plasmid. These Ara-A(r) AK+ mutants displayed reduced adenosine uptake capability, implying a defect in adenosine transport. Sequences flanking the transgene integration point in one mutant were rescued from a genomic library of Ara-A(r) AK+ DNA, and Southern blot analysis revealed that all Ara-A(r) AK+ mutants were disrupted at the same locus. Probes derived from this locus, designated TgAT, were employed to isolate genomic and cDNA clones from wild-type libraries. Conceptual translation of the TgAT cDNA open reading frame predicts a 462 amino acid protein containing 11 transmembrane domains, a primary structure and membrane topology similar to members of the mammalian equilibrative nucleoside transporter family. Expression of TgAT cRNA in Xenopus laevis oocytes increased adenosine uptake capacity in a saturable manner, with an apparent K(m) value of 114 microM. Uptake was inhibited by various nucleosides, nucleoside analogs, hypoxanthine, guanine, and dipyridamole. The combination of genetic and biochemical studies demonstrates that TgAT is the sole functional adenosine transporter in T. gondii and a rational target for therapeutic intervention.     

Peroxisomal metabolic function is required for appressorium-mediated plant infection by Colletotrichum lagenarium

Peroxisomes are organelles that perform a wide range of metabolic functions in eukaryotic cells. However, their role in fungal pathogenesis is poorly understood. Here we report that ClaPEX6, an ortholog of PEX6, is required for the fungus Colletotrichum lagenarium to infect host plants. ClaPEX6 was identified in random insertional mutagenesis experiments aimed at elucidating genes involved in pathogenesis. Functional analysis, using a green fluorescent protein cassette containing the peroxisomal targeting signal1 (PTS1), revealed that import of PTS1-containing proteins is impaired in clapex6 mutants generated by targeted gene disruption. Failure of growth on fatty acids shows an inability of fatty acid beta-oxidation in these mutants. These results indicate that disruption of ClaPEX6 impairs peroxisomal metabolism, even though clapex6 mutants show normal growth and conidiation in nutrient-rich conditions. The clapex6 mutants formed small appressoria with severely reduced melanization that failed to form infectious hyphae. These data indicate that peroxisomes are necessary for appressorium-mediated penetration of host plants. The addition of glucose increased the pathogenicity of clapex6 mutants, suggesting that the glucose metabolic pathway can compensate partially for peroxisomes in phytopathogenicity.     

The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli.

Mutants of Escherichia coli K12 have been isolated that grow on media containing pyruvate of proline as sole carbon sources despite the presence of 10 or 50 mM-sodium fluoroacetate. Such mutants lack either acetate kinase [ATP: acetate phosphotransferase; EC 2.7.2.1] or phosphotransacetylase [acetyl-CoA: orthophosphate acetyltransferase; EC 2.3.1.8] activity. Unlike wild-type E. coli, phosphotransacetylase mutants do not excrete acetate when growing aerobically or anaerobically on glucose; their anaerobic growth on this sugar is slow. The genes that specify acetate kinase (ack) and phosphotransacetylase (pta) activities are cotransducible with each other and with purF and are thus located at about min 50 on the E. coli linkage map. Although Pta- and Ack- mutants are greatly impaired in their growth on acetate, they incorporate [2-14C]acetate added to cultures growing on glycerol, but not on glucose. An inducible acetyl-CoA synthetase [acetate: CoA ligase (AMP-forming); EC 6.2.1.1] effects this uptake of acetate.     

Sterptomyces rochei4089的L基因阻断变株的构建及功能研究

运用同源重组技术破坏了一株格尔德霉素产生菌Sterptomyces rochei 4089的L基因,该基因编码氧化还原酶.以Sterptomyces rochei 4089基因组总DNA为模板,PCR扩增AHBA-KLM基因簇,采取Red/ET重组技术,构建L基因阻断质粒pKC1139-KLM-KmR.采用大肠杆菌与链霉菌的结合转移将阻断质粒含AHBA-KLM基因簇和Kan表达单元的3.0 kb线性片段转化Sterptomyces rochei 4089菌株,在卡纳霉素的平板上筛选卡纳霉素抗性转化子,经PCR检测分离到L基因阻断突变菌株.对原、变株的发酵液进行TLC和HPLC分析显示,Sterptomyces rochei 4089基因组中的L基因失活后,导致该菌株不能合成安莎类抗生素格尔德霉素.通过阻断L基因,为筛查这类放线菌产生安莎类抗生素提供了明确的组分指示作用.     

Propionic acid fermentation of lactose by Propionibacterium acidipropionici: effects of pH

Batch propionic acid fermentation of lactose by Propionibacterium acidipropionici were studied at various pH values ranging from 4.5 to 7.12. The optimum pH range for cell growth was between 6.0 and 7.1, where the specific growth rate was approximately 0.23 h(-1). The specific growth rate decreased with the pH in the acids have been identified as the two major fermentation products from lactose. The production of propionic acid was both growth and nongrowth associated, while acetic acid formation was closely associated with cell growth. The propionic acid yield increased with decreasing pH; It changed from approximately 33% (w/w) at pH 6.1-7.1 to approximately 63% at pH 4.5-5.0. In contrast, the acetic acid yield was not significantly affected by the pH; it remained within the range of 9%-12% at all pH values. Significant amounts of succinic and pyruvic acids were also formed during propionic acid fermentation of lactose. However, pyruvic acid was reconsumed and disappeared toward the end of the fermentation. The succinic acid yield generally decreased with the pH, from a high value of 17% at pH 7.0 to a low 8% at pH 5.0 Effects of growth nutrients present in yeast ex-tract on the fermentation were also studied. In general, the same trend of pH effects was found for fermentations with media containing 5 to 10 g/L yeast extract. However, More growth nutrients would be required for fermentations to be carried out efficienytly at acidic pH levels.     

An adenosine kinase mutation in baby hamster kidney cells causing increased sensitivity to adenosine

A class of aravinosyladenine (araA)-resistant mutants of baby hamster kidney (BHK 21/C13) cells exhibits multiple phenotypes: resistance to araA and deoxyadenosine, extreme sensitivity to adenosine (Ado) and varying degrees of deficiency in adenosine kinase (AK) activity. One of these Ado^s/araA^r strains, ara-S10d, was isolated without mutagenesis and was shown to possess about 59% level of the wild-type AK activity. The AK from ara-S10d had an altered K_m and pH optimum and was stimulated by K⁺ cations. A number of Ado^s to Ado^r revertants were isolated from araS10d, and in all of the 7 examined, the AK activity was reduced to a nondetectable level. The altered kinetic parameters of the AK enzyme in ara-S10d cells suggest a mutation of the AK gene that leads to the synthesis of an altered enzyme. The loss of AK activity in the Ado^r revertants suggests an association of the enhanced Ado sensitivity to the AK mutation.     

Overproduction of glycogen in Escherichia coli blocked in the acetate pathway improves cell growth

Excessive production of acetate is a problem frequently encountered in aerobic high-cell-density fermentations of Escherichia coli. Here, we have examined genetic alterations resulting in glycogen overproduction as a possible means to direct the flux of carbon away from the acetate pool. Glycogen overaccumulation was achieved either by using a regulatory glgQ mutation or by transforming cells with a plasmid containing the glycogen biosynthesis genes glgC (encoding ADPG pyrophosphorylase) and glgA (encoding glycogen synthase) under their native promoter. Both strategies resulted in an approximately five-fold increase in glycogen levels but had no significant effect on acetate excretion. The glgC and glgA genes were then placed under the control of the isopropyl---D-thiogalactopyranoside (IPTG) inducible tac promoter, and this construct was used to stimulate glycogen production in a mutant defective in acetate biosynthesis due to deletion of the ack (acetate kinase) and pta (phosphotransacetylase) genes. If glycogen overproduction in the ack pta strain was induced during the late log phase, biomass production increased by 15 to 20% relative to uninduced controls. Glycogen overaccumulation had a significant influence on carbon partitioning: The output of carbon dioxide peaked earlier than in the control strain, and the levels of an unusual fermentation byproduct, pyruvate, were reduced. Exogenous pyruvate was metabolized more rapidly, suggesting higher activity of gluconeogenesis or the tricarboxylic acid (TCA) cycle as a result of glycogen overproduction. Potential mechanisms of the observed metabolic alterations are discussed. Our results suggest that ack pta mutants over producing glycogen may be a suitable starting point for constructing E. coli strains with improved characteristics in high-cell-density fermentations. (c) 1994 John Wiley & Sons, Inc.     

黑曲霉pepD基因阻断突变菌株的构建及功能分析

运用同源重组技术破坏了黑曲霉基因组中的pepD基因,该基因编码一种类subtilisin的胞外蛋白酶PEPD。实验以黑曲霉GICC2773基因组DNA为模板,PCR扩增pepD基因,并在此基因中间插入潮霉素抗性基因(hph)表达单元,由此产生了3.7kb的pepD阻断基因片段。将此阻断基因片段与载体pBS连接,构建成pepD基因阻断质粒pBSDH。采用原生质体-CaCl2/PEG法将酶切阻断质粒得到的含pepD基因和hph表达单元的3.7kb线性片段转化AspergillusnigerGICC2773菌株,在含潮霉素的平板上筛选潮霉素抗性转化子,从这些抗性转化子中经PCR检测分离到到1个pepD基因阻断突变菌株?pepD66。外源漆酶分泌活性分析显示,黑曲霉pepD基因的破坏使其外源漆酶的分泌表达有所提高。     

嗜水气单胞菌合成含3-羟基戊酸单体的聚羟基脂肪酸共聚酯的研究

分别利用葡萄糖或葡萄糖酸钠与十一碳酸、月桂酸与十一碳酸为混合碳源进行嗜水气单孢菌 (Aeromonashydrophila)菌株 4AK4的摇瓶培养 ,实现了含有 3 羟基戊酸 (3HV)单体的聚羟基脂肪酸酯的微生物合成。当使用葡萄糖或葡萄糖酸钠与十一碳酸为混合碳源时 ,野生型A .hydrophila 4AK4及含有 3 羟基丁酸辅酶A合成基因phaA和phaB的重组A .hydrophila 4AK4 (pTG01)能够合成-3-羟基丁酸(3HB)与-3HV的共聚物 ,且葡萄糖或葡萄糖酸钠与十一碳酸比例为 1∶1时最利于细胞生长和PHA的积累。当使用月桂酸和十一碳酸为混合碳源时 ,A .hydrophila4AK4能够合成-3HB、3HV与 β-羟基己酸 (3HHx)的共聚物 ,且随着混合碳源中十一碳酸的含量增加 ,A .hydrophila4AK4合成的PHA中-3HV的比例增加 ,而-3HB和-3HHx的比例降低.     

地衣芽胞杆菌FLP/FRT基因编辑系统的构建及验证

地衣芽胞杆菌有效的基因编辑工具有限,为了拓展和丰富其基因编辑手段,在地衣芽胞杆菌中构建一个抗性标记可重复使用的FLP/FRT基因编辑系统,并通过敲除α-淀粉酶基因amyL、蛋白酶基因aprE及敲入外源透明颤菌血红蛋白基因vgb对该系统进行初步验证。首先以温敏质粒pNZT1为载体分别构建amyL和aprE基因敲除质粒pNZTT-AFKF和pNZTT-EFKF,两个敲除质粒各自包含针对目标基因的同源臂、抗性基因及同向的FRT位点;将敲除质粒转化地衣芽胞杆菌并经过两次同源交换过程实现目标基因的敲除;最后导入一个FLP重组酶表达质粒通过FLP/FRT系统的重组作用介导抗性基因的回收。为进一步验证本系统的实用性及编辑效率,构建了包含透明颤菌血红蛋白编码基因vgb表达盒及基因组丙酮酸甲酸裂解酶编码基因pflB敲除盒的重组质粒pNZTK-PFTF-vgb,并以此进行外源基因vgb在基因组上的定向敲入。结果显示,成功敲除amyL及aprE并回收了抗性标记卡那霉素基因,敲除后淀粉酶活和蛋白酶活分别减少95.3%和80.4%;vgb基因成功整合入基因组pflB位点并回收了抗性标记四环素基因,并利用荧光定量PCR技术检测到vgb的整合表达。文中首次建立了一个适用于地衣芽胞杆菌的、抗性标记可重复使用的FLP/FRT基因编辑系统,并进行了基因敲除及基因敲入验证,为地衣芽胞杆菌遗传改造提供了良好的方法参考。     

pSH-CUP重组质粒的构建及面包酵母酸性海藻糖 酶基因的敲除

设计含有与面包酵母(Saccharomyces cerevisiae BY-6)编码酸性海藻糖酶ATH基因内部部分序列同源的长引物,以质粒pUG6为模板进行PCR构建带有Cre/loxP系统的敲除单元,转化面包酵母获得G418阳性克隆.将铜抗性基因(cuP1-MT1)导入Cre重组酶表达质粒pSH47,得到重组质粒pSH-CUZ,并转化阳性克隆,以铜抗性筛选面包酵母转化子.半乳糖诱导表达Cre酶切除Kanr基因.重组质粒pSH-CUP的构建,不仅解决了酵母转化子筛选标记问题和非酵母基因的引入,而且使LoxP-kanMX-loxP基因敲除体系在进行真核生物基因敲除时更加方便可行.     

Propionic acid production in an in situ cell retention bioreactor

Continuous fermentations were conducted with in situ cell retention using spin filters (pore size 5 microm and 10 microm) for propionic acid production by Propionibacterium acidipropionici. Continuous fermentation with a 5-microm pore size spin filter resulted in 50% cell retention. Propionic acid productivity was enhanced (0.9 g l(-1) h(-1)) by approximately four-fold compared to conventional batch fermentation (0.25 g l(-1) h(-1)). The in situ cell retention (5-microm pore size spin filter) bioreactor was operated continuously and smoothly for 8 days at a dilution rate of D=0.05 h(-1).