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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 Q1 Engineering 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
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 Q1 Engineering 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 Q1 Engineering 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 Q1 Engineering 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 Q1 Engineering 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
Engineering Ustilago maydis for production of tailor-made mannosylerythritol lipids 工程黑穗病菌用于生产量身定制的甘露糖赤藓糖醇脂
IF 5.2 Q1 Engineering Pub Date : 2021-06-01 DOI: 10.1016/j.mec.2021.e00165
Fabienne Becker , Thorsten Stehlik , Uwe Linne , Michael Bölker , Johannes Freitag , Björn Sandrock

Mannosylerythritol lipids (MELs) are surface active glycolipids secreted by various fungi. MELs can be used as biosurfactants and are a biodegradable resource for the production of detergents or pharmaceuticals. Different fungal species synthesize a unique mixture of MELs differing in acetyl- and acyl-groups attached to the sugar moiety. Here, we report the construction of a toolbox for production of glycolipids with predictable fatty acid side chains in the basidiomycete Ustilago maydis. Genes coding for acyl-transferases involved in MEL production (Mac1 and Mac2) from different fungal species were combined to obtain altered MEL variants with distinct physical properties and altered antimicrobial activity. We also demonstrate that a U. maydis paralog of the acyltransferase Mac2 with a different substrate specificity can be employed for the biosynthesis of modified MEL variants. In summary, our data showcase how the fungal repertoire of Mac enzymes can be used to engineer tailor-made MELs according to specific biotechnological or pharmaceutical requirements.

甘露糖赤四醇脂是多种真菌分泌的具有表面活性的糖脂。mel可以用作生物表面活性剂,是生产洗涤剂或药品的可生物降解资源。不同的真菌种类合成一种独特的mel混合物,这些mel的乙酰基和酰基与糖部分的连接不同。在这里,我们报告了一个工具箱的构建,用于生产糖脂具有可预测的脂肪酸侧链的担子菌黑穗病菌。研究人员将来自不同真菌物种的参与MEL生产的酰基转移酶(Mac1和Mac2)的编码基因组合在一起,获得了具有不同物理特性和抗菌活性的改变的MEL变体。我们还证明,具有不同底物特异性的U. maydis酰基转移酶Mac2类似物可用于修饰MEL变体的生物合成。总之,我们的数据展示了如何根据特定的生物技术或制药要求,利用Mac酶的真菌库来设计定制的mel。
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引用次数: 14
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 Q1 Engineering 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 Q1 Engineering 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
Metabolic engineering of Synechocystis sp. PCC 6803 for improved bisabolene production 聚囊藻(Synechocystis sp. PCC 6803)代谢工程提高双abolene产量
IF 5.2 Q1 Engineering Pub Date : 2021-06-01 DOI: 10.1016/j.mec.2020.e00159
João S. Rodrigues, Pia Lindberg

Terpenoids are a wide class of organic compounds with industrial relevance. The natural ability of cyanobacteria to produce terpenoids via the methylerythritol 4-phosphate (MEP) pathway makes these organisms appealing candidates for the generation of light-driven cell factories for green chemistry. Here we address the improvement of the production of (E)-α-bisabolene, a valuable biofuel feedstock, in Synechocystis sp. PCC 6803 via sequential heterologous expression of bottleneck enzymes of the native pathway. Expression of the bisabolene synthase is sufficient to complete the biosynthetic pathway of bisabolene. Expression of a farnesyl-pyrophosphate synthase from Escherichia coli did not influence production of bisabolene, while enhancement of the MEP pathway via additional overexpression of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and IPP/DMAPP isomerase (IDI) significantly increased production per cell. However, in the absence of a carbon sink, the overexpression of DXS and IDI leads to significant growth impairment. The final engineered strain reached a volumetric titre of 9 ​mg ​L−1 culture of bisabolene after growing for 12 days. When the cultures were grown in a high cell density (HCD) system, we observed an increase in the volumetric titres by one order of magnitude for all producing-strains. The strain with improved MEP pathway presented an increase twice as much as the remaining engineered strains, yielding more than 180 ​mg ​L−1 culture after 10 days of cultivation. Furthermore, the overexpression of these two MEP enzymes prevented the previously reported decrease in the bisabolene specific titres when grown in HCD conditions, where primary metabolism is usually favoured. We conclude that fine-tuning of the cyanobacterial terpenoid pathway is crucial for the generation of microbial platforms for terpenoid production on industrial-scale.

萜类化合物是一类广泛的具有工业意义的有机化合物。蓝藻通过甲基赤藓糖醇4-磷酸(MEP)途径产生萜类物质的天然能力使这些生物成为绿色化学产生光驱动细胞工厂的有吸引力的候选者。本研究通过序列异源表达Synechocystis sp. PCC 6803的瓶颈酶,提高了生物燃料原料(E)-α-双abolene的产量。双abolene合成酶的表达足以完成双abolene的生物合成途径。来自大肠杆菌的法尼基焦磷酸合成酶的表达不影响双abolene的产量,而通过额外过表达1-脱氧-d -木醛糖-5-磷酸合成酶(DXS)和IPP/DMAPP异构酶(IDI)来增强MEP途径,显著增加了每个细胞的产量。然而,在缺乏碳汇的情况下,DXS和IDI的过表达会导致显著的生长损害。最终的工程菌株在生长12天后达到9 mg L−1双abolene培养液的体积滴度。当培养物在高细胞密度(HCD)系统中生长时,我们观察到所有生产菌株的体积滴度增加了一个数量级。改良MEP途径的菌株比其余工程菌株的产量增加了两倍,培养10天后产量超过180 mg L−1。此外,这两种MEP酶的过表达阻止了先前报道的在HCD条件下生长时双abolene特异性滴度的下降,而HCD条件通常有利于初级代谢。我们得出结论,蓝藻萜类途径的微调对于在工业规模上产生萜类生产的微生物平台至关重要。
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引用次数: 33
Trans-4-hydroxy-L-proline production by the cyanobacterium Synechocystis sp. PCC 6803 聚囊蓝细菌pcc6803生产反式4-羟基- l -脯氨酸的研究
IF 5.2 Q1 Engineering Pub Date : 2021-06-01 DOI: 10.1016/j.mec.2020.e00155
Fabian Brandenburg , Eleni Theodosiou , Carolin Bertelmann, Marcel Grund, Stephan Klähn, Andreas Schmid, Jens O. Krömer

Cyanobacteria play an important role in photobiotechnology. Yet, one of their key central metabolic pathways, the tricarboxylic acid (TCA) cycle, has a unique architecture compared to most heterotrophs and still remains largely unexploited. The conversion of 2-oxoglutarate to succinate via succinyl-CoA is absent but is by-passed by several other reactions. Overall, fluxes under photoautotrophic growth conditions through the TCA cycle are low, which has implications for the production of chemicals. In this study, we investigate the capacity of the TCA cycle of Synechocystis sp PCC 6803 for the production of trans-4-hydroxy-L-proline (Hyp), a valuable chiral building block for the pharmaceutical and cosmetic industries. For the first time, photoautotrophic Hyp production was achieved in a cyanobacterium expressing the gene for the L-proline-4-hydroxylase (P4H) from Dactylosporangium sp. strain RH1. Interestingly, while elevated intracellular Hyp concentrations could be detected in the recombinant Synechocystis strains under all tested conditions, detectable Hyp secretion into the medium was only observed when the pH of the medium exceeded 9.5 and mostly in the late phases of the cultivation. We compared the rates obtained for autotrophic Hyp production with published sugar-based production rates in E. coli. The land-use efficiency (space-time yield) of the phototrophic process is already in the same order of magnitude as the heterotrophic process considering sugar farming as well. But, the remarkable plasticity of the cyanobacterial TCA cycle promises the potential for a 23–55 fold increase in space-time yield when using Synechocystis. Altogether, these findings contribute to a better understanding of bioproduction from the TCA cycle in photoautotrophs and broaden the spectrum of chemicals produced in metabolically engineered cyanobacteria.

蓝藻在光生物技术中发挥着重要作用。然而,与大多数异养生物相比,它们的关键中心代谢途径之一三羧酸(TCA)循环具有独特的结构,并且在很大程度上仍未被开发。没有通过琥珀酰辅酶a将2-氧戊二酸转化为琥珀酸盐,但有几个其他反应绕过。总的来说,在光自养生长条件下通过三羧酸循环的通量很低,这对化学品的生产有影响。在这项研究中,我们研究了聚囊藻sp PCC 6803的TCA循环生产反式4-羟基- l -脯氨酸(Hyp)的能力,这是一种有价值的手性成分,用于制药和化妆品工业。首次在表达Dactylosporangium sp.菌株RH1的l -脯氨酸-4-羟化酶(P4H)基因的蓝藻中实现了光自养hypp的产生。有趣的是,虽然在所有测试条件下,重组聚胞菌菌株的细胞内Hyp浓度都有所升高,但只有当培养基的pH超过9.5时,培养基中才有可检测到的Hyp分泌,而且大部分是在培养后期。我们比较了在大肠杆菌中获得的自养产氢酶的速率和已发表的糖基产氢酶的速率。考虑到食糖种植,光养过程的土地利用效率(时空产量)已与异养过程处于同一数量级。但是,蓝藻TCA循环的显著可塑性保证了使用聚囊藻时时空产量增加23-55倍的潜力。总之,这些发现有助于更好地理解光自养生物中TCA循环的生物生产,并拓宽了代谢工程蓝藻产生的化学物质的范围。
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引用次数: 7
期刊
Metabolic Engineering Communications
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