Increase of lycopene production by supplementing auxiliary carbon sources in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

In the fed-batch culture of glycerol using a metabolically engineered strain of Escherichia coli, supplementation with glucose as an auxiliary carbon source increased lycopene production due to a significant increase in cell mass, despite a reduction in specific lycopene content. l-Arabinose supplementation increased lycopene production due to increases in cell mass and specific lycopene content. Supplementation with both glucose and l-arabinose increased lycopene production significantly due to the synergistic effect of the two sugars. Cell growth by the consumption of carbon sources was related to endogenous metabolism in the host E. coli. Supplementation with l-arabinose stimulated only the mevalonate pathway for lycopene biosynthesis and supplementation with both glucose and l-arabinose stimulated synergistically only the mevalonate pathway. In the fed-batch culture of glycerol with 10 g l⁻¹ glucose and 7.5 g l⁻¹ l-arabinose, the cell mass, lycopene concentration, specific lycopene content, and lycopene productivity after 34 h were 42 g l⁻¹, 1,350 mg l⁻¹, 32 mg g cells⁻¹, and 40 mg l⁻¹ h⁻¹, respectively. These values were 3.9-, 7.1-, 1.9-, and 11.7-fold higher than those without the auxiliary carbon sources, respectively. This is the highest reported concentration and productivity of lycopene.     

Stability of Lycopene in Oil‐in‐Water Emulsions

Lycopene can be dissolved within the oil phase of oil‐in‐water emulsions to increase bioavailability in water‐dispersible systems. It is sensitive to oxidative conditions and easily undergoes isomerization at high temperatures. Degradation kinetics and isomerization of lycopene in oil‐in‐water‐emulsions were investigated as a function of thermal treatment and oxygen content. Lycopene degradation was found to follow a first‐order kinetics and rate constants were determined. Higher temperatures are directly correlated with increasing lycopene losses. Moreover, thermal treatment leads to a significant decrease of the concentrations of all‐trans and 13‐cis isomer, while the concentration of the 9‐cis isomer increased. Oxygen‐free conditions reduce lycopene losses significantly.     

Enhancing the carotenoid content of <Emphasis Type="Italic">Brassica napus</Emphasis> seeds by downregulating lycopene epsilon cyclase

The accumulation of carotenoids in higher plants is regulated by the environment, tissue type and developmental stage. In Brassica napus leaves, beta-carotene and lutein were the main carotenoids present while petals primarily accumulated lutein and violaxanthin. Carotenoid accumulation in seeds was developmentally regulated with the highest levels detected at 35-40 days post anthesis. The carotenoid biosynthesis pathway branches after the formation of lycopene. One branch forms carotenoids with two beta rings such as beta-carotene, zeaxanthin and violaxanthin, while the other introduces both beta- and epsilon-rings in lycopene to form alpha-carotene and lutein. By reducing the expression of lycopene epsilon-cyclase (epsilon-CYC) using RNAi, we investigated altering carotenoid accumulation in seeds of B. napus. Transgenic seeds expressing this construct had increased levels of beta-carotene, zeaxanthin, violaxanthin and, unexpectedly, lutein. The higher total carotenoid content resulting from reduction of epsilon-CYC expression in seeds suggests that this gene is a rate-limiting step in the carotenoid biosynthesis pathway. epsilon-CYC activity and carotenoid production may also be related to fatty acid biosynthesis in seeds as transgenic seeds showed an overall decrease in total fatty acid content and minor changes in the proportions of various fatty acids.     

沉默lycB基因对雨生红球藻类胡萝卜素合成代谢的影响

雨生红球藻是一种淡水浮游单细胞绿藻, 逆境条件下可积累大量的类胡萝卜素。番茄红素是类胡萝卜素中的一种, 是类胡萝卜素合成代谢中的一个重要中间产物。番茄红素β-环化酶(LycB)是催化番茄红素形成β-胡萝卜素的关键酶。文章以杜氏盐藻lycB基因为干扰序列, 构建了含卡那霉素与阿特拉津双抗性的RNAi载体p1301-BS-RNAi。将其电转化进雨生红球藻细胞, 经抗性筛选、基因组PCR及RT-PCR筛选, 获得了16个独立的干扰株系。选取生长良好的7个进行高光诱导, 发现其番茄红素含量增加了99.4%, β-胡萝卜素含量减少了48.4%, 即通过异源的lycB-RNAi基因沉默可抑制番茄红素向β-胡萝卜素的转化。对比分析发现, 番茄红素增加量仅是β-胡萝卜素减少量的5%, 表明因lycB-RNAi抑制而产生的番茄红素的95%又被其他通路转换成了其他代谢产物, 因此要实现雨生红球藻番茄红素含量的大幅增长, 需协同调控其他代谢通路。     

Identification of novel quantitative trait loci for increased lycopene content and other fruit quality traits in a tomato recombinant inbred line population

Epidemiological and clinical studies indicate that a steady dietary intake of bioavailable lycopene, a C₄₀ carotenoid and potent natural antioxidant, may be associated with a decreased incidence of prostate cancer in humans. Since fresh tomatoes and processed tomato products represent approximately 85% of the average human??s dietary lycopene intake, the identification of novel genetic factors which regulate high fruit lycopene content in tomato is imperative for the improvement of nutritional quality in this commercially valuable specialty crop. To understand the genetic control of the extraordinarily high fruit lycopene content in an accession (LA2093) of the tomato wild species Solanum pimpinellifolium, a quantitative trait locus (QTL) mapping study was conducted using a recombinant inbred line (RIL) population of a cross between LA2093 and a cultivated tomato (S. lycopersicum) breeding line, NCEBR-1. The parental lines, F1 progeny, and F7-F10 RIL populations were grown in replicated field trials in four successive years and evaluated for lycopene content as well as several other traits, including fruit fresh weight, soluble solids content, pH of puree, and plant maturity. The lycopene content of ripe fruit was estimated using three methods: high-performance liquid chromatography (HPLC), spectrophotometry, and colorimetric assays. Based on these measurements, QTL were identified and compared across generations. Among the QTL identified for lycopene, two QTL, located on chromosomes 7 and 12, had very large effects and were consistent across generations. The genomic intervals in which these two QTL reside do not correspond to known map positions of carotenoid biosynthetic genes, indicating that these QTL may represent novel alleles with potentially important implications for tomato breeding as well as increased understanding of carotenoid accumulation in tomato. Several QTL were also identified for fruit weight, soluble solids content and plant maturity. The potential implications of these results for tomato crop improvement are discussed.     

Engineering the lycopene synthetic pathway in <Emphasis Type="Italic">E. coli</Emphasis> by comparison of the carotenoid genes of <Emphasis Type="Italic">Pantoea agglomerans</Emphasis> and <Emphasis Type="Italic">Pantoea ananatis</Emphasis>

The lycopene synthetic pathway was engineered in Escherichia coli using the carotenoid genes (crtE, crtB, and crtI) of Pantoea agglomerans and Pantoea ananatis. E. coli harboring the P. agglomerans crt genes produced 27 mg/l of lycopene in 2YT medium without isopropyl-beta-d-thiogalactopyranoside (IPTG) induction, which was twofold higher than that produced by E. coli harboring the P. ananatis crt genes (12 mg/l lycopene) with 0.1 mM IPTG induction. The crt genes of P. agglomerans proved better for lycopene production in E. coli than those of P. ananatis. The crt genes of the two bacteria were also compared in E. coli harboring the mevalonate bottom pathway, which was capable of providing sufficient carotenoid building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), with exogenous mevalonate supplementation. Lycopene production significantly increased using the mevalonate bottom pathway and 60 mg/l of lycopene was obtained with the P. agglomerans crt genes, which was higher than that obtained with the P. ananatis crt genes (35 mg/l lycopene). When crtE among the P. ananatis crt genes was replaced with P. agglomerans crtE or Archaeoglobus fulgidus gps, both lycopene production and cell growth were similar to that obtained with P. agglomerans crt genes. The crtE gene was responsible for the observed difference in lycopene production and cell growth between E. coli harboring the crt genes of P. agglomerans and P. ananatis. As there was no significant difference in lycopene production between E. coli harboring P. agglomerans crtE and A. fulgidus gps, farnesyl diphosphate (FPP) synthesis was not rate-limiting in E. coli. Sang-Hwal Yoon and Ju-Eun Kim: These authors contributed equally to this work.     

鲜食番茄果实中番茄红素含量的主基因－多基因混合遗传分析

选择2个番茄红素含量显著不同的鲜食番茄品系,通过P₁、P₂、F₁、F₂、B₁和B₂6世代联合分析法,研究分析了番茄红素的遗传规律。结果表明：番茄红素的遗传符合一个主基因和加性－显性－上位性多基因模型,主基因遗传力在B₁、B₂和F₂分别为6.85%、34.78％和58.33％,多基因遗传力在B₁、B₂和F₂分别为58.48％、30.69％和0。      

Effect of two ergosterol biosynthesis inhibitors on lycopene production by Blakeslea trispora

Limiting ergosterol accumulation through metabolic control increased lycopene production by Blakeslea trispora. Lycopene and ergosterol are both biosynthesized from a common precursor, farnesyl diphosphate (FPP). The effects of two ergosterol biosynthesis inhibitors, terbinafine hydrochloride (TH) and ketoconazole, on the production of lycopene by B. trispora were investigated. TH at 0.7 mg/l and ketoconazole at 30 mg/l added to the medium at 48 h of fermentation caused an increase in lycopene content of 23% or 277%, respectively. The timing of addition for both inhibitors at 48 h resulted in the most optimal lycopene productivity, however, compared with TH, ketoconazole was superior in enhancing lycopene production by inhibiting ergosterol biosynthesis.     

Expression profile of genes coding for carotenoid biosynthetic pathway during ripening and their association with accumulation of lycopene in tomato fruits

Fruit ripening process is associated with change in carotenoid profile and accumulation of lycopene in tomato (Solanum lycopersicum L.). In this study, we quantified the β-carotene and lycopene content at green, breaker and red-ripe stages of fruit ripening in eight tomato genotypes by using high-performance liquid chromatography. Among the genotypes, lycopene content was found highest in Pusa Rohini and lowest in VRT-32-1. To gain further insight into the regulation of lycopene biosynthesis and accumulation during fruit ripening, expression analysis of nine carotenoid pathway-related genes was carried out in the fruits of high lycopene genotype—Pusa Rohini. We found that expression of phytoene synthase and β-carotene hydroxylase-1 was four and thirty-fold higher, respectively, at breaker stage as compared to red-ripe stage of fruit ripening. Changes in the expression level of these genes were associated with a 40% increase in lycopene content at red-ripe stage as compared with breaker stage. Thus, the results from our study suggest the role of specific carotenoid pathway-related genes in accumulation of high lycopene during the fruit ripening processes.     

The role of exogenous lipids in lycopene synthesis in the mucoraceous fungus Blakeslea trispora

The addition of plant oils to the growth medium stimulated growth and lipid synthesis in the fungus Blakeslea trispora. However, only oils with high content of linoleic and especially linolenic acid enhanced lycopene formation. The increase in lycopene formation was accompanied by accumulation in the neutral lipid fraction of the fatty acids prevailing in plant oils. In contrast, the influence of exogenous lipids on the fatty acid composition of bulk fungal phospholipids was insignificant. Nonetheless, a marked increase in the content of membrane lipids and of their phosphatidylethanolamine content was revealed. Presumably, the main mechanism of stimulation of lycopene formation by the oils involves an increase in the solubility of lycopene in the triacylglycerols of the lipid bodies, which is due to an increase in the desaturation degree of their fatty acids. The predominance of linoleic and especially of linolenic fatty acid in plant oils is regarded as a criterion for selecting the oil species for the purpose of intensifying lycopene synthesis.     

Ubiquinone and carotene production in the <Emphasis Type="Italic">Mucorales Blakeslea</Emphasis> and <Emphasis Type="Italic">Phycomyces</Emphasis>

The filamentous fungi Phycomyces blakesleeanus and Blakeslea trispora (Zygomycota, Mucorales) are actual or potential industrial sources of β-carotene and lycopene. These chemicals and the large terpenoid moiety of ubiquinone derive from geranylgeranyl pyrophosphate. We measured the ubiquinone and carotene contents of wild-type and genetically modified strains under various conditions. Light slightly increased the ubiquinone content of Blakeslea and had no effect on that of Phycomyces. Oxidative stress modified ubiquinone production in Phycomyces and carotene production in both fungi. Sexual interaction and mutations in both organisms made the carotene content vary from traces to 23 mg/g dry mass, while the ubiquinone content remained unchanged at 0.3 mg/g dry mass. We concluded that the biosyntheses of ubiquinone and carotene are not coregulated. The specific regulation for carotene biosynthesis does not affect even indirectly the production of ubiquinone, as would be expected if terpenoids were synthesized through a branched pathway that could divert precursor flows from one branch to another.     

Precise precursor rebalancing for isoprenoids production by fine control of gapA expression in Escherichia coli

Biosynthesis of isoprenoids via the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway requires equimolar glyceraldehyde 3-phosphate and pyruvate to divert carbon flux toward the products of interest. Here, we demonstrate that precursor balancing is one of the critical steps for the production of isoprenoids in Escherichia coli. First, the implementation of the synthetic lycopene production pathway as a model system and the amplification of the native DXP pathway were accomplished using synthetic constitutive promoters and redesigned 5′-untranslated regions (5′-UTRs). Next, fine-controlled precursor balancing was investigated by tuning phosphoenolpyruvate synthase (PpsA) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The results showed that tuning-down of gapA improved the specific lycopene content by 45% compared to the overexpression of ppsA. The specific lycopene content in the strains with down-regulated gapA increased by 97% compared to that in the parental strain. Our results indicate that gapA is the best target for precursor balancing to increase biosynthesis of isoprenoids.     

Generation of lycopene-overproducing strains of the fungus <Emphasis Type="Italic">Mucor circinelloides</Emphasis> reveals important aspects of lycopene formation and accumulation

Objectives

To generate lycopene-overproducing strains of the fungus Mucor circinelloides with interest for industrial production and to gain insight into the catalytic mechanism of lycopene cyclase and regulatory process during lycopene overaccumulation.

Results

Three lycopene-overproducing mutants were generated by classic mutagenesis techniques from a β-carotene-overproducing strain. They carried distinct mutations in the carRP gene encoding lycopene cyclase that produced loss of enzymatic activity to different extents. In one mutant (MU616), the lycopene cyclase was completely destroyed, and a 43.8% (1.1 mg/g dry mass) increase in lycopene production was observed in comparison to that by the previously existing lycopene overproducer. In addition, feedback regulation of the end product was suggested in lycopene-overproducing strains.

Conclusions

A lycopene-overaccumulating strain of the fungus M. circinelloides was generated that could be an alternative for the industrial production of lycopene. Vital catalytic residues for lycopene cyclase activity and the potential mechanism of lycopene formation and accumulation were identified.

     

果实特异性RNAi介导的Lcy基因沉默来增加番茄中番茄红素的含量

根据GenBank中番茄的番茄红素β-环化酶(Lcy)基因序列和八氢番茄红素去饱和酶基因(Pds)启动子序列设计特异引物从番茄基因组DNA中分别扩增出了Lcy基因的高度保守的长302bp的DNA片段和长1790的Pds启动子片段。根据RNAi的原理,将Lcy基因的DNA片段以正反两个方向通过一段内含子序列连接在一起形成RNAi片段,将该片段与Pds启动子一起插入到pVCT2020的表达载体中,通过农杆菌介导的方法转化番茄,获得转基因植株5棵,PCR检测证实外源片段已成功导入番茄基因组中。收获转色期后20d左右的完全成熟的番茄果实提取番茄红素进行含量分析,结果显示转基因番茄果实中番茄红素的含量极大的增加了。上述结果表明通过RNAi果实特异性的抑制类胡萝卜素代谢途径中生物合成酶基因的表达能够极大的增加番茄果实中番茄红素的含量。这为通过基因工程手段提高番茄果实中的营养价值提供了参考。     

High versus low level expression of the lycopene biosynthesis genes from <Emphasis Type="Italic">Pantoea ananatis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The heterologous synthesis of lycopene in non-carotenogenic Escherichia coli required the introduction of the biosynthesis genes crtE, crtB, and crtI. Recombinant E. coli strains, expressing each lycopene biosynthesis gene from Pantoea ananatis using multi-copy plasmid or single-copies after stable chromosomal integration, were cultivated and the formation of lycopene was investigated. The different expression conditions significantly influenced the lycopene formation as well as the growth behaviour. High plasmid expression levels of crtI with a single copy background of crtE and crtB in E. coli led to a predominate synthesis of tetradehydrolycopene at 253 μg g⁻¹ (cdw).     

Photoprotective potential of lycopene, β-carotene, vitamin E, vitamin C and carnosic acid in UVA-irradiated human skin fibroblasts

The photoprotective potential of the dietary antioxidants vitamin C, vitamin E, lycopene, β-carotene, and the rosemary polyphenol, carnosic acid, was tested in human dermal fibroblasts exposed to ultraviolet-A (UVA) light. The carotenoids were prepared in special nanoparticle formulations together with vitamin C and/or vitamin E. Nanoparticle formulations, in contrast to dimethylsulphoxide, stablized lycopene in the cell culture medium and allowed efficient cellular uptake. The presence of vitamin E in the formulation further increased the stability and cellular uptake of lycopene. UVA irradiation of the human skin fibroblasts led to a 10–15-fold rise in metalloproteinase 1 (MMP-1) mRNA. This rise was suppressed in the presence of low μM concentrations of vitamin E, vitamin C, or carnosic acid but not with β-carotene or lycopene. Indeed, in the presence of 0.5–1.0 μM β-carotene or lycopene, the UVA-induced MMP-1 mRNA was further increased by 1.5–2-fold. This increase was totally suppressed when vitamin E was included in the nanoparticle formulation. Heme-oxygenase 1 (HO-1) mRNA expression was strongly induced by UVA irradiation but none of the antioxidants inhibited this effect at the concentrations used in this study. Indeed, β-carotene or lycopene (0.5–1.0 μM) led to a further 1.5-fold rise in the UVA-induced HO-1 mRNA levels. In conclusion, vitamin C, vitamin E, and carnosic acid showed photoprotective potential. Lycopene and β-carotene did not protect on their own but in the presence of vitamin E, their stability in culture was improved and the rise in MMP-1 mRNA expression was suppressed, suggesting a requirement for antioxidant protection of the carotenoids against formation of oxidative derivatives that can influence the cellular and molecular responses.     

Changes in carotenoid and chlorophyll content of black tomatoes (Lycopersicone sculentum L.) during storage at various temperatures

Black tomatoes have a unique color and higher lycopene content than typical red tomatoes. Here, black tomatoes were investigated how maturation stage and storage temperature affected carotenoid and chlorophyll accumulation. Immature fruits were firmer than mature fruits, but failed to develop their distinctive color and contained less lycopene when stored at 8 °C. Hunter a^* values of black tomatoes increased with storage temperature and duration; storage of immature fruits at high temperature favored lycopene accumulation. Chlorophyll levels of black tomatoes declined during storage, but differences between mature and immature tomatoes stored at 12 °C were minimal. β-Carotene levels of black tomatoes increased during early storage, but rapidly declined beginning 13 d post-harvest. The highest lycopene and chlorophyll levels were observed in mature black tomatoes stored at 12 °C for 13 d; these conditions also yielded the best quality fruit. Thus, the unique pigmentation properties of black tomatoes can be precisely controlled by standardizing storage conditions.