Redesign,Reconstruction, and Directed Extension of the Brevibacterium linens C40 Carotenoid Pathway in Escherichia coli

In this study, the carotenoid biosynthetic pathways of Brevibacterium linens DSMZ 20426 were reconstructed, redesigned, and extended with additional carotenoid-modifying enzymes of other sources in a heterologous host Escherichia coli. The modular lycopene pathway synthesized an unexpected carotenoid structure, 3,4-didehydrolycopene, as well as lycopene. Extension of the novel 3,4-didehydrolycopene pathway with the mutant Pantoea lycopene cyclase CrtY₂ and the Rhodobacter spheroidene monooxygenase CrtA generated monocyclic torulene and acyclic oxocarotenoids, respectively. The reconstructed β-carotene pathway synthesized an unexpected 7,8-dihydro-β-carotene in addition to β-carotene. Extension of the β-carotene pathway with the B. linens β-ring desaturase CrtU and Pantoea β-carotene hydroxylase CrtZ generated asymmetric carotenoid agelaxanthin A, which had one aromatic ring at the one end of carotene backbone and one hydroxyl group at the other end, as well as aromatic carotenoid isorenieratene and dihydroxy carotenoid zeaxanthin. These results demonstrate that reconstruction of the biosynthetic pathways and extension with promiscuous enzymes in a heterologous host holds promise as a rational strategy for generating structurally diverse compounds that are hardly accessible in nature.Carotenoids, which are produced by many microorganisms and plants, belong to a class of pigment chemicals found in nature. These structurally diverse pigments have different biological functions such as coloration, photo protection, light-harvesting, and precursors for many hormones (3, 22). Carotenoids are commercially used as food colorants, animal feed supplements and, more recently, as nutraceuticals and as cosmetic and pharmaceutical compounds (19). Currently, only a few carotenoids can be produced commercially by chemical synthesis, fermentation, or isolation from a few abundant natural sources (13). The increasing industrial importance of carotenoids has led to renewed efforts to develop bioprocesses for large-scale production of a range of carotenoids, including lycopene, β-carotene, and more structurally diverse carotenoids (17, 21, 30, 31, 34). Interestingly, a recent study showed that carotenoids with more diverse structures tend to have higher biological activity than simple structures (1).Previously, in vitro evolution altered the catalytic functions of the carotenoid enzymes phytoene desaturase CrtI and lycopene cyclase CrtY (Fig. ​(Fig.1)1) and produced novel carotenoid structures of tetradehydrolycopene and torulene in Escherichia coli (27). Furthermore, these in vitro evolved pathways and redesigned C₃₀ carotenoid biosynthetic pathways were successfully extended with additional, wild-type carotenoid modifying enzymes and evolved enzymes (21), generating novel carotenoid structures (26).Open in a separate windowFIG. 1.Reconstructed and redesigned B. linens carotenoid biosynthetic pathway in the heterologous host E. coli. Carotenogenic enzymes of B. linens, P. ananatis, and R. capsulatus, which were used for the biosynthetic pathway reconstruction, are indicated by boldface letters. Idi (IPP isomerase), IspA (FPP synthase), CrtE (GGPP synthase), CrtB (phytoene synthase), CrtI (phytoene desaturase), CrtYcYd (lycopene cyclase), CrtU (β-carotene desaturase), CrtZ (β-carotene hydrolase), CrtY₂ (mutant lycopene cyclase), and CrtA (spheroidene monooxygenase). B. linens 3,3′-dihydroxyisorenieratene biosynthesis is indicated by dashed arrows.Beside in vitro evolution (23, 34), combinatorial biosynthesis with carotenoid-modifying enzymes in a heterologous host has often been used to generate structurally novel carotenoids (24, 32). This combinatorial biosynthetic approach basically relies on the functional coordination of pathway enzymes from different sources in a heterologous host (5, 19, 35). Carotenogenic enzymes tend to be promiscuous in their substrate specificity (33) and show unexpected/hidden activities (20) when expressed in heterologous host microorganisms. One example is the unusual activity of diapophytoene desaturase CrtN in E. coli, which resulted in structurally novel compounds (20). Therefore, utilizing the promiscuity of carotenogenic enzymes makes combinatorial biosynthesis one of the most powerful strategies to generate structurally novel carotenoids that cannot be accessed in nature.Yellow colored Brevibacterium linens is commonly used as a food colorant by the cheese industry (15). Interestingly, B. linens is known to synthesize aromatic ring-containing carotenoids, isorenieratene and its hydroxy derivatives (6, 7, 16). They are produced by seven carotenogenic enzymes expressed in B. linens: GGPP synthase CrtE, phytoene synthase CrtE, phytoene desaturase CrtI, lycopene cyclase CrtYcYd, β-carotene desaturase CrtU, and the cytochrome P450 (Fig. ​(Fig.1).1). Even though the carotenoid biosynthetic pathways of B. linens have been recently studied (6, 10), there have been no systematic functional study of downstream enzymes such as lycopene cyclase CrtYcYd in the biosynthetic pathway of B. linens in a heterologous environment.Therefore, in the present study, for the first time we reconstructed, redesigned, and rationally extended the B. linens carotenoids biosynthetic pathway in E. coli to investigate the flexibility of the pathway enzymes in a heterologous host. Using this approach, we obtained an unexpected structure 3,4-didehydrolycopene, 7,8-dihydro-β-carotene, torulene, and the asymmetric carotenoid, agelaxanthin A, from engineered B. linens carotenoid pathways in E. coli.     

Identification and Characterization of Pseudomonas syringae pv. syringae,a Causative Bacterium of Apple Canker in Korea

In the present investigation, bacterial isolates from infected apple trees causing apple canker during winter were studied in the northern Gyeongbuk Province, Korea. The pathogen was identified as Pseudomonas syringae pv. syringae (Pss) through various physiological and biochemical characterization assays such as BIOLOG, gas chromatography of fatty acid methyl esters, and 16S rRNA. Bioassays for the production of phytotoxins were positive for syringopeptin and syringomycin against Bacillus megaterium and Geotrichum candidum, respectively. The polymerase chain reaction (PCR) method enabled the detection of toxin-producing genes, syrB1, and sypB in Pss. The differentiation of strains was performed using LOPAT and GATTa tests. Pss further exhibited ice nucleation activity (INA) at a temperature of −0.7°C, indicating an INA⁺ bacterium. The ice-nucleating temperature was −4.7°C for a non-treated control (sterilized distilled water), whereas it was −9.6°C for an INA⁻ bacterium Escherichia coli TOP10. These methods detected pathogenic strains from apple orchards. Pss might exist in an apple tree during ice injury, and it secretes a toxin that makes leaves yellow and cause canker symptoms. Until now, Korea has not developed antibiotics targeting Pss. Therefore, it is necessary to develop effective disease control to combat Pss in apple orchards. Pathogenicity test on apple leaves and stems showed canker symptoms. The pathogenic bacterium was re-isolated from symptomatic plant tissue and confirmed as original isolates by 16S rRNA. Repetitive element sequence-based PCR and enterobacterial repetitive intergenic consensus PCR primers revealed different genetic profiles within P. syringae pathovars. High antibiotic susceptibility results showed the misreading of mRNA caused by streptomycin and oxytetracycline.     

Glycoengineering of the methylotrophic yeast Hansenula polymorpha for the production of glycoproteins with trimannosyl core N-glycan by blocking core oligosaccharide assembly

The initial lipid-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-dolichyl pyrophosphate (Dol-PP) for N-glycan is synthesized and assembled at the membrane of the endoplasmic reticulum (ER) and subsequently transferred to a nascent polypeptide by the oligosaccharide transferase complex. We have identified an ALG3 homolog (HpALG3) coding for a dolichyl-phosphate-mannose dependent alpha-1,3-mannosyltransferase in the methylotrophic yeast Hansenula polymorpha. The detailed analysis of glycan structure by linkage-specific mannosidase digestion showed that HpALG3 is responsible for the conversion of Man5GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP, the first step to attach a mannose to the lipid-linked oligosaccharide in the ER. The N-glycosylation pathway of H. polymorpha has been remodeled by deleting the HpALG3 gene in the Hpoch1 null mutant strain blocked in the yeast-specific outer mannose chain synthesis and by introducing an ER-targeted Aspergillus saitoi alpha-1,2-mannosidase gene. This glycoengineered H. polymorpha strain produced glycoproteins mainly containing trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core backbone of various human-type N-glycans. The results demonstrate the high potential of H. polymorpha to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.     

Synthesis and pharmacological profile of 1-aryl-3-substituted pyrrolo[3,2-c]quinolines.

A series of 1-aryl-3-substituted pyrrolo[3,2-c]quinolines were synthesized and evaluated for their anti-ulcer activity. While 3-substituents of pyrrolo[3,2-c]quinolines mostly affected the in vitro H+/K+ ATPase activity, 1-aryl substituents of pyrrolo[3,2-c]quinolines affected the in vivo gastric acid secretion. In addition, the compounds with good in vivo activity protected from ethanol-induced ulcer.     

The effect of barley yellow dwarf virus on field populations of the cereal aphids, Sitobion avenae and Metopolophium dirhodum

The numbers of cereal aphids, especially Metopolophium dirhodum in 1979, and Sitobion avenae in 1980, were significantly increased on BYDV infected wheat and oats in 1979, and wheat, barley and oats in 1980. The differences were probably caused by attraction of alates of each species to virus infected plants which had changed colour as a result of their infection. Significantly more alates of M. dirhodum were found on virus infected oats in 1979, and of S. avenae on oats and barley in 1980, although not on wheat in either year. probably because the colour contrast in wheat was less intense than in the other crops. Flight chamber experiments with alates of both species confirmed their visual attraction to virus-infected leaves. The interaction between virus, vector and host plants is discussed with reference to the ecology of virus spread.     

Cloning and characterization ofMannheimia succiniciproducens MBEL55E phosphoenolpyruvate carboxykinase (pckA) gene

ApckA gene encoding phosphoenolpyruvate carboxykinase (PEPCK) was cloned and sequenced from the succinic acid producing bacteriumMannheimia succiniciproducens MBEL55E. The gene encoded a 538 residue polypeptide with a calculated molecular mass of 58.8 kDa and a calculated pI of 5.03. The deduced amino acid sequence of theM. succiniciproducens MBEL55E PEPCK was similar to those of all known ATP-dependent PEPCKs.     

A new species of the `Copidognathus tricorneatus' group (Acari: Halacaridae) from India

A new species of the Copidognatus tricorneatus group, C. andhraensis is described on the basis of the specimens collected among the phytal samples from Visakhapatnam, India (Bay of Bengal). Similarities and dissimilarities with the related species are discussed. This is the first record of C. tricorneatus group from India.     

Effects of a synthetic juvenile hormone and some analogues on Ephestia spp. (Lepidoptera: Phycitidae)

Depending on their stage of development, treatment of mature larvae of Ephestia kühniella with a synthetic juvenile hormone resulted in the production of super larvae (which invariably prolonged larval life) and larval-pupal intermediates. When migrating last-instar larvae were treated with the juvenile hormone analogues (JHA) ethyl-3,7, n-trimethyldodeca-2,4-dienoate (ZR512) and isopropyl ii-methoxy-3,7, ii-trimethyldodeca-2,4-dienoate (ZR515), larval-pupal intermediates and pupal mortality were induced. However, when applied topically, ZR515 appeared more effective than ZR512. Both analogues prevented adult emergence when topically applied to the migrating larvae at doses between 28–52 ng. One-day-old pupae were most susceptible while older individuals became less sensitive with age. When larvae pupated in corrugated cardboard rolls were treated with ZR512 those of both E. cautella and E. kiihniella failed to emerge. At an estimated dose of 179 ng cm^-2, ZR515 prevented 77^-6%E. cautella and 100%E. kühniella larvae from emerging as adults. The control of Ephestia by JHA treatment of the pupation sites is discussed.     

Binding affinities of 3-(3-phenylisoxazol-5-yl)methylidene-1-azabicycles to acetylcholine receptors.

A series of 3-(3-phenylisoxazol-5-yl)methylidene-1-azabicycles synthesized showed different binding characteristics to acetylcholine receptors depending on the substituents on the phenyl ring. Small polar substituents gave preferential binding affinity to nicotinic receptors, and large hydrophobic substituents to muscarinic receptors.