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Metabolic engineering of Acinetobacter baylyi ADP1 for naringenin production 利用代谢工程改造贝氏不动杆菌 ADP1 以生产柚皮苷
IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-10-31 DOI: 10.1016/j.mec.2024.e00249
Naringenin, a flavanone and a precursor for a variety of flavonoids, has potential applications in the health and pharmaceutical sectors. The biological production of naringenin using genetically engineered microbes is considered as a promising strategy. The naringenin synthesis pathway involving chalcone synthase (CHS) and chalcone isomerase (CHI) relies on the efficient supply of key substrates, malonyl-CoA and p-coumaroyl-CoA. In this research, we utilized a soil bacterium, Acinetobacter baylyi ADP1, which exhibits several characteristics that make it a suitable candidate for naringenin biosynthesis; the strain naturally tolerates and can uptake and metabolize p-coumaric acid, a primary compound in alkaline-pretreated lignin and a precursor for naringenin production. A. baylyi ADP1 also produces intracellular lipids, such as wax esters, thereby being able to provide malonyl-CoA for naringenin biosynthesis. Moreover, the genomic engineering of this strain is notably straightforward. In the course of the construction of a naringenin-producing strain, the p-coumarate catabolism was eliminated by a single gene knockout (ΔhcaA) and various combinations of plant-derived CHS and CHI were evaluated. The best performance was obtained by a novel combination of genes encoding for a CHS from Hypericum androsaemum and a CHI from Medicago sativa, that enabled the production of 17.9 mg/L naringenin in batch cultivations from p-coumarate. Furthermore, the implementation of a fed-batch system led to a 3.7-fold increase (66.4 mg/L) in naringenin production. These findings underscore the potential of A. baylyi ADP1 as a host for naringenin biosynthesis as well as advancement of lignin-based bioproduction.
柚皮苷是一种黄烷酮,也是多种黄酮类化合物的前体,在健康和医药领域具有潜在的应用价值。利用基因工程微生物进行柚皮苷的生物生产被认为是一种前景广阔的策略。涉及查尔酮合成酶(CHS)和查尔酮异构酶(CHI)的柚皮苷合成途径依赖于关键底物丙二酰-CoA 和对香豆酰-CoA 的有效供应。在这项研究中,我们利用了一种土壤细菌--Acinetobacter baylyi ADP1,它所表现出的一些特性使其成为柚皮苷生物合成的合适候选菌株;该菌株天然耐受对香豆酸,并能吸收和代谢对香豆酸,对香豆酸是碱处理木质素中的一种主要化合物,也是生产柚皮苷的前体物质。A. baylyi ADP1 还能产生蜡酯等细胞内脂类,从而为柚皮苷的生物合成提供丙二酰-CoA。此外,该菌株的基因组工程也非常简单。在构建柚皮苷生产菌株的过程中,通过单基因敲除(ΔhcaA)消除了对香豆酸的分解作用,并对植物来源的 CHS 和 CHI 的各种组合进行了评估。来自金丝桃的 CHS 和来自美智子的 CHI 的新型编码基因组合获得了最佳性能,能够在批量培养中利用对香豆酸生产出 17.9 mg/L 的柚皮苷。此外,采用饲料批处理系统后,柚皮苷的产量增加了 3.7 倍(66.4 毫克/升)。这些发现凸显了巴氏杀菌杆菌 ADP1 作为柚皮苷生物合成宿主的潜力,并推动了基于木质素的生物生产。
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引用次数: 0
PEZy-miner: An artificial intelligence driven approach for the discovery of plastic-degrading enzyme candidates PEZy-miner:发现候选塑料降解酶的人工智能驱动方法
IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-05 DOI: 10.1016/j.mec.2024.e00248

Plastic waste has caused a global environmental crisis. Biocatalytic depolymerization mediated by enzymes has emerged as an efficient and sustainable alternative for plastic treatment and recycling. However, it is challenging and time-consuming to discover novel plastic-degrading enzymes using conventional cultivation-based or omics methods. There is a growing interest in developing effective computational methods to identify new enzymes with desirable plastic degradation functionalities by exploring the ever-increasing databases of protein sequences. In this study, we designed an innovative machine learning-based framework, named PEZy-Miner, to mine for enzymes with high potential in degrading plastics of interest. Two datasets integrating information from experimentally verified enzymes and homologs with unknown plastic-degrading activity were created respectively, covering eleven types of plastic substrates. Protein language models and binary classification models were developed to predict enzymatic degradation of plastics along with confidence and uncertainty estimation. PEZy-Miner exhibited high prediction accuracy and stability when validated on experimentally verified enzymes. Furthermore, by masking the experimentally verified enzymes and blending them into homolog dataset, PEZy-Miner effectively concentrated the experimentally verified entries by 14∼30 times while shortlisting promising plastic-degrading enzyme candidates. We applied PEZy-Miner to 0.1 million putative sequences, out of which 27 new sequences were identified with high confidence. This study provided a new computational tool for mining and recommending promising new plastic-degrading enzymes.

塑料废物已引发全球环境危机。由酶介导的生物催化解聚已成为塑料处理和回收的一种高效、可持续的替代方法。然而,使用传统的基于培养或 omics 方法发现新型塑料降解酶具有挑战性且耗时。开发有效的计算方法,通过探索不断增加的蛋白质序列数据库来识别具有理想塑料降解功能的新酶,引起了越来越多的兴趣。在这项研究中,我们设计了一个基于机器学习的创新框架,名为 PEZy-Miner,用于挖掘在降解相关塑料方面具有高潜力的酶。我们创建了两个数据集,分别整合了实验验证的酶和具有未知塑料降解活性的同源物的信息,涵盖了 11 种塑料底物。开发了蛋白质语言模型和二元分类模型来预测塑料的酶降解以及置信度和不确定性估计。PEZy-Miner 在经过实验验证的酶上表现出了很高的预测准确性和稳定性。此外,通过屏蔽实验验证的酶并将其混合到同源数据集中,PEZy-Miner 有效地将实验验证的条目集中了 14∼30 倍,同时筛选出了有潜力的塑料降解酶候选者。我们将 PEZy-Miner 应用于 10 万个推定序列,其中有 27 个新序列被鉴定为高置信度序列。这项研究为挖掘和推荐有潜力的新型塑料降解酶提供了一种新的计算工具。
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引用次数: 0
Production of (R)-citramalate by engineered Saccharomyces cerevisiae 利用工程酿酒酵母生产(R)-柠檬酸盐
IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-10 DOI: 10.1016/j.mec.2024.e00247

The budding yeast, Saccharomyces cerevisiae, has a high tolerance to organic acids and alcohols, and thus grows well under toxic concentrations of various compounds in the culture medium, potentially allowing for highly efficient compound production. (R)-citramalate is a raw material for methyl methacrylate and can be used as a metabolic intermediate in the biosynthesis of higher alcohols. (R)-citramalate is synthesized from pyruvate and acetyl-CoA. Unlike Escherichia coli, S. cerevisiae has organelles, and its intracellular metabolites are compartmentalized, preventing full use of intracellular acetyl-CoA. Therefore, in this study, to increase the amount of cytosolic acetyl-CoA for highly efficient production of (R)-citramalate, we inhibited the transport of cytosolic acetyl-CoA and pyruvate to the mitochondria. We also constructed a heterologous pathway to supply cytosolic acetyl-CoA. Additionally, we attempted to export (R)-citramalate from cells by expressing a heterologous dicarboxylate transporter gene. We evaluated the effects of these approaches on (R)-citramalate production and constructed a final strain by combining these positive approaches. The resulting strain produced 16.5 mM (R)-citramalate in batch culture flasks. This is the first report of (R)-citramalate production by recombinant S. cerevisiae, and the (R)-citramalate production by recombinant yeast achieved in this study was the highest reported to date.

芽殖酵母(Saccharomyces cerevisiae)对有机酸和酒精有很强的耐受性,因此在培养基中各种化合物浓度有毒的情况下也能很好地生长,从而有可能实现高效的化合物生产。(R)-柠檬醛酸酯是甲基丙烯酸甲酯的原料,可用作高级醇类生物合成的代谢中间体。(R)-柠檬醛酸由丙酮酸和乙酰-CoA 合成。与大肠杆菌不同,酿酒酵母具有细胞器,其胞内代谢物被分隔开来,无法充分利用胞内乙酰-CoA。因此,在本研究中,为了增加细胞内乙酰-CoA 的含量以高效生产(R)-柠檬酸,我们抑制了细胞内乙酰-CoA 和丙酮酸向线粒体的运输。我们还构建了一条异源途径来提供细胞质乙酰-CoA。此外,我们还尝试通过表达异源二羧酸盐转运体基因从细胞中输出 (R)-citramalate 。我们评估了这些方法对(R)-柠檬醛酸生产的影响,并结合这些积极的方法构建了最终菌株。由此产生的菌株在批量培养瓶中产生了 16.5 mM (R)-柠檬醛酸。这是重组酿酒酵母生产(R)-柠檬醛酸的首次报道,本研究中重组酵母的(R)-柠檬醛酸产量是迄今为止报道的最高产量。
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引用次数: 0
Engineering thioesterase as a driving force for novel itaconate production via its degradation scheme 工程硫酯酶是通过其降解方案生产新型伊塔康酸的驱动力
IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-05 DOI: 10.1016/j.mec.2024.e00246

Incorporation of irreversible steps in pathway design enhances the overall thermodynamic favorability and often leads to better bioconversion yield given functional enzymes. Using this concept, here we constructed the first non-natural itaconate biosynthesis pathway driven by thioester hydrolysis. Itaconate is a commercially valuable platform chemical with wide applications in the synthetic polymer industry. Production of itaconate has long relied on the decarboxylation of TCA cycle intermediate cis-aconitate as the only biosynthetic route. Inspired by nature's design of itaconate detoxification, here we engineered a novel itaconate producing pathway orthogonal to native metabolism with no requirement of auxotrophic knock-out. The reversed degradation pathway initiates with pyruvate and acetyl-CoA condensation forming (S)-citramalyl-CoA, followed by its dehydration and isomerization into itaconyl-CoA then hydrolysis into itaconate. Phenylacetyl-CoA thioesterase (PaaI) from Escherichia coli was identified via screening to deliver the highest itaconate formation efficiency when coupled to the reversible activity of citramalate lyase and itaconyl-CoA hydratase. The preference of PaaI towards itaconyl-CoA hydrolysis over acetyl-CoA and (S)-citramalyl-CoA also minimized the inevitable precursor loss due to enzyme promiscuity. With acetate recycling, acetyl-CoA conservation, and condition optimization, we achieved a final itaconate titer of 1 g/L using the thioesterase driven pathway, which is a significant improvement compared to the original degradation pathway based on CoA transferase. This study illustrates the significance of thermodynamic favorability as a design principle in pathway engineering.

在途径设计中加入不可逆步骤可提高整体热力学的有利性,在功能性酶的作用下,往往可获得更高的生物转化产率。利用这一概念,我们在此构建了首个由硫酯水解驱动的非天然衣康酸生物合成途径。衣康酸是一种具有商业价值的平台化学品,在合成聚合物行业有着广泛的应用。长期以来,衣康酸的生产一直依赖于 TCA 循环中间体顺式-乌头酸的脱羧作用,这是唯一的生物合成途径。受大自然中依他康酸解毒设计的启发,我们在此设计了一种与原生代谢正交的新型依他康酸生产途径,无需敲除辅助营养体。这种逆向降解途径以丙酮酸和乙酰-CoA 缩合形成 (S)-citramalyl-CoA 为起点,然后脱水并异构化为 itaconyl-CoA,最后水解为 itaconate。通过筛选确定了大肠杆菌中的苯乙酰-CoA 硫代酯酶(PaaI),当与柠檬醛酸裂解酶和衣康酰-CoA 水合酶的可逆活性结合时,衣康酸的形成效率最高。与乙酰-CoA 和 (S)-citramalyl-CoA 相比,PaaI 更倾向于水解 itaconyl-CoA,这也最大程度地减少了由于酶的杂交性而不可避免的前体损失。通过乙酸酯循环、乙酰-CoA 保护和条件优化,我们利用硫酯酶驱动的途径使伊塔康酸的最终滴度达到了 1 克/升,这与原来基于 CoA 转移酶的降解途径相比有了显著改善。这项研究说明了热力学有利性作为途径工程设计原则的重要性。
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引用次数: 0
A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae: Production of d-xylitol from d-xylose as a case study 酿酒酵母中 NADPH 供应策略的比较分析:以从二木糖生产二木糖醇为例进行研究
IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-07-05 DOI: 10.1016/j.mec.2024.e00245
Priti Regmi , Melanie Knesebeck , Eckhard Boles , Dirk Weuster-Botz , Mislav Oreb

Enhancing the supply of the redox cofactor NADPH in metabolically engineered cells is a critical target for optimizing the synthesis of many product classes, such as fatty acids or terpenoids. In S. cerevisiae, several successful approaches have been developed in different experimental contexts. However, their systematic comparison has not been reported. Here, we established the reduction of xylose to xylitol by an NADPH-dependent xylose reductase as a model reaction to compare the efficacy of different NADPH supply strategies in the course of a batch fermentation, in which glucose and ethanol are sequentially used as carbon sources and redox donors. We show that strains overexpressing the glucose-6-phosphate dehydrogenase Zwf1 perform best, producing up to 16.9 g L−1 xylitol from 20 g L−1 xylose in stirred tank bioreactors. The beneficial effect of increased Zwf1 activity is especially pronounced during the ethanol consumption phase. The same notion applies to the deletion of the aldehyde dehydrogenase ALD6 gene, albeit at a quantitatively lower level. Reduced expression of the phosphoglucose isomerase Pgi1 and heterologous expression of the NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase Gdp1 from Kluyveromyces lactis acted synergistically with ZWF1 overexpression in the presence of glucose, but had a detrimental effect after the diauxic shift. Expression of the mitochondrial NADH kinase Pos5 in the cytosol likewise improved the production of xylitol only on glucose, but not in combination with enhanced Zwf1 activity. To demonstrate the generalizability of our observations, we show that the most promising strategies – ZWF1 overexpression and deletion of ALD6 - also improve the production of l-galactonate from d-galacturonic acid. Therefore, we expect that these findings will provide valuable guidelines for engineering not only the production of xylitol but also of diverse other pathways that require NADPH.

提高代谢工程细胞中氧化还原辅助因子 NADPH 的供应量是优化脂肪酸或萜类化合物等多种产品合成的关键目标。在 S. cerevisiae 中,人们在不同的实验环境中开发出了几种成功的方法。但是,还没有对这些方法进行系统比较的报道。在这里,我们将依赖 NADPH 的木糖还原酶将木糖还原成木糖醇的反应作为一个模型反应,以比较在批量发酵过程中不同 NADPH 供应策略的功效,在批量发酵过程中,葡萄糖和乙醇依次被用作碳源和氧化还原供体。我们的研究表明,过量表达葡萄糖-6-磷酸脱氢酶 Zwf1 的菌株表现最佳,在搅拌罐生物反应器中可从 20 克/升木糖中产生高达 16.9 克/升的木糖醇。在乙醇消耗阶段,Zwf1 活性增加的有利影响尤其明显。醛脱氢酶 ALD6 基因的缺失也是同样的道理,尽管其数量水平较低。磷酸葡萄糖异构酶 Pgi1 的表达量减少,以及来自乳酸克鲁维酵母菌的依赖 NADP+ 的甘油醛-3-磷酸脱氢酶 Gdp1 的异源表达,在葡萄糖存在的情况下与 ZWF1 的过量表达具有协同作用,但在二重转移后则产生了不利影响。同样,在细胞质中表达线粒体 NADH 激酶 Pos5 也只能在葡萄糖条件下提高木糖醇的产量,但不能与 Zwf1 活性的增强相结合。为了证明我们的观察结果具有普遍性,我们发现最有前景的策略--ZWF1 的过表达和 ALD6 的缺失--也能改善由 d-半乳糖醛酸产生 l-半乳糖醛酸的情况。因此,我们希望这些发现不仅能为木糖醇的生产工程提供有价值的指导,也能为需要 NADPH 的其他各种途径的生产工程提供有价值的指导。
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引用次数: 0
CFSA: Comparative flux sampling analysis as a guide for strain design CFSA:作为应变设计指南的通量取样比较分析
IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-06-24 DOI: 10.1016/j.mec.2024.e00244
R.P. van Rosmalen , S. Moreno-Paz , Z.E. Duman-Özdamar, M. Suarez-Diez

Genome-scale metabolic models of microbial metabolism have extensively been used to guide the design of microbial cell factories, still, many of the available strain design algorithms often fail to produce a reduced list of targets for improved performance that can be implemented and validated in a step-wise manner. We present Comparative Flux Sampling Analysis (CFSA), a strain design method based on the extensive comparison of complete metabolic spaces corresponding to maximal or near-maximal growth and production phenotypes. The comparison is complemented by statistical analysis to identify reactions with altered flux that are suggested as targets for genetic interventions including up-regulations, down-regulations and gene deletions. We applied CFSA to the production of lipids by Cutaneotrichosporon oleaginosus and naringenin by Saccharomyces cerevisiae identifying engineering targets in agreement with previous studies as well as new interventions. CFSA is an easy-to-use, robust method that suggests potential metabolic engineering targets for growth-uncoupled production that can be applied to the design of microbial cell factories.

微生物新陈代谢的基因组尺度代谢模型已被广泛用于指导微生物细胞工厂的设计,但许多现有的菌株设计算法往往无法产生一个可逐步实施和验证的性能改进目标缩减列表。我们提出的比较通量取样分析(CFSA)是一种菌株设计方法,它基于对最大或接近最大生长和生产表型对应的完整代谢空间进行广泛比较。统计分析对这种比较进行了补充,以确定通量发生变化的反应,并建议将这些反应作为基因干预的目标,包括上调、下调和基因缺失。我们将 CFSA 应用于油菜酵母菌(Cutaneotrichosporon oleaginosus)生产脂类和柚皮苷的过程,确定了与以往研究一致的工程目标以及新的干预措施。CFSA 是一种易于使用且稳健的方法,可为生长不耦合生产提出潜在的代谢工程目标,并可应用于微生物细胞工厂的设计。
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引用次数: 0
Expression and characterization of monofunctional alcohol dehydrogenase enzymes in Clostridium thermocellum 热梭菌中单功能醇脱氢酶的表达和特性分析
IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-06-20 DOI: 10.1016/j.mec.2024.e00243
Daniela Prates Chiarelli , Bishal Dev Sharma , Shuen Hon , Luana Walravens Bergamo , Lee R. Lynd , Daniel G. Olson

Clostridium thermocellum is a thermophilic anaerobic bacterium that could be used for cellulosic biofuel production due to its strong native ability to consume cellulose, however its ethanol production ability needs to be improved to enable commercial application. In our previous strain engineering work, we observed a spontaneous mutation in the native adhE gene that reduced ethanol production. Here we attempted to complement this mutation by heterologous expression of 18 different alcohol dehydrogenase (adh) genes. We were able to express all of them successfully in C. thermocellum. Surprisingly, however, none of them increased ethanol production, and several actually decreased it. Our findings contribute to understanding the correlation between C. thermocellum ethanol production and Adh enzyme cofactor preferences. The identification of a set of adh genes that can be successfully expressed in this organism provides a foundation for future investigations into how the properties of Adh enzymes affect ethanol production.

嗜热梭菌是一种嗜热厌氧细菌,由于其具有很强的消耗纤维素的原生能力,可用于纤维素生物燃料的生产。在我们之前的菌株工程工作中,我们观察到原生 adhE 基因发生了自发突变,从而降低了乙醇产量。在此,我们尝试通过异源表达 18 种不同的乙醇脱氢酶(adh)基因来补充这一突变。我们成功地在热菌中表达了所有这些基因。但令人惊讶的是,这些基因都没有提高乙醇产量,其中几个基因实际上还降低了乙醇产量。我们的发现有助于理解热菌乙醇产量与 Adh 酶辅因子偏好之间的相关性。确定了一组可在该生物体内成功表达的 Adh 基因,为今后研究 Adh 酶的特性如何影响乙醇产量奠定了基础。
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引用次数: 0
13C-metabolic flux analysis reveals metabolic rewiring in HL-60 neutrophil-like cells through differentiation and immune stimulation 13C 代谢通量分析揭示了 HL-60 中性粒细胞样细胞在分化和免疫刺激过程中的代谢重构
IF 5.2 Q1 Engineering Pub Date : 2024-06-01 DOI: 10.1016/j.mec.2024.e00239
Takeo Taniguchi , Nobuyuki Okahashi , Fumio Matsuda

Neutrophils are innate immune cells and the first line of defense for the maintenance of homeostasis. However, our knowledge of the metabolic rewiring associated with their differentiation and immune stimulation is limited. Here, quantitative 13C-metabolic flux analysis was performed using HL-60 cells as the neutrophil model. A metabolic model for 13C-metabolic flux analysis of neutrophils was developed based on the accumulation of 13C in intracellular metabolites derived from 13C-labeled extracellular carbon sources and intracellular macromolecules. Aspartate and glutamate in the medium were identified as carbon sources that enter central carbon metabolism. Furthermore, the breakdown of macromolecules, estimated to be fatty acids and nucleic acids, was observed. Based on these results, a modified metabolic model was used for 13C-metabolic flux analysis of undifferentiated, differentiated, and lipopolysaccharide (LPS)-activated HL-60 cells. The glucose uptake rate and glycolytic flux decreased with differentiation, whereas the tricarboxylic acid (TCA) cycle flux remained constant. The addition of LPS to differentiated HL-60 cells activated the glucose uptake rate and pentose phosphate pathway (PPP) flux levels, resulting in an increased rate of total NADPH regeneration, which could be used to generate reactive oxygen species. The flux levels of fatty acid degradation and synthesis were also increased in LPS-activated HL-60 cells. Overall, this study highlights the quantitative metabolic alterations in multiple pathways via the differentiation and activation of HL-60 cells using 13C-metabolic flux analysis.

中性粒细胞是先天性免疫细胞,也是维持体内平衡的第一道防线。然而,我们对与中性粒细胞分化和免疫刺激相关的新陈代谢重新布线了解有限。在此,我们以 HL-60 细胞为中性粒细胞模型进行了 13C 代谢通量定量分析。中性粒细胞 13C 代谢通量分析的代谢模型是基于 13C 标记的细胞外碳源和细胞内大分子在细胞内代谢产物中的积累而建立的。培养基中的天冬氨酸和谷氨酸被确定为进入中枢碳代谢的碳源。此外,还观察到大分子的分解,估计是脂肪酸和核酸。在这些结果的基础上,对未分化、已分化和脂多糖(LPS)活化的 HL-60 细胞进行了 13C 代谢通量分析。葡萄糖摄取率和糖酵解通量随细胞分化而降低,而三羧酸(TCA)循环通量则保持不变。向分化的 HL-60 细胞中添加 LPS 会激活葡萄糖摄取率和磷酸戊糖途径通量水平,导致 NADPH 总再生率增加,从而产生活性氧。在 LPS 激活的 HL-60 细胞中,脂肪酸降解和合成的通量水平也有所增加。总之,本研究通过 13C 代谢通量分析,强调了 HL-60 细胞分化和活化过程中多种途径的定量代谢变化。
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引用次数: 0
Engineering a carbon source-responsive promoter for improved biosynthesis in the non-conventional yeast Kluyveromyces marxianus 在非常规酵母 Kluyveromyces marxianus 中设计碳源响应型启动子以提高生物合成能力
IF 5.2 Q1 Engineering Pub Date : 2024-06-01 DOI: 10.1016/j.mec.2024.e00238
Shane Bassett, Nancy A. Da Silva

Many desired biobased chemicals exhibit a range of toxicity to microbial cell factories, making industry-level biomanufacturing more challenging. Separating microbial growth and production phases is known to be beneficial for improving production of toxic products. Here, we developed a novel synthetic carbon-responsive promoter for use in the rapidly growing, stress-tolerant yeast Kluyveromyces marxianus, by fusing carbon-source responsive elements of the native ICL1 promoter to the strong S. cerevisiae TDH3 or native NC1 promoter cores. Two hybrids, PIT350 and PIN450, were validated via EGFP fluorescence and demonstrated exceptional strength, partial repression during growth, and late phase activation in glucose- and lactose-based medium, respectively. Expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for synthesis of the polyketide triacetic acid lactone (TAL) under the control of PIN450 increased TAL more than 50% relative to the native NC1 promoter, and additional promoter engineering further increased TAL titer to 1.39 g/L in tube culture. Expression of the Penicillium griseofulvum 6-methylsalicylic acid synthase (6-MSAS) under the control of PIN450 resulted in a 6.6-fold increase in 6-MSA titer to 1.09 g/L and a simultaneous 1.5-fold increase in cell growth. Finally, we used PIN450 to express the Pseudomonas savastanoi IaaM and IaaH proteins and the Salvia pomifera sabinene synthase protein to improve production of the auxin hormone indole-3-acetic acid and the monoterpene sabinene, respectively, both extremely toxic to yeast. The development of carbon-responsive promoters adds to the synthetic biology toolbox and available metabolic engineering strategies for K. marxianus, allowing greater control over heterologous protein expression and improved production of toxic metabolites.

许多理想的生物基化学品对微生物细胞工厂有不同程度的毒性,这使得工业级生物制造更具挑战性。众所周知,分离微生物生长和生产阶段有利于提高有毒产品的产量。在这里,我们通过将本地 ICL1 启动子的碳源响应元件与强大的 S. cerevisiae TDH3 或本地 NC1 启动子核心融合,开发出一种新型合成碳响应启动子,用于快速生长、耐受压力的酵母 Kluyveromyces marxianus。两个杂交种 PIT350 和 PIN450 通过 EGFP 荧光进行了验证,并分别在基于葡萄糖和乳糖的培养基中表现出卓越的强度、生长过程中的部分抑制和晚期激活。在 PIN450 的控制下表达非洲菊 2-吡喃酮合成酶(2-PS)以合成多酮三乙酸内酯(TAL),与原生 NC1 启动子相比,TAL 增加了 50%以上,额外的启动子工程进一步将试管培养中的 TAL 滴度提高到 1.39 克/升。在 PIN450 的控制下,表达青霉 6-甲基水杨酸合成酶(6-MSAS)使 6-MSA 滴度增加了 6.6 倍,达到 1.09 克/升,细胞生长也同时增加了 1.5 倍。最后,我们利用 PIN450 表达了 Pseudomonas savastanoi IaaM 和 IaaH 蛋白以及丹参桧烯合成酶蛋白,分别提高了对酵母有剧毒的辅助激素吲哚-3-乙酸和单萜桧烯的产量。碳响应启动子的开发增加了 K. marxianus 的合成生物学工具箱和可用的代谢工程策略,从而能够更好地控制异源蛋白的表达,并提高有毒代谢物的产量。
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引用次数: 0
Sustainable biosynthesis of squalene from waste cooking oil by the yeast Yarrowia lipolytica 利用脂肪分解酵母菌从废弃食用油中可持续地生物合成角鲨烯
IF 5.2 Q1 Engineering Pub Date : 2024-06-01 DOI: 10.1016/j.mec.2024.e00240
Shuhui Wang , Xu Sun , Yuqing Han , Zhuo Li , Xiaocong Lu , Hongrui Shi , Cui-ying Zhang , Adison Wong , Aiqun Yu

Squalene is a highly sought-after triterpene compound in growing demand, and its production offers a promising avenue for circular economy practices. In this study, we applied metabolic engineering principles to enhance squalene production in the nonconventional yeast Yarrowia lipolytica, using waste cooking oil as a substrate. By overexpressing key enzymes in the mevalonate pathway — specifically ERG9 encoding squalene synthase, ERG20 encoding farnesyl diphosphate synthase, and HMGR encoding hydroxy-methyl-glutaryl-CoA reductase — we achieved a yield of 779.9 mg/L of squalene. Further co-overexpression of DGA1, encoding diacylglycerol acyltransferase, and CAT2, encoding carnitine acetyltransferase, in combination with prior metabolic enhancements, boosted squalene production to 1381.4 mg/L in the engineered strain Po1g17. To enhance the supply of the precursor acetyl-CoA and inhibit downstream squalene conversion, we supplemented with 6 g/L pyruvic acid and 0.7 mg/L terbinafine, resulting in an overall squalene titer of 2594.1 mg/L. These advancements underscore the potential for sustainable, large-scale squalene production using Y. lipolytica cell factories, contributing to circular economy initiatives by valorizing waste materials.

角鲨烯是一种广受欢迎的三萜类化合物,其需求量与日俱增,它的生产为循环经济实践提供了一条前景广阔的途径。在这项研究中,我们运用代谢工程原理,以废弃食用油为底物,提高了非常规酵母亚罗酵母(Yarrowia lipolytica)的角鲨烯产量。通过过表达甲羟戊酸途径中的关键酶,特别是编码角鲨烯合成酶的 ERG9、编码二磷酸法呢基合成酶的 ERG20 和编码羟甲基-戊二酰-CoA 还原酶的 HMGR,我们获得了 779.9 mg/L 的角鲨烯产量。进一步联合表达编码二酰甘油酰基转移酶的 DGA1 和编码肉碱乙酰转移酶的 CAT2,并结合之前的代谢强化,使工程菌株 Po1g17 的角鲨烯产量提高到 1381.4 mg/L。为了增加前体乙酰-CoA 的供应并抑制下游角鲨烯的转化,我们补充了 6 克/升丙酮酸和 0.7 毫克/升特比萘芬,结果角鲨烯的总滴度达到了 2594.1 毫克/升。这些进展凸显了利用脂溶性角鲨烯细胞工厂进行可持续、大规模角鲨烯生产的潜力,通过对废弃材料进行估值,为循环经济倡议做出了贡献。
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引用次数: 0
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Metabolic Engineering Communications
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