heterologous production

异源生产
  • 文章类型: Journal Article
    柚皮素是一种植物多酚,由于其有趣的生物活性而被广泛探索,即抗癌,抗氧化剂,和抗炎。由于其潜在的应用和克服工业需求的尝试,对其异源生产的兴趣增加。产生柚皮素的微生物生物合成途径由酪氨酸氨裂解酶(TAL)组成,4-香豆酸辅酶A连接酶(4CL),查耳酮合成酶(CHS),和查尔酮异构酶(CHI)。在这里,我们通过对途径进行逐步验证和优化,以在大肠杆菌中从头高效生产柚皮素为目标.为此,我们首先通过在三种不同的大肠杆菌菌株中表达来自不同来源的两种TAL基因开始。在酪氨酸过量生产的M-PAR-121菌株中获得了最高的对香豆酸产量(2.54g/L),该菌株携带来自约翰逊黄杆菌(FjTAL)的TAL。之后,该平台菌株用于表达来自不同来源的4CL和CHS基因的不同组合。通过表达FjTAL与拟南芥4CL(At4CL)和南瓜CHS(CmCHS)的结合,可以获得最高的柚皮素查尔酮产量(560.2mg/L)。最后,对不同的CHIs进行了测试和验证,通过表达紫花苜蓿(MsCHI)的CHI与其他先前选择的基因结合,产生765.9mg/L的柚皮素。据我们所知,该滴度对应于迄今在大肠杆菌中报道的柚皮素的最高从头生产。关键点:•选择最佳酶和菌株组合用于从头生产柚皮素。•在遗传和运营优化之后,产生765.9mg/L的柚皮素。•这种从头生产是迄今为止在大肠杆菌中报道的最高的。
    Naringenin is a plant polyphenol, widely explored due to its interesting biological activities, namely anticancer, antioxidant, and anti-inflammatory. Due to its potential applications and attempt to overcome the industrial demand, there has been an increased interest in its heterologous production. The microbial biosynthetic pathway to produce naringenin is composed of tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Herein, we targeted the efficient de novo production of naringenin in Escherichia coli by performing a step-by-step validation and optimization of the pathway. For that purpose, we first started by expressing two TAL genes from different sources in three different E. coli strains. The highest p-coumaric acid production (2.54 g/L) was obtained in the tyrosine-overproducing M-PAR-121 strain carrying TAL from Flavobacterium johnsoniae (FjTAL). Afterwards, this platform strain was used to express different combinations of 4CL and CHS genes from different sources. The highest naringenin chalcone production (560.2 mg/L) was achieved by expressing FjTAL combined with 4CL from Arabidopsis thaliana (At4CL) and CHS from Cucurbita maxima (CmCHS). Finally, different CHIs were tested and validated, and 765.9 mg/L of naringenin was produced by expressing CHI from Medicago sativa (MsCHI) combined with the other previously chosen genes. To our knowledge, this titer corresponds to the highest de novo production of naringenin reported so far in E. coli. KEY POINTS: • Best enzyme and strain combination were selected for de novo naringenin production. • After genetic and operational optimizations, 765.9 mg/L of naringenin was produced. • This de novo production is the highest reported so far in E. coli.
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  • 文章类型: Journal Article
    由于二萜类化合物具有多样的化学结构和优异的生物活性,越来越受到人们的广泛关注。并已发展成为临床药物或消费品。绝大多数的二萜系来源于植物。随着植物药的长远发展,许多植物二萜的自然资源正在减少,关键活性成分的生物合成机理日益成为研究热点。使用合成生物学将微生物改造成“细胞工厂”以生产所需的化合物是解决这些问题的重要手段。在这次审查中,我们从化学结构描述了植物来源的二萜,生物活动,和生物合成途径。我们以具有代表性的植物二萜为例,阐述了其生物合成的研究进展,并总结了近年来植物二萜在微生物中的异源生产,希望为今后植物二萜的开发和应用奠定基础。
    More and more diterpenoids have attracted extensive attention due to the diverse chemical structures and excellent biological activities, and have been developed into clinical drugs or consumer products. The vast majority of diterpenoids are derived from plants. With the long-term development of plant medicinal materials, the natural resources of many plant diterpenoids are decreasing, and the biosynthetic mechanism of key active components has increasingly become a research hotspot. Using synthetic biology to engineer microorganisms into \"cell factories\" to produce the desired compounds is an essential means to solve these problems. In this review, we depict the plant-derived diterpenoids from chemical structure, biological activities, and biosynthetic pathways. We use representative plant diterpenes as examples to expound the research progress on their biosynthesis, and summarize the heterologous production of plant diterpenoids in microorganisms in recent years, hoping to lay the foundation for the development and application of plant diterpenoids in the future.
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  • 文章类型: Journal Article
    真菌聚酮化合物是一大类次生代谢产物,由于其不同的药理活性谱而有价值。丝状真菌中的聚酮化合物生物合成提出了一些挑战:产量小和纯度低。为了解决这些问题,我们改用解脂酵母,一种容易培养的异源宿主。作为一种含油酵母,Y.Lipolytica显示出脂质合成中使用的高通量乙酰基-和丙二酰-CoA前体。同样,乙酰和丙二酰辅酶A是许多天然聚酮化合物的组成部分,我们探索了将这种通量重定向到聚酮化合物生产的可能性。尽管前景看好,迄今为止,Y.Lipolytica仅用于从植物中异源表达简单的III型聚酮合酶(PKS)。因此,我们决定通过靶向I型PKSs合成的更复杂的真菌聚酮来评估Y.Lipolytica的潜力。我们采用了CRISPR-Cas9介导的基因组编辑方法,实现了负责枯萎病镰刀菌(fsr1,fsr2和fsr3)和6-甲基水杨酸(6-MSA)生物合成的基因的无标记基因整合。此外,我们尝试通过过表达两种酶的代谢工程来优化滴度,TGL4和AOX2参与脂质β-氧化,但是我们没有观察到对聚酮化合物生产的影响。最大效价为403mg/L6-MSA和35mg/Lbostrycoidin,后者大大高于我们以前在酿酒酵母中的结果(2.2mg/L),这项工作证明了Y。lipolytica作为异源生产复杂真菌聚酮的平台的潜力。
    Fungal polyketides are a large group of secondary metabolites, valuable due to their diverse spectrum of pharmacological activities. Polyketide biosynthesis in filamentous fungi presents some challenges: small yield and low-purity titers. To tackle these issues, we switched to the yeast Yarrowia lipolytica, an easily cultivable heterologous host. As an oleaginous yeast, Y. lipolytica displays a high flux of acetyl- and malonyl-CoA precursors used in lipid synthesis. Likewise, acetyl- and malonyl-CoA are the building blocks of many natural polyketides, and we explored the possibility of redirecting this flux toward polyketide production. Despite its promising prospect, Y. lipolytica has so far only been used for heterologous expression of simple type III polyketide synthases (PKSs) from plants. Therefore, we decided to evaluate the potential of Y. lipolytica by targeting the more complex fungal polyketides synthesized by type I PKSs. We employed a CRISPR-Cas9-mediated genome editing method to achieve markerless gene integration of the genes responsible for bostrycoidin biosynthesis in Fusarium solani (fsr1, fsr2, and fsr3) and 6-methylsalicylic acid (6-MSA) biosynthesis in Aspergillus hancockii (6MSAS). Moreover, we attempted titer optimization through metabolic engineering by overexpressing two enzymes, TGL4 and AOX2, involved in lipid β-oxidation, but we did not observe an effect on polyketide production. With maximum titers of 403 mg/L 6-MSA and 35 mg/L bostrycoidin, the latter being substantially higher than our previous results in Saccharomyces cerevisiae (2.2 mg/L), this work demonstrates the potential of Y. lipolytica as a platform for heterologous production of complex fungal polyketides.
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  • 文章类型: Journal Article
    建立用于生产商品化学品的可持续过程是当今生物技术行业的主要挑战之一。CO2的化学自养固定以及随后通过厌氧气体发酵由产乙酸细菌产生乙酸盐代表了通过顺序发酵过程在生态上可持续生产高价值生物商品的有希望的平台。在这项研究中,研究了在建立良好的非致病性生产菌株P.putidaKT2440中,含乙酸盐的无细胞废培养基作为生长和重组生产铜绿假单胞菌PAO1单鼠李糖脂的饲养菌株的适用性。此外,通过在含有细胞的A.woodii培养肉汤中培养恶臭假单胞菌,阐明了简化生产过程的潜在可能性,而无需必要分离饲养菌株细胞。对于这些文化,通过qPCR检查菌株排他性基因的相对定量来研究两个菌株的含量。对于无细胞和含细胞的A.woodii用过的培养基,以大约360-400mg/L的最大滴度成功地实现了单鼠李糖脂的重组生产。因此,所报道的方法代表了气体发酵衍生的乙酸盐作为由恶臭假单胞菌KT2440进行的未来重组鼠李糖脂生产工艺的潜在可持续碳源的原理的成功证明。
    The establishment of sustainable processes for the production of commodity chemicals is one of today\'s central challenges for biotechnological industries. The chemo-autotrophic fixation of CO2 and the subsequent production of acetate by acetogenic bacteria via anaerobic gas fermentation represents a promising platform for the ecologically sustainable production of high-value biocommodities via sequential fermentation processes. In this study, the applicability of acetate-containing cell-free spent medium of the gas-fermenting acetogenic bacterium A. woodii WP1 as the feeder strain for growth and the recombinant production of P. aeruginosa PAO1 mono-rhamnolipids in the well-established nonpathogenic producer strain P. putida KT2440 were investigated. Additionally, the potential possibility of a simplified production process without the necessary separation of feeder strain cells was elucidated via the cultivation of P. putida in cell-containing A. woodii culture broth. For these cultures, the content of both strains was investigated by examining the relative quantification of strain-exclusive genes via qPCR. The recombinant production of mono-rhamnolipids was successfully achieved with maximum titers of approximately 360-400 mg/L for both cell-free and cell-containing A. woodii spent medium. The reported processes therefore represent a successful proof of principle for gas fermentation-derived acetate as a potential sustainable carbon source for future recombinant rhamnolipid production processes by P. putida KT2440.
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  • 文章类型: Journal Article
    我们介绍了新分离的sungeiensis链霉菌SD3菌株,作为一种有前途的微生物底盘,用于异源生产次生代谢产物。S.sungeiensisSD3作为微生物底盘表现出几种有利的特征,包括遗传的可操作性,快速增长,对抗生素的敏感性,和支持次生代谢的代谢能力。基因组和转录组测序揭示了S.sungeiensisSD3的主要代谢能力和次要生物合成途径,包括以前未知的负责链塔区B1生物合成的途径。sungeiensisSD3在系统发育树中的独特位置将其指定为类型菌株,将其与其他经常使用的链霉菌底盘区分开来。这种区别使其成为表达生物合成基因簇(BGC)的优选底盘,所述生物合成基因簇源自相同的系统发育或相邻的系统发育进化枝的菌株。来自密切相关但生长缓慢的菌株的二级生物合成途径的成功表达强调了S.sungeiensisSD3作为异源表达底盘的实用性。CRISPR/Cas9辅助的染色体缺失和插入遗传工具的验证为通过合理的基因组编辑进一步的菌株改进和BGC重构铺平了道路。将S.sungeiensisSD3添加到异源底盘工具包中将有助于次生代谢产物的发现和生产。
    We present the newly isolated Streptomyces sungeiensis SD3 strain as a promising microbial chassis for heterologous production of secondary metabolites. S. sungeiensis SD3 exhibits several advantageous traits as a microbial chassis, including genetic tractability, rapid growth, susceptibility to antibiotics, and metabolic capability supporting secondary metabolism. Genomic and transcriptomic sequencing unveiled the primary metabolic capabilities and secondary biosynthetic pathways of S. sungeiensis SD3, including a previously unknown pathway responsible for the biosynthesis of streptazone B1. The unique placement of S. sungeiensis SD3 in the phylogenetic tree designates it as a type strain, setting it apart from other frequently employed Streptomyces chassis. This distinction makes it the preferred chassis for expressing biosynthetic gene clusters (BGCs) derived from strains within the same phylogenetic or neighboring phylogenetic clade. The successful expression of secondary biosynthetic pathways from a closely related yet slow-growing strain underscores the utility of S. sungeiensis SD3 as a heterologous expression chassis. Validation of CRISPR/Cas9-assisted genetic tools for chromosomal deletion and insertion paved the way for further strain improvement and BGC refactoring through rational genome editing. The addition of S. sungeiensis SD3 to the heterologous chassis toolkit will facilitate the discovery and production of secondary metabolites.
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  • 文章类型: Journal Article
    微藻生物技术受到高生产成本和大量种植过程中大量使用水的阻碍。这些缺点可以通过高价值化合物的生产和通过采用代谢工程策略来改善其性能和生产率来缓解。今天,最可持续的方法是利用工业废水进行微藻培养,将有价值的生物质生产与水资源回收相结合。在食品加工部门中,乳制品行业通过制造过程产生的废水量最大。这些流出物通常富含溶解的有机物和营养物质,这使得它成为公司管理的具有挑战性和昂贵的废物流。然而,这些丰富的废水代表了微藻生物技术的吸引力资源。在这项研究中,我们提出了一种可持续的方法,通过蓝藻从乳制品废水中生产高价值的化合物。该策略基于2-苯基乙醇(2-PE)的模型蓝细菌SynechococcuselongatusPCC7942(已在其他地方发表)的代谢工程菌株。2-PE是一种高价值的芳香化合物,由于其令人愉悦的花香,被广泛用作食品和化妆品行业的香料。首先,我们定性评估了四种乳品废水对蓝藻生长的影响,以确定最有希望的底物。洗罐水和废污泥的液体流出物都是合适的营养源。因此,我们创造了一个理想的缓冲系统,通过结合两种废水,同时提供均衡的营养,完全避免了对淡水的需要。耗尽的污泥的75%液态流出物和25%的洗罐水与微调铵的补充相结合,在10天内产生了180mgL-1的2-PE和0.6gDWL-1的生物量浓度。90%的废污泥和10%的洗涤水的混合物产生了最高的2-PE产量(205mgL-1)和生物量积累(0.7gDWL-1),虽然在16天。通过这些治疗,磷酸盐被完全消耗,和氮气在74%-77%的范围内被去除。总的来说,我们的方法显着提高了水循环利用价值,并利用有价值的废水通过微藻生物技术循环生产适销对路的化合物,为后续实施和扩大规模奠定了有希望的基础。
    Microalgae biotechnology is hampered by the high production costs and the massive usage of water during large-volume cultivations. These drawbacks can be softened by the production of high-value compounds and by adopting metabolic engineering strategies to improve their performances and productivity. Today, the most sustainable approach is the exploitation of industrial wastewaters for microalgae cultivation, which couples valuable biomass production with water resource recovery. Among the food processing sectors, the dairy industry generates the largest volume of wastewaters through the manufacturing process. These effluents are typically rich in dissolved organic matter and nutrients, which make it a challenging and expensive waste stream for companies to manage. Nevertheless, these rich wastewaters represent an appealing resource for microalgal biotechnology. In this study, we propose a sustainable approach for high-value compound production from dairy wastewaters through cyanobacteria. This strategy is based on a metabolically engineered strain of the model cyanobacterium Synechococcus elongatus PCC 7942 (already published elsewhere) for 2-phenylethanol (2-PE). 2-PE is a high-value aromatic compound that is widely employed as a fragrance in the food and cosmetics industry thanks to its pleasant floral scent. First, we qualitatively assessed the impact of four dairy effluents on cyanobacterial growth to identify the most promising substrates. Both tank-washing water and the liquid effluent of exhausted sludge resulted as suitable nutrient sources. Thus, we created an ideal buffer system by combining the two wastewaters while simultaneously providing balanced nutrition and completely avoiding the need for fresh water. The combination of 75% liquid effluent of exhausted sludge and 25% tank-washing water with a fine-tuning ammonium supplementation yielded 180 mg L-1 of 2-PE and a biomass concentration of 0.6 gDW L-1 within 10 days. The mixture of 90% exhausted sludge and 10% washing water produced the highest yield of 2-PE (205 mg L-1) and biomass accumulation (0.7 gDW L-1), although in 16 days. Through these treatments, the phosphates were completely consumed, and nitrogen was removed in a range of 74%-77%. Overall, our approach significantly valorized water recycling and the exploitation of valuable wastewaters to circularly produce marketable compounds via microalgae biotechnology, laying a promising groundwork for subsequent implementation and scale-up.
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  • 文章类型: Journal Article
    藏红花素是我国药用植物藏红花中最有价值的成分之一,化妆品,和制药行业。C.sativus的传统种植无法满足全球市场对crocin日益增长的需求,然而,因此,研究人员将注意力转移到各种宿主中藏花素的异源生产上。目前,有报道在大肠杆菌中成功异源生产藏红花素,酿酒酵母,微藻,和不自然产生藏红花素的植物。其中,微藻盐藻,产生高水平的β-胡萝卜素,藏红花素生物合成的底物,值得关注。这篇文章描述了crocin的生物合成,比较每个异源宿主的特征,并阐明了在微藻中高效生产藏红素的要求。
    Crocin is one of the most valuable components of the Chinese medicinal plant Crocus sativus and is widely used in the food, cosmetics, and pharmaceutical industries. Traditional planting of C. sativus is unable to fulfill the increasing demand for crocin in the global market, however, such that researchers have turned their attention to the heterologous production of crocin in a variety of hosts. At present, there are reports of successful heterologous production of crocin in Escherichia coli, Saccharomyces cerevisiae, microalgae, and plants that do not naturally produce crocin. Of these, the microalga Dunaliella salina, which produces high levels of β-carotene, the substrate for crocin biosynthesis, is worthy of attention. This article describes the biosynthesis of crocin, compares the features of each heterologous host, and clarifies the requirements for efficient production of crocin in microalgae.
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  • 文章类型: Journal Article
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  • 文章类型: Journal Article
    链霉菌是重要的工业细菌,其产生药学上有价值的聚酮。然而,工业规模的大规模生产受到生产率低的限制,可以通过代谢工程和宿主菌株的合成生物学来克服。最近,根据微生物生理状态引入自动诱导表达系统已被建议作为工业规模生产聚酮化合物的重要工具。在这项研究中,通过增强聚酮化合物生产所需的CoA衍生前体的集合来提高滴度是以群体感应(QS)依赖性方式驱动的。一个自我维持和独立于诱导者的监管体系,命名为基于QS的前体池代谢工程(QMP)系统,建造,其中参与前体生物合成的基因的表达受QS应答启动子的调控,scbAp.将QMP系统应用于异源宿主中的新荧光素生产,天色链霉菌M1152,生产率提高了4倍。特别是,工程化的超级生产者产生了高水平的新氧素,而不会对细胞生长产生不利影响。总的来说,这项研究表明,由QS介导的自我调节代谢工程具有工程菌株改善聚酮化合物滴度的潜力。
    Streptomyces are important industrial bacteria that produce pharmaceutically valuable polyketides. However, mass production on an industrial scale is limited by low productivity, which can be overcome through metabolic engineering and the synthetic biology of the host strain. Recently, the introduction of an auto-inducible expression system depending on microbial physiological state has been suggested as an important tool for the industrial-scale production of polyketides. In this study, titer improvement by enhancing the pool of CoA-derived precursors required for polyketide production was driven in a quorum sensing (QS)-dependent manner. A self-sustaining and inducer-independent regulatory system, named the QS-based metabolic engineering of precursor pool (QMP) system, was constructed, wherein the expression of genes involved in precursor biosynthesis was regulated by the QS-responsive promoter, scbAp. The QMP system was applied for neoaureothin production in a heterologous host, Streptomyces coelicolor M1152, and productivity increased by up to 4-fold. In particular, the engineered hyperproducers produced high levels of neoaureothin without adversely affecting cell growth. Overall, this study showed that self-regulated metabolic engineering mediated by QS has the potential to engineer strains for polyketide titer improvement.
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  • 文章类型: Journal Article
    MicrocinJ25(MccJ25)和microcinY(MccY)是套索肽,被认为是抗生素和有害防腐剂的潜在替代品。这两种微生物的组合可以提供针对食源性沙门氏菌的广泛的抗微生物谱。目前,MccJ25和MccY是使用大肠杆菌表达系统生产的;然而,整个生产过程都伴随着内毒素的负面影响。在这项研究中,我们确定枯草芽孢杆菌是MccJ25和MccY生产的合适宿主。通过启动子优化实现了微生物的高水平生产,宿主菌株选择,和重组表达。工程菌株产生2.827μMCcJ25和1.481μMCcY的最大产量。这是第一个证明MccJ25和MccY在枯草芽孢杆菌中表达的研究,它提供了一些没有抗生素抗性标记的工程菌株,无诱导物,孢子形成缺陷,并且没有内毒素对抗菌治疗和食品保存的负面影响。
    Microcin J25 (MccJ25) and microcin Y (MccY) are lasso peptides and considered potential alternatives to antibiotics and harmful preservatives. The combination of these two microcins can provide a wide antimicrobial spectrum against food-borne Salmonella. Currently, MccJ25 and MccY are produced using Escherichia coli expression systems; however, the entire production process is accompanied by negative effects from endotoxins. In this study, we identified Bacillus subtilis as a suitable host for MccJ25 and MccY production. High-level production of microcins was achieved by promoter optimization, host strain selection, and recombinant expression. The engineered strains produced maximum yields of 2.827 μM MccJ25 and 1.481 μM MccY. This is the first study to demonstrate the expression of MccJ25 and MccY in B. subtilis, and it offers a few engineered strains that are without antibiotic resistance markers, inducer-free, sporulation-deficient, and free of the negative effects of endotoxins for antibacterial therapy and food preservation.
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