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Analysis of metabolic network disruption in engineered microbial hosts due to enzyme promiscuity 酶乱交导致工程微生物宿主代谢网络中断的分析
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2021-06-01 DOI: 10.1016/j.mec.2021.e00170
Vladimir Porokhin , Sara A. Amin , Trevor B. Nicks , Venkatesh Endalur Gopinarayanan , Nikhil U. Nair , Soha Hassoun

Increasing understanding of metabolic and regulatory networks underlying microbial physiology has enabled creation of progressively more complex synthetic biological systems for biochemical, biomedical, agricultural, and environmental applications. However, despite best efforts, confounding phenotypes still emerge from unforeseen interplay between biological parts, and the design of robust and modular biological systems remains elusive. Such interactions are difficult to predict when designing synthetic systems and may manifest during experimental testing as inefficiencies that need to be overcome. Transforming organisms such as Escherichia coli into microbial factories is achieved via several engineering strategies, used individually or in combination, with the goal of maximizing the production of chosen target compounds. One technique relies on suppressing or overexpressing selected genes; another involves introducing heterologous enzymes into a microbial host. These modifications steer mass flux towards the set of desired metabolites but may create unexpected interactions. In this work, we develop a computational method, termed Metabolic Disruption Workflow (MDFlow), for discovering interactions and network disruptions arising from enzyme promiscuity – the ability of enzymes to act on a wide range of molecules that are structurally similar to their native substrates. We apply MDFlow to two experimentally verified cases where strains with essential genes knocked out are rescued by interactions resulting from overexpression of one or more other genes. We demonstrate how enzyme promiscuity may aid cells in adapting to disruptions of essential metabolic functions. We then apply MDFlow to predict and evaluate a number of putative promiscuous reactions that can interfere with two heterologous pathways designed for 3-hydroxypropionic acid (3-HP) production. Using MDFlow, we can identify putative enzyme promiscuity and the subsequent formation of unintended and undesirable byproducts that are not only disruptive to the host metabolism but also to the intended end-objective of high biosynthetic productivity and yield. As we demonstrate, MDFlow provides an innovative workflow to systematically identify incompatibilities between the native metabolism of the host and its engineered modifications due to enzyme promiscuity.

对微生物生理学基础上的代谢和调控网络的日益了解,使越来越复杂的合成生物系统能够用于生化、生物医学、农业和环境应用。然而,尽管尽了最大的努力,混淆表型仍然出现在不可预见的生物部分之间的相互作用,稳健和模块化的生物系统的设计仍然难以捉摸。这种相互作用在设计合成系统时很难预测,并可能在实验测试中表现为需要克服的低效率。将大肠杆菌等生物转化为微生物工厂是通过几种工程策略来实现的,这些策略可以单独使用,也可以组合使用,目的是使选定的目标化合物的产量最大化。一种技术依赖于抑制或过度表达选定的基因;另一种方法是将异源酶引入微生物宿主。这些修饰将质量通量导向所需的代谢物集,但可能产生意想不到的相互作用。在这项工作中,我们开发了一种称为代谢破坏工作流(MDFlow)的计算方法,用于发现酶滥交引起的相互作用和网络破坏-酶作用于结构上与其天然底物相似的广泛分子的能力。我们将MDFlow应用于两个经过实验验证的案例,其中必需基因被敲除的菌株通过一个或多个其他基因过表达引起的相互作用而获救。我们展示了酶乱交如何帮助细胞适应基本代谢功能的破坏。然后,我们应用MDFlow来预测和评估一些可能干扰3-羟基丙酸(3-HP)生产的两种异源途径的假定混杂反应。使用MDFlow,我们可以识别假定的酶乱交和随后形成的意外和不希望的副产物,这些副产物不仅破坏宿主代谢,而且破坏高生物合成生产力和产量的预期最终目标。正如我们所展示的,MDFlow提供了一种创新的工作流程,可以系统地识别宿主的天然代谢与其由于酶混杂而引起的工程修饰之间的不兼容性。
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引用次数: 5
Enhanced limonene production in a fast-growing cyanobacterium through combinatorial metabolic engineering 通过组合代谢工程提高快速生长蓝藻的柠檬烯产量
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2021-06-01 DOI: 10.1016/j.mec.2021.e00164
Po-Cheng Lin , Fuzhong Zhang , Himadri B. Pakrasi

Terpenoids are a large and diverse group of natural products with commercial applications. Microbial production of terpenes is considered as a feasible approach for the stable supply of these complex hydrocarbons. Cyanobacteria, photosynthetic prokaryotes, are attractive hosts for sustainable bioproduction, because these autotrophs require only light and CO2 for growth. Despite cyanobacteria having been engineered to produce a variety of compounds, their productivities of terpenes are generally low. Further research is needed to determine the bottleneck reactions for enhancing terpene production in cyanobacteria. In this study, we engineered the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce a commercially-used terpenoid, limonene. We identified a beneficial mutation in the gene encoding geranylgeranyl pyrophosphate synthase crtE, leading to a 2.5-fold increase in limonene production. The engineered strain produced 16.4 ​mg ​L−1 of limonene at a rate of 8.2 ​mg ​L−1 day−1, which is 8-fold higher than limonene productivities previously reported in other cyanobacterial species. Furthermore, we employed a combinatorial metabolic engineering approach to optimize genes involved in the upstream pathway of limonene biosynthesis. By modulating the expression of genes encoding the enzymes in the MEP pathway and the geranyl pyrophosphate synthase, we showed that optimization of the expression level is critical to enhance limonene production in cyanobacteria.

萜类化合物是一大类具有商业用途的天然产物。微生物生产萜烯被认为是稳定供应这些复杂碳氢化合物的可行途径。蓝藻,光合原核生物,是可持续生物生产的有吸引力的宿主,因为这些自养生物只需要光和二氧化碳来生长。尽管蓝藻经过改造可以产生多种化合物,但它们的萜烯产量通常很低。需要进一步的研究来确定提高蓝藻中萜烯产量的瓶颈反应。在这项研究中,我们设计了快速生长的蓝藻长聚球菌UTEX 2973,以生产一种商业用途的萜类化合物柠檬烯。我们在编码香叶基焦磷酸合成酶crtE的基因中发现了一个有益的突变,导致柠檬烯产量增加2.5倍。该工程菌株以8.2 mg L−1 day−1的速率产生16.4 mg L−1柠檬烯,比以前报道的其他蓝藻物种的柠檬烯产量高8倍。此外,我们采用组合代谢工程的方法来优化参与柠檬烯生物合成上游途径的基因。通过调节MEP通路和香叶基焦磷酸合成酶编码基因的表达,我们发现优化表达水平对提高蓝藻柠檬烯的产量至关重要。
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引用次数: 36
The identification of novel promoters and terminators for protein expression and metabolic engineering applications in Kluyveromyces marxianus 马氏克鲁维菌蛋白表达新启动子和终止子的鉴定及其代谢工程应用
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2021-06-01 DOI: 10.1016/j.mec.2020.e00160
Pradeep Kumar , Debendra Kumar Sahoo , Deepak Sharma

The K. marxianus has emerged as a potential yeast strain for various biotechnological applications. However, the limited number of available genetic tools has hindered the widespread usage of this yeast. In the current study we have expanded the molecular tool box by identifying novel sets of promoters and terminators for increased recombinant protein expression in K. marxianus. The previously available transcriptomic data were analyzed to identify top 10 promoters of highest gene expression activity. We further characterized and compared strength of these identified promoters using eGFP as a reporter protein, at different temperatures and carbon sources. To examine the regulatory region driving protein expression, serially truncated shorter versions of two selected strong promoters were designed, and examined for their ability to drive eGFP protein expression. The activities of these two promoters were further enhanced using different combinations of native transcription terminators of K. marxianus. We further utilized the identified DNA cassette encoding strong promoter in metabolic engineering of K. marxianus for enhanced β-galactosidase activity. The present study thus provides novel sets of promoters and terminators as well as engineered K. marxianus strain for its wider utility in applications requiring lactose degradation such as in cheese whey and milk.

马氏酵母菌已成为一种潜在的生物技术应用酵母菌株。然而,可用的遗传工具数量有限,阻碍了这种酵母的广泛使用。在目前的研究中,我们通过鉴定新的启动子和终止子,扩大了分子工具箱,以增加K. marxianus中重组蛋白的表达。通过分析已有的转录组学数据,鉴定出基因表达活性最高的前10个启动子。我们使用eGFP作为报告蛋白,在不同温度和碳源下进一步表征和比较了这些鉴定的启动子的强度。为了检测驱动蛋白表达的调控区域,设计了两个选定的强启动子的连续截短版本,并检测了它们驱动eGFP蛋白表达的能力。这两个启动子的活性通过不同组合的方式进一步增强。我们进一步利用鉴定出的编码强启动子的DNA盒在马氏酵母代谢工程中增强β-半乳糖苷酶活性。因此,本研究提供了一套新的启动子和终止子,以及改造的马氏乳杆菌菌株,使其在奶酪乳清和牛奶等需要乳糖降解的应用中得到更广泛的应用。
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引用次数: 3
Surfactin, a quorum sensing signal molecule, globally affects the carbon metabolism in Bacillus amyloliquefaciens 表面蛋白是一种群体感应信号分子,对解淀粉芽孢杆菌的碳代谢具有全局影响
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2021-06-01 DOI: 10.1016/j.mec.2021.e00174
Jiahong Wen, Xiuyun Zhao, Fengmei Si, Gaofu Qi

Surfactin, a quorum sensing signal molecule, is correlated with carbon metabolism in Bacillus amyloliquefaciens. In the present work, we found that mutation of srfAsrfA) led to an obviously changed carbon metabolism in B. amyloliquefaciens. Firstly, the PTS-glucose system was significantly increased as a feedback to glucose exhaustion. Secondly, the basic carbon metabolism such as glycolysis and TCA cycle was obviously weakened in ΔsrfA. Thirdly, the global regulator of CcpA (carbon catabolite protein A) and P ~ Ser46-HPr (seryl-phosphorylated form of histidine-containing protein) to mediate the CcpA-dependent CCR (carbon catabolite repression) were not increased, but the ability to use extracellular non- and less-preferred carbon sources was down-regulated in ΔsrfA. Fourthly, the carbon overflow metabolism such as biosynthesis of acetate was enhanced while biosynthesis of acetoin/2,3-butanediol and branched-chain amino acids were weakened in ΔsrfA. Finally, ΔsrfA could use most of non- and less-preferred carbon sources except for fatty acids, branched chain amino acids, and some organic acids (e.g. pyruvate, citrate and glutamate) after glucose exhaustion. Collectively, surfactin showed a global influence on carbon metabolism in B. amyloliquefaciens. Our studies highlighted a way to correlate quorum sensing with carbon metabolism via surfactin in Bacillus species.

表面蛋白是一种群体感应信号分子,与解淀粉芽孢杆菌的碳代谢有关。在本研究中,我们发现srfA (ΔsrfA)突变导致解淀粉芽孢杆菌的碳代谢发生明显变化。首先,pts -葡萄糖系统作为葡萄糖耗竭的反馈显著增加。其次,糖酵解、TCA循环等基础碳代谢在ΔsrfA中明显减弱。第三,在ΔsrfA中,CcpA(碳分解代谢蛋白A)和P ~ Ser46-HPr(含组氨酸蛋白的丝氨酸磷酸化形式)介导CcpA依赖的CCR(碳分解代谢抑制)的全局调节因子没有增加,但使用细胞外非和不太受欢迎的碳源的能力被下调。第四,在ΔsrfA中,碳溢出代谢如醋酸酯的生物合成增强,而醋酸酯/2,3-丁二醇和支链氨基酸的生物合成减弱。最后,ΔsrfA在葡萄糖耗尽后,除了脂肪酸、支链氨基酸和一些有机酸(如丙酮酸、柠檬酸和谷氨酸)外,可以使用大多数非和不太优选的碳源。总的来说,表面素对解淀粉芽孢杆菌的碳代谢具有全局影响。我们的研究强调了一种在芽孢杆菌中通过表面蛋白将群体感应与碳代谢联系起来的方法。
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引用次数: 9
Engineering precursor pools for increasing production of odd-chain fatty acids in Yarrowia lipolytica 增脂耶氏菌奇链脂肪酸的工程前体池
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2021-06-01 DOI: 10.1016/j.mec.2020.e00158
Young-Kyoung Park , Florence Bordes , Fabien Letisse , Jean-Marc Nicaud

Microbial production of lipids is one of the promising alternatives to fossil resources with increasing environmental and energy concern. Odd-chain fatty acids (OCFA), a type of unusual lipids, are recently gaining a lot of interest as target compounds in microbial production due to their diverse applications in the medical, pharmaceutical, and chemical industries. In this study, we aimed to enhance the pool of precursors with three-carbon chain (propionyl-CoA) and five-carbon chain (β-ketovaleryl-CoA) for the production of OCFAs in Yarrowia lipolytica. We evaluated different propionate-activating enzymes and the overexpression of propionyl-CoA transferase gene from Ralstonia eutropha increased the accumulation of OCFAs by 3.8 times over control strain, indicating propionate activation is the limiting step of OCFAs synthesis. It was shown that acetate supplement was necessary to restore growth and to produce a higher OCFA contents in total lipids, suggesting the balance of the precursors between acetyl-CoA and propionyl-CoA is crucial for OCFA accumulation. To improve β-ketovaleryl-CoA pools for further increase of OCFA production, we co-expressed the bktB encoding β-ketothiolase in the producing strain, and the OCFA production was increased by 33% compared to control. Combining strain engineering and the optimization of the C/N ratio promoted the OCFA production up to 1.87 ​g/L representing 62% of total lipids, the highest recombinant OCFAs titer reported in yeast, up to date. This study provides a strong basis for the microbial production of OCFAs and its derivatives having high potentials in a wide range of applications.

随着环境和能源的日益关注,微生物生产的脂质是化石资源的有前途的替代品之一。奇链脂肪酸(OCFA)是一种特殊的脂类,由于其在医疗、制药和化学工业中的广泛应用,近年来作为微生物生产的目标化合物受到了广泛的关注。在这项研究中,我们的目的是增加三碳链(丙炔- coa)和五碳链(β-酮戊酰- coa)的前体池,用于在脂性耶氏菌中生产OCFAs。我们对不同的丙酸激活酶进行了评价,结果表明,过表达丙酰coa转移酶基因的富菌OCFAs的积累量比对照菌株增加了3.8倍,表明丙酸激活是OCFAs合成的限制步骤。结果表明,为了恢复生长和提高总脂质中OCFA的含量,补充乙酸是必要的,这表明乙酰辅酶a和丙酰辅酶a前体之间的平衡对OCFA的积累至关重要。为了改善β-酮戊酰辅酶a库,进一步提高OCFA产量,我们在产菌中共表达了编码β-酮硫酶的bktB, OCFA产量比对照提高了33%。结合菌株工程和优化C/N比,使OCFA产量达到1.87 g/L,占总脂质的62%,是迄今为止在酵母中报道的最高重组OCFA滴度。该研究为微生物生产OCFAs及其衍生物提供了强有力的基础,具有广泛的应用潜力。
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引用次数: 16
Integrated laboratory evolution and rational engineering of GalP/Glk-dependent Escherichia coli for higher yield and productivity of L-tryptophan biosynthesis GalP/ glk依赖性大肠杆菌的综合实验室进化和合理工程,提高l -色氨酸生物合成的产量和生产力
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2021-06-01 DOI: 10.1016/j.mec.2021.e00167
Chen Minliang, Ma Chengwei, Chen Lin, An-Ping Zeng

L-Tryptophan (Trp) is a high-value aromatic amino acid with diverse applications in food and pharmaceutical industries. Although production of Trp by engineered Escherichia coli has been extensively studied, the need of multiple precursors for its synthesis and the complex regulations of the biosynthetic pathways make the achievement of a high product yield still very challenging. Metabolic flux analysis suggests that the use of a phosphoenolpyruvate:sugar phosphotransferase system (PTS) independent glucose uptake system, i.e. the galactose permease/glucokinase (GalP/Glk) system, can theoretically double the Trp yield from glucose. To explore this possibility, a PTS and GalP/Glk-dependent E. coli strain was constructed from a previously rationally developed Trp producer strain S028. However, the growth rate of the S028 mutant was severely impaired. To overcome this problem, promoter screening for modulated gene expression of GalP/Glk was carried out, following by a batch mode of adaptive laboratory evolution (ALE) which resulted in a strain K3 with a similar Trp yield and concentration as S028. In order to obtain a more efficient Trp producer, a novel continuous ALE system was developed by combining CRISPR/Cas9-facilitated in vivo mutagenesis with real-time measurement of cell growth and online monitoring of Trp-mediated fluorescence intensity. With the aid of this automatic system (auto-CGSS), a promising strain T5 was obtained and fed-batch fermentations showed an increase of Trp yield by 19.71% with this strain compared with that obtained by the strain K3 (0.164 vs. 0.137 ​g/g). At the same time, the specific production rate was increased by 52.93% (25.28 vs. 16.53 ​mg/g DCW/h). Two previously engineered enzyme variants AroGD6G−D7A and AnTrpCR378F were integrated into the strain T5, resulting in a highly productive strain T5AA with a Trp yield of 0.195 ​g/g and a specific production rate of 28.83 ​mg/g DCW/h.

l -色氨酸(Trp)是一种高价值的芳香氨基酸,在食品和制药工业中有着广泛的应用。尽管利用工程大肠杆菌生产色氨酸已经得到了广泛的研究,但合成色氨酸需要多种前体,生物合成途径的复杂调控使得实现高产量仍然非常具有挑战性。代谢通量分析表明,使用磷酸烯醇丙酮酸:糖磷酸转移酶系统(PTS)独立的葡萄糖摄取系统,即半乳糖渗透酶/葡萄糖激酶(GalP/Glk)系统,理论上可以使葡萄糖的色氨酸产量翻倍。为了探索这种可能性,我们利用先前合理开发的色氨酸产生菌S028构建了依赖PTS -和GalP/ glk的大肠杆菌菌株。然而,S028突变体的生长速率严重受损。为了克服这一问题,我们对GalP/Glk调控基因表达的启动子进行了筛选,然后进行了批量适应实验室进化(ALE)模式,结果菌株K3具有与S028相似的Trp产量和浓度。为了获得更高效的色氨酸产生物,我们将CRISPR/ cas9介导的体内诱变与实时测量细胞生长和在线监测色氨酸介导的荧光强度相结合,开发了一种新型的连续ALE系统。在该自动系统(auto-CGSS)的辅助下,获得了一个很有前途的菌株T5,与菌株K3相比,该菌株的色氨酸产量提高了19.71% (0.164 g/g比0.137 g/g)。同时,比产率提高52.93%(25.28比16.53 mg/g DCW/h)。将先前设计的两种酶变体AroGD6G−D7A和AnTrpCR378F整合到菌株T5中,得到了Trp产量为0.195 g/g,比产率为28.83 mg/g DCW/h的高产菌株T5AA。
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引用次数: 14
Engineering of a new Escherichia coli strain efficiently metabolizing cellobiose with promising perspectives for plant biomass-based application design 一株高效代谢纤维素二糖的新型大肠杆菌的工程设计及其在植物生物量应用设计中的前景
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2021-06-01 DOI: 10.1016/j.mec.2020.e00157
Romain Borne , Nicolas Vita , Nathalie Franche , Chantal Tardif, Stéphanie Perret, Henri-Pierre Fierobe

The necessity to decrease our fossil energy dependence requests bioprocesses based on biomass degradation. Cellobiose is the main product released by cellulases when acting on the major plant cell wall polysaccharide constituent, the cellulose. Escherichia coli, one of the most common model organisms for the academy and the industry, is unable to metabolize this disaccharide. In this context, the remodeling of E. coli to catabolize cellobiose should thus constitute an important progress for the design of such applications. Here, we developed a robust E. coli strain able to metabolize cellobiose by integration of a small set of modifications in its genome. Contrary to previous studies that use adaptative evolution to achieve some growth on this sugar by reactivating E. coli cryptic operons coding for cellobiose metabolism, we identified easily insertable modifications impacting the cellobiose import (expression of a gene coding a truncated variant of the maltoporin LamB, modification of the expression of lacY encoding the lactose permease) and its intracellular degradation (genomic insertion of a gene encoding either a cytosolic β-glucosidase or a cellobiose phosphorylase). Taken together, our results provide an easily transferable set of mutations that confers to E. coli an efficient growth phenotype on cellobiose (doubling time of 2.2 ​h in aerobiosis) without any prior adaptation.

减少我们对化石能源依赖的必要性要求基于生物质降解的生物过程。纤维素二糖是纤维素酶作用于植物细胞壁的主要多糖成分纤维素时所释放的主要产物。大肠杆菌是学术界和工业界最常见的模式生物之一,它无法代谢这种双糖。在这种情况下,重塑大肠杆菌以分解纤维素糖应该是设计此类应用的重要进展。在这里,我们开发了一种强大的大肠杆菌菌株,能够通过整合其基因组中的一小组修饰来代谢纤维二糖。与先前的研究相反,通过重新激活编码纤维素二糖代谢的大肠杆菌隐操作子,利用适应性进化来实现这种糖的一些生长,我们发现了容易插入的修饰,影响纤维素二糖的进口(编码麦芽糖蛋白LamB的截断变体的基因的表达)。编码乳糖渗透酶的lacY表达的修饰及其细胞内降解(编码胞质β-葡萄糖苷酶或纤维素二糖磷酸化酶的基因的基因组插入)。综上所述,我们的研究结果提供了一组易于转移的突变,这些突变赋予了大肠杆菌在纤维素二糖上的高效生长表型(在有氧状态下翻倍时间为2.2 h),而无需任何预先适应。
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引用次数: 2
Engineered cyanobacteria with additional overexpression of selected Calvin-Benson-Bassham enzymes show further increased ethanol production 工程蓝藻与额外的过表达选定的卡尔文-本森-巴萨姆酶显示进一步增加乙醇产量
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2021-06-01 DOI: 10.1016/j.mec.2021.e00161
Stamatina Roussou, Alessia Albergati, Feiyan Liang, Peter Lindblad

Cyanobacteria are one of the most promising microorganisms to produce biofuels and renewable chemicals due to their oxygenic autotrophic growth properties. However, to rely on photosynthesis, which is one of the main reasons for slow growth, low carbon assimlation rate and low production, is a bottleneck. To address this challenge, optimizing the Calvin-Benson-Bassham (CBB) cycle is one of the strategies since it is the main carbon fixation pathway. In a previous study, we showed that overexpression of either aldolase (FBA), transketolase (TK), or fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), enzymes responsible for RuBP regeneration and vital for controlling the CBB carbon flux, led to higher production rates and titers in ethanol producing strains of Synechocystis PCC 6803. In the present study, we investigated the combined effects of the above enzymes on ethanol production in Synechocystis PCC 6803.

The ethanol production of the strains overexpressing two CBB enzymes (FBA ​+ ​TK, FBP/SBPase ​+ ​FBA or FBP/SBPase ​+ ​TK) was higher than the respective control strains, overexpressing either FBA or TK. The co-overexpression of FBA and TK led to more than 9 times higher ethanol production compared to the overexpression of FBA. Compared to TK the respective increase is 4 times more ethanol production. Overexpression of FBP/SBPase in combination with FBA showed 2.5 times higher ethanol production compared to FBA. Finally, co-overexpression of FBP/SBPase and TK reached about twice the production of ethanol compared to overexpression of only TK. This study clearly demonstrates that overexpression of two selected CBB enzymes leads to significantly increased ethanol production compared to overexpression of a single CBB enzyme.

蓝藻由于其富氧自养的生长特性,是生产生物燃料和可再生化学品最有前途的微生物之一。然而,依赖光合作用是一个瓶颈,这是生长缓慢,低碳同化率和低产量的主要原因之一。为了应对这一挑战,优化Calvin-Benson-Bassham (CBB)循环是一种策略,因为它是主要的碳固定途径。在之前的一项研究中,我们发现醛缩酶(FBA)、转酮醇酶(TK)或果糖-1,6/sedoheptulose-1,7-双磷酸酶(FBP/SBPase)的过表达导致产乙醇菌株PCC 6803的产率和滴度更高。这些酶负责RuBP再生,对控制CBB碳通量至关重要。在本研究中,我们研究了上述酶对聚囊藻pcc6803乙醇生产的综合影响。过表达两种CBB酶(FBA + TK、FBP/SBPase + FBA或FBP/SBPase + TK)的菌株乙醇产量均高于过表达FBA和过表达TK的对照菌株。与FBA过表达相比,FBA和TK共过表达导致乙醇产量增加9倍以上。与TK相比,乙醇产量分别增加了4倍。FBP/SBPase与FBA联合过表达的乙醇产量是FBA的2.5倍。最后,FBP/SBPase和TK共过表达的乙醇产量约为仅过表达TK的两倍。该研究清楚地表明,与过表达单一CBB酶相比,过表达两种选定的CBB酶可显著增加乙醇产量。
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引用次数: 18
NetFlow: A tool for isolating carbon flows in genome-scale metabolic networks NetFlow:一个在基因组尺度代谢网络中分离碳流的工具
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2021-06-01 DOI: 10.1016/j.mec.2020.e00154
Sean G. Mack, Ganesh Sriram

Genome-scale stoichiometric models (GSMs) have been widely utilized to predict and understand cellular metabolism. GSMs and the flux predictions resulting from them have proven indispensable to fields ranging from metabolic engineering to human disease. Nonetheless, it is challenging to parse these flux predictions due to the inherent size and complexity of the GSMs. Several previous approaches have reduced this complexity by identifying key pathways contained within the genome-scale flux predictions. However, a reduction method that overlays carbon atom transitions on stoichiometry and flux predictions is lacking. To fill this gap, we developed NetFlow, an algorithm that leverages genome-scale carbon mapping to extract and quantitatively distinguish biologically relevant metabolic pathways from a given genome-scale flux prediction. NetFlow extends prior approaches by utilizing both full carbon mapping and context-specific flux predictions. Thus, NetFlow is uniquely able to quantitatively distinguish between biologically relevant pathways of carbon flow within the given flux map. NetFlow simulates 13C isotope labeling experiments to calculate the extent of carbon exchange, or carbon yield, between every metabolite in the given GSM. Based on the carbon yield, the carbon flow to or from any metabolite or between any pair of metabolites of interest can be isolated and readily visualized. The resulting pathways are much easier to interpret, which enables an in-depth mechanistic understanding of the metabolic phenotype of interest. Here, we first demonstrate NetFlow with a simple network. We then depict the utility of NetFlow on a model of central carbon metabolism in E. coli. Specifically, we isolated the production pathway for succinate synthesis in this model and the metabolic mechanism driving the predicted increase in succinate yield in a double knockout of E. coli. Finally, we describe the application of NetFlow to a GSM of lycopene-producing E. coli, which enabled the rapid identification of the mechanisms behind the measured increases in lycopene production following single, double, and triple knockouts.

基因组尺度化学计量模型(GSMs)已被广泛用于预测和了解细胞代谢。事实证明,从代谢工程到人类疾病等领域都离不开GSMs及其通量预测。然而,由于gsm固有的规模和复杂性,解析这些通量预测是具有挑战性的。先前的几种方法通过确定基因组尺度通量预测中包含的关键途径来降低这种复杂性。然而,缺乏一种覆盖碳原子跃迁的化学计量学和通量预测的还原方法。为了填补这一空白,我们开发了NetFlow算法,该算法利用基因组尺度的碳映射,从给定的基因组尺度通量预测中提取并定量区分生物学相关的代谢途径。NetFlow通过利用全碳映射和特定环境的通量预测扩展了先前的方法。因此,NetFlow是唯一能够定量区分给定通量图中碳流的生物学相关途径的方法。NetFlow模拟13C同位素标记实验,以计算给定GSM中每种代谢物之间的碳交换程度或碳产量。基于碳产量,碳流或从任何代谢物或任何对感兴趣的代谢物之间可以分离和容易地可视化。由此产生的途径更容易解释,这使得对感兴趣的代谢表型有深入的机制理解。在这里,我们首先用一个简单的网络演示NetFlow。然后,我们描述了NetFlow在大肠杆菌中心碳代谢模型上的效用。具体来说,我们在该模型中分离了琥珀酸盐合成的生产途径,以及在大肠杆菌双敲除中驱动琥珀酸盐产量预测增加的代谢机制。最后,我们描述了NetFlow在产生番茄红素的大肠杆菌的GSM中的应用,它能够快速识别在单次、双次和三次敲除后测量的番茄红素产量增加背后的机制。
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引用次数: 4
Synthetic production of prenylated naringenins in yeast using promiscuous microbial prenyltransferases 利用混杂的微生物戊烯基转移酶在酵母中合成制备戊烯基化柚皮素
IF 5.2 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2021-06-01 DOI: 10.1016/j.mec.2021.e00169
Shota Isogai , Nobuyuki Okahashi , Ririka Asama , Tomomi Nakamura , Tomohisa Hasunuma , Fumio Matsuda , Jun Ishii , Akihiko Kondo

Reconstitution of prenylflavonoids using the flavonoid biosynthetic pathway and prenyltransferases (PTs) in microbes can be a promising attractive alternative to plant-based production or chemical synthesis. Here, we demonstrate that promiscuous microbial PTs can be a substitute for regiospecific but mostly unidentified botanical PTs. To test the prenylations of naringenin, we constructed a yeast strain capable of producing naringenin from l-phenylalanine by genomic integration of six exogenous genes encoding components of the naringenin biosynthetic pathway. Using this platform strain, various microbial PTs were tested for prenylnaringenin production. In vitro screening demonstrated that the fungal AnaPT (a member of the tryptophan dimethylallyltransferase family) specifically catalyzed C-3′ prenylation of naringenin, whereas SfN8DT-1, a botanical PT, specifically catalyzed C-8 prenylation. In vivo, the naringenin-producing strain expressing the microbial AnaPT exhibited heterologous microbial production of 3′-prenylnaringenin (3′-PN), in contrast to the previously reported in vivo production of 8-prenylnaringenin (8-PN) using the botanical SfN8DT-1. These findings provide strategies towards expanding the production of a variety of prenylated compounds, including well-known prenylnaringenins and novel prenylflavonoids. These results also suggest the opportunity for substituting botanical PTs, both known and unidentified, that display relatively strict regiospecificity of the prenyl group transfer.

利用类黄酮生物合成途径和微生物中戊烯基转移酶(PTs)重组戊烯基类黄酮是一种有前途的有吸引力的替代植物生产或化学合成的方法。在这里,我们证明了混杂的微生物PTs可以替代区域特异性但大多数未知的植物PTs。为了测试柚皮素的前置化,我们通过基因组整合编码柚皮素生物合成途径成分的6个外源基因,构建了一株能够从l-苯丙氨酸生产柚皮素的酵母菌。利用该平台菌株,对不同微生物PTs进行了产丙烯基柚皮素的试验。体外筛选表明,真菌AnaPT(色氨酸二甲基烯丙基转移酶家族成员)特异性催化柚皮素的C-3 '烯酰化,而植物PT SfN8DT-1特异性催化C-8烯酰化。在体内,表达微生物AnaPT的柚皮素生产菌株表现出异源微生物生产3 ' -烯丙基柚皮素(3 ' -PN),与之前报道的使用植物SfN8DT-1在体内生产8-烯丙基柚皮素(8-PN)形成对比。这些发现为扩大各种烯丙基化化合物的生产提供了策略,包括众所周知的烯丙基柚皮素和新型烯丙基类黄酮。这些结果也表明,有机会取代植物PTs,无论是已知的还是未知的,显示相对严格的区域特异性的戊烯基转移。
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引用次数: 9
期刊
Metabolic Engineering Communications
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