Improved conditions for production of recombinant plant sesquiterpene synthases in Escherichia coli

Amorpha-4,11-diene synthase (ADS) from Artemisia annua and (+)-germacrene synthase (GDS) from Zingiber officinale were expressed in Escherichia coli under different conditions to optimize the yield of active soluble protein. The cDNAs of these enzymes were inserted into the pET28 vector (Novagen) and expressed in four different bacterial strains; BL21 (DE3), BL21 (DE3) Tuner, BL21 (DE3) pLysS and BL21 (DE3) pLysS Tuner using different inducing agents (IPTG, The Inducer). The effects of induction under osmotic stress in the presence of glycine betaine and sorbitol were investigated. Although background expression for ADS was reduced when using pLysS strains, no significant difference was noted for ADS activity in soluble whole cell lysates after induction with either IPTG or The Inducer. For GDS, on the other hand, the change between BL21 (DE3) cells and BL21 (DE3) Tuner, induced with IPTG, leads to a twofold increase in enzyme activity in the soluble fraction while a reduction in activity is observed when using the pLysS strains. The same doubling of activity is observed for GDS when the commonly used BL.21 (DE3) is induced with The Inducer. Addition of 2.5 mM glycine betaine and 660 mM sorbitol to the bacterial growth media resulted in reduction of growth rate and biomass yield but under these conditions the best overall protein production, for both enzymes, was obtained. Compared to the standard conditions previously used in our laboratory the yield of soluble active protein was increased 7- and 2.5-fold for ADS and GDS, using BL21 (DE3) pLysS Tuner and BL21 (DE3), respectively.     

Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli

The murein (peptidoglycan) sacculus is an essential polymer embedded in the bacterial envelope. The Escherichia coli class B penicillin-binding protein (PBP) 3 is a murein transpeptidase and essential for cell division. In an affinity chromatography experiment, the bifunctional transglycosylase-transpeptidase murein synthase PBP1B was retained by PBP3-sepharose when a membrane fraction of E. coli was applied. The direct protein-protein interaction between purified PBP3 and PBP1B was characterized in vitro by surface plasmon resonance. The interaction was confirmed in vivo employing two different methods: by a bacterial two-hybrid system, and by cross-linking/co-immunoprecipitation. In the bacterial two-hybrid system, a truncated PBP3 comprising the N-terminal 56 amino acids interacted with PBP1B. Both synthases could be cross-linked in vivo in wild-type cells and in cells lacking FtsW or FtsN. PBP1B localized diffusely and in foci at the septation site and also at the side wall. Statistical analysis of the immunofluorescence signals revealed that the localization of PBP1B at the septation site depended on the physical presence of PBP3, but not on the activity of PBP3. These studies have demonstrated, for the first time, a direct interaction between a class B PBP (PBP3) and a class A PBP (PBP1B) in vitro and in vivo, indicating that different murein synthases might act in concert to enlarge the murein sacculus during cell division.     

Heterologous production of polyketides by modular type I polyketide synthases in Escherichia coli

Heterologous production of polyketide compounds, an important class of natural products with complex chemical structures, was first demonstrated with Streptomyces parvulus in 1984. Although Streptomyces strains are good first options for heterologous polyketide biosynthesis, their slow growth kinetics prompt other hosts to also be considered. Escherichia coli provides key elements of an ideal host in terms of the growth rate, culture conditions, and available recombinant DNA tools. Here we review the current status and potential for metabolic engineering of polyketides in E. coli.     

Variability of peptidoglycan surface density in Escherichia coli

Abstract Murein synthesis in Escherichia coli can be partially inhibited by d-methionine without concomitant alterations in growth and morphology. d-Methionine-treated cultures grow steadily for an indefinite time, therefore murein surface density should be reduced. Determination of this parameter in control and d-methionine-treated cells showed a severe reduction in the latter. Murein surface density increases drastically in resting cells, irrespective of the presence of d-methionine. Mutants in ponB are hypersensitive to d-methionine. Analysis of ponB strains revealed an important reduction in murein surface density. An approximately two-fold reduction in average surface density is apparently compatible with normal growth and division.     

A mecillinam-sensitive peptidoglycan crosslinking reaction in Escherichia coli

The amidinopenicillin, mecillinam, induces the formation of spherical cells of Escherichia coli by inactivation of penicillin-binding protein 2 (PBP2). A mecillinam-sensitive peptidoglycan crosslinking reaction has been demonstrated in particulate membrane preparations from this organism. The activity was detected in membranes that contained elevated levels of PBP2 and in which crosslinking reactions due to all other PBPs had been inactivated with the cephamycin antibiotic, cefmetazole. The particulate membrane preparation catalyzed synthesis of peptidoglycan that was up to 20% crosslinked from nucleotide precursors. Crosslinkage of the peptidoglycan was inhibited 50% by 0.2 μg mecillinam per ml but was not inhibited by much higher concentrations of cephamycins, which have very low affinity for PBP2. The crosslinking reaction appears to be due to the transpeptidase activity of PBP2, which is implicated in the mechanism of cell shape determination, and is the killing target for mecillinam.     

Escherichia coli ghost production by expression of lysis gene E and Staphylococcal nuclease

The production of bacterial ghosts from Escherichia coli is accomplished by the controlled expression of phage phiX174 lysis gene E and, in contrast to other gram-negative bacterial species, is accompanied by the rare detection of nonlysed, reproductive cells within the ghost preparation. To overcome this problem, the expression of a secondary killing gene was suggested to give rise to the complete genetic inactivation of the bacterial samples. The expression of staphylococcal nuclease A in E. coli resulted in intracellular accumulation of the protein and degradation of the host DNA into fragments shorter than 100 bp. Two expression systems for the nuclease are presented and were combined with the protein E-mediated lysis system. Under optimized conditions for the coexpression of gene E and the staphylococcal nuclease, the concentration of viable cells fell below the lower limit of detection, whereas the rates of ghost formation were not affected. With regard to the absence of reproductive cells from the ghost fractions, the reduction of viability could be determined as being at least 7 to 8 orders of magnitude. The lysis process was characterized by electrophoretic analysis and absolute quantification of the genetic material within the cells and the culture supernatant via real-time PCR. The ongoing degradation of the bacterial nucleic acids resulted in a continuous quantitative clearance of the genetic material associated with the lysing cells until the concentrations fell below the detection limits of either assay. No functional, released genetic units (genes) were detected within the supernatant during the lysis process, including nuclease expression.     

Extraction and identification by mass spectrometry of undecaprenyl diphosphate-MurNAc-pentapeptide-GlcNAc from Escherichia coli

Undecaprenyl diphosphate-MurNAc-pentapeptide-GlcNAc (lipid II) is extracted from Escherichia coli cells by utilizing its unusual pH-dependent solubility property in a Bligh-Dyer system, and identified by electrospray ionization mass spectrometry in conjunction with a novel 15N mass shift analysis. The described approach will facilitate the structural characterization of lipid II variants from diverse bacteria, including antibiotic-resistant mutants, as well as the numerous minor uncharacterized lipids present in all biological systems.     

Activation of the cryptic PhnE permease promotes rapid adaptive evolution in a population of Escherichia coli K-12 starved for phosphate

Escherichia coli K-12 suffers acetic acid stress during prolonged incubation in glucose minimal medium containing a limiting concentration of inorganic phosphate (0.1 mM P(i)), which decreases the number of viable cells from 6 × 10(8) to ≤10 CFU/ml between days 6 and 14 of incubation. Here we show that following two serial transfers into P(i)-limiting medium, evolved mutants survived prolonged incubation (≈10(7) CFU/ml on day 14 of incubation). The evolved strains that overtook the populations were generally PhnE(+), whereas the ancestral K-12 strain carries an inactive phnE allele, which prevents the transport of phosphonates. The switching in phnE occurred with a high frequency as a result of the deletion of an 8-bp repeated sequence. In a mixed culture starved for P(i) that contained the K-12 ancestral strain in majority, evolved strains grew through PhnE-dependent scavenging of probably organic phosphate esters (not phosphonates or P(i)) released by E. coli K-12 between days 1 and 3, before acetic acid excreted by E. coli K-12 reached toxic levels. The growth yield of phnE(+) strains in mixed culture was dramatically enhanced by mutations that affect glucose metabolism, such as an rpoS mutation inactivating the alternative sigma factor RpoS. The long-term viability of evolved populations was generally higher when the ancestral strain carried an inactive rather than an active phnE allele, which indicates that cross-feeding of phosphorylated products as a result of the phnE polymorphism may be essential for the spread of mutants which eventually help populations to survive under P(i) starvation conditions.     

Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli.

The cellular pool levels of most of the cytoplasmic precursors of peptidoglycan synthesis were determined for normally growing cells of Escherichia coli K-12. In particular, a convenient method for analyzing the uridine nucleotide precursor contents was developed by associating gel filtration and reverse-phase high-pressure liquid chromatography techniques. The enzymatic parameters of the four synthetases which catalyze the stepwise addition of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine to uridine diphosphate-N-acetylmuramic acid were determined. It was noteworthy that the pool levels of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine were much higher than the Km values determined for these substrates, whereas the molar concentrations of the uridine nucleotide precursors were lower than or about the same order of magnitude as the corresponding Km values. Taking into consideration the data obtained, an attempt was made to compare the in vitro activities of the D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine adding enzymes with their in vivo functioning, expressed by the amounts of peptidoglycan synthesized. The results also suggested that these adding activities were not in excess in the cell under normal growth conditions, but their amounts appeared adjusted to the requirements of peptidoglycan synthesis. Under the different in vitro conditions considered, only low levels of L-alanine adding activity were observed.     

Product distribution and pre-steady-state kinetic analysis of Escherichia coli undecaprenyl pyrophosphate synthase reaction

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C(55) undecaprenyl pyrophosphate. We investigated the kinetics and mechanism of this reaction pathway using Escherichia coli UPPs. With a variety of different ratios of enzyme to substrate and FPP to IPP in the presence or absence of Triton, different product distributions were found. In the presence of excess FPP, the intermediates (C(25)-C(50)) accumulated. Under a condition with enzyme and FPP in excess of IPP, instead of C(20)-geranylgeranyl pyrophosphate, C(20), C(25), and C(30) were the major products. The UPPs steady-state k(cat) value (2.5 s(-1)) in the presence of 0.1% Triton was 190-fold larger than in the absence of Triton (0.013 s(-1)). The k(cat) value matched the rate constant of each IPP condensation obtained from the enzyme single-turnover experiments. This suggested that the IPP condensation rather than product release was the rate-limiting step in the presence of Triton. In the absence of Triton, the intermediates formed and disappeared in a similar manner under enzyme single turnover in contrast to the slow steady-state rate, which indicated a step after product generation was rate limiting. This was further supported by a burst product formation. Judging from the accumulation level of C(55), C(60), and C(65), their dissociation from the enzyme cannot be too slow and an even slower enzyme conformational change with a rate of 0.001 s(-1) might govern the UPPs reaction rate under the steady-state condition in the absence of Triton.     

High-level expression of Escherichia coli and Bacillus subtilis thymidylate synthases

Procedures are described for the preparation of highly purified thymidylate synthases from Escherichia coli and Bacillus subtilis. The yields in each case are quite high with about 350 mg of pure protein obtained from 1 liter of cells. Basically all that is required to obtain pure enzyme is an induction step from a high-expression vector, followed by a DE-52 column elution. Both enzymes appeared to be fairly stable in that incubation at 43 degrees C for 10 min resulted in the loss of 50% of the E. coli thymidylate synthase activity, while 50 degrees C for 10 min was required to obtain the same effect with the B. subtilis enzyme. In the presence of the substrate, dUMP, each protein was stabilized further by 6 to 7 degrees C, which was increased to 9 to 10 degrees C on addition of dihydrofolate. It was shown also that the E. coli thymidylate synthase could be maintained at 4 degrees C for at least 4 months with little or no loss in activity provided that mercaptoethanol was not present. The presence of the latter led to a progressive loss in activity until little activity could be detected after 18 weeks, which was due, in part, to the formation of a disulfide bond with the active site cysteine. Addition of dithiothreitol restored the enzyme activity to its original state.     

Autolysis-resistant peptidoglycan of anomalous composition in amino-acid-starved Escherichia coli.

Nongrowing Escherichia coli deprived of an essential amino acid continued to produce peptidoglycan at a rate approximately 30% of that of growing cells. The composition of this peptidoglycan was very different from that of growing cells and resembled that of peptidoglycan left undegraded during partial autolysis of the bacteria. Synthesis of this peptidoglycan of anomalous composition began at once upon the removal of the amino acid from the medium. Fifteen minutes of amino acid deprivation was sufficient to virtually completely prevent penicillin-induced autolytic wall degradation in vivo. During this time, although the specific activities of soluble and membrane-bound hydrolytic transglycosylases and endopeptidases remained high, the peptidoglycan produced showed decreased sensitivity to degradation in vitro. After more extensive (2-h) starvation, triggering of autolysis by chaotropic agents was also blocked. Autolysis in growing cells may be selective for peptidoglycan representing the cylindrical portion of the sacculus. It is suggested that at least part of the mechanism of the well-known lysis resistance of nongrowing E. coli is related to the deposition of structurally anomalous and relatively autolysin-resistant peptidoglycan at some strategically located sites on the bacterial surface.     

Substrate specificity and membrane topology of Escherichia coli PgpB, an undecaprenyl pyrophosphate phosphatase

The synthesis of the lipid carrier undecaprenyl phosphate (C(55)-P) requires the dephosphorylation of its precursor, undecaprenyl pyrophosphate (C(55)-PP). The latter lipid is synthesized de novo in the cytosol and is also regenerated after its release from the C(55)-PP-linked glycans in the periplasm. In Escherichia coli the dephosphorylation of C(55)-PP was shown to involve four integral membrane proteins, BacA, and three members of the type 2 phosphatidic acid phosphatase family, PgpB, YbjG, and YeiU. Here, the PgpB protein was purified to homogeneity, and its phosphatase activity was examined. This enzyme was shown to catalyze the dephosphorylation of C(55)-PP with a relatively low efficiency compared with diacylglycerol pyrophosphate and farnesyl pyrophosphate (C(15)-PP) lipid substrates. However, the in vitro C(55)-PP phosphatase activity of PgpB was specifically enhanced by different phospholipids. We hypothesize that the phospholipids are important determinants to ensure proper conformation of the atypical long axis C(55) carrier lipid in membranes. Furthermore, a topological analysis demonstrated that PgpB contains six transmembrane segments, a large periplasmic loop, and the type 2 phosphatidic acid phosphatase signature residues at a periplasmic location.     

Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli

Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The ¹³C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0 g/L isoprene with a yield of 0.267 g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms.     

The phosphate pool in Escherichia coli THU

The phosphate pool of Escherichia coli was determined as a fraction of the total cell phosphate. This relative pool size was found to be essentially independent of cell age.     

Daughter cell separation by penicillin-binding proteins and peptidoglycan amidases in Escherichia coli

As one of the final steps in the bacterial growth cycle, daughter cells must be released from one another by cutting the shared peptidoglycan wall that separates them. In Escherichia coli, this delicate operation is performed by several peptidoglycan hydrolases, consisting of multiple amidases, lytic transglycosylases, and endopeptidases. The interactions among these enzymes and the molecular mechanics of how separation occurs without lysis are unknown. We show here that deleting the endopeptidase PBP 4 from strains lacking AmiC produces long chains of unseparated cells, indicating that PBP 4 collaborates with the major peptidoglycan amidases during cell separation. Another endopeptidase, PBP 7, fulfills a secondary role. These functions may be responsible for the contributions of PBPs 4 and 7 to the generation of regular cell shape and the production of normal biofilms. In addition, we find that the E. coli peptidoglycan amidases may have different substrate preferences. When the dd-carboxypeptidase PBP 5 was deleted, thereby producing cells with higher levels of pentapeptides, mutants carrying only AmiC produced a higher percentage of cells in chains, while mutants with active AmiA or AmiB were unaffected. The results suggest that AmiC prefers to remove tetrapeptides from peptidoglycan and that AmiA and AmiB either have no preference or prefer pentapeptides. Muropeptide compositions of the mutants corroborated this latter conclusion. Unexpectedly, amidase mutants lacking PBP 5 grew in long twisted chains instead of straight filaments, indicating that overall septal morphology was also defective in these strains.