Biosynthesis and incorporation into protein of norleucine by Escherichia coli

The methionine analog norleucine was produced during the synthesis of bovine somatotropin by Escherichia coli strain W3110G containing the recombinant plasmid pBGH1. Norleucine was generated by the leucine biosynthetic pathway from pyruvate or alpha-ketobutyrate in place of alpha-ketoisovalerate as the initial substrate. The intracellular level of norleucine was high enough to permit the analog to compete successfully with methionine for incorporation into protein. Two ways were found to prevent either the formation of norleucine or its incorporation into protein. The endogenous synthesis of norleucine was eliminated by deleting the leucine operon. The addition of sufficient methionine or 2-hydroxy-4-methylthiobutanoic acid, a precursor of methionine, to the culture medium prevented any norleucine from being incorporated into protein.     

Identification of unusual replacement of methionine by norleucine in recombinant interleukin-2 produced by E. coli

Moderate amounts of norleucine incorporation into recombinant interleukin-2 (IL-2) produced in E. coli have been detected. Incorporation of norleucine occurs both at the amino terminal and internal methionines as confirmed by the isolation of norleucine-containing tryptic peptides which eluted later than the respective methionine-containing peptides by reverse-phase HPLC. The occurrence of norleucine in intact protein and modified peptides was determined by amino acid analysis and amino acid sequencing including Edman degradation and fast atom bombardment mass spectrometry. In the subsequent paper, we determined that norleucine incorporation is caused by the endogenous synthesis of norleucine in E. coli.     

Relieving effects of glycine and methionine from acetic acid inhibition in Escherichia coli fermentation

Among amino acids screened for their potential to relieve wild and recombinant Escherichia coli from the negative effects of acetic acid, glycine, and methionine showed a sparing effect. In the presence of 2 g/L of acetic acid, addition of 0.5 g/L of glycine or methionine resulted in either a complete recovery or a further enhancement in the specific growth rate, while the enhancement was significant but not fully complete in the presence of 4 g/L of acetic acid. The addition of 0.5 g/L of methionine alleviated the negative effect of acetic acid on recombinant E. Coli growth to produce more beta-lactamase, which was encoded by plasmid pUC18. In continuous fermentation the methionine effect on recombinant. E. coli metabolism depended on dilution rate; at high dilution rates, above 0.4 h(-1), the methionine addition enhanced beta-lactamase production and reduced acetic acid formation, while at low dilution rates, below 0.3 h (-1), the effect was reversed. In def-batch fermentation with wild-type E. Coli, cell growth rate and cell yield from glucose were enhanced with methionine addition, while the acetic acid concentration reached over 4 g/L. (c) 1993 John Wiley & Sons, Inc.     

Assessment of physiological conditions in E. coli fermentations by epifluorescent microscopy and image analysis

The development of monitoring methods for assessing the physiological state of microorganisms during recombinant fermentation processes has been encouraged by the need to evaluate the influence of processing conditions in recombinant protein production. In this work, a technique based on microscopy and image analysis was developed that allows the simultaneous quantification of parameters associated with viability and fluorescent protein production in recombinant Escherichia coli fermentations. Images obtained from light microscopy with phase contrast are used to assess the total number of cells in a given sample and, from epifluorescence microscopy, both protein producing and injured cells are evaluated using two different fluorochromes: propidium iodide and enhanced yellow fluorescent protein. This technique revealed the existence of different cell populations in the recombinant E. coli fermentation broth that were evaluated along four batch fermentations, complementing information obtained with standard techniques to study the effects of the temperature and induction time in recombinant protein production processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Formate accumulation due to DNA release in aerobic cultivations of Escherichia coli.

Three different aerobic fed-batch processes of Escherichia coli were studied, two for the production of a recombinant protein and one process with a wild-type E. coli strain. In all three processes, an accumulation of formate could be observed in the latter part of the process. Analysis of the concentration of DNA in the medium revealed that the release of DNA coincided with the accumulation of formate. It was found that increasing concentrations of DNA correlated in almost linearly increasing concentrations of formate. Formate accumulation is caused by mixed acid fermentation, although no oxygen limitation was measured with the DO electrode. It is proposed that extracellular DNA restrained mass transfer between the bulk medium and the cell. To investigate if the DNA accumulation caused formate production, DNA was removed by continuous feeding of a DNA binding polymer to the medium. The addition of the polymer decreased the content of free DNA in the broth and the formate was reassimilated. Furthermore, additional DNA early in the process resulted in early formate accumulation.     

重组大肠杆菌高密度发酵研究进展

重组大肠杆菌的高密度发酵是提高基因工程产品产量的一个非常有效的手段,是现代发酵工程研究的一个热点。本文就高密度发酵中影响重组大肠杆菌发酵产率的几个因素,包括宿主菌、培养基、培养条件、补料方法以及高密度发酵过程中存在的问题和对策加以讨论,着重探讨了高密度下大肠杆菌产生的有害代谢副产物———乙酸的产生机制、抑制作用机理,以及控制乙酸积累的技术方法 。     

Conservative replacement of methionine by norleucine in Escherichia coli adenylate kinase

Escherichia coli grown in limited methionine and excess norleucine media accumulate cyanogen bromide-resistant species of proteins after the methionine supply is exhausted. Bacteria, transformed by recombinant plasmid pIPD37 carrying the adk gene and grown under limiting methionine and excess norleucine, synthesize 16-20% of adenylate kinase molecules having all 6 methionine residues replaced by norleucine. Species showing only partial replacement of methionine residues by norleucine are identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after cyanogen bromide treatment of pure enzyme. Norleucine-substituted adenylate kinase shows structural and catalytic properties similar to the wild-type protein as indicated by circular dichroism spectroscopy and kinetic experiments but exhibits a much higher resistance to hydrogen peroxide inactivation under denaturing conditions.     

Effect of norleucine on growth,protein and catechol oxidase synthesis in tobacco suspension cultures

Tobacco cells were grown in artificial media with defined amino acid composition. In such media, the addition of methionine or norleucine caused increases in the specific activity of the catechol oxidase, while in the normal medium norleucine depressed it. The differences of the effect of norleucine on synthesis of catechol oxidase and on cell growth is demonstrated, as is the reversibility of the norleucine effect by methionine. The incorporation of norleucine into a purified enzyme fraction is shown. The change in the electrophoretic patterns of the enzyme during growth in the absence and presence of norleucine was followed. [¹⁴C]-Leucine incorporation by control and norleucine treated cells was examined and it was shown that protein synthesis in the norleucine treated cells was markedly changed and total incorporation reduced. Incorporation into soluble protein was reduced, but increased in the 20 000 g precipitate fraction. Nevertheless use of autoradiography indicates that some catechol oxidase is apparently synthesised in the presence of norleucine.     

Influences of yeast extract on specific cellular yield of Ovine growth hormone during fed-batch fermentation of E. coli

In most cases of E. coli high cell density fermentation process, maximizing cell concentration helps in increasing the volumetric productivity of recombinant proteins usually at the cost of lower specific cellular protein yield. In this report, we describe a process for maintaining the specific cellular yield of Ovine growth hormone (oGH) from E. coli by optimal feeding of yeast extract during high cell density fermentation process. Recombinant oGH was produced as inclusion bodies in Escherichia coli. Specific cellular yield of recombinant oGH was maintained by feeding yeast extract along with glucose during fed-batch fermentation. Glucose to yeast extract ratio of 0.75 was found to be optimum for maintaining the specific cellular oGH yield of 66 mg/g of E. coli cells. Continuous feeding of yeast extract along with glucose helped in reducing acetic acid secretion and promoted higher cell growth during fed-batch fermentation. High cell growth of E. coli and high specific yield of recombinant oGH thus helped in achieving high volumetric productivity of the expressed protein. A maximum of 2 g/l of ovine growth hormone was expressed as inclusion bodies in 12 h of fed-batch fermentation.     

The fate of norleucine as a replacement for methionine in protein synthesis.

In vivo synthesised protein with norleucine occupying one half of the normal methionine loci was prepared using a methionine auxotroph of Escherichia coli K12. The extent of charging of the analogue onto both tRNA_met species and subsequent incorporation into soluble protein was monitored with a double-labelling system comprising [G-³H]norleucine and [³⁵S]methionine. Further experiments established that norleucine can be formylated in vivo once charged onto the initiator tRNA^f_met. An N-terminal analysis of the crude soluble protein revealed that formylnorleucyl-tRNA^f_met can initiate protein synthesis and that the formyl group is then removed from the nascent polypeptide. We were also led to conclude that the N-terminal methionine-amino peptidase does not recognise the analogue in this position. Slow growth rates on the methionine analogue have been partly attributed to limiting levels of charged tRNA^m_met, resulting in turn from the inefficiency of norleucine charging by methionyl-tRNA synthetase. Finally no evidence has been found for the production of aberrant protein as a result of norleucine incorporation, implying that limited growth on the analogue is due to its inability to replace methionine as the precursor of S-adenosyl methionine.     

Application of fed-batch fermentation to the preparation of isotopically labeled or selenomethionyl-labeled proteins.

An increasing demand for isotopically labeled samples for spectroscopic and crystallographic studies has led to a corresponding need for effective and efficient methods for producing these samples. The present work is based on the strategy of using an isotopically labeled compound as the growth-limiting nutrient during protein expression in Escherichia coli (DE3) strains. By using dissolved O2 and agitation rate data, the cell growth, feeding of the isotopic label, induction of protein expression, and the harvest of cells can be coordinated in a feedback controlled fermenter in a simple, easily defined manner. This approach is demonstrated for the nutrient-limited production of [U-15N]- and [U-13C, U-15N]-labeled toluene 4-monooxygenase effector protein in E. coli BL21(DE3) with isotopic abundance identical to that of the labeled precursors. For selective labeling, demonstrated with selenomethionine using methionine auxotroph E. coli B834(DE3), approximately 80-85% incorporation was obtained from methionine-dependent growth of the auxotroph followed by selenomethionine feeding and protein induction upon methionine depletion. This selective labeling is accomplished in a single culture, does not require washing or resuspension, minimizes costly incorporation of label into host cell mass prior to induction, and can be easily adapted to selective labeling with other amino acids. Moreover, cell mass yield from these experiments can be readily optimized to provide the desired level of protein for a given investigation from a single growth and purification. This combination provides an efficient, controllable option for isotopic labeling experiments.     

Attenuation of the editing activity of the Escherichia coli leucyl-tRNA synthetase allows incorporation of novel amino acids into proteins in vivo

The fidelity of translation is dependent on the specificity of the aminoacyl-tRNA synthetases (aaRSs). The aaRSs that activate the hydrophobic amino acids leucine, isoleucine, and valine employ a proofreading mechanism that hydrolyzes noncognate aminoacyl adenylates and misaminoacylated tRNAs. Discrimination between structurally similar amino acids by these AARSs is believed to operate by a double-sieve principle, wherein a separate editing domain governs hydrolysis on the basis of the size and hydrophilicity of the amino acid side chain. Leucyl-tRNA synthetase (LeuRS) relies on its editing function to correct misaminoacylation of tRNA(Leu) by isoleucine and methionine. Thr252 of Escherichia coli LeuRS has been shown previously to be important in defining the size of the editing cavity. Here we report the isolation and characterization of three LeuRS mutants with point mutations at this position (T252Y, T252L, and T252F). The proofreading activity of the synthetase is significantly impaired when an amino acid bulkier than threonine is introduced. The rate of misaminoacylation of tRNA(Leu) by isoleucine and valine increases with the increasing size of the amino acid substituent at position 252, and the noncognate amino acids norvaline and norleucine are inserted efficiently at the leucine sites of recombinant proteins under conditions of constitutive overexpression of the T252Y mutant in E. coli. In addition, the unsaturated amino acids allylglycine, homoallylglycine, homopropargylglycine, and 2-butynylalanine all support protein synthesis in E. coli hosts harboring the mutant synthetase. These results demonstrate that programmed manipulation of the editing cavity can allow in vivo incorporation of novel protein building blocks.     

Methionine production by fermentation

Fermentation processes have been developed for producing most of the essential amino acids. Methionine is one exception. Although microbial production of methionine has been attempted, no commercial bioproduction exists. Here, we discuss the prospects of producing methionine by fermentation. A detailed account is given of methionine biosynthesis and its regulation in some potential producer microorganisms. Problems associated with isolation of methionine overproducing strains are discussed. Approaches to selecting microorganism having relaxed and complex regulatory control mechanisms for methionine biosynthesis are examined. The importance of fermentation media composition and culture conditions for methionine production is assessed and methods for recovering methionine from fermentation broth are considered.     

Methionine control of cephalosporin C formation

DL -Norleucine, a nonsulfur analogue of methionine was found to markedly stimulate synthesis of cephalosporin C by Cephalosporium acremonium strain CW19 in three different chemically defined media. Methionine, but not norleucine, stimulated cephalosporin C biosynthesis in a crude medium. The lack of stimulation by norleucine in complex medium was shown to be due to lack of uptake of this amino acid by mycelia growing in such a medium. In defined media containing a suboptimal methionine concentration, norleucine stimulated antibiotic production up to the level reached by optimal methionine. At an optimal dose of methionine, norleucine elicited no further increase in cephalosporin C production, indicating that these two amino acids act by the same mechanism. The data strongly indicate that stimulation by methionine is not a function of its ability to donate sulfur for antibiotic formation. Methionine was found to neither repress nor inhibit cysteine metabolism.     

表达酿酒酵母ALAS的重组大肠杆菌胞外5-氨基乙酰丙酸的产量和纯化

5-氨基乙酰丙酸(5-aminolevulinate,ALA)由5-氨基乙酰丙酸合酶(5-aminolevulinate synthase,ALAS)催化产生。利用重组细菌在大肠杆菌合成ALA已有不少研究。重组真核生物ALAS在大肠杆菌合成ALA的研究没有报道。酿酒酵母ALAS在大肠杆菌重组表达,在摇瓶培养条件下,分析了胞外ALA的产量,重组菌的生长状况和细胞中ALAS的活性,利用两种国产树脂纯化ALA,毛细管电泳分析确定ALA纯度在LB培养基中,初始pH6.5,含有20mmol/L的酮戊酸、20mmol/L琥珀酸和20mmol/L的甘氨酸,37℃下诱导培养12h,胞外ALA的产量为162mg/L培养基。纯化的ALA纯度达到90%。     

大肠杆菌I型分泌表达系统研究进展及提高蛋白表达量的策略

大肠杆菌是表达重组蛋白最常用的宿主之一。利用大肠杆菌分泌途径胞外表达重组蛋白具有可促进蛋白正确折叠,有效减少包涵体形成,简化纯化工序等诸多优势,近年来备受关注。其中,大肠杆菌I型分泌途径具有分泌表达速度快,蛋白活性高,对宿主代谢无影响等特点,是目前应用最广泛的分泌途径之一。综述了大肠杆菌I型分泌系统的元件组成和分泌机理及提高I型分泌系统蛋白表达量的有效策略,为重组蛋白生产应用提供了理论依据。     

Increasing recombinant protein production in <Emphasis Type="Italic">Escherichia coli</Emphasis> through metabolic and genetic engineering

Different hosts have been used for recombinant protein production, ranging from simple bacteria, such as Escherichia coli and Bacillus subtilis, to more advanced eukaryotes as Saccharomyces cerevisiae and Pichia pastoris, to very complex insect and animal cells. All have their advantages and drawbacks and not one seems to be the perfect host for all purposes. In this review we compare the characteristics of all hosts used in commercial applications of recombinant protein production, both in the area of biopharmaceuticals and industrial enzymes. Although the bacterium E. coli remains a very often used organism, several drawbacks limit its possibility to be the first-choice host. Furthermore, we show what E. coli strains are typically used in high cell density cultivations and compare their genetic and physiological differences. In addition, we summarize the research efforts that have been done to improve yields of heterologous protein in E. coli, to reduce acetate formation, to secrete the recombinant protein into the periplasm or extracellular milieu, and to perform post-translational modifications. We conclude that great progress has been made in the incorporation of eukaryotic features into E. coli, which might allow the bacterium to regain its first-choice status, on the condition that these research efforts continue to gain momentum.     

Similarity of the Escherichia coli proteome upon completion of different biopharmaceutical fermentation processes

A comprehensive view of the physiological state of Escherichia coli cells at the completion of fermentation processes for biopharmaceutical production was attained via two-dimensional gel electrophoretic analysis of cellular proteins. For high cell density fermentations in which phosphate is depleted to induce recombinant protein expression from the alkaline phosphatase promoter, proteome analysis confirms that phosphate limitation occurs. Known phosphate starvation inducible proteins are observed at high levels; these include the periplasmic phosphate binding protein and the periplasmic phosphonate binding protein. The phn (EcoK) locus of these E. coli K-12 strains remains cryptic, as demonstrated by failure to grow with phosphonate as the sole phosphorus source. Proteome analysis also provided evidence that cells utilize alternative carbon and energy sources during these fermentation processes. To address regulatory issues in the biopharmaceutical industry, comparative electrophoretic analyses were conducted on a qualitative basis for four different fermentation processes. Using this approach, the protein profiles for these processes were found to be highly similar, with the vast majority (85-90%) of proteins detected in all profiles. The observed similarity in proteomes suggests that multiproduct host cell protein immunoassays are a feasible means of quantifying host-derived polypeptides from a variety of biopharmaceutical fermentation processes.     

基因重组Echistatin发酵工艺的优化

目的:利用基因工程方法对一种蛇毒锯鳞蝰素蛋白的发酵纯化工艺进行优化,以提高目的蛋白的产量和纯度。方法:对工程菌进行发酵培养并诱导表达,研究不同的培养基、不同补料方式、溶解氧浓度、培养和诱导时间对工程菌产量和目的蛋白表达量的影响,利用几丁质亲和层析纯化Ecs融合蛋白,通过合适温度和pH裂解融合蛋白得到Ecs纯品,并鉴定和检测Ecs活性。结果:经过高密度发酵优化后,菌体湿重可达110g/L,目的蛋白表达量约占总蛋白的40%;亲和层析纯化后,得到Ecs单体,得率为68mg/L发酵液。生物学活性分析显示,重组Ecs能有效抑制血小板的聚集,其活性与天然Ecs相似。结论:通过发酵和纯化工艺优化,大大提高了目的蛋白产量,为进一步规模化研究和生产奠定了基础。     

Improved production of human type II procollagen in the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> in shake flasks by a wireless-controlled fed-batch system

Background  

Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control.