fed-batch fermentation

分批补料发酵
  • 文章类型: Journal Article
    1,3-二羟基丙酮(DHA)是一种重要的商业化学品,广泛用于化妆品中,制药,和食品工业,因为它可以防止水分过度蒸发,并提供抗紫外线保护和抗氧化活性。目前,DHA的工业生产基于使用氧化葡糖杆菌的生物技术合成路线。然而,实现更高的产量需要在合成过程中进行更多的改进。在这项研究中,我们比较了五种工业野生型葡糖杆菌菌株的DHA合成水平,之后,选择氧化银菌WSH-003菌株。然后,16个脱氢酶基因,与DHA合成无关,被单独击倒,一个菌株显着提高DHA的产量,达到89.49gL-1,比野生型菌株高42.27%。通过优化文化媒介,包括种子培养和发酵培养基,DHA产量进一步提高。最后,使用已建立的补料分批发酵系统,在5升生物反应器中,DHA产量达到198.81gL-1,甘油转化率为82.84%。
    1,3-Dihydroxyacetone (DHA) is a commercially important chemical and widely used in cosmetics, pharmaceuticals, and food industries as it prevents excessive water evaporation, and provides anti-ultraviolet radiation protection and antioxidant activity. Currently, the industrial production of DHA is based on a biotechnological synthetic route using Gluconobacter oxydans. However, achieving higher production requires more improvements in the synthetic process. In this study, we compared DHA synthesis levels in five industrial wild-type Gluconobacter strains, after which the G. oxydans WSH-003 strain was selected. Then, 16 dehydrogenase genes, unrelated to DHA synthesis, were individually knocked out, with one strain significantly enhancing DHA production, reaching 89.49 g L-1 and 42.27% higher than the wild-type strain. By optimizing the culture media, including seed culture and fermentation media, DHA production was further enhanced. Finally, using an established fed-batch fermentation system, DHA production reached 198.81 g L-1 in a 5 L bioreactor, with a glycerol conversion rate of 82.84%.
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  • 文章类型: Journal Article
    维生素B12是由微生物合成的复杂化合物。维生素B12的工业生产依赖于特定的微生物发酵过程。大肠杆菌已被用作从头生物合成维生素B12的宿主,掺入了大约30个异源基因。然而,复杂途径中的代谢失衡显著限制了维生素B12的产生。在这项研究中,我们采用多变量模块化代谢工程,通过操纵两个模块,在维生素B12生物合成途径中包含总共10个基因,来提高大肠杆菌中维生素B12的产量.这两个模块被整合到底盘细胞的染色体中,由T7、J23119和J23106启动子调控,实现组合途径优化。通过对由J23119和T7启动子控制的两个模块进行工程改造来获得最高的维生素B12滴度。在发酵培养基中掺入酵母粉可使维生素B12滴度提高至1.52mg/L。这种增强归因于酵母粉对提高氧转移速率和增强菌株的异丙基-β-d-1-硫代半乳糖苷(IPTG)耐受性的影响。最终,通过在5升发酵罐中放大发酵,维生素B12滴度达到2.89mg/L。本文报道的策略将加速利用大肠杆菌的工业规模维生素B12生产的发展。
    Vitamin B12 is a complex compound synthesized by microorganisms. The industrial production of vitamin B12 relies on specific microbial fermentation processes. E. coli has been utilized as a host for the de novo biosynthesis of vitamin B12, incorporating approximately 30 heterologous genes. However, a metabolic imbalance in the intricate pathway significantly limits vitamin B12 production. In this study, we employed multivariate modular metabolic engineering to enhance vitamin B12 production in E. coli by manipulating two modules comprising a total of 10 genes within the vitamin B12 biosynthetic pathway. These two modules were integrated into the chromosome of a chassis cell, regulated by T7, J23119, and J23106 promoters to achieve combinatorial pathway optimization. The highest vitamin B12 titer was attained by engineering the two modules controlled by J23119 and T7 promoters. The inclusion of yeast powder to the fermentation medium increased the vitamin B12 titer to 1.52 mg/L. This enhancement was attributed to the effect of yeast powder on elevating the oxygen transfer rate and augmenting the strain\'s isopropyl-β-d-1-thiogalactopyranoside (IPTG) tolerance. Ultimately, vitamin B12 titer of 2.89 mg/L was achieved through scaled-up fermentation in a 5-liter fermenter. The strategies reported herein will expedite the development of industry-scale vitamin B12 production utilizing E. coli.
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  • 文章类型: Journal Article
    虾青素,一种多功能的C40类胡萝卜素因其在食品中的应用而备受赞誉,化妆品,和健康,是一种具有强大抗氧化性能的鲜红色颜料。为了提高谷氨酸棒杆菌的虾青素产量,我们采用了合理的道路工程策略,专注于提高前体的可用性和优化末端氧官能化C40类胡萝卜素的生物合成。我们的努力导致虾青素前体的供应增加,而菌株BETA6(18mgg-1CDW)的β-胡萝卜素产量提高了1.5倍。通过微调β-胡萝卜素羟化酶基因crtZ和β-胡萝卜素酮酶基因crtW的表达,进一步促进了虾青素的生产。虾青素(菌株ASTA**)增加近五倍,虾青素占总类胡萝卜素的72%。ASTA**已成功转移到2L补料分批发酵中,滴度提高了103mgL-1虾青素,体积生产率为1.5mgL-1h-1。基于该菌株,在糖基转移酶基因crtX的异源表达下,实现了针对糖基化C40类胡萝卜素的途径扩展。据我们所知,这是第一次用谷氨酸棒杆菌生产虾青素-β-D-二葡萄糖苷,达到39mgL-1的高滴度的微生物C40葡萄糖苷。这项研究展示了途径工程解锁新的C40类胡萝卜素变体用于各种工业应用的潜力。
    Astaxanthin, a versatile C40 carotenoid prized for its applications in food, cosmetics, and health, is a bright red pigment with powerful antioxidant properties. To enhance astaxanthin production in Corynebacterium glutamicum, we employed rational pathway engineering strategies, focused on improving precursor availability and optimizing terminal oxy-functionalized C40 carotenoid biosynthesis. Our efforts resulted in an increased astaxanthin precursor supply with 1.5-fold higher β-carotene production with strain BETA6 (18 mg g-1 CDW). Further advancements in astaxanthin production were made by fine-tuning the expression of the β-carotene hydroxylase gene crtZ and β-carotene ketolase gene crtW, yielding a nearly fivefold increase in astaxanthin (strain ASTA**), with astaxanthin constituting 72% of total carotenoids. ASTA** was successfully transferred to a 2 L fed-batch fermentation with an enhanced titer of 103 mg L-1 astaxanthin with a volumetric productivity of 1.5 mg L-1 h-1. Based on this strain a pathway expansion was achieved towards glycosylated C40 carotenoids under heterologous expression of the glycosyltransferase gene crtX. To the best of our knowledge, this is the first time astaxanthin-β-D-diglucoside was produced with C. glutamicum achieving high titers of microbial C40 glucosides of 39 mg L-1. This study showcases the potential of pathway engineering to unlock novel C40 carotenoid variants for diverse industrial applications.
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  • 文章类型: Journal Article
    2'-岩藻糖基乳糖(2'-FL)是母乳中重要的功能性低聚糖之一。到目前为止,通过救助途径生物合成2'-FL的尝试很少报道。在这里,将救助途径酶基因导入大肠杆菌BL21star(DE3),合成2'-FL。通过删除竞争途径上的几个内源基因,2'-FL滴度从1.56g/L增加到2.13g/L。选择α-1,2-岩藻糖基转移酶(WbgL),并将2'-FL滴度提高到2.88g/L。此外,通过优化质粒拷贝数来调整通路酶基因的表达水平。此外,通过与MinDC标签融合,WbgL的空间分布得到增强。优化发酵条件后,2'-FL滴度达到7.13g/L最终菌株在5L发酵罐中产生59.22g/L的2'-FL,乳糖摩尔转化率为95%,岩藻糖摩尔转化率为92%。这些发现将有助于构建高效的微生物细胞工厂以产生2'-FL或其他HMO。
    2\'-Fucosyllactose (2\'-FL) is one of the important functional oligosaccharides in breast milk. So far, few attempts on biosynthesis of 2\'-FL by the salvage pathway have been reported. Herein, the salvage pathway enzyme genes were introduced into the E. coli BL21star(DE3) for synthesis of 2\'-FL. The 2\'-FL titer increased from 1.56 to 2.13 g/L by deleting several endogenous genes on competitive pathways. The α-1,2-fucosyltransferase (WbgL) was selected, and improved the 2\'-FL titer to 2.88 g/L. Additionally, the expression level of pathway enzyme genes was tuned through optimizing the plasmid copy number. Furthermore, the spatial distribution of WbgL was enhanced by fusing with the MinD C-tag. After optimizing the fermentation conditions, the 2\'-FL titer reached to 7.13 g/L. The final strain produced 59.22 g/L of 2\'-FL with 95% molar conversion rate of lactose and 92% molar conversion rate of fucose in a 5 L fermenter. These findings will contribute to construct a highly efficient microbial cell factory to produce 2\'-FL or other HMOs.
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  • 文章类型: Journal Article
    农业食品残留物作为通过微生物发酵生产L-乳酸的原料具有巨大的潜力。威兹曼尼亚凝结菌,以前被称为凝结芽孢杆菌,是一种孢子形成,生产乳酸,革兰氏阳性,具有已知的益生菌和益生元特性。本研究旨在评估利用未经处理的柑橘废物作为可持续原料的一步法生产L-乳酸的可行性,通过使用菌株W.凝结物MA-13。通过使用嗜热酶混合物(CellicCTec2)结合MA-13的水解能力,生物质降解提高了62%。此外,分批和补料分批发酵实验证明了葡萄糖完全发酵为L-乳酸,达到高达44.8g/L的浓度这些结果表明,MA-13是一步生产L-乳酸的微生物细胞工厂,通过将具有成本效益的糖化与MA-13发酵性能相结合,农业食品废物。此外,这种方法对农业废物流的可持续增值的潜力已得到成功证明。关键点:•柑橘废料的估价,在地中海国家有丰富的残留物。•在一步法中可持续生产L-(+)-乳酸。•酶预处理是使用化学品的有价值的替代方法。
    Agri-food residues offer significant potential as a raw material for the production of L-lactic acid through microbial fermentation. Weizmannia coagulans, previously known as Bacillus coagulans, is a spore-forming, lactic acid-producing, gram-positive, with known probiotic and prebiotic properties. This study aimed to evaluate the feasibility of utilizing untreated citrus waste as a sustainable feedstock for the production of L-lactic acid in a one-step process, by using the strain W. coagulans MA-13. By employing a thermophilic enzymatic cocktail (Cellic CTec2) in conjunction with the hydrolytic capabilities of MA-13, biomass degradation was enhanced by up to 62%. Moreover, batch and fed-batch fermentation experiments demonstrated the complete fermentation of glucose into L-lactic acid, achieving a concentration of up to 44.8 g/L. These results point to MA-13 as a microbial cell factory for one-step production of L-lactic acid, by combining cost-effective saccharification with MA-13 fermentative performance, on agri-food wastes. Moreover, the potential of this approach for sustainable valorization of agricultural waste streams is successfully proven. KEY POINTS: • Valorization of citrus waste, an abundant residue in Mediterranean countries. • Sustainable production of the L-( +)-lactic acid in one-step process. • Enzymatic pretreatment is a valuable alternative to the use of chemical.
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  • 文章类型: Journal Article
    L-半胱氨酸是一种与药物相关的氨基酸,食物,饲料,和化妆品行业。其化学生产的环境和社会影响导致开发了更可持续的发酵L-半胱氨酸生产工艺,其中基于葡萄糖和硫代硫酸盐作为硫源的工程大肠杆菌。尽管如此,大多数已发布的工艺都显示出较低的产量。为了进一步确定代谢工程目标,工程大肠杆菌细胞从分批补料生产过程中撤出,然后根据短期扰动实验的数据进行体内代谢控制分析(MCA),代谢组学(LC-MS),和热力学通量分析(TFA)。体内MCA表明,从生产过程中取出的细胞的L-半胱氨酸合酶的活性可能受到限制,我们假设L-半胱氨酸前体O-乙酰丝氨酸(OAS)可能比将OAS转化为L-半胱氨酸更快地从细胞中输出。通过增加L-半胱氨酸合酶的表达,磺基半胱氨酸合酶或L-半胱氨酸合酶,将OAS转化为L-半胱氨酸,在标准化补料分批工艺中实现了比L-半胱氨酸生产率的高达70%和最终L-半胱氨酸浓度的高达47%的提高,从而使葡萄糖的产率提高了超过85%至9.2%(w/w)。关键点:•应用代谢对照分析来分析大肠杆菌的L-半胱氨酸生产•OAS输出比其向L-半胱氨酸的转化更快•L-半胱氨酸合酶的过表达提高了L-半胱氨酸生产率和产量。
    L-cysteine is an amino acid with relevance to the pharmaceutical, food, feed, and cosmetic industry. The environmental and societal impact of its chemical production has led to the development of more sustainable fermentative L-cysteine production processes with engineered E. coli based on glucose and thiosulfate as sulphur source. Still, most of the published processes show low yields. For the identification of further metabolic engineering targets, engineered E. coli cells were withdrawn from a fed-batch production process, followed by in vivo metabolic control analysis (MCA) based on the data of short-term perturbation experiments, metabolomics (LC-MS), and thermodynamic flux analysis (TFA). In vivo MCA indicated that the activities of the L-cysteine synthases of the cells withdrawn from the production process might be limiting, and we hypothesised that the L-cysteine precursor O-acetylserine (OAS) might be exported from the cells faster than it took to transform OAS into L-cysteine. By increasing the expression of the L-cysteine synthases, either sulfocysteine synthase or L-cysteine synthase, which transform OAS into L-cysteine, an improvement of up to 70% in specific L-cysteine productivity and up to 47% in the final L-cysteine concentration was achieved in standardised fed-batch processes thereby increasing the yield on glucose by more than 85 to 9.2% (w/w). KEY POINTS: • Metabolic control analysis was applied to analyse L-cysteine production with E. coli • OAS export was faster than its transformation to L-cysteine • Overexpression of L-cysteine synthases improved L-cysteine productivity and yield.
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  • 文章类型: Journal Article
    生物乙醇是一种可再生燃料,广泛用于公路运输,通常被视为清洁能源。尽管发酵是生物乙醇生产的主要过程之一,关于通过运营设计提高效率的研究有限,特别是与其他步骤(预处理和水解/糖化)相比。在这项研究中,两种适应的喂养策略,其中调整饲料培养基添加(糖输送)以增加可发酵糖的供应,被开发用于提高酿酒酵母5-L补料分批发酵中的乙醇生产率。具体来说,基于细胞生物量的变化,建立了线性适应的饲喂策略,并基于细胞生物量积累开发了指数适应的饲喂策略。通过实施这两种喂养策略,总乙醇生产率达到0.88±0.04和0.87±0.06g/L/h,分别。与固定的脉冲进料操作相比,这对应于乙醇生产率增加~20%。此外,发酵结束时没有残留的葡萄糖,最终乙醇含量在线性适应操作下达到95±3g/L,在指数适应喂养策略下达到104±3g/L。固定和适应喂养策略之间的总乙醇产率(乙醇与糖的比率)未观察到统计学差异(〜91%)。这些结果表明,通过适应的饲喂策略控制的糖输送比固定饲喂操作更有效。导致更高的乙醇生产率。总的来说,这项研究提供了新的适应喂养策略,以提高糖输送和乙醇生产率。整合到乙醇工业的当前实践中可以提高生产率并降低发酵过程的生产成本。
    Bioethanol is a renewable fuel widely used in road transportation and is generally regarded as a clean energy source. Although fermentation is one of the major processes in bioethanol production, studies on improving its efficiency through operational design are limited, especially compared to other steps (pretreatment and hydrolysis/saccharification). In this study, two adapted feeding strategies, in which feed medium addition (sugar delivery) was adjusted to increase the supply of fermentable sugar, were developed to improve ethanol productivity in 5-L fed-batch fermentation by Saccharomyces cerevisiae. Specifically, a linear adapted feeding strategy was established based on changes in cell biomass, and an exponential adapted feeding strategy was developed based on cell biomass accumulation. By implementing these two feeding strategies, the overall ethanol productivity reached 0.88±0.04 and 0.87±0.06 g/L/h, respectively. This corresponded to ~20% increases in ethanol productivity compared to fixed pulsed feeding operations. Additionally, there was no residual glucose at the end of fermentation, and final ethanol content reached 95±3 g/L under the linear adapted operation and 104±3 g/L under the exponential adapted feeding strategy. No statistical difference was observed in the overall ethanol yield (ethanol-to-sugar ratio) between fixed and adapted feeding strategies (~91%). These results demonstrate that sugar delivery controlled by adapted feeding strategies was more efficient than fixed feeding operations, leading to higher ethanol productivity. Overall, this study provides novel adapted feeding strategies to improve sugar delivery and ethanol productivity. Integration into the current practices of the ethanol industry could improve productivity and reduce production costs of fermentation processes.
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  • 文章类型: Journal Article
    化合物K(C-K),最具生物活性的人参皂苷之一,通过水解人参提取物中原人参二醇(PPD)型糖基化人参皂苷的糖苷部分而产生。目的增强西洋参提取物(AGE)中PPD型人参皂苷向C-K的生物转化,饲料类型的优化,浓度,碳源蔗糖和反应物AGE的周期是使用发酵罐在管曲霉的补料分批发酵中进行的。补料分批发酵优化后的C-K浓度(3.94g/L)和生产率(27.4mg/L/h)与分批发酵(1.29g/L和8.96mg/L/h)相比增加了3.1倍。并且实现100%的摩尔转化率。据我们所知,这是首次将人参提取物转化为去糖基化人参皂苷的补料分批发酵试验,也是通过发酵使用人参提取物报道的最高C-K浓度和生产率。乙醇和树脂处理后,从发酵液中获得纯度为59%和96%的C-K固体,作为食品和药物级产品,分别。
    Compound K (C-K), one of the most bioactive ginsenoside, is produced by hydrolyzing the glycoside moieties of protopanaxadiol (PPD)-type glycosylated ginsenosides in the ginseng extract. To enhance the biotransformation of PPD-type ginsenosides in American ginseng extract (AGE) into C-K, the optimization of the feed type, concentration, and period for the carbon source sucrose and the reactant AGE was performed in fed-batch fermentation of Aspergillus tubingensis using a fermenter. The concentration (3.94 g/L) and productivity (27.4 mg/L/h) of C-K after feed optimization in fed-batch fermentation increased 3.1-fold compared to those (1.29 g/L and 8.96 mg/L/h) in batch fermentation, and a molar conversion of 100% was achieved. To the best of our knowledge, this is the first trial of fed-batch fermentation to convert ginseng extract into deglycosylated ginsenoside and the highest reported C-K concentration and productivity using ginseng extract via fermentation. After ethanol and resin treatments, C-K solids with purities of 59% and 96% were obtained from the fermentation broth as food- and pharmaceutical-grade products, respectively.
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  • 文章类型: Journal Article
    紫杉二烯是生物合成高效抗癌药物紫杉醇的重要前体,但其微生物生物合成产率很低。在这项研究中,我们使用Yarrowialipolytica作为微生物宿主来生产紫杉二烯。首先,采用“推拉”策略,使紫杉二烯产量增加234%。然后将紫杉二烯合酶分别与五个增溶标签融合,与SUMO融合时,紫杉二烯产量最大增加62.3%。随后,采用多拷贝迭代积分法进一步提高紫杉二烯滴度,经三轮整合后,摇瓶培养达到23.7mg/L的最大滴度。最后,通过优化分批发酵条件,将紫杉二烯滴度提高到101.4mg/L。这是在Y.lipolytica中完成的紫杉二烯生物合成的第一份报告,作为在这种含油酵母中可持续生产紫杉二烯和其他萜类化合物的一个很好的例子。
    Taxadiene is an important precursor for the biosynthesis of highly effective anticancer drug paclitaxel, but its microbial biosynthesis yield is very low. In this study, we employed Yarrowia lipolytica as a microbial host to produce taxadiene. First, a \"push-pull\" strategy was adopted to increase taxadiene production by 234%. Then taxadiene synthase was fused with five solubilizing tags respectively, leading a maximum increase of 62.3% in taxadiene production when fused with SUMO. Subsequently, a multi-copy iterative integration method was used to further increase taxadiene titer, achieving the maximum titer of 23.7 mg/L in shake flask culture after three rounds of integration. Finally, the taxadiene titer was increased to 101.4 mg/L by optimization of the fed-batch fermentation conditions. This is the first report of taxadiene biosynthesis accomplished in Y. lipolytica, serving as a good example for the sustainable production of taxadiene and other terpenoids in this oleaginous yeast.
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  • 文章类型: Journal Article
    吩嗪-1-羧酸(PCA)是具有预防和控制作物病害能力的生物活性物质。它于2011年被中国农业部认证为农药,并被命名为“参苯霉素”。“Lzh-T5是一种在西红柿根际中发现的绿鱼假单胞菌菌株。该菌株只能产生230mg/L的PCA。我们使用LDA-4,它生产大量的吩嗪合成中间体反式-2,3-二氢-3-羟基邻氨基苯甲酸,作为起始应变。通过恢复phzF和敲除phzO,我们实现了PCA积累。此外,剔除负调节剂后,PCA的产量得到了提高,增强莽草酸途径,进行补料分批发酵,从而产生10,653mg/L的PCA。这表明P.chlooraphisLzh-T5具有成为生物活性物质的高效细胞工厂的潜力。
    Phenazine-1-carboxylic acid (PCA) is a biologically active substance with the ability to prevent and control crop diseases. It was certified as a pesticide by the Ministry of Agriculture of China in 2011 and was named \"Shenzimycin.\" Lzh-T5 is a Pseudomonas chlororaphis strain found in the rhizosphere of tomatoes. This strain can produce only 230 mg/L of PCA. We used LDA-4, which produces the phenazine synthetic intermediate trans-2,3-dihydro-3-hydroxyanthranilic acid in high amounts, as the starting strain. By restoring phzF and knocking out phzO, we achieved PCA accumulation. Moreover, PCA production was enhanced after knocking out negative regulators, enhancing the shikimate pathway, and performing fed-batch fermentation, thus resulting in the production of 10,653 mg/L of PCA. It suggested that P. chlororaphis Lzh-T5 has the potential to become an efficiency cell factory of biologically active substances.
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