重组大肠杆菌生物转化甘油生产3-羟基丙酸

目的:以甘油为底物构建高效的3-羟基丙酸生产菌株。方法:以自身携带乙醛脱氢酶的E.coli BL21(DE3)plysS作为宿主,异源表达源自Klebsiella pneumoniae的甘油脱水酶基因dhaB。结果:重组菌E.coli HP获得的甘油脱水酶比活力在1.0mmol/L IPTG的诱导下达到了77.2 U/mg,摇瓶条件下,3-HP的最大产量为5.44 g/L,摩尔转化率为53%,该产量比目前报道的最高水平(4.4 g/L)提高了23.6%。结论:重组菌株E.coli HP实现了甘油向3-羟基丙酸(3-HP)的高效生物转化。     

以甘油为底物利用重组大肠杆菌生产聚3-羟基丙酸

【目的】解决前期研究中所构建的以甘油为底物合成聚3-羟基丙酸(P3HP)的代谢途径中存在两个主要的问题——细胞内还原力不平衡和质粒丢失,以提高P3HP的产量。【方法】克隆来源于肺炎克雷伯氏菌的1,3-丙二醇(1,3-PDO)氧化还原酶基因,构建P3HP和1,3-PDO联产的菌株,解决细胞内还原力不平衡的问题。利用自杀性载体系统介导的同源重组技术,将甘油脱水酶及其激活因子的基因整合到大肠杆菌基因组中,提高质粒的稳定性。同时,对发酵条件进行优化。【结果】菌种改造和发酵条件优化显著提高了P3HP产量,在摇瓶条件下到达2.7 g/L,比以前的报道提高2倍,并可同时得到2.4 g/L 1,3-PDO。【结论】该重组大肠杆菌合成P3HP的产量得到提高,具有较好的工业化生产前景。     

甘油脱水酶再激活因子提高重组大肠杆菌3-羟基丙酸合成能力

甘油脱水酶是甘油转化3-羟基丙酸生物合成途径中的关键性限速酶,然而底物甘油的存在会抑制该酶的活性,从而引起3-羟基丙酸合成量的下降.因此解除底物甘油对甘油脱水酶活性的抑制作用,是提高生物合成3-羟基丙酸产量的方法之一.克隆来源于克雷伯氏菌(Klebsiella pneumoniae)的甘油脱水酶编码基因dhaB、甘油脱...     

产1,3-丙二醇新型重组大肠杆菌的构建

利用PCR技术从大肠杆菌(Escherichia coli )中扩增出1.16 kb的编码1,3-丙二醇氧化还原酶同工酶的基因yqhD,将其连接到表达载体pEtac,得到重组载体pEtac-yqhD,重组载体在大肠杆菌JM109中得到高效表达。SDS_PAGE分析显示融合表达产物的分子量均为43 kD,同核酸序列测定所推导的值相符。对含有yqh-D的基因工程菌进行表达研究表明：37 ℃,以1.0 mmol /L IPTG诱导4 h,1,3-丙二醇氧化还原酶同工酶的酶活力达到120 u/mg蛋白,而对照菌株的酶活力为0.5 u/mg蛋白。再将含甘油脱水酶基因dhaB和含1,3-丙二醇氧化还原酶同工酶基因yqhD的重组质粒共转化大肠杆菌JM109得到重组大肠杆菌JM109（pUCtac-dhaB, pEtac-yqhD）,该菌株在好氧条件下,以1.0mmol/L IPTG诱导可将50 g/L甘油转化为38.0 g/L 1,3-丙二醇。首次发现1,3-丙二醇氧化还原酶同工酶在好氧条件下表现出较高的活性。     

重组大肠杆菌多基因串联表达合成反式-4-羟基-L-脯氨酸

[目的]得到表达多个基因的重组大肠杆菌,以期实现葡萄糖为碳源生产反式-4-羟基-L-脯氨酸。[方法]以菌液为模板,PCR得到不同来源的proB、proA、proC、p4h基因,重叠PCR串联相邻基因,所得片段通过无缝组装与p ET-28a载体连接,并在大肠杆菌BL21中表达,筛选阳性表达菌株,电泳及测序验证,重组菌在30 ml MCG培养基摇瓶中发酵,分光光度法和HPLC检测产量。[结果]0.5 mmol/L IPTG诱导24 h,5组串联基因成功在大肠杆菌中表达,并以葡萄糖为碳源,获得产物反式-4-羟基-L-脯氨酸,培养基不添加L-脯氨酸时E.coli BL21/p ET-28a-BAHHbs、E.coli BL21/p ET-28a-HBACkp和E.coli BL21/p ET-28a-HBAmg产量达到0.28 g/L、0.16 g/L和0.29 g/L,添加4 mmol/L的L-脯氨酸时,分别为0.64 g/L、0.55 g/L和0.74 g/L。[结论]proB、proA、proC及p4h基因成功在大肠杆菌中表达,5株重组菌在30℃摇瓶中诱导表达24 h得到产物反式-4-羟基-L-脯氨酸,其中E.coli BL21/p ET-28a-HBAmg产量达到0.74 g/L,为今后在发酵罐中生产奠定基础。     

dhaBCE基因与yqhD基因串联表达载体的构建及其生物转化甘油

将来自于肺炎克雷伯氏杆菌的甘油脱水酶基因插入到质粒pET28(a+) -yqhD的上游,并用SD序列隔开,串联构建重组质粒pET28(a+)dhaBCE-yqhD,转化到大肠杆菌E.coli novablue中进行共表达。结果显示：含有pET28(a+) dhaBCE-yqhD的重组菌在28℃条件下,IPTG诱导16h后,甘油脱水酶和yqhD氧化还原酶的酶活力分别达到35 U/ mg和 82 U/ mg ,而对照组检测不到甘油脱水酶酶活;当甘油浓度为55g/L,产物1,3-PD的产量可达39g/L;甘油浓度过量不利于产物合成,且产物1,3-丙二醇对合成反应具有一定的抑制作用。     

重组肺炎克雷伯氏菌转化甘油为聚3-羟基丙酸

聚3-羟基丙酸[Poly(3-hydroxypropionate),P3HP]是一种生物可降解及生物相容的新型聚羟基脂肪酸酯。目前已鉴定的生物均不能天然合成P3HP。采用PCR克隆鼠伤寒沙门氏菌的丙醛脱氢酶(Pdu P)基因及罗尔斯通氏菌的聚羟基脂肪酸酯合成酶(Pha C)基因,构建共表达载体,转化肺炎克雷伯氏菌后获得两株重组菌。以甘油为唯一碳源进行摇瓶发酵,pdu P和pha C共用tac启动子的工程菌K.p(p ET-tac-pdu P-pha C)产生0.054 g/L的P3HP,而pdu P和pha C各自独用tac启动子的工程菌K.p(p ET-tac-pdu P-tac-pha C)产生0.091 g/L的P3HP。     

利用甘油发酵耦联生产3-羟基丙酸及1,3-丙二醇重组菌的构建及筛选

在肺炎克雷伯杆菌(Klebsiella pneumoniae)代谢甘油生产1,3-丙二醇(1,3-PD)的过程中,为了减少有毒中间产物3-羟基丙醛(3-HPA)的积累,可将其转化为3-羟基丙酸(3-HP),从而实现1,3-丙二醇和3-羟基丙酸的联产。克隆来自于酿酒酵母的NAD+依赖型的乙醛脱氢酶(ALDH)的基因aldh4,构建了表达载体pKP-aldh,转化K.pneumoniae,得到了有效表达乙醛脱氢酶的重组肺炎克雷伯杆菌(K.pneumoniae A+)。在此基础上,使用紫外诱变联合菌种驯化的方法对K.pneumoniae A+进行筛选,获得了可耐受较高3-HP浓度(≥35 g/L)的重组肺炎克雷伯杆菌K.pneumoniae A+5-3。发酵实验结果表明,K.pneumoniae A+5-3可将3-HPA转化为3-HP,能够同时利用甘油耦联生产3-HP和1,3-PD,产量分别达到5.0 g/L和74.5 g/L。     

产1,3-丙二醇基因工程菌发酵条件的研究

利用途径工程的方法,将来源于克雷伯氏菌(Klebsiella pneumoniae)的甘油脱水酶基因dhaB和1,3-丙二醇氧化还原酶基因dhaT构建成多顺反子重组质粒pSE-dhaB-dhaT并在大肠杆菌JM 109中进行表达,在大肠杆菌中构建一条新的产1,3-丙二醇代谢途径。研究表明,重组菌株JM 109/pSE-dhaB-dhaT在微好氧条件下,尝试用廉价的乳糖为诱导物、维生素B12为辅酶,可以将甘油转化为1,3-丙二醇,产量达15.34 g/L,甘油转化率为35.7%,对低成本生产1,3-丙二醇作了有益的探索。     

重组大肠杆菌合成聚(3-巯基丙酸)和聚(3-羟基丁酸-3-巯基丙酸)的研究

利用Clostridium acetobutylicum的丁酸激酶基因 (buk) 和磷酸转丁酰基酶基因(ptb),以及Thiocapsa pfennigii的PHA合成酶基因,设计了一条能够合成多种聚羟基烷酸的代谢途径,用构建的质粒转化大肠杆菌,获得了重组大肠杆菌菌株.前期的研究表明,在合适的前体物条件下,该重组大肠杆菌能够合成包括聚羟基丁酸、聚(羟基丁酸-戊酸)等多种生物聚酯[Liu and Steinbüchel, Appl. Environ. Microbiol. 66739-743].利用该重组大肠杆菌,通过生物催化作用合成了3-巯基丙酸的同型共聚酯,同时利用该重组大肠杆菌还获得了含3-巯基丙酸单体的多种异型共聚物.实验首先研究了3-巯基丙酸对大肠杆菌生长的影响,在此基础上优化了培养过程中添加3-巯基丙酸的时机和浓度,结果表明,在实验的条件下,细胞合成聚(3-巯基丙酸)可达6.7%(占细胞干重),合成聚(3-羟基丁酸-3-巯基丙酸)(分子中3-巯基丙酸3-羟基丁酸=31)可达24.3%.实验进一步研究了同时或分别表达以上3个基因的重组大肠杆菌合成聚合物的能力,结果表明只有当3个基因同时表达时才能合成聚合物,说明3个基因对合成过程是必须的,从而表明了合成途径是按照设计的路线进行的.还通过GC/MS、GPC、IR等手段对合成的化合物进行了定性的研究.聚(3-巯基丙酸)或聚(3-羟基丁酸-3-巯基丙酸)等聚酯属于一类新型生物聚合物,它在分子骨架中含有硫酯键,不同于聚羟基烷酸酯的氧酯键,从而具有显著不同的物理、化学、光学等性质和具有重要的潜在应用价值.     

Investigation of the mechanism of temperature sensitivity of the electroreceptors of ampullae of lorenzini

In experiments on Black Sea skates (Raja clavata), the potential of the receptor epithelium of the ampullae of Lorenzini and spike activity of single nerve fibers connected to them were investigated during electrical and temperature stimulation. Usually the potential within the canal was between 0 and –2 mV, and the input resistance of the ampulla 250–400 k. Heating of the region of the receptor epithelium was accompanied by a negative wave of potential, an increase in input resistance, and inhibition of spike activity. With worsening of the animal's condition the transepithelial potential became positive (up to +10 mV) but the input resistance of the ampulla during stimulation with a positive current was nonlinear in some cases: a regenerative spike of positive polarity appeared in the channel. During heating, the spike response was sometimes reversed in sign. It is suggested that fluctuations of the transepithelial potential and spike responses to temperature stimulation reflect changes in the potential difference on the basal membrane of the receptor cells, which is described by a relationship of the Nernst's or Goldman's equation type.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov, Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Pacific Institute of Oceanology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 67–74, January–February, 1980.     

Principles of organization and evolution of systems of regulation of functions

Evolution of living organisms is closely connected with evolution of structure of the system of regulations and its mechanisms. The functional ground of regulations is chemical signalization. As early as in unicellular organisms there is a set of signal mechanisms providing their life activity and orientation in space and time. Subsequent evolution of ways of chemical signalization followed the way of development of delivery pathways of chemical signal and development of mechanisms of its regulation. The mechanism of chemical regulation of the signal interaction is discussed by the example of the specialized system of transduction of signal from neuron to neuron, of effect of hormone on the epithelial cell and modulation of this effect. These mechanisms are considered as the most important ways of the fine and precise adaptation of chemical signalization underlying functioning of physiological systems and organs of the living organism