基于PTS缺陷型大肠杆菌构建莽草酸生产菌

对大肠杆菌芳香族氨基酸合成途径进行代谢流改造, 以实现高效的生物制备莽草酸。以磷酸烯醇式丙酮酸-糖磷酸转移酶系统(PTS系统)敲除菌DH5α△ptsHIcrr (DHP)为基础, 特异性敲除aroL、ydiB基因并转入受阿拉伯糖诱导表达的T7-RNA聚合酶基因, 最终构建一系列产莽草酸宿主菌。再将aroE、aroB、tktA、glk、aroFfbr组成的系列基因串联起来置于质粒上, 在T7启动子控制下表达, 经摇瓶培养检测得知, 不同重组菌产莽草酸能力与对照相比均有明显提高, 其中DHPYA-T7/pAOC-TGEFB菌株产量最高, 可达到392 mg/L。为进一步构建高表达莽草酸工程菌奠定基础。     

外源基因在苏云金杆菌染色体上的定点整合及表达

[目的]研究构建稳定表达外源基因、无抗性标记基因的苏云金杆菌(Bacillus thuringiensis简称Bt)工程菌的方法.在构建Bt工程菌时,高拷贝外源质粒的转入导致Bt芽孢数量减少,芽孢形成期延滞,影响Bt菌株的杀虫活力.而且,外源质粒在Bt中的稳定性较差,外源基因容易丢失.将基因整合人染色体是一种构建遗传性状稳定、杀虫活力高的Bt工程菌的有效方法.[方法]本研究采用PCR技术,分两段扩增定位于Bt无晶体突变株XBU001染色体上的trigger factor基因片段作为同源臂,克隆入温度敏感型载体pKSV7,构建了定点整合载体pKTF12.并利用pKTF12质粒将crylAc基因定点整合入XBU001染色体上.[结果]利用载体pKTF12将crylAc定点插入triggerfactor位点,对宿主菌XBU001的正常生长没有影响.重组菌株KCTF12中的crylAc基因能够稳定遗传、表达并形成菱形晶体.与携带高拷贝外源质粒的Bt菌株HTX42相比较,KCTF12具有芽孢数量增多、芽孢形成期提前的优势.[结论]定点整合法是一种构建稳定表达外源基因、无抗性标记基因Bt工程菌的有效方法.     

A simple and reliable method to conduct and monitor expression cassette integration into the Escherichia coli chromosome

We report a method for the integration of expression cassettes into the Escherichia coli chromosome using rare and dispensable sugar degradation gene loci as sites for integration. Clones carrying successfully recombined DNA fragments in the chromosome are easily screened using a solid differential medium containing the respective sugar compound. As an example for the heterologous expression of a complex natural product biosynthesis pathway, we show the stepwise chromosomal integration of the zeaxanthin biosynthesis pathway from Pantoea ananatis into E. coli.     

Site-specific integration of genes into hot spots for recombination flanking aadA in Tn21 transposons.

Summary Tn21-related transposons are widespread among bacteria and carry various resistance determinants at preferential sites, hs1 and hs2. In an in vivo integrative recombination assay it was demonstrated that these hot spots direct the integration of aminoglycoside resistance genes like aadB from Klebsiella pneumoniae and aacAI from Serratia marcescens, in a recA ^– background. The maximum required recognition sequence which must be present in both the donor and recipient plasmids is 5 CTAAAACAAAGTTA 3 (hs2). The double-site-specific recombination occurred with a frequency of 10^–5–10^–6. The resulting structures include not only replicon fusion products but also more complex structures carrying two copies of the donor plasmid or simply the donor gene flanked by hs elements. hs1 and hs2 are thought to act as recognition sites for a trans-acting site-specific recombinase. By the use of Tn21 deletion derivatives, it has been shown that the recombinase is not encoded by Tn21. This new integrative recombination system is involved in the acquisition of new genes by Tn21-related transposons and their spread among bacterial populations.     

Incorporation of radioactive shikimic acid into N-(γ-Lglutamyl)-4-hydroxyaniline in Agaricus bisporus

Agaricus bisporus contains novel aromatic compounds. By incubation of the mushroom with [G-¹⁴ C] shikimic acid, the radioactivity was incorporated into tyrosine, phenylalanine and several unidentified metabolites. The most radioactive metabolite in the stipe and the cap was identified as $\text{N-(γ-}\text{L}\text{-glutamyl}\text{)-4-}\text{hyrroxyaniline}$. The radioactivity was proved to be localized in the 4-hydroxyaniline moiety of this compound.     

使用动态分子开关调控大肠杆菌生产莽草酸

莽草酸是大肠杆菌合成芳香族氨基酸的中间代谢物,也是抗流感药物"达菲"的重要合成前体。合成莽草酸需要截断莽草酸途径,导致芳香族氨基酸无法合成,因此面临细胞生长受到抑制的问题。使用动态调控策略通过将细胞生长和莽草酸的合成相互分离,可以提高菌株的生产性能。通过使用生长偶联型启动子和降解决定子(Degrons),组建动态分子开关。利用该动态分子开关实现细胞生长与莽草酸合成分离,在5L发酵罐中经过72h发酵得到了14.33g/L的莽草酸。结果表明,这种动态分子开关可以通过调控靶蛋白丰度来改变碳流量平衡,使菌株获得更优秀的生产性能。     

Site-specific integration of light chain and heavy chain genes of antibody into CHO-K1 stable hot spot and detection of antibody and fusion protein expression level

Abstract

Expression cell line constructed by random integration method will often meet with unstable expression problem because target genes may be integrated into unstable region of chromatin. Rational cell line construction can overcome this shortcoming by inserting target gene into stable region of chromatin specifically. Here, we successfully got one knock-in cell line where light chain and heavy chain genes of antibody was site specifically integrated into stable hot spot reported before via homologous dependent recombination method mediated by CRISPR/Cas9. The targeting efficiency was around 1.35%. This cell line together with other three pre-established targeting cell lines (targeting with glucagon-like peptide 1 with human serum albumin fusion protein gene, or NGGH) were all undergoing protein expression level detection. In adherent cell mode, the amount of antibody expressed per cell per day were all around 0.006?pg/cell/day over passage 3, 12, 23, 35 and 50 while the amount of NGGH expressed per cell per day of 3 cell lines were all around 1.2?pg/cell/day over passage 3, 12, 23, 35 and 50. In batch mode, the antibody concentration within supernatant were around 2.5?µg/L over passage 1, 25, and 50 while the NGGH fusion protein concentration within supernatant were around 17?mg/L over passage 1, 25, and 50.     

Carbon dioxide increases acid resistance in Escherichia coli

AIMS: To investigate how carbon dioxide affects the acid resistance of Escherichia coli. METHODS AND RESULTS: Escherichia coli W3110 was grown in minimal EG medium at pH 7.5, and cells were adapted at pH 5.5 at 37 degrees C with and without supply of carbon dioxide and nitrogen gases. The number of colonies grown on LB medium was measured after cells were challenged in minimal EG medium of pH 2.5 at 37 degrees C under various conditions. When carbon dioxide was supplied at both the acid adaptation and challenge stages, 94% of cells survived after the acid challenge for 1 h, while the survival rates were 50 and 67% when nitrogen gas and glutamate were supplied respectively. After the acid challenge for 3 h, the survival rate observed with the carbon dioxide gas supply was again 2.5-fold higher than those with the nitrogen gas supply. CONCLUSION: Carbon dioxide was shown to participate in the maintenance of high viability under acidic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information for research into bacterial pathogenesis, fermentation and food preservation.     

Metabolic engineering for microbial production of shikimic acid

Shikimic acid is a high valued compound used as a key starting material for the synthesis of the neuramidase inhibitor GS4104, which was developed under the name Tamiflu for treatment of antiviral infections. An excellent alternative to the isolation of shikimic acid from fruits of the Illicium plant is the fermentative production by metabolic engineered microorganisms. Fermentative production of shikimic acid was most successfully carried out by rational designed Escherichia coli strains by blocking the aromatic amino acid pathway after the production of shikimic acid. An alternative is to produce shikimic acid as a result of dephosphorylation of shikimate-3-phosphate. Engineering the uptake of carbon, the regulatory circuits, central metabolism and the common aromatic pathway including shikimic acid import that have all been targeted to effect higher productivities and lower by-product formation are discussed.     

理性设计和构建过量合成莽草酸的大肠杆菌代谢工程菌

利用代谢工程手段理性改造野生大肠杆菌的莽草酸(Shikimic acid,SA)合成途径及相关代谢节点,以构建高产莽草酸的工程菌株.根据细胞代谢网络分析,利用Red-Xer重组系统连续删除了野生型大肠杆菌CICIMB0013的莽草酸激酶基因(aroL、aroK),葡萄糖磷酸转移酶系统(PTS)的关键组分EIICBglc的编码基因(ptsG)以及奎宁酸/莽草酸脱氢酶基因(ydiB)并系统评价了基因删除对细胞的生长、葡萄糖代谢和莽草酸积累的影响.aroL、aroK的删除阻断了莽草酸进一步转化成为莽草酸-3-磷酸,初步提高莽草酸的累积.删除ptsG基因使大肠杆菌PTS系统部分缺失,细胞通过GalP-glk(半乳糖透性酶-葡萄糖激酶)途径,利用ATP将葡萄糖磷酸化后进入细胞.利用该途径运输葡萄糖能够减少PEP的消耗,使得更多的碳代谢流进入莽草酸合成途径,从而显著提高了莽草酸的产量.在此基础上删除ydiB基因,阻止了莽草酸合成的前体物质3-脱氢奎宁酸转化为副产物奎宁酸(Quinic acid,QA),进一步提高了莽草酸的累积.初步发酵显示4个基因缺失的大肠杆菌代谢工程菌生产莽草酸的能力比原始菌提高了90多倍.     

Production of shikimic acid

Shikimic acid is a key intermediate for the synthesis of the antiviral drug oseltamivir (Tamiflu®). Shikimic acid can be produced via chemical synthesis, microbial fermentation and extraction from certain plants. An alternative production route is via biotransformation of the more readily available quinic acid. Much of the current supply of shikimic acid is sourced from the seeds of Chinese star anise (Illicium verum). Supply from star anise seeds has experienced difficulties and is susceptible to vagaries of weather. Star anise tree takes around six-years from planting to bear fruit, but remains productive for long. Extraction and purification from seeds are expensive. Production via fermentation is increasing. Other production methods are too expensive, or insufficiently developed. In the future, production in recombinant microorganisms via fermentation may become established as the preferred route. Methods for producing shikimic acid are reviewed.     

The cyclohexane moiety of rapamycin is derived from shikimic acid inStreptomyces hygroscopicus

Summary Although the addition of shikimic acid to the medium had no effect on the level of production of rapamycin byStreptomyces hygroscopicus,¹⁴C-shikimic acid was incorporated into rapamycin to a very high degree.¹³C-Shikimic acid was successfully prepared from 1-[¹³C]-glucose using a mutant ofKlebsiella pneumoniae, and used to label rapamycin. It was found that¹³C-shikimic acid was incorporated into the cyclohexane moiety of rapamycin, thereby establishing the shikimic acid pathway origin of the seven-carbon starter unit.     

Differential expression of over 60 chromosomal genes in Escherichia coli by constitutive expression of MarA

In Escherichia coli, the MarA protein controls expression of multiple chromosomal genes affecting resistance to antibiotics and other environmental hazards. For a more-complete characterization of the mar regulon, duplicate macroarrays containing 4,290 open reading frames of the E. coli genome were hybridized to radiolabeled cDNA populations derived from mar-deleted and mar-expressing E. coli. Strains constitutively expressing MarA showed altered expression of more than 60 chromosomal genes: 76% showed increased expression and 24% showed decreased expression. Although some of the genes were already known to be MarA regulated, the majority were newly determined and belonged to a variety of functional groups. Some of the genes identified have been associated with iron transport and metabolism; other genes were previously known to be part of the soxRS regulon. Northern blot analysis of selected genes confirmed the results obtained with the macroarrays. The findings reveal that the mar locus mediates a global stress response involving one of the largest networks of genes described.     

丁醇合成途径关键酶基因在大肠杆菌中的克隆和表达

【目的】克隆丙酮丁醇梭状芽胞杆菌(Clostridium acetobutylicum)ATCC824丁醇合成途径关键酶基因,构建产丁醇的工程大肠杆菌。【方法】以C.acetobutylicum ATCC824基因组为模板,分别扩增丁醇合成途径关键酶基因thil,adhE2和BCS operon(crt-bcd-etfB-etfA-hbd)基因序列,构建BCS operon-adhE2-thil/pTrc99a/MG1655(pBAT)。重组菌E.coli pBAT采用0.1 mmol异丙基-β-硫代半乳糖苷(IPTG)诱导5 h,测定乙酰基转移酶(THL)、3-羟基丁酰辅酶A脱氢酶(HBD)、3-羟基丁酰辅酶A脱水酶(CRT)、丁酰辅酶A脱氢酶(BCD)、醛醇脱氢酶(BYDH/BDH)的酶活。并以该基因工程菌作为发酵菌种,采用好氧、厌氧和微好氧三种培养方式,检测丁醇产量。【结果】酶活测定结果显示:THL酶活达到0.160 U/mg protein,酶活力提高了近30倍;HBD酶活力提高了近5倍;CRT酶活达到1.53 U/mg protein,野生菌株无此酶活;BCD酶活力提高了32倍;BYDH/BDH酶活力无显著提高。3种发酵培养结果显示在微好氧和厌氧条件下,均有丁醇产生,且丁醇的最大产量约为84 mg/L。【结论】本实验通过构建产丁醇基因工程大肠杆菌,实现了丁醇关键酶基因在大肠杆菌中的活性表达以及发酵产丁醇,为发酵法生产丁醇开辟了一条新的途径。     

Organization of transfer ribonucleic acid genes in the Escherichia coli chromosome.

The arrangement of transfer ribonucleic acid (RNA) genes in the chromosome of Escherichia coli K-12 (C600) was examined with the techniques of restriction endonuclease digestion and Southern blotting. The number and size of restriction fragments containing transfer or ribosomal RNA sequences or both were estimated by a variety of restriction endonucleases, including EcoRI, BglI, SmaI, SalI, BamHI, and PstI. EcoRI liberated a minimum of 27 fragments which hybridized to transfer RNA and 16 which hybridized to ribosomal RNA. Enzymes which did not cut within the ribosomal RNA operons (PstI and BamHI) liberated 16 and 13 fragments, respectively, which hybridized to transfer RNA. Five PstI and six BamHi fragments also hybridized to ribosomal RNA, suggesting that there may be at least 11 chromosomal locations distinct from ribosomal RNA operons which encode transfer RNA genes. In addition, our data indicated that several transfer RNA genes may be very close to the 5' proximal ends of certain ribosomal RNA operons and close to the 3' distal ends of all seven ribosomal RNA operons. Similar studies have been carried out with 22 purified species of transfer RNA, and we report here the number and size of EcoRI restriction fragments which hybridize to these transfer RNA species.     

Improved growth,productivity and quality of tomato (Solanum lycopersicum L.) plants through application of shikimic acid

A field experiment was conducted to investigate the effect of seed presoaking of shikimic acid (30, 60 and 120 ppm) on growth parameters, fruit productivity and quality, transpiration rate, photosynthetic pigments and some mineral nutrition contents of tomato plants. Shikimic acid at all concentrations significantly increased fresh and dry weights, fruit number, average fresh and dry fruit yield, vitamin C, lycopene, carotenoid contents, total acidity and fruit total soluble sugars of tomato plants when compared to control plants. Seed pretreatment with shikimic acid at various doses induces a significant increase in total leaf conductivity, transpiration rate and photosynthetic pigments (Chl. a, chl. b and carotenoids) of tomato plants. Furthermore, shikimic acid at various doses applied significantly increased the concentration of nitrogen, phosphorus and potassium in tomato leaves as compared to control non-treated tomato plants. Among all doses of shikimic acid treatment, it was found that 60 ppm treatment caused a marked increase in growth, fruit productivity and quality and most studied parameters of tomato plants when compared to other treatments. On the other hand, no significant differences were observed in total photosynthetic pigments, concentrations of nitrogen and potassium in leaves of tomato plants treated with 30 ppm of shikimic acid and control plants. According to these results, it could be suggested that shikimic acid used for seed soaking could be used for increasing growth, fruit productivity and quality of tomato plants growing under field conditions.