Metabolic engineering of plant secondary products

Plants interact with their environment by producing a diverse array of secondary metabolites. Many of these compounds are valued for their medicinal, industrial or agricultural properties. Other secondary products are toxic or otherwise undesirable and can reduce the commercial value of crops. Gene transfer technology offers new opportunities to modify directly plant secondary product synthesis through metabolic engineering. This article reviews some of the strategies which have been used to increase or decrease the synthesis of specific plant metabolites, as well as methods for expanding the biosynthetic capabilities of individual species.     

Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures

Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing -galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50. Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium. Specific -galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (3×10⁶ cells mL^–1). In cultures infected at later growth stages, -galactosidase yields could only be maintained by medium replacement. The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined. Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM. Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase. Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific -galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein. Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium.     

Microbial transformation of ethylpyridines

Pyridine and its derivatives have been found as pollutants in the environment. Although alkylpyridines constitute the largest class of pyridines contaminating the environment, little information is available concerning the fate and transformation of these compounds. In this investigation ethylpyridines have been used as model compounds for investigating the biodegradability of alkylpyridines. A mixed culture of ethylpyridine-degrading microorganisms was obtained from a soil that had been exposed to a variety of pyridine derivatives for several decades. The enrichment culture was able to degrade 2-, 3-, and 4-ethylpyridine (100 mg/L) at 28° C and pH 7 within two weeks under aerobic conditions. The degradation rate was greatest for 2-ethylpyridine and least for 3-ethylpyridine. Transformation of ethylpyridines was dependent on substrate concentration, pH, and incubation temperature. Studies on the metabolic pathway of 4-ethylpyridine revealed two products; these chemicals were identified by MS and NMR analyses as 4-ethyl-2(1H)-pyridone and 4-ethyl-2-piperidone. 6-Ethyl-2(1H)-pyridone was determined to be a product of 2-ethylpyridine degradation. These results indicate that the transformation mechanism of ethylpyridines involves hydroxylation and reduction of the aromatic ring before ring cleavage.     

Metabolic engineering of Clostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon

The ability to genetically alter the product-formation capabilities of Clostridium acetobutylicum is necessary for continued progress toward industrial production of the solvents butanol and acetone by fermentation. Batch fermentations at pH 4.5, 5.5, or 6.5 were conducted using C. acetobutylicum ATCC 824 (pFNK6). Plasmid pFNK6 contains a synthetic operon (the "ace operon") in which the three homologous acetone-formation genas (adc, ctfA, and ctfB) are transcribed from the adc promoter. The corresponding enzymes (acetoacetate decarboxylase and CoA-transferase) were best expressed in pH 4.5 fermentations. However, the highest levels of solvents were attained at pH 5.5. Relative to the plasmid-free control strain at pH 5.5, ATCC 824 (pFNK6) produced 95%, 37%, and 90% higher final concentrations of acetone, butanol, and ethanol, respectively; a 50% higher yield (g/g) of solvents on glucose; and a 22-fold lower mass of residual carboxylic acids. At all pH values, the acetone-formation enzymes were expressed earlier with ATCC 824 (pFNK6) than in control fermentations, leading to earlier induction of acetone formation. Furthermore, strain ATCC 824 (pFNK6) produced butanol significantly earlier in the fermentation and produced significant levels of solvents at pH 6.5. Only trace levels of solvents were produced by strain ATCC 824 at pH 6.5. Compared with ATCC 824, a plasmid-control strain containing a vector without the ace operon also produced higher levels of solvents [although lower than those of strain ATCC 824 (pFNK6)] and lower levels of acids. Strains containing plasmid-borne derivatives of the ace operon, in which either the acetoacetate decarboxylase or CoA-transferase alone were expressed at elevated levels, produced acids and solvents at levels similar to those of the plasmid-control strain. (c) 1993 John Wiley & Sons, Inc.     

Proteomics analysis of meclofenamic acid-treated small cell lung carcinoma cells revealed changes in cellular energy metabolism for cancer cell survival

Small cell lung carcinoma (SCLC) is a highly aggressive cancer with low survival rate. Although initial response to chemotherapy in SCLC patients is well-rated, the treatments applied after the disease relapses are not successful. Drug resistance is accepted to be one of the main reasons for this failure. Therefore, there is an urgent need for new treatment strategies for SCLC. Meclofenamic acid, a nonsteroidal anti-inflammatory drug, has been shown to have anticancer effects on various types of cancers via different mechanisms. The aim of this study was to investigate the alterations that meclofenamic acid caused on a SCLC cell line, DMS114 using the tools of proteomics namely two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF and nHPLC coupled to LC-MS/MS. Among the proteins identified by both methods, those showing significantly altered expression levels were evaluated using bioinformatics databases, PANTHER and STRING. The key altered metabolism upon meclofenamic acid treatment appeared to the cellular energy metabolism. Glycolysis was suppressed, whereas mitochondrial activity and oxidative phosphorylation were boosted. The cells underwent metabolic reprogramming to adapt into their new environment for survival. Metabolic reprogramming is known to cause drug resistance in several cancer types including SCLC. The identified differentially regulated proteins in here associated with energy metabolism hold value as the potential targets to overcome drug resistance in SCLC treatment.     

Suppression of mitochondrial alternative oxidase can result in upregulation of the ROS scavenging network: some possible mechanisms underlying the compensation effect

Mitochondrial alternative oxidase is an important protein involved in maintaining cellular metabolic and energy balance, especially under stress conditions. AOX genes knockout is aimed at revealing the functions of AOX genes. Under unfavourable conditions, AOX-suppressed plants (mainly based on Arabidopsis AOX1a-knockout lines) usually experience strong oxidative stress. However, a compensation effect, which consists of the absence of AOX1a leading to an increase in defence response mechanisms, concomitant with a decrease in ROS content, has also been demonstrated. This review briefly describes the possible mechanisms underlying the compensation effect upon the suppression of AOX1a. Information about mitochondrial retrograde regulation of AOX is given. The importance of ROS and mitochondrial membrane potential in triggering the signal transmission from mitochondria in the absence of AOX or disturbance of mitochondrial electron transport chain functions is indicated. The few available data on the response of the cell to the absence of AOX at the level of changes in the hormonal balance and the reactions of chloroplasts are presented. The decrease in the relative amount of reduced ascorbate at stable ROS levels as a result of compensation in AOX1a-suppressed plants is proposed as a sign of stress development. Obtaining direct evidence on the mechanisms and signalling pathways involved in AOX modulation in the genome should facilitate a deeper understanding of the role of AOX in the integration of cellular signalling pathways.     

枯草芽孢杆菌聚谷氨酸合成途径相关基因功能研究

聚谷氨酸(polyglutamic acid, PGA)作为一种天然多功能的聚合物,近年来成为研究的热点。由于很难通过化学方法合成,微生物发酵是目前生产聚谷氨酸的有效途径。【目的】从基因水平探究枯草芽孢杆菌聚谷氨酸合成途径中degS、degQ、degU、swrA、rocA、putM基因的功能,通过分子改造实现对代谢途径的调控。【方法】以枯草芽孢杆菌为出发菌株,通过对代谢途径中相关基因进行敲除或过表达,分别构建degS、degQ和degU基因缺失的重组菌,swrA、rocA和putM基因过表达的重组菌,借助菌株胞外聚谷氨酸积累的变化分析影响途径的关键节点。【结果】在摇瓶发酵条件下,重组菌Bacillus subtilis 168-swrA、Bacillus subtilis 168-rocA、Bacillus subtilis 168-putM的胞外聚谷氨酸含量分别是原始菌株的1.28倍、1.47倍和1.37倍。重组菌Bacillus subtilis 168-ΔdegS、Bacillus subtilis 168-ΔdegQ、Bacillus subtilis 168-ΔdegU的胞外...     

嗜黏蛋白阿克曼氏菌治疗疾病的潜力与作用机制研究进展

嗜黏蛋白阿克曼氏菌(Akkermansia muciniphila, AKK)可促进肠道黏液分泌,维持肠道黏液动态平衡,调节肠黏膜屏障功能,在机体代谢调节、免疫应答中发挥重要作用。AKK对肠道炎症、神经炎症、机体代谢紊乱和癌症等疾病具有显著改善作用,被视为极具潜力的下一代益生菌。本文分别从消化系统、神经系统、代谢性紊乱和癌症等角度入手,系统概述AKK在疾病治疗中的潜力及作用分子机制。     

Phytopathogenic bacteria utilize host glucose as a signal to stimulate virulence through LuxR homologues

Chemical signal-mediated biological communication is common within bacteria and between bacteria and their hosts. Many plant-associated bacteria respond to unknown plant compounds to regulate bacterial gene expression. However, the nature of the plant compounds that mediate such interkingdom communication and the underlying mechanisms remain poorly characterized. Xanthomonas campestris pv. campestris (Xcc) causes black rot disease on brassica vegetables. Xcc contains an orphan LuxR regulator (XccR) which senses a plant signal that was validated to be glucose by HPLC-MS. The glucose concentration increases in apoplast fluid after Xcc infection, which is caused by the enhanced activity of plant sugar transporters translocating sugar and cell-wall invertases releasing glucose from sucrose. XccR recruits glucose, but not fructose, sucrose, glucose 6-phosphate, and UDP-glucose, to activate pip expression. Deletion of the bacterial glucose transporter gene sglT impaired pathogen virulence and pip expression. Structural prediction showed that the N-terminal domain of XccR forms an alternative pocket neighbouring the AHL-binding pocket for glucose docking. Substitution of three residues affecting structural stability abolished the ability of XccR to bind to the luxXc box in the pip promoter. Several other XccR homologues from plant-associated bacteria can also form stable complexes with glucose, indicating that glucose may function as a common signal molecule for pathogen–plant interactions. The conservation of a glucose/XccR/pip-like system in plant-associated bacteria suggests that some phytopathogens have evolved the ability to utilize host compounds as virulence signals, indicating that LuxRs mediate an interkingdom signalling circuit.