共查询到20条相似文献，搜索用时 0 毫秒
1.
David Skelding Samuel F M Hart Thejas Vidyasagar Alexander E Pozhitkov Wenying Shou 《Quantitative Biology.》2018,6(2):129
Background: Multiplexed milliliter-scale chemostats are useful for measuring cell physiology under various degrees of nutrient limitation and for carrying out evolution experiments. In each chemostat, fresh medium containing a growth rate-limiting metabolite is pumped into the culturing chamber at a constant rate, while culture effluent exits at an equal rate. Although such devices have been developed by various labs, key parameters — the accuracy, precision, and operational range of flow rate — are not explicitly characterized. Methods: Here we re-purpose a published multiplexed culturing device to develop a multiplexed milliliter-scale chemostat. Flow rates for eight chambers can be independently controlled to a wide range, corresponding to population doubling times of 3~13 h, without the use of expensive feedback systems. Results: Flow rates are precise, with the maximal coefficient of variation among eight chambers being less than 3%. Flow rates are accurate, with average flow rates being only slightly below targets, i.e., 3%–6% for 13-h and 0.6%–1.0% for 3-h doubling times. This deficit is largely due to evaporation and should be correctable. We experimentally demonstrate that our device allows accurate and precise quantification of population phenotypes. Conclusions: We achieve precise control of cellular growth in a low-cost milliliter-scale chemostat array, and show that the achieved precision reduces the error when measuring biological processes. 相似文献
2.
This review is devoted to the problems of the physiology and cell biology of microorganisms in relation to metabolic engineering.
The latter is considered as a branch of fundamental and applied biotechnology aimed at controlling microbial metabolism by
methods of genetic engineering and classical genetics and based on intimate knowledge of cell metabolism. Attention is also
given to the problems associated with the metabolic limitation of microbial biosyntheses, analysis and control of metabolic
fluxes, rigidity of metabolic pathways, the role of pleiotropic (global) regulatory systems in the control of metabolic fluxes,
and prospects of physiological and evolutionary approaches in metabolic engineering. 相似文献
3.
The methionine salvage pathway (MSP) regenerates methionine from 5′-methylthioadenosine (MTA). Aerobic MSP consists of six enzymatic steps. The mug14+ and adi1+ genes that are involved in the third and fifth steps of the pathway are repressed when Schizosaccharomyces pombe undergoes a transition from high- to low-iron conditions. Results consistently show that methionine auxotrophic cells (met6Δ) require iron for growth in the presence of MTA as the sole source of methionine. Inactivation of the iron-using protein Adi1 leads to defects in the utilization of MTA. In the case of the third step of the pathway, co-expression of two distinct proteins, Mta3 and Mde1, is required. These proteins are interdependent to rescue MTA-dependent growth deficit of met6Δ cells. Coimmunoprecipitation experiments showed that Mta3 is a binding partner of Mde1. Meiotic met6Δ cells co-expressing mta3+ and mde1+ or mta3+ and mug14+ produce comparable levels of spores in the presence of MTA, revealing that Mde1 and Mug14 share a common function when co-expressed with Mta3 in sporulating cells. In sum, our findings unveil several novel features of MSP, especially with respect to its regulation by iron and the discovery of a non-canonical third enzymatic step in the fission yeast. 相似文献
4.
Isoprenoids are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages to isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway. 相似文献
5.
6.
白藜芦醇是一种有价值的植物多酚类化合物,目前主要通过植物提取法获得,受到天然原料的限制,不易长期大量获取,因此有必要开发一种更加简便、经济的白藜芦醇生产方法。本研究以树干毕赤酵母的野生型菌株为宿主,首先通过引入编码白藜芦醇合成的关键酶基因(HaTAL1、AtPAL2、AtC4H、At4CL2以及VvSTS)得到菌株Ss05,合成白藜芦醇55.28 mg/L。随后通过过表达3-脱氧-d-阿拉伯庚糖酮酸-7-磷酸(3-deoxy-d-arabino-heptulonate-7-phosphate, DAHP)合成酶突变体SsARO4K221L及分支酸变位酶突变体SsARO7G139S、敲除丙酮酸脱羧酶基因(PDC1)、过表达乙酰辅酶A羧化酶突变体SsACC1S650A,S1152A,增强对香豆酸及丙二酰辅酶A的供应。在此基础上,进一步增加关键基因的拷贝数得到工程菌株Ss17,白藜芦醇产量提高到150.56 mg/L。最后将工程菌株Ss17在5 L发酵罐中通过分批补料葡萄糖发酵128 h,得到了558.40 mg/L白藜芦醇。本研究运用合成生物学思路,构建利用简单碳源合成白藜芦醇的工程菌株,同时利用生物反应器放大微生物发酵规模,为树干毕赤酵母中芳香族化合物的生物合成提供了重要参考。 相似文献
7.
8.
Christina Schmidt;Marco Aras;Oliver Kayser; 《Biotechnology journal》2024,19(2):2300507
Phytocannabinoids are natural products with highly interesting pharmacological properties mainly produced by plants. The production of cannabinoids in a heterologous host system has gained interest in recent years as a promising alternative to production from plant material. However, the systems reported so far do not achieve industrially relevant titers, highlighting the need for alternative systems. Here, we show the production of the cannabinoids cannabigerolic acid and cannabigerol from glucose and hexanoic acid in a heterologous yeast system using the aromatic prenyltransferase NphB from Streptomyces sp. strain CL190. The production was significantly increased by introducing a fusion protein consisting of ERG20WW and NphB. Furthermore, we improved the production of the precursor olivetolic acid to a titer of 56 mg L−1. The implementation of the cannabinoid synthase genes enabled the production of Δ9-tetrahydrocannabinolic acid, cannabidiolic acid as well as cannabichromenic acid, where the heterologous biosynthesis of cannabichromenic acid in a yeast system was demonstrated for the first time. In addition, we found that the product spectrum of the cannabinoid synthases localized to the vacuoles of the yeast cells was highly dependent on extracellular pH, allowing for easy manipulation. Finally, using a fed-batch approach, we showed cannabigerolic acid and olivetolic acid titers of up to 18.2 mg L−1 and 117 mg L−1, respectively. 相似文献
9.
Metabolic engineering of yeast: the perils of auxotrophic hosts 总被引：3，自引：0，他引：3
Auxotrophic mutants may have physiological alterations and sensitivities which are not generally recognized. Such features are shown here by observations that final cell densities attained by several leucine-auxotrophic Saccharomyces cerevisiae strains depend differently on the initial leucine concentration in the medium. Furthermore, complementing such auxotrophic strains with the plasmid-based LEU2 selection marker resulted in different final cell densities than chromosomal expression of LEU2 in the otherwise isogenic, prototrophic strains. These results warn that auxotrophic host-related physiological influences overlay any metabolic effect of a cloned gene expressed in such a host, clearly complicating interpretation of the effect of that gene's product in scientific or metabolic engineering research. 相似文献
10.
11.
Shun Shimazaki;Ryosuke Yamada;Yoshiki Yamamoto;Takuya Matsumoto;Hiroyasu Ogino; 《Biotechnology journal》2024,19(1):2300285
Simultaneous modification of the expression levels of many metabolic enzyme genes results in diverse expression ratios of these genes; however, the relationship between gene expression levels and chemical productivity remains unclear. However, clarification of this relationship is expected to improve the productivity of useful chemicals. Supervised machine learning is considered to be an effective means to clarify this relationship. In this study, to improve the productivity of carotenoids in yeast Saccharomyces cerevisiae, we aimed to build a machine-learning model that can predict the optimal gene expression level for carotenoid production. First, we obtained data on the expression levels of mevalonate pathway enzyme genes and carotenoid production. Then, based on these data, we built a machine-learning model to predict carotenoid productivity based on gene expression levels. The prediction accuracy of 0.6292 (coefficient of determination) was achieved using the test data. The maximum predicted carotenoid productivity was 4.3 times higher in the engineered strain than in the parental strain, suggesting that the expression levels of the mevalonate pathway enzyme genes tHMG1 and ERG8 have a particularly large impact on carotenoid productivity. This study could be one of the important achievements in addressing the uncertainty of genotype-phenotype correlations, which is one of the challenges facing metabolic engineering strategies. 相似文献
12.
《Critical reviews in biotechnology》2013,33(1-2):65-86
AbstractA decade or so ago, there was considerable interest in developing single cell protein production from raw materials. Many factors have influenced the development of fodder yeast technology, notably the biochemistry and physiology of the yeast.It is shown that those considerations have led to the choice of a continuous fermentation technology. 相似文献
13.
充分利用木质纤维素中的糖分是提高以此类生物质为原料生产二代燃料乙醇经济盈利性的基本要求,也是实现其他生物基化学品规模化生产的基础。传统的乙醇生产微生物酿酒酵母Saccharomyces cerevisiae具有独特的生产性能及内在优势,是备受关注的底盘细胞,但其不能有效地利用戊糖。利用代谢工程、合成生物学策略,对二代燃料乙醇生产专用酿酒酵母的精准构制持续研究了30余年,已明显改善了其对木糖/葡萄糖的乙醇共发酵能力。近年来关注点集中在早期忽略的限速步骤即糖转运环节的研究上,以期实现不同糖分各行其道、高效专一性转运蛋白各行其责的二代燃料乙醇生产特种酿酒酵母所需的糖转运理想状态。文中主要综述了酿酒酵母戊糖转运蛋白的研究进展,及酿酒酵母的木糖和L-阿拉伯糖代谢工程的研究现状。 相似文献
14.
丁二酸(又称琥珀酸)是一种重要的C4平台化合物,可作为1,4-丁二醇、四氢呋喃以及生物可降解塑料聚丁二酸丁二醇酯(polybutylene succinate, PBS)的生产原料。与传统的以顺酐为原料的石化基路线相比,采用微生物发酵法生产丁二酸不仅具有更高的经济可持续性,同时也展现出更佳的环境友好性。酵母具有良好的耐酸性,能够实现丁二酸的低pH发酵,从而大幅降低产物提取成本。因此,通过代谢工程改造构建高产丁二酸酵母菌株受到越来越多的关注。本文系统介绍了丁二酸的应用价值及其市场规模,总结了微生物中参与丁二酸合成的途径及其关键酶,详细阐述了利用酵母细胞工厂合成丁二酸的最新研究进展,同时还展示了酵母工程菌株以甘油、乙酸、木质纤维素水解液等非粮原料为底物进行丁二酸合成的现状,最后对基于酵母细胞工厂的低pH丁二酸生物制造进行了展望。 相似文献
15.
16.
以选育低pH条件下高产L-乳酸的酵母菌为目的,从自然样品中筛选分离得到一株能在pH 2.5 (乳酸调节) 的培养基中生长且不利用乳酸的酵母 (初步鉴定为木兰假丝酵母Candida magnolia);进一步将来源于米根霉As3.819的乳酸脱氢酶编码基因 (ldhA) 插入含有G418抗性基因的酵母穿梭载体,构建了重组质粒pYX212-kanMX-ldhA,电转化入野生型C. magnolia中,筛选获得了一株具有产L-乳酸能力的重组菌株C. magnolia-2;通过发酵实验表明,该重组菌产L-乳酸的最 相似文献
17.
Aims: The aim of this study was to determine sulphite tolerance for a large number of Dekkera bruxellensis isolates and evaluate the relationship between this phenotype and previously assigned genotype markers. Methods and Results: A published microplate‐based method for evaluation of yeast growth in the presence of sulphite was benchmarked against culturability following sulphite treatment, for the D. bruxellensis type strain (CBS 74) and a reference wine isolate (AWRI 1499). This method was used to estimate maximal sulphite tolerance for 41 D. bruxellensis isolates, which was found to vary over a fivefold range. Significant differences in sulphite tolerance were observed when isolates were grouped according to previously assigned genotypes and ribotypes. Conclusions: Variable sulphite tolerance for the wine spoilage yeast D. bruxellensis can be linked to genotype markers. Significance and Impact of the Study: Strategies to minimize risk of wine spoilage by D. bruxellensis must take into account at least a threefold range in effective sulphite concentration that is dependent upon the genotype group(s) present. The isolates characterized in this study will be a useful resource for establishing the mechanisms conferring sulphite tolerance for this industrially important yeast species. 相似文献
18.
Ahmed Zahoor Felix T. F. Küttner Lars M. Blank Birgitta E. Ebert 《Engineering in Life Science》2019,19(10):711-720
Dicarboxylic acids are important bio‐based building blocks, and Saccharomyces cerevisiae is postulated to be an advantageous host for their fermentative production. Here, we engineered a pyruvate decarboxylase‐negative S. cerevisiae strain for succinic acid production to exploit its promising properties, that is, lack of ethanol production and accumulation of the precursor pyruvate. The metabolic engineering steps included genomic integration of a biosynthesis pathway based on the reductive branch of the tricarboxylic acid cycle and a dicarboxylic acid transporter. Further modifications were the combined deletion of GPD1 and FUM1 and multi‐copy integration of the native PYC2 gene, encoding a pyruvate carboxylase required to drain pyruvate into the synthesis pathway. The effect of increased redox cofactor supply was tested by modulating oxygen limitation and supplementing formate. The physiologic analysis of the differently engineered strains focused on elucidating metabolic bottlenecks. The data not only highlight the importance of a balanced activity of pathway enzymes and selective export systems but also shows the importance to find an optimal trade‐off between redox cofactor supply and energy availability in the form of ATP. 相似文献
19.
Saccharomyces cerevisiae was able to produce 20% (v/v) of ethanol in 45 h in a fully aerated fed-batch process recently developed in our laboratory. A notable feature of this process was a production phase uncoupled to growth, the extent of which was critical for high-level ethanol production. As the level of production was found to be highly variable, we investigated on this high variability by means of a detailed physiological analysis of yeast cells in two fed-batch fermentations showing the most extreme behaviour. We found a massive leakage of intracellular metabolites into the growth medium which correlated with the drop of cell viability. The loss of viability was also found to be proportional to the reduction of plasma membrane phospholipids. Finally, the fed-batch processes with the longest uncoupling phase were characterized by induction of storage carbohydrates at the onset of this phase, whereas this metabolic event was not seen in processes with a short uncoupling phase. Taken together, our results suggested that reproducible high-level bioethanol production in aerated fed-batch processes may be linked to the ability of yeast cells to impede ethanol toxicity by triggering a metabolic remodelling reminiscent to that of cells entering a quiescent GO/G1 state. 相似文献
20.