Genetic engineering to contain the Vitreoscilla hemoglobin gene enhances degradation of benzoic acid by Xanthomonas maltophilia

Xanthomonas maltophilia was transformed with the gene encoding Vitreoscilla (bacterial) hemoglobin, vgb, and the growth of the engineered strain was compared with that of the untransformed strain using benzoic acid as the sole carbon source. In general, growth of the engineered strain was greater than that of the untransformed strain; this was true for experiments using both overnight cultures and log phase cells as inocula, but particularly for the latter. In both cases the engineered strain was also more efficient than the untransformed strain in converting benzoic acid into biomass. (c) 1996 John Wiley & Sons, Inc.     

Cyclic mechanical strain regulates the development of engineered smooth muscle tissue.

We show that the appropriate combinations of mechanical stimuli and polymeric scaffolds can enhance the mechanical properties of engineered tissues. The mechanical properties of tissues engineered from cells and polymer scaffolds are significantly lower than the native tissues they replace. We hypothesized that application of mechanical stimuli to engineered tissues would alter their mechanical properties. Smooth muscle tissue was engineered on two different polymeric scaffolds and subjected to cyclic mechanical strain. Short-term application of strain increased proliferation of smooth muscle cells (SMCs) and expression of collagen and elastin, but only when SMCs were adherent to specific scaffolds. Long-term application of cyclic strain upregulated elastin and collagen gene expression and led to increased organization in tissues. This resulted in more than an order of magnitude increase in the mechanical properties of the tissues.     

Increased ethanol production from glycerol by Saccharomyces cerevisiae strains with enhanced stress tolerance from the overexpression of SAGA complex components

During the industrial production of ethanol using yeast, the cells are exposed to stresses that affect their growth and productivity; therefore, stress-tolerant yeast strains are highly desirable. To increase ethanol production from glycerol, a greater tolerance to osmotic and ethanol stress was engineered in yeast strains that were impaired in endogenous glycerol production by the overexpression of both SPT3 and SPT15, components of the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex. The engineered strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupSpt3.15Cas) formed significantly more biomass compared to the strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupCas), and both engineered strains displayed increased biomass when compared to the control YPH499 fps1Δgpd2Δ (pESC-TRP) strain. The trehalose accumulation and ergosterol content of these strains were 2.3-fold and 1.6-fold higher, respectively, than the parent strains, suggesting that levels of cellular membrane components were correlated with the enhanced stress tolerance of the engineered strains. Consequently, the ethanol production of the engineered strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupSpt3.15Cas) was 1.8-fold more than that of strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupCas), with about 8.1g/L ethanol produced. In conclusion, we successfully established that the co-expression of SPT3 and SPT15 that improved the fermentation performance of the engineered yeast strains which produced higher ethanol yields than stress-sensitive yeast strains.     

Genetically Engineered Erwinia carotovora: Survival, Intraspecific Competition, and Effects upon Selected Bacterial Genera

Environmental use of genetically engineered microorganisms has raised concerns about potential ecological impact. This research evaluated the survival, competitiveness, and effects upon selected bacterial genera of wild-type and genetically engineered Erwinia carotovora subsp. carotovora to ascertain if differences between the wild-type and genetically engineered strains exist in soil microcosms. The engineered strain contained a chromosomally inserted gene for kanamycin resistance. No significant differences in survival in nonsterile soil over 2 months or in the competitiveness of either strain were observed when the strains were added concurrently to microcosms. For reasons that remain unclear, the engineered strain did survive longer in sterilized soil. The effects of both strains on total bacteria, Pseudomonas and Staphylococcus strains, and actinomycetes were observed. While some apparent differences were observed, they were not statistically significant. A better understanding of the microbial ecology of engineered bacteria, especially pathogens genetically altered for use as biological control agents, is essential before commercial applications can be accomplished.     

Construction and hyperproductivity of engineered strain QE79 bearing recombinant plasmid containing penicillin G acylase gene from Escherichia coli strain AS1.76

Summary An engineered strain QE79 bearing a recombinant plasmid containing the penicillin G acylase gene from E.coli strain AS1.76 was constructed. Formation conditions of the penicillin G acylase were studied. The activity of the enzyme reached over 200 units per 100ml of the culture when the strain QE79 was grown in the medium consisting of mineral salts with supplementary glucose or sucrose at 28°C for 32 hr on shaker. The productivity of the engineered strain QE79 was nearly nine times higher than that of the original strain.     

Scaffolds for engineering smooth muscle under cyclic mechanical strain conditions

Cyclic mechanical strain has been demonstrated to enhance the development and function of engineered smooth muscle (SM) tissues, but appropriate scaffolds for engineering tissues under conditions of cyclic strain are currently lacking. These scaffolds must display elastic behavior, and be capable of inducing an appropriate smooth muscle cell (SMC) phenotype in response to mechanical signals. In this study, we have characterized several scaffold types commonly utilized in tissue engineering applications in order to select scaffolds that exhibit elastic properties under appropriate cyclic strain conditions. The ability of the scaffolds to promote an appropriate SMC phenotype in engineered SM tissues under cyclic strain conditions was subsequently analyzed. Poly(L-lactic acid)-bonded polyglycolide fiber-based scaffolds and type I collagen sponges exhibited partially elastic mechanical properties under cyclic strain conditions, although the synthetic polymer scaffolds demonstrated significant permanent deformation after extended times of cyclic strain application. SM tissues engineered with type I collagen sponges subjected to cyclic strain were found to contain more elastin than control tissues, and the SMCs in these tissues exhibited a contractile phenotype. In contrast, SMCs in control tissues exhibited a structure more consistent with the nondifferentiated, synthetic phenotype. These studies indicate the appropriate choice of a scaffold for engineering tissues in a mechanically dynamic environment is dependent on the time frame of the mechanical stimulation, and elastic scaffolds allow for mechanically directed control of cell phenotype in engineered tissues.     

Generating Embryonic Stem Cells from the Inbred Mouse Strain DBA/2J,a Model of Glaucoma and Other Complex Diseases

Mouse embryonic stem (ES) cells are derived from the inner cell mass of blastocyst stage embryos and are used primarily for the creation of genetically engineered strains through gene targeting. While some inbred strains of mice are permissive to the derivation of embryonic stem cell lines and are therefore easily engineered, others are nonpermissive or recalcitrant. Genetic engineering of recalcitrant strain backgrounds requires gene targeting in a permissive background followed by extensive backcrossing of the engineered allele into the desired strain background. The inbred mouse strain DBA/2J is a recalcitrant strain that is used as a model of many human diseases, including glaucoma, deafness and schizophrenia. Here, we describe the generation of germ-line competent ES cell lines derived from DBA/2J mice. We also demonstrate the utility of DBA/2J ES cells with the creation of conditional knockout allele for Endothelin-2 (Edn2) directly on the DBA/2J strain background.     

Space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b and screening of higher yielding strains

The aim of this study was to investigate the space mutagenesis of genetically engineered bacteria expressing recombinant human interferon α1b. The genetically engineered bacteria expressing the recombinant interferon α1b were sent into outer space on the Chinese Shenzhou VIII spacecraft. After the 17 day space flight, mutant strains that highly expressed the target gene were identified. After a series of screening of spaceflight-treated bacteria and the quantitative comparison of the mutant strains and original strain, we found five strains that showed a significantly higher production of target proteins, compared with the original strain. Our results support the notion that the outer space environment has unique effects on the mutation breeding of microorganisms, including genetically engineered strains. Mutant strains that highly express the target protein could be obtained through spaceflight-induced mutagenesis.     

产顺式-4-L-羟脯氨酸工程菌的构建及转化条件的优化

【目的】构建产顺式-4-L-羟脯氨酸(cis-4-Hyp)的工程菌并优化其转化条件。【方法】通过调整大肠杆菌的密码子偏好性以及mRNA二级结构对顺式-4-L-脯氨酸羟化酶(cis-P4H)基因进行优化,构建该基因的表达菌株。采用Ni-NTA亲和层析柱分离纯化cis-P4H,测定cis-P4H的酶活和稳定性。然后采用全细胞催化法制备cis-4-Hyp,通过单因素试验和正交试验对相关的转化条件进行优化。【结果】构建了一株产cis-4-Hyp的工程菌,cis-P4H的比活为2.65 U/mg,半衰期为2.32 h。经过条件优化后,采用OD600为0.9时加入IPTG获得的工程菌菌体构建转化体系,在转化体系pH 6.5,转化温度为31°C,转化时间为60 h时,L-脯氨酸转化率最高达到83.33%。【结论】研究获得的工程菌及转化条件具有良好的工业应用前景。     

Homofermentative lactate production cannot sustain anaerobic growth of engineered Saccharomyces cerevisiae: possible consequence of energy-dependent lactate export

Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.     

提高大肠杆菌通过MVA途径合成异戊二烯

异戊二烯是橡胶合成的重要前体物质。为了提高菌株的异戊二烯产量,本实验室在研究中构建了一株异戊二烯产气的菌株BW-01,基于蛋白质预算理论的指导,理性设计通过改变质粒拷贝数、增加稀有密码子等合成生物学手段调控关键限速酶编码基因表达,从而提高大肠杆菌外源MVA代谢途径的异戊二烯产量。摇瓶发酵实验中我们构建的新产气菌株BW-07比原有的产气菌株BW-01的产量提高了73%,达到了761.1 mg/L。为后续菌株改造及进行发酵罐实验奠定了基础。     

Field performance of a genetically engineered strain of pink bollworm

Pest insects harm crops, livestock and human health, either directly or by acting as vectors of disease. The Sterile Insect Technique (SIT)--mass-release of sterile insects to mate with, and thereby control, their wild counterparts--has been used successfully for decades to control several pest species, including pink bollworm, a lepidopteran pest of cotton. Although it has been suggested that genetic engineering of pest insects provides potential improvements, there is uncertainty regarding its impact on their field performance. Discrimination between released and wild moths caught in monitoring traps is essential for estimating wild population levels. To address concerns about the reliability of current marking methods, we developed a genetically engineered strain of pink bollworm with a heritable fluorescent marker, to improve discrimination of sterile from wild moths. Here, we report the results of field trials showing that this engineered strain performed well under field conditions. Our data show that attributes critical to SIT in the field--ability to find a mate and to initiate copulation, as well as dispersal and persistence in the release area--were comparable between the genetically engineered strain and a standard strain. To our knowledge, these represent the first open-field experiments with a genetically engineered insect. The results described here provide encouragement for the genetic control of insect pests.     

Homofermentative Lactate Production Cannot Sustain Anaerobic Growth of Engineered Saccharomyces cerevisiae: Possible Consequence of Energy-Dependent Lactate Export

Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.     

Production of isopropanol by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

A genetically engineered strain of Escherichia coli JM109 harboring the isopropanol-producing pathway consisting of five genes encoding four enzymes, thiolase, coenzyme A (CoA) transferase, acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824, and primary–secondary alcohol dehydrogenase from C. beijerinckii NRRL B593, produced up to 227 mM of isopropanol from glucose under aerobic fed-batch culture conditions. Acetate production by the engineered strain was approximately one sixth that produced by a control E. coli strain bearing an expression vector without the clostridial genes. These results demonstrate a functional isopropanol-producing pathway in E. coli and consequently carbon flux from acetyl-CoA directed to isopropanol instead of acetate. This is the first report on isopropanol production by genetically engineered microorganism under aerobic culture conditions.     

杀虫遗传工程荧光假单胞菌IPP202部分生物学特性

对遗传工程荧光假单胞菌IPP202进行了质粒稳定性检 测、抑菌活性测定、在棉花根部和叶面定殖能力分析、杀虫蛋白抗紫外能力检测及田间杀虫 活性测定等试验。结果表明,工程菌与出发菌株P303相比,其抑菌、定殖等有益于植物的优 良特性未发生显著变化;经过连续培养和连续稀释培养后工程菌的质粒都非常稳定;广波长 紫外线照射2 h后,苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)由于裸露的伴 孢晶体杀虫蛋白受紫外线破坏因而杀虫活性大大下降,而荧光假单胞菌工程菌的活性变化不 大;IPP202田间杀虫活性与Bt野生菌株接近。工程菌有望解决Bt本身存在的杀虫蛋白多以裸 露晶体的形式存在而易受紫外线破坏的弱点,同时也发挥了P303菌株在多种植物上定殖能力 的优点,使其具有在植物周围大量繁殖而直到杀虫作用的优势。通过进一步研究,将有望构 建成更有实用价值的工程菌。     

酰胺水解酶基因工程菌的构建及其对棘白菌素B的转化

棘白菌素B0(ECB)去侧链母核为重要抗真菌药物阿尼芬净的半合成前体.本实验室从保存的犹他游动放线菌(Actinoplanes utahensis)SIPI-A.2001 基因组中克隆到ECB酰胺水解酶及其上下游基因,并将其构建入表达质粒pTGV2,通过接合转移的方法将此表达质粒导入到变铅青链霉菌(Streptomyc...     

Construction of an engineered strain free of antibiotic resistance gene markers for simultaneous mineralization of methyl parathion and ortho-nitrophenol

Pseudomonas sp. strain NyZ402, a native soil organism that grows on para-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of methyl parathion (MP) and ortho-nitrophenol (ONP) by integrating mph (methyl parathion hydrolase gene) from Pseudomonas sp. strain WBC-3 and onpAB (ONP 2-monooxygenase and ONP o-benzoquinone reductase genes) from Alcaligenes sp. strain NyZ215 into the genome of strain NyZ402. Methyl parathion hydrolase (MPH), ONP 2-monooxygenase (OnpA) and o-benzoquinone reductase (OnpB) were constitutively expressed in the engineered strain NyZ-MO. Strain NyZ-MO was free of exogenous antibiotic resistance gene markers and the introduced genes were genetically stable. Degradation experiments showed that strain NyZ-MO could utilize MP or ONP as the sole carbon and energy source, and mineralize 0.1 mM MP–0.1 mM ONP simultaneously. This method may serve as a useful strategy for the construction of engineered strains in the degradation of multiple environmental pollutants.     

Effect of cycling on the stability of plasmid-bearing microorganisms in continuous culture

The implication of the possible existence of differences in the times required for plasmid-bearing and non-plasmid-bearing microorganisms to adjust their metabolic activities to step changes in their environment is examined. This adaptability difference suggests the possibility of maintaining an engineered strain in continuous culture by transient operation. It is shown for the case where adaptability is neglected that no cycling strategy will prevent the washout of the engineered strain, but the addition to the model of a time delay in substrate utilization can result in coexistence upon cycling. Numerical simulations of cycling in feed substrate concentration are carried out to illustrate the concept Operating diagrams are also constructed to indicate the conditions under which washout of the plasmid bearing strain can be prevented.     

Evolution of a Saccharomyces cerevisiae metabolic pathway in Escherichia coli

The Saccharomyces cerevisiae glycerol pathway (GPD1 and GPP2) was evolved in vivo in Escherichia coli. The central metabolism of E. coli was engineered to link glucose consumption and glycerol production. The engineered strain was evolved in a chemostat culture and a high glycerol producer was rapidly obtained. The evolution of the strain was associated to a deletion between GPD1 and GPP2, resulting in the production of a fusion protein with both glycerol-3-P dehydrogenase and glycerol-3-P phosphatase activities. The higher efficiency of the fusion protein was due to partial glycerol-3-P channeling between the two active sites. The evolved strain produces glycerol from glucose at high yield, concentration and productivity.     

Sweetening up yeast glycoprotein synthesis

An engineered yeast strain is capable of carrying out the full range of human protein glycosylation reactions.