Engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the synthesis of <Emphasis Type="Italic">N</Emphasis>-hydroxycinnamoyl tryptamine and serotonin

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC–tryptamines and HC–serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.     

Production of caffeoylmalic acid from glucose in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve biosynthesis of caffeoylmalic acid from glucose in engineered Escherichia coli.

Results

We constructed the biosynthetic pathway of caffeoylmalic acid in E. coli by co-expression of heterologous genes RgTAL, HpaBC, At4CL2 and HCT2. To enhance the production of caffeoylmalic acid, we optimized the tyrosine metabolic pathway of E. coli to increase the supply of the substrate caffeic acid. Consequently, an E. coli–E. coli co-culture system was used for the efficient production of caffeoylmalic acid. The final titer of caffeoylmalic acid reached 570.1 mg/L.

Conclusions

Microbial production of caffeoylmalic acid using glucose has application potential. In addition, microbial co-culture is an efficient tool for producing caffeic acid esters.

     

Biosynthesis of poly(2-hydroxybutyrate-co-lactate) in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have previously reported in vivo biosynthesis of polyhydroxyalkanoates containing 2-hydroxyacid monomers such as lactate and 2-hydroxybutyrate in recombinant Escherichia coli strains by the expression of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 _Ps6-19). Here, we report the biosynthesis of poly(2-hydroxybutyrate-co-lactate)[P(2HB-co-LA)] by direct fermentation of metabolically engineered E. coli strain. Among E. coli strains WL3110, XL1-Blue, and BL21(DE3), recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 produced P(76.4mol%2HB-co-23.6mol%LA) to the highest content of 88 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 2-hydroxybutyrate. When recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 was cultured in a chemically defined medium containing 20 g/L of glucose and varying concentration of sodium 2-hydroxybutyrate, 2HB monomer fraction in P(2HB-co-LA) increased proportional to the concentration of sodium 2-hydroxybutyrate added to the culture medium. P(2HB-co-LA)] could also be produced from glucose as a sole carbon source without sodium 2-hydroxybutyrate into the culture medium. Recombinant E. coli XL1-Blue strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene, successfully produced P(23.5mol%2HB-co-76.5mol%LA)] to the polymer content of 19.4 wt% when it cultured in a chemically defined medium containing 20 g/L of glucose. The metabolic engineering strategy reported here should be useful for the production of novel copolymer P(2HB-co-LA)].     

Phosphoenolpyruvate:glucose phosphotransferase system modification increases the conversion rate during <Emphasis Type="SmallCaps">l</Emphasis>-tryptophan production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli FB-04(pta1), a recombinant l-tryptophan production strain, was constructed in our laboratory. However, the conversion rate (l-tryptophan yield per glucose) of this strain is somewhat low. In this study, additional genes have been deleted in an effort to increase the conversion rate of E. coli FB-04(pta1). Initially, the pykF gene, which encodes pyruvate kinase I (PYKI), was inactivated to increase the accumulation of phosphoenolpyruvate, a key l-tryptophan precursor. The resulting strain, E. coli FB-04(pta1)ΔpykF, showed a slightly higher l-tryptophan yield and a higher conversion rate in fermentation processes. To further improve the conversion rate, the phosphoenolpyruvate:glucose phosphotransferase system (PTS) was disrupted by deleting the ptsH gene, which encodes the phosphocarrier protein (HPr). The levels of biomass, l-tryptophan yield, and conversion rate of this strain, E. coli FB-04(pta1)ΔpykF/ptsH, were especially low during fed-batch fermentation process, even though it achieved a significant increase in conversion rate during shake-flask fermentation. To resolve this issue, four HPr mutations (N12S, N12A, S46A, and S46N) were introduced into the genomic background of E. coli FB-04(pta1)ΔpykF/ptsH, respectively. Among them, the strain harboring the N12S mutation (E. coli FB-04(pta1)ΔpykF-ptsHN12S) showed a prominently increased conversion rate of 0.178 g g^?1 during fed-batch fermentation; an increase of 38.0% compared with parent strain E. coli FB-04(pta1). Thus, mutation of the genomic of ptsH gene provided an alternative method to weaken the PTS and improve the efficiency of carbon source utilization.     

Cyclohexanone-induced stress metabolism of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Solvent stress occurs during whole-cell biocatalysis of organic chemicals. Organic substrates and/or products may accumulate in the cellular membranes of whole cells, causing structural destabilization of the membranes, which leads to disturbances in cellular carbon and energy metabolism. Here, we investigate the effect of cyclohexanone on carbon metabolism in Escherichia coli BL21 and Corynebacterium glutamicum ATCC13032. Adding cyclohexanone to the culture medium (i.e., glucose mineral medium) resulted in a decreased specific growth rate and increased cellular maintenance energy in both strains of bacteria. Notably, carbon metabolism, which is mainly involved to increase cellular maintenance energy, was very different between the bacteria. Carbon flux into the acetic acid fermentation pathway was dominantly enhanced in E. coli, whereas the TCA cycle appeared to be activated in C. glutamicum. In fact, carbon flux into the TCA cycle in E. coli appeared to be reduced with increasing amounts of cyclohexanone in the culture medium. Metabolic engineering of E. coli cells to maintain or improve TCA cycle activity and, presumably, that of the electron transport chain, which are involved in regeneration of cofactors (e.g., NAD(P)H and ATP) and formation of toxic metabolites (e.g., acetic acid), may be useful in increasing solvent tolerance and biotransformation of organic chemicals (e.g., cyclohexanone).     

Characterization of the lipopolysaccharide of <Emphasis Type="Italic">Escherichia coli</Emphasis> 126

The lipopolysaccharide (LPS) of Escherichia coli 126 was isolated and studied. The lipid A fatty acid composition of the investigated LPS was similar to that of other members of the family Enterobacteriaceae. The E. coli 126 LPS was more toxic than the LPSs of previously studied E. coli strains and of other members of the Enterobacteriaceae (Budvicia aquatica and Pragia fontium), and was less pyrogenic than pyrogenal. SDS-PAG electrophoresis showed a bimodal distribution typical of S-form LPSs. The LPS of E. coli 126 decreased the adhesive index indicating a possible competition between LPS molecules of E. coli 126 and adhesins of E. coli F-50 on rabbit erythrocytes. The LPS of E. coli 126 in a homologous system showed antigenic activity in the reactions of double immunodiffusion in agar by Ouchterlony. No serological cross-reaction of the LPS of other E. coli strains, as well as of that of the B. aquatica type strain, with the antiserum to E. coli 126 was observed. The structural components of the lipopolysaccharide obtained by mild acid hydrolysis were lipid A, the core oligosaccharide, and the O-specific polysaccharide. Based on the data of monosaccharide analysis and ¹H and ¹³C NMR spectroscopy it was found that the O-specific polysaccharide had the structure characteristic of the representatives of E. coli serogroup O15.     

Identification and Mutational Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> Sorbitol-Enhanced Glucose-Repressed <Emphasis Type="Italic">srlA</Emphasis> Promoter Expressed in LB Medium by Using Homologous Recombination and One-Round PCR Products

Escherichia coli has been used for recombinant protein production for many years. However, no native E. coli promoters have been found for constitutive expression in LB medium. To obtain high-expression E. coli promoters active in LB medium, we inserted various promoter regions upstream of eEmRFP that encodes a red fluorescent protein. Among the selected promoters, only colonies of srlA promoter transformants turned red on LB plate. srlA is a gene that regulates sorbitol utilization. The addition of sorbitol enhanced eEmRFP expression but glucose and other sugars repressed, indicating that srlAp is a sorbitol-enhanced glucose-repressed promoter. To analyze the srlAp sequence, a novel site-directed mutagenesis method was developed. Since we demonstrated that homologous recombination in E. coli could occur between 12-bp sequences, 12-bp overlapping sequences were attached to the set of primers that were designed to produce a full-length plasmid, denoted “one-round PCR product.” Using this method, we identified that the srlA promoter region was 100 bp. Further, the sequence adjacent to the start codon was found to be essential for high expression, suggesting that the traditionally used restriction enzyme sites for cloning in the promoter region have hindered expression. The srlA-driven expression system and DNA manipulation with one-round PCR products are useful tools in E. coli genetic engineering.     

<Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> RecX and <Emphasis Type="Italic">Escherichia coli</Emphasis> RecX proteins are capable to replace each other in vivo and in vitro

A plasmid carrying the Deinococcus radiodurans recX gene under the control of a lactose promoter decreases the Escherichia coli cell resistance to UV irradiation and γ irradiation and also influences the conjugational recombination process. The D. radiodurans RecX protein functions in the Escherichia coli cells similarly to the E. coli RecX protein. Isolated and purified D. radiodurans RecX and E. coli RecX proteins are able to replace each other interacting with the E. coli RecA and D. radiodurans RecA proteins in vitro. Data obtained demonstrated that regulatory interaction of RecA and RecX proteins preserves a high degree of conservatism despite all the differences in the recombination reparation system between E. coli and D. radiodurans.     

Chromosomal Sil system contributes to silver resistance in <Emphasis Type="Italic">E. coli</Emphasis> ATCC 8739

The rise of antibiotic resistance in pathogenic bacteria is endangering the efficacy of antibiotics, which consequently results in greater use of silver as a biocide. Chromosomal mapping of the Cus system or plasmid encoded Sil system and their relationship with silver resistance was studied for several gram-negative bacteria. However, only few reports investigated silver detoxification mediated by the Sil system integrated in Escherichia coli chromosome. Accordingly, this work aimed to study the Sil system in E. coli ATCC 8739 and to produce evidence for its role in silver resistance development. Silver resistance was induced in E. coli ATCC 8739 by stepwise passage in culture media containing increasing concentrations of AgNO₃. The published genome of E. coli ATCC 8739 contains a region showing strong homology to the Sil system genes. The role of this region in E. coli ATCC 8739 was assessed by monitoring the expression of silC upon silver stress, which resulted in a 350-fold increased expression. De novo sequencing of the whole genome of a silver resistant strain derived from E. coli ATCC 8739 revealed mutations in ORFs putative for SilR and CusR. The silver resistant strain (E. coli AgNO₃R) showed constitutive expression of silC which posed a cost of fitness resulting in retarded growth. Furthermore, E. coli AgNO₃R exhibited cross-resistance to ciprofloxacin and a slightly increased tolerance to ampicillin. This study demonstrates that E. coli is able to develop resistance to silver, which may pose a threat towards an effective use of silver compounds as antiseptics.     

Virulence genes and antimicrobial susceptibility of lactose-negative and lactose-positive strains of <Emphasis Type="Italic">Escherichia coli</Emphasis> isolated from pregnant women and neonates

Escherichia coli can cause serious infections in the neonates and pregnant women. Although E. coli is widely studied, E. coli lactose-negative (lac?) strains have been rarely described before. So, the aim of this study was to compare lac? and lactose-positive (lac+) E. coli strains in respect of antimicrobial susceptibility and the frequency of virulence genes (VGs). The study included 58 lac+ and 58 lac? E. coli strains isolated from pregnant women and neonates. Culture and the results of biochemical reactions were conducted for lac? and lac+ E. coli identification and differentiation. Disc diffusion test was performed to study the antimicrobial susceptibility of the isolates, and PCR was used to detect VGs. Resistance to at least one of the tested antibiotics was found among 14 (25.9%) E. coli lac+ and in 26 (44.9%) E. coli lac? strains. Both lac+ and lac? E. coli strains were mostly resistant to ampicillin (22.4 and 39.7%) and ticarcillin (20.7 and 39.7%). None of the tested strains produced extended-spectrum β-lactamases (ESBLs). Genes fimH, fimA, iutA, sfa/foc, neuC, ibeA, and hlyF were detected, respectively, in 96.6, 82.8, 32.8, 24.1, 22.4, 12.1, and 6.9% of lac+ E. coli strains and in 94.8, 86.2, 48.3, 19.0, 8.6, 8.6, and 1.7% of lac? strains. The antimicrobial susceptibility and the pathogenic potential of both tested groups of E. coli strains are similar. Therefore, omitting E. coli lac? strains as a potential etiological agent of infections may pose a threat to the health and life of both mothers and neonates.     

Engineering the growth pattern and cell morphology for enhanced PHB production by <Emphasis Type="Italic">Escherichia coli</Emphasis>

E. coli JM109?envC?nlpD deleted with genes envC and nlpD responsible for degrading peptidoglycan (PG) led to long filamentous cell shapes. When cell fission ring location genes minC and minD of Escherichia coli were deleted, E. coli JM109?minCD changed the cell growth pattern from binary division to multiple fissions. Bacterial morphology can be further engineered by overexpressing sulA gene resulting in inhibition on FtsZ, thus generating very long cellular filaments. By overexpressing sulA in E. coli JM109?envC?nlpD and E. coli JM109?minCD harboring poly(3-hydroxybutyrate) (PHB) synthesis operon phbCAB encoded in plasmid pBHR68, respectively, both engineered cells became long filaments and accumulated more PHB compared with the wild-type. Under same shake flask growth conditions, E. coli JM109?minCD (pBHR68) overexpressing sulA grown in multiple fission pattern accumulated approximately 70 % PHB in 9 g/L cell dry mass (CDM), which was significantly higher than E. coli JM109?envC?nlpD and the wild type, that produced 7.6 g/L and 8 g/L CDM containing 64 % and 51 % PHB, respectively. Results demonstrated that a combination of the new division pattern with elongated shape of E. coli improved PHB production. This provided a new vision on the enhanced production of inclusion bodies.     

Heterologous expression of <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> serotype 1 O-antigen analog in <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 W3110 by transferring a minimum number of genes involved in O-polysaccharide biosynthesis

Objective

To heterologously produce the Shigella dysenteriae serotype 1 O-polysaccharide (O-PS, O-antigen) in Escherichia coli by transferring the minimum number of genes instead of the entire O-PS gene cluster.

Results

The three glycosyltransferase genes (rfbR, rfbQ and rfp) responsible for the formation of the O-repeat unit were introduced into E. coli K-12 W3110 to synthesize S. dysenteriae 1 O-PS. The specific O-antigen ladder type with different chain lengths of O-repeat units was observed in the recombinant E. coli strain by SDS-PAGE silver staining and western blotting using S. dysenteriae 1 lipopolysaccharide antiserum. Analysis by mass spectrometry and ion chromatography suggested generation of the specific S. dysenteriae 1 O-repeat unit structure with an extra glucose residue attached.

Conclusions

Recombinant E. coli expressing specific glycosyltransferase genes can generate the O-PS of S. dysenteriae 1 and might be able to synthesize heterologous O-antigens of various pathogenic bacteria for vaccine preparation.

     

Conjugative plasmid transfer from <Emphasis Type="Italic">Escherichia coli</Emphasis> is a versatile approach for genetic transformation of thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Geobacillus</Emphasis> species

We previously demonstrated efficient transformation of the thermophile Geobacillus kaustophilus HTA426 using conjugative plasmid transfer from Escherichia coli BR408. To evaluate the versatility of this approach to thermophile transformation, this study examined genetic transformation of various thermophilic Bacillus and Geobacillus spp. using conjugative plasmid transfer from E. coli strains. E. coli BR408 successfully transferred the E. coli–Geobacillus shuttle plasmid pUCG18T to 16 of 18 thermophiles with transformation efficiencies between 4.1 × 10^?7 and 3.8 × 10^?2/recipient. Other E. coli strains that are different from E. coli BR408 in intracellular DNA methylation also generated transformants from 9 to 15 of the 18 thermophiles, including one that E. coli BR408 could not transform, although the transformation efficiencies of these strains were generally lower than those of E. coli BR408. The conjugation was performed by simple incubation of an E. coli donor and a thermophile recipient without optimization of experimental conditions. Moreover, thermophile transformants were distinguished from abundant E. coli donor only by high temperature incubation. These observations suggest that conjugative plasmid transfer, particularly using E. coli BR408, is a facile and versatile approach for plasmid introduction into thermophilic Bacillus and Geobacillus spp., and potentially a variety of other thermophiles.     

An engineered non-oxidative glycolysis pathway for acetone production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To find new metabolic engineering strategies to improve the yield of acetone in Escherichia coli.

Results

Results of flux balance analysis from a modified Escherichia coli genome-scale metabolic network suggested that the introduction of a non-oxidative glycolysis (NOG) pathway would improve the theoretical acetone yield from 1 to 1.5 mol acetone/mol glucose. By inserting the fxpk gene encoding phosphoketolase from Bifidobacterium adolescentis into the genome, we constructed a NOG pathway in E.coli. The resulting strain produced 47 mM acetone from glucose under aerobic conditions in shake-flasks. The yield of acetone was improved from 0.38 to 0.47 mol acetone/mol glucose which is a significant over the parent strain.

Conclusions

Guided by computational analysis of metabolic networks, we introduced a NOG pathway into E. coli and increased the yield of acetone, which demonstrates the importance of modeling analysis for the novel metabolic engineering strategies.

     

Heterologous biosynthesis of triterpenoid dammarenediol-II in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve heterologous biosynthesis of dammarenediol-II, which is the precursor of dammarane-type tetracyclic ginsenosides, by reconstituting the 2,3-oxidosqualene-derived triterpenoid biosynthetic pathway in Escherichia coli.

Results

By the strategy of synthetic biology, dammarenediol-II biosynthetic pathway was reconstituted in E. coli by co-expression of squalene synthase (SS), squalene epoxidase (SE), NADPH-cytochrome P450 reductase (CPR) from Saccharomyces cerevisiae, and SE from Methylococcus capsulatus (McSE), NADPH-cytochrome P450 reductase (CPR) from Arabidopsis thaliana. Sequences of transmembrane domains were truncated if necessary in each of the genes. Different sources of SE/CPR combinations were tested, during which two CPRs were detected to be new reductase partners of McSE. When the gene encoding dammarenediol-II synthase was co-expressed with the 2,3-oxidosqualene expression modules, dammarenediol-II was detected and the production was 8.63 mg l^?1 in E. coli under the shake-flask conditions.

Conclusions

Two E. coli chassis for production of dammarenediol-II were established which could be potentially applied in other triterpenoid production in E. coli when different oxidosqualene cyclases (OSCs) introduced into the system.

     

Improved acid-stress tolerance of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> NZ9000 and <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 by overexpression of the anti-acid component <Emphasis Type="Italic">recT</Emphasis>

Acid accumulation caused by carbon metabolism severely affects the fermentation performance of microbial cells. Here, different sources of the recT gene involved in homologous recombination were functionally overexpressed in Lactococcus lactis NZ9000 and Escherichia coli BL21, and their acid-stress tolerances were investigated. Our results showed that L. lactis NZ9000 (ERecT and LRecT) strains showed 1.4- and 10.4-fold higher survival rates against lactic acid (pH 4.0), respectively, and that E. coli BL21 (ERecT) showed 16.7- and 9.4-fold higher survival rates than the control strain against lactic acid (pH 3.8) for 40 and 60 min, respectively. Additionally, we found that recT overexpression in L. lactis NZ9000 improved their growth under acid-stress conditions, as well as increased salt- and ethanol-stress tolerance and intracellular ATP concentrations in L. lactis NZ9000. These findings demonstrated the efficacy of recT overexpression for enhancing acid-stress tolerance and provided a promising strategy for insertion of anti-acid components in different hosts.     

Improved Tolerance of <Emphasis Type="Italic">Escherichia coli</Emphasis> to Propionic Acid by Overexpression of Sigma Factor RpoS

Propionic acid (PA) is an economically important compound, but large-scale microbial production of PA confronts obstacle such as acid stress on microbial cells. Here, we show that overexpressing sigma factor RpoS improves the acid tolerance of Escherichia coli. Four genes including rpoS, fur, pgi and dnaK (encoding RNA polymerase sigma factor, ferric uptake regulator, phosphoglucoisomerase, and chaperone, respectively) were independently overexpressed in E. coli. The recombinant E. coli overexpressing rpoS showed the highest PA tolerance. This strain could grow in M9 medium at pH 4.62, whereas wild type E. coli survived only at pHs above 5.12. Moreover, in the shake-flask cultivation, the E. coli strain overexpressing rpoS grew faster than wild type. Notably, the minimum inhibitory concentration of PA for this recombinant strain was 7.81 mg/mL, which was 2-fold higher in comparison with wild type. Overall these results indicated that overexpression of sigma factor rpoS significantly enhanced E. coli tolerance to PA.     

2D [<Superscript>1</Superscript>H,<Superscript>13</Superscript>C] NMR study of carbon fluxes during glucose utilization by <Emphasis Type="Italic">Escherichia coli</Emphasis> MG1655

Carbon fluxes through main pathways of glucose utilization in Escherichia coli cells-glycolysis, pentose phosphate pathway (PPP), and Enther-Doudoroff pathway (EDP)—were studied. Their ratios were analyzed in E. coli strains MG1655, MG1655Δ(edd-eda), MG1655Δ(zwf, edd-eda), and MG1655Δ(pgi, edd-eda). It was shown that the carbon flux through glycolysis was the main route of glucose utilization, averaging ca. 80%. Inactivation of EDP did not affect growth parameters. Nevertheless, it altered carbon fluxes through the tricarboxylic acid cycles and energy metabolism in the cell. Inactivation of PPP decreased growth rate to a lesser degree than glycolysis inactivation.