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RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus RNA聚合酶ii驱动的CRISPR-Cas9系统高效非生长偏倚克卢维酵母代谢工程
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00208
Danielle Bever , Ian Wheeldon , Nancy Da Silva

The thermotolerant yeast Kluyveromyces marxianus has gained significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. Despite several contributions to the expanding molecular toolbox for gene expression and metabolic engineering of K. marxianus, there remains a need for a more efficient and versatile genome editing platform. To address this, we developed a CRISPR-based editing system that enables high efficiency marker-less gene disruptions and integrations using only 40 bp homology arms in NHEJ functional and non-functional K. marxianus strains. The use of a strong RNA polymerase II promoter allows efficient expression of gRNAs flanked by the self-cleaving RNA structures, tRNA and HDV ribozyme, from a single plasmid co-expressing a codon optimized Cas9. Implementing this system resulted in nearly 100% efficiency of gene disruptions in both NHEJ-functional and NHEJ-deficient K. marxianus strains, with donor integration efficiencies reaching 50% and 100% in the two strains, respectively. The high gRNA targeting performance also proved instrumental for selection of engineered strains with lower growth rate but improved polyketide biosynthesis by avoiding an extended outgrowth period, a common method used to enrich for edited cells but that fails to recover advantageous mutants with even slightly impaired fitness. Finally, we provide the first demonstration of simultaneous, markerless integrations at multiple loci in K. marxianus using a 2.6 kb and a 7.6 kb donor, achieving a dual integration efficiency of 25.5% in a NHEJ-deficient strain. These results highlight both the ease of use and general robustness of this system for rapid and flexible metabolic engineering in this non-conventional yeast.

近年来,耐热酵母菌马氏克鲁维菌(Kluyveromyces marxianus)作为一种有前景的工业生物制造微生物候选者受到了极大的关注。尽管对K. marxianus的基因表达和代谢工程的分子工具箱有了一些贡献,但仍然需要一个更高效、更通用的基因组编辑平台。为了解决这个问题,我们开发了一种基于crispr的编辑系统,该系统可以在NHEJ功能性和非功能性马氏k.m arxianus菌株中使用仅40 bp的同源臂进行高效的无标记基因破坏和整合。使用强大的RNA聚合酶II启动子,可以从一个共表达密码子优化的Cas9的质粒上有效地表达带有自切割RNA结构、tRNA和HDV核酶的gRNAs。该系统对nhej功能菌株和nhej缺陷菌株的基因破坏效率均接近100%,两株菌株的供体整合效率分别达到50%和100%。高gRNA靶向性能也被证明有助于选择生长速度较低但通过避免延长生长周期而改善聚酮生物合成的工程菌株,这是一种用于富集编辑细胞的常用方法,但无法恢复适应性略有受损的有利突变体。最后,我们首次展示了利用2.6 kb和7.6 kb的供体在马氏K. marxianus的多个位点上同时进行无标记整合,在缺乏nhej的菌株中实现了25.5%的双整合效率。这些结果突出了该系统的易用性和总体稳健性,可用于这种非常规酵母的快速和灵活的代谢工程。
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引用次数: 2
Genome-scale reconstruction and metabolic modelling of the fast-growing thermophile Geobacillus sp. LC300 快速生长的嗜热细菌Geobacillus sp. LC300的基因组重建和代谢模型
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00212
Emil Ljungqvist, Martin Gustavsson

Thermophilic microorganisms show high potential for use as biorefinery cell factories. Their high growth temperatures provide fast conversion rates, lower risk of contaminations, and facilitated purification of volatile products. To date, only a few thermophilic species have been utilized for microbial production purposes, and the development of production strains is impeded by the lack of metabolic engineering tools. In this study, we constructed a genome-scale metabolic model, an important part of the metabolic engineering pipeline, of the fast-growing thermophile Geobacillus sp. LC300. The model (iGEL604) contains 604 genes, 1249 reactions and 1311 metabolites, and the reaction reversibility is based on thermodynamics at the optimum growth temperature. The growth phenotype is analyzed by batch cultivations on two carbon sources, further closing balances in carbon and degree-of-reduction. The predictive ability of the model is benchmarked against experimentally determined growth characteristics and internal flux distributions, showing high similarity to experimental phenotypes.

嗜热微生物在生物炼制细胞工厂中具有很高的应用潜力。它们的高生长温度提供了快速的转化率,较低的污染风险,并促进了挥发性产物的净化。迄今为止,只有少数嗜热菌株被用于微生物生产目的,并且由于缺乏代谢工程工具,生产菌株的开发受到阻碍。在本研究中,我们构建了快速生长的嗜热菌Geobacillus sp. LC300的基因组尺度代谢模型,这是代谢工程管道的重要组成部分。该模型(iGEL604)包含604个基因,1249个反应和1311个代谢物,在最佳生长温度下的反应可逆性基于热力学。通过对两种碳源的批量培养来分析生长表型,进一步接近碳和还原度的平衡。该模型的预测能力以实验确定的生长特性和内部通量分布为基准,显示出与实验表型的高度相似性。
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引用次数: 1
Metabolomics and modelling approaches for systems metabolic engineering 代谢组学和系统代谢工程建模方法
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00209
Jasmeet Kaur Khanijou , Hanna Kulyk , Cécilia Bergès , Leng Wei Khoo , Pnelope Ng , Hock Chuan Yeo , Mohamed Helmy , Floriant Bellvert , Wee Chew , Kumar Selvarajoo

Metabolic engineering involves the manipulation of microbes to produce desirable compounds through genetic engineering or synthetic biology approaches. Metabolomics involves the quantitation of intracellular and extracellular metabolites, where mass spectrometry and nuclear magnetic resonance based analytical instrumentation are often used. Here, the experimental designs, sample preparations, metabolite quenching and extraction are essential to the quantitative metabolomics workflow. The resultant metabolomics data can then be used with computational modelling approaches, such as kinetic and constraint-based modelling, to better understand underlying mechanisms and bottlenecks in the synthesis of desired compounds, thereby accelerating research through systems metabolic engineering. Constraint-based models, such as genome scale models, have been used successfully to enhance the yield of desired compounds from engineered microbes, however, unlike kinetic or dynamic models, constraint-based models do not incorporate regulatory effects. Nevertheless, the lack of time-series metabolomic data generation has hindered the usefulness of dynamic models till today. In this review, we show that improvements in automation, dynamic real-time analysis and high throughput workflows can drive the generation of more quality data for dynamic models through time-series metabolomics data generation. Spatial metabolomics also has the potential to be used as a complementary approach to conventional metabolomics, as it provides information on the localization of metabolites. However, more effort must be undertaken to identify metabolites from spatial metabolomics data derived through imaging mass spectrometry, where machine learning approaches could prove useful. On the other hand, single-cell metabolomics has also seen rapid growth, where understanding cell-cell heterogeneity can provide more insights into efficient metabolic engineering of microbes. Moving forward, with potential improvements in automation, dynamic real-time analysis, high throughput workflows, and spatial metabolomics, more data can be produced and studied using machine learning algorithms, in conjunction with dynamic models, to generate qualitative and quantitative predictions to advance metabolic engineering efforts.

代谢工程涉及通过基因工程或合成生物学方法操纵微生物以产生所需的化合物。代谢组学涉及细胞内和细胞外代谢物的定量,其中经常使用质谱和核磁共振分析仪器。在这里,实验设计、样品制备、代谢物淬火和提取是定量代谢组学工作流程的关键。由此产生的代谢组学数据可以与计算建模方法一起使用,例如动力学和基于约束的建模,以更好地了解所需化合物合成的潜在机制和瓶颈,从而通过系统代谢工程加速研究。基于约束的模型,如基因组规模模型,已经成功地用于提高工程微生物所需化合物的产量,然而,与动力学或动态模型不同,基于约束的模型不包含调节作用。然而,直到今天,缺乏时间序列代谢组学数据的生成阻碍了动态模型的有用性。在这篇综述中,我们表明自动化、动态实时分析和高通量工作流程的改进可以通过时间序列代谢组学数据生成来驱动动态模型生成更多高质量的数据。空间代谢组学也有可能被用作传统代谢组学的补充方法,因为它提供了关于代谢物定位的信息。然而,必须付出更多努力,从通过成像质谱法获得的空间代谢组学数据中识别代谢物,在这方面机器学习方法可能被证明是有用的。另一方面,单细胞代谢组学也得到了快速发展,了解细胞-细胞异质性可以为微生物的有效代谢工程提供更多的见解。展望未来,随着自动化、动态实时分析、高通量工作流程和空间代谢组学的潜在改进,可以使用机器学习算法和动态模型产生和研究更多数据,以生成定性和定量预测,以推进代谢工程工作。
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引用次数: 6
Isobutanol production by combined in vivo and in vitro metabolic engineering 体内体外联合代谢工程生产异丁醇的研究
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00210
Mamta Gupta , Matthew Wong , Kamran Jawed , Kamil Gedeon , Hannah Barrett , Marcelo Bassalo , Clifford Morrison , Danish Eqbal , Syed Shams Yazdani , Ryan T. Gill , Jiaqi Huang , Marc Douaisi , Jonathan Dordick , Georges Belfort , Mattheos A.G. Koffas

The production of the biofuel, isobutanol, in E. coli faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both in vivo with in vitro metabolic engineering to produce isobutanol. The in vivo production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in E. coli and inhibition of competitive valine pathway using CRISPRi technology. The subsequent in vitro conversion to isobutanol was carried out with engineered enzymes for 2-ketoacid decarboxylase (KIVD) and alcohol dehydrogenase (ADH). For the in vivo production of KIV and subsequent in vitro production of isobutanol, this two-step serial approach resulted in yields of 56% and 93%, productivities of 0.62 and 0.074 g L−1 h−1, and titers of 5.6 and 1.78 g L−1, respectively. Thus, this combined biosynthetic system can be used as a modular approach for producing important metabolites, like isobutanol, without the limitations associated with in vivo production using a consolidated bioprocess.

在大肠杆菌中生产生物燃料异丁醇面临着酒精毒性、产品抑制、产品回收和长期工业可行性的限制。在这里,我们展示了一种结合体内和体外代谢工程来生产异丁醇的方法。α-酮异戊酸(KIV)的体内生成是通过CRISPR介导的大肠杆菌双胞设计(BCD)中KIV通路的整合和CRISPRi技术对竞争缬氨酸通路的抑制来实现的。随后用2-酮酸脱羧酶(KIVD)和醇脱氢酶(ADH)工程酶进行体外异丁醇转化。对于KIV的体内生产和随后的体外异丁醇生产,这种两步连续方法的产率分别为56%和93%,产率分别为0.62和0.074 g L−1 h−1,滴度分别为5.6和1.78 g L−1。因此,这种组合的生物合成系统可以作为一种模块化的方法来生产重要的代谢物,如异丁醇,而不受体内使用统一生物过程生产的限制。
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引用次数: 2
Characterization and engineering of the xylose-inducible xylP promoter for use in mold fungal species 木糖诱导型霉菌真菌启动子的鉴定与工程
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00214
Annie Yap , Irene Glarcher , Matthias Misslinger, Hubertus Haas

Conditional promoters allowing both induction and silencing of gene expression are indispensable for basic and applied research. The xylP promoter (pxylP) from Penicillium chrysogenum was demonstrated to function in various mold species including Aspergillus fumigatus. pxylP allows high induction by xylan or its degradation product xylose with low basal activity in the absence of an inducer. Here we structurally characterized and engineered pxylP in A. fumigatus to optimize its application. Mutational analysis demonstrated the importance of the putative TATA-box and a pyrimidine-rich region in the core promoter, both copies of a largely duplicated 91-bp sequence (91bpDS), as well as putative binding sites for the transcription factor XlnR and a GATA motif within the 91bpDS. In agreement, pxylP activity was found to depend on XlnR, while glucose repression appeared to be indirect. Truncation of the originally used 1643-bp promoter fragment to 725 bp largely preserved the promoter activity and the regulatory pattern. Integration of a third 91bpDS significantly increased promoter activity particularly under low inducer concentrations. Truncation of pxylP to 199 bp demonstrated that the upstream region including the 91bpDSs mediates not only inducer-dependent activation but also repression in the absence of inducer. Remarkably, the 1579-bp pxylP was found to act bi-bidirectionally with a similar regulatory pattern by driving expression of the upstream-located arabinofuranosidase gene. The latter opens the possibility of dual bidirectional use of pxylP. Comparison with a doxycycline-inducible TetOn system revealed a significantly higher dynamic range of pxylP. Taken together, this study identified functional elements of pxylP and opened new methodological opportunities for its application.

诱导和沉默基因表达的条件启动子在基础和应用研究中是不可或缺的。从青霉菌中提取的xyylp启动子(pxylP)已被证明在包括烟曲霉在内的多种霉菌中起作用。在没有诱导剂的情况下,木聚糖或其降解产物木糖对pxyylp的诱导作用很高,但基础活性很低。本文对烟曲霉中的pxylP进行了结构表征和工程化,以优化其应用。突变分析证明了核心启动子中假定的TATA-box和富含嘧啶的区域、大量重复的91-bp序列(91bpDS)的两个拷贝以及91bpDS中转录因子XlnR和GATA基序的假定结合位点的重要性。与此一致的是,pxylP活性依赖于XlnR,而葡萄糖抑制似乎是间接的。将最初使用的1643-bp的启动子片段截断为725 bp,在很大程度上保留了启动子活性和调控模式。第三个91bpDS的整合显著提高了启动子活性,特别是在低诱导剂浓度下。截断到199 bp的pxylP表明,包括91bpdp在内的上游区域不仅介导依赖诱导剂的激活,而且在没有诱导剂的情况下介导抑制。值得注意的是,1579-bp的pxylP被发现以类似的调控模式双向作用,通过驱动位于上游的阿拉伯糖醛酸苷酶基因的表达。后者开启了pxyylp双向使用的可能性。与强力霉素诱导的TetOn体系相比,pxylP的动态范围显著提高。综上所述,本研究确定了pxylP的功能元素,为其应用开辟了新的方法学机遇。
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引用次数: 5
Biosynthesis of value-added bioproducts from hemicellulose of biomass through microbial metabolic engineering 利用微生物代谢工程从生物质半纤维素中合成增值生物产品
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00211
Biao Geng , Xiaojing Jia , Xiaowei Peng , Yejun Han

Hemicellulose is the second most abundant carbohydrate in lignocellulosic biomass and has extensive applications. In conventional biomass refinery, hemicellulose is easily converted to unwanted by-products in pretreatment and therefore can't be fully utilized. The present study aims to summarize the most recent development of lignocellulosic polysaccharide degradation and fully convert it to value-added bioproducts through microbial and enzymatic catalysis. Firstly, bioprocess and microbial metabolic engineering for enhanced utilization of lignocellulosic carbohydrates were discussed. The bioprocess for degradation and conversion of natural lignocellulose to monosaccharides and organic acids using anaerobic thermophilic bacteria and thermostable glycoside hydrolases were summarized. Xylose transmembrane transporting systems in natural microorganisms and the latest strategies for promoting the transporting capacity by metabolic engineering were summarized. The carbon catabolite repression effect restricting xylose utilization in microorganisms, and metabolic engineering strategies developed for co-utilization of glucose and xylose were discussed. Secondly, the metabolic pathways of xylose catabolism in microorganisms were comparatively analyzed. Microbial metabolic engineering for converting xylose to value-added bioproducts based on redox pathways, non-redox pathways, pentose phosphate pathway, and improving inhibitors resistance were summarized. Thirdly, strategies for degrading lignocellulosic polysaccharides and fully converting hemicellulose to value-added bioproducts through microbial metabolic engineering were proposed.

半纤维素是木质纤维素生物质中含量第二丰富的碳水化合物,具有广泛的应用。在传统的生物质精炼厂中,半纤维素在预处理过程中容易转化为不需要的副产物,不能得到充分利用。本研究旨在总结木质纤维素多糖降解的最新进展,并通过微生物和酶催化将其充分转化为增值生物产品。首先,讨论了提高木质纤维素碳水化合物利用率的生物工艺和微生物代谢工程。综述了利用厌氧嗜热细菌和耐热糖苷水解酶将天然木质纤维素降解转化为单糖和有机酸的生物过程。综述了天然微生物木糖跨膜转运系统的研究进展以及利用代谢工程提高木糖转运能力的最新策略。讨论了限制微生物利用木糖的碳分解代谢抑制效应,以及开发葡萄糖和木糖共同利用的代谢工程策略。其次,对微生物木糖分解代谢的代谢途径进行了比较分析。综述了基于氧化还原途径、非氧化还原途径、戊糖磷酸途径和提高抑制剂耐药性的木糖转化为增值生物制品的微生物代谢工程。第三,提出了通过微生物代谢工程降解木质纤维素多糖和将半纤维素充分转化为高附加值生物产品的策略。
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引用次数: 4
Metabolic engineering of Pseudomonas taiwanensis VLB120 for rhamnolipid biosynthesis from biomass-derived aromatics 台湾假单胞菌VLB120代谢工程对生物质衍生芳烃合成鼠李糖脂的影响
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2022-12-01 DOI: 10.1016/j.mec.2022.e00202
Vaishnavi Sivapuratharasan , Christoph Lenzen , Carina Michel , Anantha Barathi Muthukrishnan , Guhan Jayaraman , Lars M. Blank

Lignin is a ubiquitously available and sustainable feedstock that is underused as its depolymerization yields a range of aromatic monomers that are challenging substrates for microbes. In this study, we investigated the growth of Pseudomonas taiwanensis VLB120 on biomass-derived aromatics, namely, 4-coumarate, ferulate, 4-hydroxybenzoate, and vanillate. The wild type strain was not able to grow on 4-coumarate and ferulate. After integration of catabolic genes for breakdown of 4-coumarate and ferulate, the metabolically engineered strain was able to grow on these aromatics. Further, the specific growth rate of the strain was enhanced up to 3-fold using adaptive laboratory evolution, resulting in increased tolerance towards 4-coumarate and ferulate. Whole-genome sequencing highlighted several different mutations mainly in two genes. The first gene was actP, coding for a cation/acetate symporter, and the other gene was paaA coding for a phenyl acetyl-CoA oxygenase. The evolved strain was further engineered for rhamnolipid production. Among the biomass-derived aromatics investigated, 4-coumarate and ferulate were promising substrates for product synthesis. With 4-coumarate as the sole carbon source, a yield of 0.27 (Cmolrhl/Cmol4-coumarate) was achieved, corresponding to 28% of the theoretical yield. Ferulate enabled a yield of about 0.22 (Cmolrhl/Cmolferulate), representing 42% of the theoretical yield. Overall, this study demonstrates the use of biomass-derived aromatics as novel carbon sources for rhamnolipid biosynthesis.

木质素是一种普遍可用和可持续的原料,由于其解聚产生一系列芳香单体,这些单体对微生物来说是具有挑战性的底物,因此未得到充分利用。本研究研究台湾假单胞菌VLB120在4-香豆酸酯、阿魏酸酯、4-羟基苯甲酸酯和香草酸酯等生物质芳香烃上的生长情况。野生型菌株不能在4-香豆酸盐和阿魏酸盐上生长。在整合了分解4-香豆酸酯和阿魏酸酯的分解代谢基因后,代谢工程菌株能够在这些芳香化合物上生长。此外,通过适应性实验室进化,该菌株的特定生长速率提高了3倍,从而增加了对4-香豆酸盐和阿魏酸盐的耐受性。全基因组测序强调了主要在两个基因中的几种不同突变。第一个基因是actP,编码一个阳离子/乙酸同调子,另一个基因是paaA,编码一个苯基乙酰辅酶a加氧酶。进化后的菌株进一步用于鼠李糖脂的生产。在研究的生物质衍生芳烃中,4-香豆酸酯和阿魏酸酯是有前景的合成底物。以4-香豆酸酯为唯一碳源,产率为0.27 (Cmolrhl/ cmol4 -香豆酸酯),相当于理论产率的28%。阿魏酸盐的产率约为0.22 (Cmolrhl/Cmolferulate),占理论产率的42%。总之,本研究证明了利用生物质衍生的芳烃作为鼠李糖脂生物合成的新型碳源。
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引用次数: 3
Heterologous phasin expression in Rhodopseudomonas palustris CGA009 for bioplastic production from lignocellulosic biomass 异源phasin在palustris红假单胞菌CGA009中的表达,用于木质纤维素生物质的生物塑料生产
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2022-06-01 DOI: 10.1016/j.mec.2021.e00191
Brandi Brown , Cheryl Immethun , Adil Alsiyabi , Dianna Long , Mark Wilkins , Rajib Saha

Rhodopseudomonas palustris CGA009 is a metabolically robust microbe that can utilize lignin breakdown products to produce polyhydroxyalkanoates (PHAs), biopolymers with the potential to replace conventional plastics. Our recent efforts suggest PHA granule formation is a limiting factor for maximum production of the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by R. palustris. The Phap1 phasin (phaP1) from the PHB-producing model bacterium Cupriavidus necator H16 was expressed in R. palustris with the aim of overproducing PHBV from the lignin breakdown product p-coumarate by fostering smaller and more abundant granules. Expression of phaP1 yielded PHBV production from R. palustris aerobically (0.7 g/L), which does not occur in the wild-type strain, and led to a significantly higher PHBV titer than wild-type anaerobic production (0.41 g/L). The 3HV fractions were also significantly increased under both anaerobic and aerobic conditions, which boosts thermomechanical properties and potential for application. Thus, heterologous phasin expression in R. palustris provides flexibility for industrial processing and could foster compositional changes in copolymers with better thermomechanical properties compared to PHB alone.

palustris红假单胞菌CGA009是一种代谢强大的微生物,可以利用木质素分解产物生产聚羟基烷酸酯(PHAs),这是一种具有替代传统塑料潜力的生物聚合物。我们最近的研究表明,PHA颗粒的形成是R. palustris最大限度地生产生物塑料聚(3-羟基丁酸酯-co-3-羟基戊酸酯)(PHBV)的限制因素。产phb模式细菌Cupriavidus necator H16中的Phap1 phasin (Phap1)在palustris中表达,目的是通过培养更小、更丰富的颗粒,从木质素分解产物p-coumarate中过量生产PHBV。phaP1的表达使palustris菌株产生PHBV (0.7 g/L),这在野生型菌株中没有发生,并且导致PHBV滴度显著高于野生型菌株(0.41 g/L)。在厌氧和好氧条件下,3HV组分也显著增加,这提高了热机械性能和应用潜力。因此,与单独PHB相比,异源phasin在R. palustris中的表达为工业加工提供了灵活性,并且可以促进共聚物的组成变化,从而具有更好的热机械性能。
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引用次数: 11
Application of metabolic engineering to enhance the content of alkaloids in medicinal plants 应用代谢工程技术提高药用植物中生物碱含量
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2022-06-01 DOI: 10.1016/j.mec.2022.e00194
Soledad Mora-Vásquez , Guillermo Gael Wells-Abascal , Claudia Espinosa-Leal , Guy A. Cardineau , Silverio García-Lara

Plants are a rich source of bioactive compounds, many of which have been exploited for cosmetic, nutritional, and medicinal purposes. Through the characterization of metabolic pathways, as well as the mechanisms responsible for the accumulation of secondary metabolites, researchers have been able to increase the production of bioactive compounds in different plant species for research and commercial applications. The intent of the current review is to describe the metabolic engineering methods that have been used to transform in vitro or field-grown medicinal plants over the last decade and to identify the most effective approaches to increase the production of alkaloids. The articles summarized were categorized into six groups: endogenous enzyme overexpression, foreign enzyme overexpression, transcription factor overexpression, gene silencing, genome editing, and co-overexpression. We conclude that, because of the complex and multi-step nature of biosynthetic pathways, the approach that has been most commonly used to increase the biosynthesis of alkaloids, and the most effective in terms of fold increase, is the co-overexpression of two or more rate-limiting enzymes followed by the manipulation of regulatory genes.

植物是生物活性化合物的丰富来源,其中许多已被用于化妆品,营养和药用目的。通过对代谢途径的表征,以及次生代谢物积累的机制,研究人员已经能够在不同的植物物种中增加生物活性化合物的生产,用于研究和商业应用。本综述的目的是描述在过去十年中用于转化体外或田间种植药用植物的代谢工程方法,并确定增加生物碱产量的最有效方法。综述的文章可分为6类:内源性酶过表达、外源性酶过表达、转录因子过表达、基因沉默、基因组编辑和共过表达。我们得出的结论是,由于生物合成途径的复杂性和多步骤性,最常用的增加生物碱生物合成的方法,以及最有效的倍增方法,是两种或两种以上限速酶的共同过表达,然后操纵调节基因。
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引用次数: 16
Control of D-lactic acid content in P(LA-3HB) copolymer in the yeast Saccharomyces cerevisiae using a synthetic gene expression system 合成基因表达系统控制酿酒酵母P(LA-3HB)共聚物中d -乳酸含量
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2022-06-01 DOI: 10.1016/j.mec.2022.e00199
Anna Ylinen , Laura Salusjärvi , Mervi Toivari , Merja Penttilä

The fully biobased polyhydroxyalkanoate (PHA) polymers provide interesting alternatives for petrochemical derived plastic materials. The mechanical properties of some PHAs, including the common poly(3-hydroxybutyrate) (PHB), are limited, but tunable by addition of other monomers into the polymer chain. In this study we present a precise synthetic biology method to adjust lactate monomer fraction of a polymer by controlling the monomer formation in vivo at gene expression level, independent of cultivation conditions. We used the modified doxycycline-based Tet-On approach to adjust the expression of the stereospecific D-lactate dehydrogenase gene (ldhA) from Leuconostoc mesenteroides to control D-lactic acid formation in yeast Saccharomyces cerevisiae. The synthetic Tet-On transcription factor with a VP16 activation domain was continuously expressed and its binding to a synthetic promoter with eight transcription factor specific binding sites upstream of the ldhA gene was controlled with the doxycycline concentration in the media. The increase in doxycycline concentration correlated positively with ldhA expression, D-lactic acid production, poly(D-lactic acid) (PDLA) accumulation in vivo, and D-lactic acid content in the poly(D-lactate-co-3-hydroxybutyrate) P(LA-3HB) copolymer. We demonstrated that the D-lactic acid content of the P(LA-3HB) copolymer can be adjusted linearly from 6 mol% to 93 mol% in vivo in S. cerevisiae. These results highlight the power of controlling gene expression and monomer formation in the tuning of the polymer composition. In addition, we obtained 5.6% PDLA and 19% P(LA-3HB) of the cell dry weight (CDW), which are over two- and five-fold higher accumulation levels, respectively, than reported in the previous studies with yeast. We also compared two engineered PHA synthases and discovered that in S. cerevisiae the PHA synthase PhaC1437Ps6-19 produced P(LA-3HB) copolymers with lower D-lactic acid content, but with higher molecular weight, in comparison to the PHA synthase PhaC1Pre.

全生物基聚羟基烷酸酯(PHA)聚合物为石油化工衍生塑料材料提供了有趣的替代品。包括常见的聚(3-羟基丁酸酯)(PHB)在内的一些pha的机械性能是有限的,但可以通过在聚合物链中加入其他单体来调节。在这项研究中,我们提出了一种精确的合成生物学方法,通过在基因表达水平上控制单体的形成来调节聚合物的乳酸单体比例,而不依赖于培养条件。我们采用改良的基于多西环素的Tet-On方法调节mesenterostoc中立体特异性d -乳酸脱氢酶基因(ldhA)的表达,以控制酿酒酵母(Saccharomyces cerevisiae)中d -乳酸的形成。具有VP16活化结构域的合成Tet-On转录因子连续表达,其与ldhA基因上游具有8个转录因子特异性结合位点的合成启动子的结合受到培养基中强力霉素浓度的控制。多西环素浓度的升高与ldhA表达、d -乳酸生成、体内聚d -乳酸(PDLA)积累以及聚d -乳酸-co-3-羟基丁酸)P(LA-3HB)共聚物中d -乳酸含量呈正相关。我们证明了P(LA-3HB)共聚物的d -乳酸含量可以在酿酒酵母体内从6 mol%到93 mol%的范围内线性调节。这些结果突出了控制基因表达和单体形成在聚合物组成调节中的作用。此外,我们获得了细胞干重(CDW)的5.6%的PDLA和19%的P(LA-3HB),分别比以前用酵母研究报道的积累水平高出2倍和5倍。我们还比较了两种工程PHA合成酶,发现在酿酒酵母中,PHA合成酶PhaC1437Ps6-19产生的P(LA-3HB)共聚物与PHA合成酶PhaC1Pre相比,d -乳酸含量更低,但分子量更高。
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引用次数: 0
期刊
Metabolic Engineering Communications
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