A two-stage refinement approach for the enhancement of excretory production of an exoglucanase from Escherichia coli

Hyper-expression of a secretory exoglucanase, Exg, encoded by the cex gene of Cellulomonas fimi was previously shown to saturate the SecYEG pathway and result in dramatic cell death of recombinant Escherichia coli (Z.B. Fu, K.L. Ng, T.L. Lam, W.K.R. Wong, Cell death caused by hyper-expression of a secretory exoglucanase in Esherichia coli, Protein Expr. Purif. 42 (2005) 67-77). We propose here that the cell lysate ratio (Pre/Mat RQ) of the unprocessed precursor Exg protein (Pre-Exg) and its processed mature product (Mat-Exg) reflects the capacity of E. coli to secrete Exg. A Pre/Mat RQ of 20/80, designated the "Critical Value," was an important threshold measurement. A rise in the Pre/Mat RQ triggered a mass killing effect. The use of various secretion signal peptides did not improve the viability of cells expressing high levels of Pre-Exg under strong tac promoter control. However, use of the weaker vegG promoter in conjunction with a change in start codon of the spa leader sequence from ATG to TTG in a pM1vegGcexL plasmid construct resulted in a high level (0.9 U ml(-1)) of excreted Exg in shake-flask cultures. This was 50% higher than the best result obtained from plasmid construct lacUV5par8cex, using the lacUV5 promoter and the ompA leader sequence. Variations in the excreted Exg activities were attributable to differences in the Pre/Mat RQ values of the induced cultures harboring pM1vegGcexL and lacUV5par8cex. These values were 18/82 and 10/90, respectively. Employing fed-batch cultivation in two-liter fermentors, an induced JM101(pM1vegGcexL) culture yielded 4.5 U ml(-1) of excreted Exg, which was over six fold greater that previously reported. Our results illustrate the successful application of the Pre/Mat RQ ratio as a guide to the attainment of a maximum level of secreted/excreted Exg.     

Cell death caused by hyper-expression of a secretory exoglucanase in Escherichia coli

Induced expression of a gene fusion between the ompA leader sequence and the Cellulomonas fimi cex gene encoding a secretory exoglucanase, Exg, engineered in the Tac-cassette excretion vector was lethal to Escherichia coli. An exponentially growing culture harboring the recombinant construct suffered slow growth and 99.9% of its cells died within 60-100 min after induction. This abnormality was found to have a close correlation with the rapid increase in the relative amount of the OmpA/Exg fusion precursor (Pre-Exg) compared to its processed product (Mat-Exg). Analysis of subcellular fractions revealed the presence of Pre-Exg in the inner membrane of cultures expressing high levels but not low levels of Pre-Exg. As only Pre-Exg but not Mat-Exg was detectable in the cytoplasm, and Exg was shown by cross-linking experiments to be physically associated with the Sec proteins, it was concluded that secretion and processing of Pre-Exg took place in the SecYEG translocation machinery. The results were in line with the previous speculation that accumulation of unprocessed precursor proteins in the cytoplasmic membrane was detrimental, and supported the idea that cell death was caused by some unusual tie-up of Pre-Exg with the SecYEG translocation machinery, thus imposing an inhibitory effect on the secretion of endogenous secretory proteins. A new model, designated "Saturated Translocation," was proposed to explain the interchangeable lethal and non-lethal properties of Pre-Exg, and to address the possible scenarios that might occur in the course of cell death triggered by secretion of Pre-Exg.     

Overexpression of bacterial hemoglobin causes incorporation of pre-beta-lactamase into cytoplasmic inclusion bodies.

The expression of Vitreoscilla hemoglobin (VHb) in Escherichia coli JM101 (pRED2) causes the incorporation of the TEM beta-lactamase precursor into cytoplasmic inclusion bodies (IBs). Less pre-beta-lactamase is translocated and processed to its mature, periplasmic form in the strain coexpressing VHb than in the control strain E. coli JM101(pUC19) not expressing VHb. When cells are grown in a special fed-batch procedure, the formation of cytoplasmic IBs consisting of pre-beta-lactamase is also inducible in the control strain. Comparative microscopic and compositional analyses of IBs generated in E. coli JM101(pUC19) and JM101(pRED2) under identical growth conditions strongly suggest that pre-beta-lactamase and VHb coaggregate into common IBs in E. coli JM101 (pRED2).     

High cell density cultivation of recombinant <Emphasis Type="Italic">E. coli</Emphasis> for hirudin variant 1 production by temperature shift controlled by pUC18-based replicative origin

The copy number of a plasmid, pUC-based vector, was previously shown to be affected by culture temperature. In this study, intracellular hirudin variant 1 (f-HV1) fused to porcine adenylate kinase protein was produced using recombinant Escherichia coli by temperature shift cultivation coupled with a high cell density cultivation technique for E. coli JM109. The optimal temperature for cellular growth suppressing f-HV1 production was 33 degrees C, resulting in a final dried cell concentration of 45.7 g/l, with a specific growth rate of 0.54 1/h. Optimizing the temperature-shift conditions (temperature shifted to an OD660 nm of 15 from 33 degrees C to 37 degrees C) resulted in the production of f-HV1 up to 4763 mg/l as an inclusion body with dried cell concentration of 44 g/l in 18 h.     

High cell density cultivation of recombinant Escherichia coli for hirudin variant 1 production

A synthetic medium, TK-25, for high cell density cultivation (HCDC) of Escherichia coli K-12 was modified to support HCDC of strain JM109. By optimizing the culture conditions, the cell concentration of 65 g/l in 14 h was obtained in the optimized medium, namely TK-10, with glucose-fed batch cultivation. When these conditions were further applied for HCDC of E. coli JM109 harboring pUC-based recombinant plasmid which expresses a hirudin variant, HV-1-fused protein under the control of trp promoter, it grew to 24 g/l of dried cells expressed as an inclusion body as 15.9% of the total protein, corresponding to 1908 mg/l hirudin-fused protein.     

A novel technique for the measurement of disruption in high-pressure homogenization: Studies on E. coli containing recombinant inclusion bodies

The high-pressure homogenization of Escherichia coli, strain JM101, containing inclusion bodies of recombinant porcine somatotropin was investigated. A novel technique employing an analytical disc centrifuge was used to monitor the disruption. This a direct technique which measures cell disintegration rather than soluble protein release. The technique is particularly suited to measurements where the disruption approaches 100%. The disk centrifuge provides a size distribution of the homogenate, and furnishes evidence for the preferential disruption of larger cells. For E. coli containing inclusion bodies, and increase in the cell feed concentration from 145 g/L (wet weight) to 330 g/L resulted is poorer homogenization. Poorer disruption was also obtained by lowering the feed temperature from 20 degrees C to 5 degrees C. Only slight variations in performance were obtained by increasing the feed pH from 7.5 to 9.0 or by storing the feed at 4 degrees C for 24 h prior to disruption. Comparison with uninduced E. coli strain JM101, showed that the disruption obtained is higher for bacteria containing a recombinant inclusion body.     

The use of fed batch cultivation for achieving high cell densities in the production of a recombinant protein in Escherichia coli

Abstract: The production of the fusion protein staphylococcal protein A/E. coli β-galactosidase in Escherichia coli was studied in batch and fed batch cultivations. Batch cultivation of a recombinant E. coli strain yielded a final cell dry weight of 16.4 g 1^-1 with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the cell dry weight. Fed batch cultivation made it possible to increase the final cell dry weight to 77.0 g 1^-1. The intracellular product concentration (25%) was lower as compared to batch cultivation resulting in a total concentration of recombinant protein of 19.2 g 1^-1.     

大肠杆菌甘油激酶基因(glpK)和甘油脱氢酶基因(gldA)的敲除及其对甘油产量的影响

利用Red重组系统构建了大肠杆菌JM109甘油激酶基因(glpK)和甘油脱氢酶基因(gldA)缺失的双突变菌株JM109B,然后将表达酿酒酵母3-磷酸甘油脱氢酶基因(GPD1)和3-磷酸甘油酯酶基因(HOR2)的质粒pSE-gpd1-hor2转化到JM109B突变菌株中,在含1%葡萄糖的摇瓶发酵培养基中37℃发酵24 h,甘油的最高产量为5.61 g/L,是原始菌株JM109/pSE-gpd1-hor2甘油产量的1.59倍;在30 L发酵罐中发酵28 h,甘油的最高产量为103.12 g/L,是原始菌株JM109/pSE-gpd1-hor2甘油产量的1.59倍,是原始菌株BL21/pSE-gpd1-hor2甘油产量的1.41倍,葡萄糖转化率为50.39%。     

Direct chemical extraction of a recombinant viral coat protein from Escherichia coli at high cell density.

The release of protein and DNA from nonrecombinant E. coli JM101 and recombinant E. coli HMS174(DE3) expressing L1 (the major viral coat protein of human papillomavirus type 16) as an inclusion body was demonstrated at high cell density (OD(600) = 160). For the nonrecombinant strain, extraction efficiency decreased significantly as cell mass increased, with a high viscosity increase in the postextraction broth. A different dependence on cell concentration was observed for the recombinant strain, with total protein extraction efficiency exceeding 85% for both uninduced and induced cells. Almost complete release of the recombinant L1 protein was achieved at high cell concentration (OD(600) = 80 approximately 160) without the use of reducing agent. This greatly extends the concentration range for chemical extraction.     

The nucleotide sequence of an α-amylase gene from an alkalopsychrotrophic Micrococcus sp

An alpha-amylase gene from Micrococcus sp. 207 was cloned into Escherichia coli JM101 using the vector pHSG399. The constructed recombinant plasmid pYK63 contained a 4.8 kb chromosomal DNA fragment derived from strain 207 DNA. The cloned amylase isolated from E. coli JM101 (pYK63) produced mainly maltotetraose from starch, and exhibited temperature and pH activity profiles closely similar to those of the enzyme from the original strain. Nucleotide sequence analysis of the cloned DNA fragment revealed one open reading frame containing the gene which consisted of 3312 bp (1104 amino acids). When compared with several other alpha-amylases, three consensus sequences were identified in the region of the active site. About 300 amino acid residues were present both upstream and downstream of the active site region.     

大肠杆菌乙酸代谢突变株的选育和特性研究

在大肠杆菌高密度培养中 ,因代谢副产物乙酸积累 ,导致抑制菌体的生长和产物表达的下降。为减小乙酸的抑制作用 ,采用60 Co诱变处理大肠杆菌JM1 0 1 ,结合连续培养 (含乙酸钠选择压力 )定向富集方法 ,选育到一株乙酸耐受性增强的菌株JL3。该菌株表现出明显的乙酸耐受性的提高 ,在含有 1 0 g/L乙酸钠的MA培养基中 ,菌体生长和葡萄糖消耗速率都有较大程度提高 ,并且具有良好的遗传稳定性     

Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system

Recombinant Escherichia coli JM101 strains harbouring plasmids pWKW2 or lacUV5par8EGF, both encoding human epidermal growth factor (hEGF), were used in fermentations to optimize levels of excreted hEGF. Medium composition, inducer level, growth stage at induction and culture conditions, were optimized with respect to volumetric production of the recombinant protein. MMBL medium, with glucose at 5 g/l and tryptone as nitrogen source, was chosen. Isopropyl-β- -thiogalactopyranoside(IPTG) concentrations of 0.1 mM for E. coli JM101[pWKW2] and 0.2 mM for E. coli K-12 JM101[lacUV5par8EGF], were found to give the best hEGF production levels. The volumetric yields of hEGF were maximal when the cultures were induced in the mid-logarithmic phase. Growth temperature had a significant effect on hEGF yield. A simple continuous fed-batch process for cultivation of E. coli JM101[pWKW2] was developed. The maximum concentration of excreted hEGF attained in continuous fed-batch cultivation was 325 mg/l, as compared to 175 mg/l, in batch cultivation. The hEGF produced from the continuous fed-batch cultivation was substantiated by SDS-PAGE and immunoblotting.     

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。     

Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations

The growth characteristics and acetate production of several Escherichia coli strains were compared by using shake flasks, batch fermentations, and glucose-feedback-controlled fed-batch fermentations to assess the potential of each strain to grow at high cell densities. Of the E. coli strains tested, including JM105, B, W3110, W3100, HB101, DH1, CSH50, MC1060, JRG1046, and JRG1061, strains JM105 and B were found to have the greatest relative biomass accumulation, strain MC1060 accumulated the highest concentrations of acetic acid, and strain B had the highest growth rates under the conditions tested. In glucose-feedback-controlled fed-batch fermentations, strains B and JM105 produced only 2 g of acetate.liter-1 while accumulating up to 30 g of biomass.liter-1. Under identical conditions, strains HB101 and MC1060 accumulated less than 10 g of biomass.liter-1 and strain MC1060 produced 8 g of acetate.liter-1. The addition of various concentrations of sodium acetate to the growth medium resulted in a logarithmic decrease, with respect to acetate concentration, in the growth rates of E. coli JM105, JM105(pOS4201), and JRG1061. These data indicated that the growth of the E. coli strains was likely to be inhibited by the acetate they produced when grown on media containing glucose. A model for the inhibition of growth of E. coli by acetate was derived from these experiments to explain the inhibition of acetate on E. coli strains at neutral pH.     

Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations.

The growth characteristics and acetate production of several Escherichia coli strains were compared by using shake flasks, batch fermentations, and glucose-feedback-controlled fed-batch fermentations to assess the potential of each strain to grow at high cell densities. Of the E. coli strains tested, including JM105, B, W3110, W3100, HB101, DH1, CSH50, MC1060, JRG1046, and JRG1061, strains JM105 and B were found to have the greatest relative biomass accumulation, strain MC1060 accumulated the highest concentrations of acetic acid, and strain B had the highest growth rates under the conditions tested. In glucose-feedback-controlled fed-batch fermentations, strains B and JM105 produced only 2 g of acetate.liter-1 while accumulating up to 30 g of biomass.liter-1. Under identical conditions, strains HB101 and MC1060 accumulated less than 10 g of biomass.liter-1 and strain MC1060 produced 8 g of acetate.liter-1. The addition of various concentrations of sodium acetate to the growth medium resulted in a logarithmic decrease, with respect to acetate concentration, in the growth rates of E. coli JM105, JM105(pOS4201), and JRG1061. These data indicated that the growth of the E. coli strains was likely to be inhibited by the acetate they produced when grown on media containing glucose. A model for the inhibition of growth of E. coli by acetate was derived from these experiments to explain the inhibition of acetate on E. coli strains at neutral pH.     

Construction of a novel suicide vector: selection for Escherichia coli HB101 recombinants carrying the DNA insert.

We constructed a new type of cloning vector, pERISH2, that transforms Escherichia coli HB101 only when a foreign DNA fragment is ligated into the cloning site of the plasmid vector. Plasmid pERISH2 carries the rcsB gene which is derived from the chromosome of E. coli HB101 and is involved in the regulation of colanic acid production. When E. coli HB101 is transformed by this vector carrying the intact rcsB gene, the gene product RcsB blocks bacterial growth. However, if the rcsB gene is inactivated by the insertion of a foreign DNA fragment, this recombinant plasmid no longer inhibits the growth of E. coli HB101. Although E. coli HB101 is not stably transformed by pERISH2, E. coli K-12 strains such as JM109 and C600 can harbor this vector. Therefore, pERISH2 can be amplified in JM109 and be prepared from this strain in a large quantity using conventional methods. A chromosomal gene library of Klebsiella pneumoniae is constructed easily and efficiently by the utilization of this new cloning vector.     

Variation of oxygen requirement with plasmid size in recombinant Escherichia coli

We have previously found an inverse relationship between certain cell growth parameters and plasmid size for a series of recombinant Escherichia coli strains containing pUC8 or one of a series of pUC8 recombinant derivatives. To extend these results we investigated whether there was a similar variation among our strains in oxygen requirement, which might be related to the differences in growth. During logarithmic growth in shake flasks, oxygen uptake by E. coli strain JM103 containing an 8.7-kb pUC8 derivative (pBS5) was 2.5 times that of JM103 harboring pUC8 (2.7 kb) and 7.5 times that of plasmid-free JM103. Supplementing the medium with acetate eliminated both the growth disadvantage of and the increased oxygen uptake by the strain harboring pBS5 compared with that containing pUC8. In all cases oxygen consumption decreased drastically as cells began and then continued into stationary phase, and no significant difference was seen among the three strains at these times. When the three strains were grown in a fermentor with continuous monitoring of oxygen levels, plasmid-free JM103 outgrew JM103 containing pUC8 or pBS5 at three levels of aeration. The latter two strains grew identically when aeration was high; their growth curves diverged, however, when aeration was low. In the fermentor experiments the point at which the growth of the three strains diverged was coincident with the point of oxygen depletion in the cultures.     

Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli

Extracellular production of recombinant proteins in Escherichia coli has several advantages over cytoplasmic or periplasmic production. However, nonpathogenic laboratory strains of E. coli generally excrete only trace amounts of proteins into the culture medium under normal growth conditions. Here we report a systematic proteome-based approach for developing a system for high-level extracellular production of recombinant proteins in E. coli. First, we analyzed the extracellular proteome of an E. coli B strain, BL21(DE3), to identify naturally excreted proteins, assuming that these proteins may serve as potential fusion partners for the production of recombinant proteins in the medium. Next, overexpression and excretion studies were performed for the 20 selected fusion partners with molecular weights below 40 kDa. Twelve of them were found to allow fused proteins to excrete into the medium at considerable levels. The most efficient excreting fusion partner, OsmY, was used as a carrier protein to excrete heterologous proteins into the medium. E. coli alkaline phosphatase, Bacillus subtilis alpha-amylase, and human leptin used as model proteins could all be excreted into the medium at concentrations ranging from 5 to 64 mg/L during the flask cultivation. When only the signal peptide or the mature part of OsmY was used as a fusion partner, no such excretion was observed; this confirmed that these proteins were truly excreted rather than released by outer membrane leakage. The recombinant protein of interest could be recovered by cleaving off the fusion partner by enterokinase as demonstrated for alkaline phosphatase as an example. High cell density cultivation allowed production of these proteins to the levels of 250-700 mg/L in the culture medium, suggesting the good potential of this approach for the excretory production of recombinant proteins.     

Comparison of recombinant Escherichia coli strains for synthesis and accumulation of poly-(3-hydroxybutyric acid) and morphological changes

A stable high-copy-number plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoic acid (PHA) biosynthesis genes was constructed. This plasmid was transferred to seven Escherichia coli strains (K12, B, W, XL1-Blue, JM109, DH5alpha, and HB101), which were subsequently compared for their ability to synthesize and accumulate ploy- (3-hydroxybutyric acid) (PHB). Growth of recombinant cells and PHB synthesis were investigated in detail in Luria-Bertani (LB) medium containing 20 g/L glucose. Cell growth, the rate of PHB synthesis, the extent of PHB accumulation, the amount of glucose utilized, and the amount of acetate formed varied from one strain to another. XL1-Blue (pSYL105) and B (pSYL105) synthesized PHB at the fastest rate, which was ca. 0.2 g PHB/g true cell mass-h, and produced PHB up to 6-7 g/L. The yields of cell mass, true cell mass, and PHB varied considerably among the strains. The PHB yield of XL1-Blue (pSYL105) in LB plus 20 g/L glucose was as high as 0.369 g PHB/g glucose. Strains W (pSYL105) and K12 (pSYL105) accumulated the least amount of PHB with the lowest PHB yield at the lowest synthesis rate. JM109 (pSYL105) accumulated PHB to the highest extent (85.6%) with relatively low true cell mass (0.77 g/L). Considerable filamentation of cells accumulating PHB was observed for all strains except for K12 and W, which seemed to be due either to the overexpression of the foreign PHA biosynthesis enzymes or to the accumulation of PHB. (c) 1994 John Wiley & Sons, Inc.     

Production host selection for asymmetric styrene epoxidation: Escherichia coli vs. solvent-tolerant Pseudomonas

Selection of the ideal microbe is crucial for whole-cell biotransformations, especially if the target reaction intensively interacts with host cell functions. Asymmetric styrene epoxidation is an example of a reaction which is strongly dependent on the host cell owing to its requirement for efficient cofactor regeneration and stable expression of the styrene monooxygenase genes styAB. On the other hand, styrene epoxidation affects the whole-cell biocatalyst, because it involves toxic substrate and products besides the burden of additional (recombinant) enzyme synthesis. With the aim to compare two fundamentally different strain engineering strategies, asymmetric styrene epoxidation by StyAB was investigated using the engineered wild-type strain Pseudomonas sp. strain VLB120ΔC, a styrene oxide isomerase (StyC) knockout strain able to accumulate (S)-styrene oxide, and recombinant E. coli JM101 carrying styAB on the plasmid pSPZ10. Their performance was analyzed during fed-batch cultivation in two-liquid phase biotransformations with respect to specific activity, volumetric productivity, product titer, tolerance of toxic substrate and products, by-product formation, and product yield on glucose. Thereby, Pseudomonas sp. strain VLB120ΔC proved its great potential by tolerating high styrene oxide concentrations and by the absence of by-product formation. The E. coli-based catalyst, however, showed higher specific activities and better yields on glucose. The results not only show the importance but also the complexity of host cell selection and engineering. Finding the optimal strain engineering strategy requires profound understanding of bioprocess and biocatalyst operation. In this respect, a possible negative influence of solvent tolerance on yield and activity is discussed.