A chemostat culture as a tool for the improvement of a recombinant E. coli strain over-producing penicillin G acylase

The recombinant strain RE3(pKA18) of Escherichia coli constitutively overproduces penicillin G acylase (PGA) from plasmid-borne gene pga. The host strain RE3 bears the same pga gene on its chromosome, the expression of which is controlled by the natural mechanism of induction with phenylacetic acid (PA). To evaluate the maximum biosynthetic capacity for PGA, induction of the chromosomal pga by PA was studied in a culture of the recombinant strain. PGA production by batch cultures of RE3(pKA18) and RE3 showed a different response to the addition of PA to the medium: while an addition of PA induces PGA in a culture of strain RE3 as expected, in recombinant cells it lowers the specific activity of PGA and a large amount of PGA is released into the culture medium. To improve the PGA production, the strain RE3(pKA18) was cultured in a carbon-limited chemostat and subjected to selection pressure in a medium supplemented with phenylacetic acid amide (PAA). Phenylacetic acid amide served as a source of nitrogen, an inducer of PGA and a factor exerting positive selection pressure on the maintenance of the recombinant plasmid. After 130 generations of growth in the presence of the inducer, no recombinant strain with constitutive expression of the chromosomal gene pga was detected in the prevailing P(+) subpopulation in the chemostat. Shake-flask experiments with the parent recombinant strain RE3(pKA18), host strain RE3, chemostat evolvant ERE3(epKA18), the cured host ERE3 alone, and its derivative after retransformation with ancestral plasmid ERE3(pKA18) showed that inactivation of the plasmid-borne pga by a frame-shift mutation (plasmid epKA18) occurred in the plasmid-bearing subpopulation accumulated in the chemostat. Marked adaptive changes evolved in the host ERE3 during a 130 generation culture: (1) the specific growth rate of the host increased by 30% in a medium without PA, (2) the copy number of plasmids pKA18 and epKA18 in the host cultured in PA-free medium dropped by about 40%, and (3) the leakage of PGA from the cell in the presence of PA found in strain RE3(pKA18) was not observed in strain ERE3(pKA18). This new recombinant strain with modified traits was constructed by means of retransformation of the evolved host ERE3 with ancestral plasmid pKA18.     

Cloning of penicillin G acylase-encoding gene from Escherichia coli high-producing strain RE3

Summary Pga gene from the industrial strain of E. coli RE3 hyperproducing penicillin G acylase (PGA) was cloned using a simple and rapid NIPAB-based chromogenic method for the detection of PGA-positive recombinant clones. Heterogeneous genetical material was prepared by subcloning pga in vectors pACYC184, pBR322-2 and pK19. The highest constitutive expression of pga was observed in a strain bearing the recombinant plasmid pKA18 synthesizing 2.5 times more of enzyme than the parent strain.     

Effect of phenylacetic acid on the growth and production of penicillin G acylase of recombinant and host strains derived from Escherichia coli W

The industrial production strain Escherichia coli RE3(pKA18) for penicillin G acylase (PGA) bears simultaneously the pga gene on the chromosome as an inducible gene pgaⁱ, (the inductor is phenylacetic acid, PAA) and on the recombinant plasmid pKA18 as a constitutively expressed gene pga^c. Under non-selective conditions, plasmid-less strains (P⁻) appeared in 17th successive batch culture. However, the population was over taken by P⁻ cells already in fourth culture if the medium was supplemented with PAA. The rate of plasmid loss from the culture depends on the PAA concentration and on the expression of pgaⁱ, not on PGA overproduction from pga^c. PAA at inducing concentration has a negative effect on PGA expression and plasmid stability in the high-expression self-cloning system RE3(pKA18) which results in the reduction of: (1) the specific growth rate of a culture and biomass concentration, (2) the synthesis of PGA (e.g. the specific activity of the strain) and (3) the copy number of the recombinant plasmid and promotion of the plasmid loss from the culture. Segregational stability of pKA18 increases in P⁺ persisting clones and in re-transformed P⁻ clones segregated during the selection in the presence of PAA.     

Indigenous plasmids in a production line of strains for penicillin G acylase derived fromEscherichia coli W

Three indigenous plasmids designated pRK1, pRK2 and pRK3 were identified among producers of penicillin G acylase, (PGA) derived from the strainEscherichia coli W ATCC 9637. Their size and copy number (CN) inE. coli W were determined (kb; CN); pRK1 (80; 3.4), pRK2 (5.1; 71), and pRK3 (4.8; 13.7). StrainE. coli RE2 harboring these plasmids was used for selection of strains with reduced number of plasmids: the strain RE3 without plasmid pRK1 and the plasmid-less strain cERE3 were isolated. Indigenous plasmids did not code for the resistance determinants against 23 antibiotics and 10 heavy metals.     

Escherichia coli growth and plasmid copy numbers in continuous cultivations

Summary AnEscherichia coli K-12 strain harbouring either the plasmid pBR322, or the recombinant plasmid pKTH1220, a 14 kb derivative of pBR322, or no plasmid was grown in a chemostat. The cultivations were continued for 300–400 bacterial generations.E. coli hosts harbouring pBR322 or no plasmid grew in a similar way, but the growth of the host containing the big recombinant plasmid was slower. The plasmid copy numbers increased up to 2–3 fold as the dilution rate was increased from 0 to ca. 1 h^–1. After this point the increase in dilution rate seemed to induce a rapid decrease in the plasmid copy numbers. High copy numbers could be maintained using dilution rates resulting in good productivity of the cell mass.     

Genotyping of recombinant Pichia pastoris strains

A simplified amplified-fragment length polymorphism (AFLP) method was used to genotype Pichia pastoris strains obtained by transformation of P. pastoris strain GS115 with a single integration vector. A total of 14 transformants and 3 control strains were analyzed, which generated 16 different band patterns. A clonal variation was obtained after the transformation process due to genetic differences generated during the transformation event of the host strain. Furthermore, the cluster analysis showed that the transformants with lesser genetic differences with respect to the P. pastoris host strain are the recombinant strains with the highest level of recombinant protein production.     

Effect of gene copy number and chaperone coexpression on recombinant hydrophobin HFBI biosurfactant production in Pichia pastoris

Hydrophobins are small highly surface-active fungal proteins with potential as biosurfactants in a wide array of applications. However, practical implementation of hydrophobins at large scale has been hindered by low recombinant yields. In this study, the effects of increasing hydrophobin gene copy number and overexpressing endoplasmic reticulum resident chaperone proteins Kar2p, Pdi1p, and Ero1p were explored as a means to enhance recombinant yields of the class II hydrophobin HFBI in the eukaryotic expression host Pichia pastoris. One-, 2-, and 3-copy-HFBI strains were attained using an in vitro multimer ligation approach, with strains displaying copy number stability following subsequent transformations as measured by quantitative polymerase chain reaction. Increasing HFBI copy number alone had no effect on increasing HFBI secretion, but increasing copy number in concert with chaperone overexpression synergistically increased HFBI secretion. Overexpression of PDI1 or ERO1 caused insignificant changes in HFBI secretion in 1- and 2-copy strains, but a statistically significant HFBI secretion increase in 3-copy strain. KAR2 overexpression consistently resulted in enhanced HFBI secretion in all copy number strains, with 3-copy-HFBI secreting 22±1.6 fold more than the 1-copy-HFBI/no chaperone strain. The highest increase was seen in 3-copy-HFBI/Ero1p overexpressing strain with 30±4.0 fold increase in HFBI secretion over 1-copy-HFBI/no chaperone strain. This corresponded to an expression level of approximately 330 mg/L HFBI in the 5 ml small-scale format used in this study.     

Production of phenylalanine and organic acids by phosphoenolpyruvate carboxylase-deficient mutants ofEscherichia coli

Summary Isogenic strains ofEscherichia coli were grown aerobically in minimal medium in a 2-liter airlift fermentor to determine whether appc (phosphoenolpyruvate carboxylase) mutation had the effect of directing glucose carbon into phenylalanine synthesis. Two host strains, YMC9 (ppc ⁺) and KB285 (ppc ^–) were used, either with (Phe^c) or without (Phe⁰) a plasmid which determines constitutive phenylalanine production. Carbon consumption and metabolic products were monitored. Phenylalanine production occurred only in strains carrying the Phe^c plasmid.ppc ^– strains produced less cell mass and more acetate, pyruvate, and phenylalanine (in the Phe^c strains) than did isogenicppc ⁺ strains. Lactate and ethanol production were not detected in any of the strains. Phe^c strains produced less acetate and pyruvate than their Phe⁰ homologs. Importantly,ppc ^–/Phe^c produced at least six times as much phenylalanine (0.32 g phenylalanine/g dry weight cells) asppc ⁺/Phe^c. Even in this case, however, phenylalanine was produced at ten-fold lower levels than acetate. Thus, although theppc ^– mutation stimulates phenylalanine production, it also stimulates the production of unwanted by-products such as acetate and pyruvate.     

Tn1000 insertional mutagenesis of cloned repressor gene of the phage L: Plasmid oligomerization in the presence of F’lac

An ampicillin resistance plasmid carrying the cloned repressor gene c_II of the L phage (Salmonella lyphimurium) was conducted by F’lac into an F^- recipient. Two types of plaamids were isolated from Ap^r transconjugants. The majority of plasmids were dimers with one copy of Tn1000 inserted, the minority being monomers with one copy of Tn1000. This proportion remained unaltered when we used the F’lac strain transformed with a monomeric form of the recombinant plasmid as a donor. An extensive oligomerization of pBR322-originating plasmids was proved in the presence of F’lac; its presumable relationship to transposition-related processes is suggested.     

PGA基因在酿酒酵母中的表达研究

本文根据GenBank 中巨大芽孢杆菌（Bacillus megaterium）的PGA基因序列设计了上下游引物,通过PCR扩增出巨大芽孢杆菌1.1741中的PGA基因。将该基因连接到T7lac启动子控制下的表达载体pYES2(amp+,ura+)上,构建了重组质粒pYES2-PGA。用LiAc/SSDNA/PEG方法将其转化进酿酒酵母(Saccharomyces cerevisiae)H158中表达,在不需要苯乙酸诱导的重组菌株发酵液中检测到了青霉素酰化酶活性,最高酶活达到0.75 U/ml。将该PGA基因测序结果与GenBank中巨大芽孢杆菌L04471.1、U07682.1和Z37542三株的PGA基因序列比对,表现出很高的同源性,分别达到97.1%、99.8% 和99.8%。     

Stability of Escherichia coli strains harboring recombinant plasmids for L-threonine production

Escherichia coli recombinant strains bearing the thr operon have been previously selected for threonine production and phenotypically classified according to antibiotic resistance properties (Nudel et al. 1987).Further analysis of those strains permitted the isolation and restriction mapping of two different plasmids of 13 kb and 18.6 kb. The smaller one, which expressed tetracycline resistance gave better results on threonine accumulation but it was rather unstable when grown without antibiotic pressure. Therefore, other hosts were transformed with those plasmids to improve stability.A threonine-auxotrophic strain was a better host for plasmid maintenance and expression of thr operon. Host influence in plasmid-mediated threonine production was studied in terms of specific yields (the ratios of threonine accumulated to biomass values) and of plasmid maintenance (percent of Ap^rTc^r clones after cultivation in non selective media).We also determined that semisynthetic media of defined composition were better than rich media for threonine expression, due to feed-back controls exerted by undesired catabolites accumulated in complex media.     

Metabolic engineering of thermophilic Bacillus methanolicus for riboflavin overproduction from methanol

The growing need of next generation feedstocks for biotechnology spurs an intensification of research on the utilization of methanol as carbon and energy source for biotechnological processes. In this paper, we introduced the methanol-based overproduction of riboflavin into metabolically engineered Bacillus methanolicus MGA3. First, we showed that B. methanolicus naturally produces small amounts of riboflavin. Then, we created B. methanolicus strains overexpressing either homologous or heterologous gene clusters encoding the riboflavin biosynthesis pathway, resulting in riboflavin overproduction. Our results revealed that the supplementation of growth media with sublethal levels of chloramphenicol contributes to a higher plasmid-based riboflavin production titre, presumably due to an increase in plasmid copy number and thus biosynthetic gene dosage. Based on this, we proved that riboflavin production can be increased by exchanging a low copy number plasmid with a high copy number plasmid leading to a final riboflavin titre of about 523 mg L⁻¹ in methanol fed-batch fermentation. The findings of this study showcase the potential of B. methanolicus as a promising host for methanol-based overproduction of extracellular riboflavin and serve as basis for metabolic engineering of next generations of riboflavin overproducing strains.     

Correlation between specific plasmids and δ-endotoxin production in Bacillus thuringiensis

Five strains of Bacillus thuringiensis that produce crystalline δ-endotoxin were used as parental strains in an effort to isolate acrystalliferous (Cry⁻) mutants: HD-2 (B. thuringiensis var. thuringiensis, flagellar serotype 1); HD-1 and HD-73 (both var. kurstaki, serotype 3ab); HD-4 (var. alesti, serotype 3a); and HD-8 (var. galleriae, serotype 5ab). The parental strains contain complex plasmid arrays that have been previously characterized (González and Carlton, 1980). The plasmid patterns of both Cry⁻ and Cry⁺ variants were analyzed and compared to the parental strains using a modified Eckhardt (1978) lysate-electrophoresis method. Most Cry⁻ mutants derived from strain HD-2 were found to exhibit a distinctive colony morphology which facilitated their isolation. Loss of crystal production was associated with loss of a 75-Md plasmid. A 50-Md plasmid of strain HD-73 was lost in the Cry⁻ mutants. Crystal production in strain HD-4 appears to be associated with a plasmid about 105 Md in size; in strain HD-1, a smaller plasmid (29 Md in size) seems to be involved. In strain HD-8, a large plasmid (˜130 Md in size) is implicated in crystal production. Direct bioassay of several of the mutant strains has confirmed the loss of δ-endotoxin activity in the acrystalliferous isolates. The evidence obtained supports the notion of a relationship between specific extrachromosomal DNA elements and δ-endotoxin production in B. thuringiensis, and suggests that in each strain only a single plasmid is involved, although the size of the implicated plasmid varies from one strain to another.     

Escherichia coli host G668 stabilizes plasmids that are unstable in otherEscherichia coli strains

CloDF13 copy mutants that have their resolution site (crl) deleted accumulate as multimeric plasmid molecules in their host cells and are lost from severalEscherichia coli stains within 60 generations. Here we demonstrate that CloDF13cop3crl ^– mutants are stably maintained in theE.coli strain G668, although the plasmid copy number is not affected. Furthermore, we show that the stable maintenance of those plasmids is achieved even in the presence of multimeric molecules. Therefore, we conclude that a complete monomerization of multimeric molecules appears not to be a prerequisite for accurate partition of the plasmid molecules over daughter cells. The G668 strain may be applied as host for the stabilization of resolution-negative, unstable CloDF13 or related replicons.     

Improved penicillin amidase production using a genetically engineered mutant of Escherichia coli ATCC 11105

Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, we constructed various recombinant E. coli HB101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic and (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by its transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the Hindlll fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene. (c) 1993 John Wiley & Sons, Inc.     

Production of cloned carboxypeptidase G2 by Escherichia coli: Genetic and environmental considerations

Summary The optimum production of cloned carboxypeptidase G₂ from plasmid pNM21 byEscherichia coli was found to be strongly strain- and temperature-dependent. The superior host was strain RV308 and the preferred growth temperature 28°C. Copy number, which decreased during exponential growth of all strains examined, did not relate in these studies to the level of enzyme production: the strain with the highest enzyme yield also having the lowest overall copy number.     

Plasmid deoxyribonucleic acid in strains of Bacillus sphaericus and in Bacillus moritai

Bacillus moritai and six strains of Bacillus sphaericus pathogenic to dipteran larvae were examined for the presence of covalently closed circular (CCC) DNA. The plasmid profiles of the bacteria were analyzed using a cleared lysate electrophoresis technique. Four of the six strains of B. sphaericus examined contained CCC DNA. Strain SSII-1 contained two plasmids (pKA1, pKA2) having molecular weights of about 8.4 and 2.0 megadaltons (MDa). Strains 1404 and 1881 each contained one plasmid, pKA3 and pKA4, respectively. pKA3 had a molecular weight of about 8.2 MDa. pKA4 had a relatively large plasmid with a molecular weight of about 33.5 MDa. Strain K contained five size classes of CCC DNA. The plasmids pKA5, pKA6, pKA7, pKA8, and pKA9 had molecular weights of about 11.4, 10.9, 7.4, 7.0, and 6.4 MDa, respectively. Strains 1593-4 and 1691 were plasmidless and could not be distinguished from each other based on their plasmid profiles. B. moritai ATCC 21042 contained two size classes of CCC duplex DNA; pRF100 had a molecular weight of about 4.6 MDa and pRF101 had a molecular weight of about 2.1 MDa. No phenotype association with any of the isolated plasmids has been determined.     

染色体DNA琼脂糖包埋法辅助的ExoCET技术克隆放线菌天然产物生物合成基因簇

【目的】本研究旨在通过将琼脂糖包埋染色体DNA的方法与ExoCET重组技术相结合,建立放线菌天然产物生物合成基因簇的捕获方法。然后将克隆基因簇导入通用底盘宿主中,实现目标生物合成基因簇的异源表达。【方法】首先,利用低熔点琼脂糖包埋技术制备菌株的染色体基因组总DNA,再用限制性内切酶消化含有染色体DNA的琼脂块,获得线性化的DNA样品;然后利用ExoCET重组技术,以p15A线性载体片段将目标基因簇线性片段进行捕获;再通过PCR-targeting的方法向目标质粒中引入所需的接合转移DNA元件。接着,将改造质粒通过接合转移导入到Streptomyces coelicolor M1252宿主中,获得不同的重组菌株。最后,对不同的菌株进行发酵并提取化合物,最后进行活性检测以及质谱检测。【结果】通过该方法,从菌株S.lincolnensisNRR2936中成功获得了林可霉素生物合成基因簇(lmb-BGC),从菌株Nonomuraea nitratireducens WYY166^T中克隆得到了2个核糖体肽类化合物的生物合成基因簇(nioblantin,niob-BGC和nitblantin,nitb-BGC),并实现了lmb-BGC在天蓝色链霉菌M1252中的成功表达。【结论】本研究通过将低熔点琼脂糖包埋技术与ExoCET重组技术进行合理整合,定向克隆得到了林可霉素以及2个新颖的羊毛硫肽类化合物的生物合成基因簇。然后,分别对重组质粒改造后,在天蓝色链霉菌M1252宿主中进行表达,分别获得重组菌株MJX01、MJX02和MJX04。最后,利用质谱以及活性测试的手段对发酵提取物进行了检测,确定了林可霉素生物合成基因簇在天蓝色链霉菌M1252中成功表达。本研究为通过基因簇克隆和异源表达发掘新化合物奠定了基础。