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Have you tried turning it off and on again? Oscillating selection to enhance fitness-landscape traversal in adaptive laboratory evolution experiments 你试过把它关掉再打开吗?自适应实验室进化实验中振荡选择增强适应度景观遍历
IF 5.2 Q1 Engineering Pub Date : 2023-07-13 DOI: 10.1016/j.mec.2023.e00227
Alexander C. Carpenter , Adam M. Feist , Fergus S.M. Harrison , Ian T. Paulsen , Thomas C. Williams

Adaptive Laboratory Evolution (ALE) is a powerful tool for engineering and understanding microbial physiology. ALE relies on the selection and enrichment of mutations that enable survival or faster growth under a selective condition imposed by the experimental setup. Phenotypic fitness landscapes are often underpinned by complex genotypes involving multiple genes, with combinatorial positive and negative effects on fitness. Such genotype relationships result in mutational fitness landscapes with multiple local fitness maxima and valleys. Traversing local maxima to find a global maximum often requires an individual or sub-population of cells to traverse fitness valleys. Traversing involves gaining mutations that are not adaptive for a given local maximum but are necessary to ‘peak shift’ to another local maximum, or eventually a global maximum. Despite these relatively well understood evolutionary principles, and the combinatorial genotypes that underlie most metabolic phenotypes, the majority of applied ALE experiments are conducted using constant selection pressures. The use of constant pressure can result in populations becoming trapped within local maxima, and often precludes the attainment of optimum phenotypes associated with global maxima. Here, we argue that oscillating selection pressures is an easily accessible mechanism for traversing fitness landscapes in ALE experiments, and provide theoretical and practical frameworks for implementation.

适应性实验室进化(ALE)是工程和理解微生物生理学的强大工具。ALE依赖于突变的选择和富集,这些突变能够在实验设置施加的选择性条件下存活或更快地生长。表型适应度景观通常由涉及多个基因的复杂基因型支撑,对适应度有积极和消极的组合影响。这种基因型关系导致了具有多个局部适应度最大值和谷的变异适应度景观。遍历局部最大值以找到全局最大值通常需要单个或子群体的细胞来遍历适应度谷。遍历涉及获得突变,这些突变对给定的局部最大值不自适应,但对于“峰移”到另一个局部最大值或最终全局最大值是必要的。尽管有这些相对广为人知的进化原理,以及大多数代谢表型背后的组合基因型,但大多数应用ALE实验都是使用恒定的选择压力进行的。恒定压力的使用可能导致种群被困在局部最大值内,并且通常妨碍实现与全局最大值相关的最佳表型。在这里,我们认为振荡选择压力是ALE实验中遍历适应度景观的一种容易获得的机制,并为实现提供了理论和实践框架。
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引用次数: 2
Engineered ketocarotenoid biosynthesis in the polyextremophilic red microalga Cyanidioschyzon merolae 10D 工程化酮类胡萝卜素在多亲性红微藻Cyanidoschyzon merolae 10D中的生物合成
IF 5.2 Q1 Engineering Pub Date : 2023-06-26 DOI: 10.1016/j.mec.2023.e00226
Mark Seger , Fakhriyya Mammadova , Melany Villegas-Valencia , Bárbara Bastos de Freitas , Clarissa Chang , Iona Isachsen , Haley Hemstreet , Fatimah Abualsaud , Malia Boring , Peter J. Lammers , Kyle J. Lauersen

The polyextremophilic Cyanidiophyceae are eukaryotic red microalgae with promising biotechnological properties arising from their low pH and elevated temperature requirements which can minimize culture contamination at scale. Cyanidioschyzon merolae 10D is a cell wall deficient species with a fully sequenced genome that is amenable to nuclear transgene integration by targeted homologous recombination. C. merolae maintains a minimal carotenoid profile and here, we sought to determine its capacity for ketocarotenoid accumulation mediated by heterologous expression of a green algal β-carotene ketolase (BKT) and hydroxylase (CHYB). To achieve this, a synthetic transgene expression cassette system was built to integrate and express Chlamydomonas reinhardtii (Cr) sourced enzymes by fusing native C. merolae transcription, translation and chloroplast targeting signals to codon-optimized coding sequences. Chloramphenicol resistance was used to select for the integration of synthetic linear DNAs into a neutral site within the host genome. CrBKT expression caused accumulation of canthaxanthin and adonirubin as major carotenoids while co-expression of CrBKT with CrCHYB generated astaxanthin as the major carotenoid in C. merolae. Unlike green algae and plants, ketocarotenoid accumulation in C. merolae did not reduce total carotenoid contents, but chlorophyll a reduction was observed. Light intensity affected global ratios of all pigments but not individual pigment compositions and phycocyanin contents were not markedly different between parental strain and transformants. Continuous illumination was found to encourage biomass accumulation and all strains could be cultivated in simulated summer conditions from two different extreme desert environments. Our findings present the first example of carotenoid metabolic engineering in a red eukaryotic microalga and open the possibility for use of C. merolae 10D for simultaneous production of phycocyanin and ketocarotenoid pigments.

多亲蓝藻科是一种真核红色微藻,由于其低pH值和高温度要求,可以最大限度地减少大规模培养污染,因此具有良好的生物技术特性。Cyanidoschyzon merolae 10D是一种细胞壁缺陷物种,具有完全测序的基因组,可通过靶向同源重组进行核转基因整合。C.merolae保持着最小的类胡萝卜素谱,在这里,我们试图确定其通过异源表达绿藻β-胡萝卜素酮症酸酶(BKT)和羟化酶(CHYB)介导的酮类胡萝卜素积累的能力。为了实现这一点,建立了一个合成的转基因表达盒系统,通过将天然的C.merolae转录、翻译和叶绿体靶向信号融合到密码子优化的编码序列中,整合和表达来源于莱茵衣藻(Cr)的酶。使用氯霉素抗性来选择将合成的线性DNA整合到宿主基因组内的中性位点中。CrBKT的表达导致角黄素和阿多尼鲁宾作为主要类胡萝卜素的积累,而CrBKT与CrCHYB的共表达则产生虾青素作为主要的类胡萝卜素。与绿藻和植物不同,酮类胡萝卜素在C.merolae中的积累并没有降低类胡萝卜素的总含量,但观察到叶绿素a的减少。光照强度影响所有色素的整体比率,但不影响单个色素组成,并且藻蓝蛋白含量在亲本菌株和转化体之间没有显著差异。连续光照可以促进生物量的积累,所有菌株都可以在模拟的夏季条件下从两种不同的极端沙漠环境中培养。我们的发现提供了在红色真核微藻中进行类胡萝卜素代谢工程的第一个例子,并为使用C.merolae 10D同时生产藻蓝蛋白和酮类胡萝卜素色素开辟了可能性。
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引用次数: 2
Engineering of increased L-Threonine production in bacteria by combinatorial cloning and machine learning 通过组合克隆和机器学习提高细菌L-苏氨酸产量
IF 5.2 Q1 Engineering Pub Date : 2023-06-16 DOI: 10.1016/j.mec.2023.e00225
Paul Hanke , Bruce Parrello , Olga Vasieva , Chase Akins , Philippe Chlenski , Gyorgy Babnigg , Chris Henry , Fatima Foflonker , Thomas Brettin , Dionysios Antonopoulos , Rick Stevens , Michael Fonstein

The goal of this study is to develop a general strategy for bacterial engineering using an integrated synthetic biology and machine learning (ML) approach. This strategy was developed in the context of increasing L-threonine production in Escherichia coli ATCC 21277. A set of 16 genes was initially selected based on metabolic pathway relevance to threonine biosynthesis and used for combinatorial cloning to construct a set of 385 strains to generate training data (i.e., a range of L-threonine titers linked to each of the specific gene combinations). Hybrid (regression/classification) deep learning (DL) models were developed and used to predict additional gene combinations in subsequent rounds of combinatorial cloning for increased L-threonine production based on the training data. As a result, E. coli strains built after just three rounds of iterative combinatorial cloning and model prediction generated higher L-threonine titers (from 2.7 g/L to 8.4 g/L) than those of patented L-threonine strains being used as controls (4–5 g/L). Interesting combinations of genes in L-threonine production included deletions of the tdh, metL, dapA, and dhaM genes as well as overexpression of the pntAB, ppc, and aspC genes. Mechanistic analysis of the metabolic system constraints for the best performing constructs offers ways to improve the models by adjusting weights for specific gene combinations. Graph theory analysis of pairwise gene modifications and corresponding levels of L-threonine production also suggests additional rules that can be incorporated into future ML models.

本研究的目标是使用综合合成生物学和机器学习(ML)方法开发细菌工程的通用策略。该策略是在增加大肠杆菌ATCC 21277中L-苏氨酸产量的背景下开发的。最初基于与苏氨酸生物合成相关的代谢途径选择一组16个基因,并用于组合克隆以构建一组385个菌株以生成训练数据(即,与每个特定基因组合相关的一系列L-苏氨酸滴度)。基于训练数据,开发了混合(回归/分类)深度学习(DL)模型,并用于预测后续几轮组合克隆中的额外基因组合,以增加L-苏氨酸产量。因此,仅经过三轮迭代组合克隆和模型预测后构建的大肠杆菌菌株产生的L-苏氨酸滴度(从2.7 g/L到8.4 g/L)高于用作对照的专利L-苏氨酸菌株(4-5 g/L)。L-苏氨酸生产中有趣的基因组合包括tdh、metL、dapA和dhaM基因的缺失以及pntAB、ppc和aspC基因的过表达。对性能最佳构建体的代谢系统约束的机制分析提供了通过调整特定基因组合的权重来改进模型的方法。成对基因修饰和相应L-苏氨酸产生水平的图论分析也提出了可以纳入未来ML模型的额外规则。
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引用次数: 0
Optimization of a hybrid bacterial/Arabidopsis thaliana fatty acid synthase system II in Saccharomyces cerevisiae 酿酒酵母中杂交细菌/拟南芥脂肪酸合成酶系统Ⅱ的优化
IF 5.2 Q1 Engineering Pub Date : 2023-06-15 DOI: 10.1016/j.mec.2023.e00224
Tatiana A. Pozdniakova , João P. Cruz , Paulo César Silva , Flávio Azevedo , Pier Parpot , Maria Rosario Domingues , Magnus Carlquist , Björn Johansson

Fatty acids are produced by eukaryotes like baker's yeast Saccharomyces cerevisiae mainly using a large multifunctional type I fatty acid synthase (FASI) where seven catalytic steps and a carrier domain are shared between one or two protein subunits. While this system may offer efficiency in catalysis, only a narrow range of fatty acids are produced. Prokaryotes, chloroplasts and mitochondria rely instead on a FAS type II (FASII) where each catalytic step is carried out by a monofunctional enzyme encoded by a separate gene. FASII is more flexible and capable of producing a wider range of fatty acid structures, such as the direct production of unsaturated fatty acids. An efficient FASII in the preferred industrial organism S. cerevisiae could provide a platform for developing sustainable production of specialized fatty acids. We functionally replaced either yeast FASI genes (FAS1 or FAS2) with a FASII consisting of nine genes from Escherichia coli (acpP, acpS and fab -A, -B, -D, -F, -G, -H, -Z) as well as three from Arabidopsis thaliana (MOD1, FATA1 and FATB). The genes were expressed from an autonomously replicating multicopy vector assembled using the Yeast Pathway Kit for in-vivo assembly in yeast. Two rounds of adaptation led to a strain with a maximum growth rate (μmax) of 0.19 h−1 without exogenous fatty acids, twice the growth rate previously reported for a comparable strain. Additional copies of the MOD1 or fabH genes resulted in cultures with higher final cell densities and three times higher lipid content compared to the control.

脂肪酸由真核生物产生,如面包酵母酿酒酵母,主要使用大型多功能I型脂肪酸合成酶(FASI),其中七个催化步骤和一个载体结构域在一个或两个蛋白质亚基之间共享。虽然该系统可以提供催化效率,但只产生一小部分脂肪酸。原核生物、叶绿体和线粒体依赖于FAS II型(FASII),其中每个催化步骤都由一个单独基因编码的单功能酶进行。FASII更灵活,能够产生更广泛的脂肪酸结构,例如直接生产不饱和脂肪酸。在优选的工业生物酿酒酵母中高效的FASII可以为开发专门脂肪酸的可持续生产提供平台。我们用FASII在功能上取代了酵母FASI基因(FAS1或FAS2),FASII由来自大肠杆菌的9个基因(acpP、acpS和fab-a、-B、-D、-F、-G、-H、-Z)以及来自拟南芥的3个基因(MOD1、FATA1和FATB)组成。这些基因是从使用酵母通路试剂盒组装的自主复制多拷贝载体中表达的,用于在酵母中的体内组装。两轮适应使菌株在没有外源性脂肪酸的情况下具有0.19 h−1的最大生长速率(μmax),是先前报道的类似菌株生长速率的两倍。与对照相比,MOD1或fabH基因的额外拷贝导致培养物具有更高的最终细胞密度和三倍高的脂质含量。
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引用次数: 0
Cell-free metabolic engineering enables selective biotransformation of fatty acids to value-added chemicals 无细胞代谢工程使脂肪酸能够选择性生物转化为增值化学品
IF 5.2 Q1 Engineering Pub Date : 2023-06-01 DOI: 10.1016/j.mec.2022.e00217
Yushi Liu , Wan-Qiu Liu , Shuhui Huang , Huiling Xu , Haofan Lu , Changzhu Wu , Jian Li

Fatty acid-derived products such as alkanes, fatty aldehydes, and fatty alcohols have many applications in the chemical industry. These products are predominately produced from fossil resources, but their production processes are often not environmentally friendly. While microbes like Escherichia coli have been engineered to convert fatty acids to corresponding products, the design and optimization of metabolic pathways in cells for high productivity is challenging due to low mass transfer, heavy metabolic burden, and intermediate/product toxicity. Here, we describe an E. coli-based cell-free protein synthesis (CFPS) platform for in vitro conversion of long-chain fatty acids to value-added chemicals with product selectivity, which can also avoid the above issues when using microbial production systems. We achieve the selective biotransformation by cell-free expression of different enzymes and the use of different conditions (e.g., light and heating) to drive the biocatalysis toward different final products. Specifically, in response to blue light, cell-free expressed fatty acid photodecarboxylase (CvFAP, a photoenzyme) was able to convert fatty acids to alkanes with approximately 90% conversion. When the expressed enzyme was switched to carboxylic acid reductase (CAR), fatty acids were reduced to corresponding fatty aldehydes, which, however, could be further reduced to fatty alcohols by endogenous reductases in the cell-free system. By using a thermostable CAR and a heating treatment, the endogenous reductases were deactivated and fatty aldehydes could be selectively accumulated (>97% in the product mixture) without over-reduction to alcohols. Overall, our cell-free platform provides a new strategy to convert fatty acids to valuable chemicals with notable properties of operation flexibility, reaction controllability, and product selectivity.

脂肪酸衍生产品,如烷烃、脂肪醛和脂肪醇,在化学工业中有许多应用。这些产品主要由化石资源生产,但其生产过程往往不环保。虽然像大肠杆菌这样的微生物已经被改造成可以将脂肪酸转化为相应的产物,但由于低传质、重代谢负担和中间体/产物毒性,设计和优化细胞中的代谢途径以获得高产率是具有挑战性的。在这里,我们描述了一种基于大肠杆菌的无细胞蛋白质合成(CFPS)平台,用于体外将长链脂肪酸转化为具有产品选择性的增值化学品,在使用微生物生产系统时也可以避免上述问题。我们通过无细胞表达不同的酶和使用不同的条件(如光照和加热)来实现选择性的生物转化,以驱动生物催化产生不同的最终产物。具体而言,响应蓝光,无细胞表达的脂肪酸光羧化酶(CvFAP,一种光酶)能够以大约90%的转化率将脂肪酸转化为烷烃。当表达的酶转换为羧酸还原酶(CAR)时,脂肪酸被还原为相应的脂肪醛,然而,在无细胞系统中,脂肪醛可以通过内源性还原酶进一步还原为脂肪醇。通过使用热稳定的CAR和加热处理,内源性还原酶被失活,并且脂肪醛可以选择性地积累(在产物混合物中>97%)而不会过度还原为醇。总的来说,我们的无细胞平台提供了一种将脂肪酸转化为有价值化学物质的新策略,具有显著的操作灵活性、反应可控性和产物选择性。
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引用次数: 4
Engineered production of isoprene from the model green microalga Chlamydomonas reinhardtii 利用莱茵衣藻工程化生产异戊二烯
IF 5.2 Q1 Engineering Pub Date : 2023-06-01 DOI: 10.1016/j.mec.2023.e00221
Razan Z. Yahya, Gordon B. Wellman, Sebastian Overmans, Kyle J. Lauersen

Isoprene is a clear, colorless, volatile 5-carbon hydrocarbon that is one monomer of all cellular isoprenoids and a platform chemical with multiple applications in industry. Many plants have evolved isoprene synthases (IspSs) with the capacity to liberate isoprene from dimethylallyl diphosphate (DMADP) as part of cellular thermotolerance mechanisms. Isoprene is hydrophobic and volatile, rapidly leaves plant tissues and is one of the main carbon emission sources from vegetation globally. The universality of isoprenoid metabolism allows volatile isoprene production from microbes expressing heterologous IspSs. Here, we compared heterologous overexpression from the nuclear genome and localization into the plastid of four plant terpene synthases (TPs) in the green microalga Chlamydomonas reinhardtii. Using sealed vial mixotrophic cultivation, direct quantification of isoprene production was achieved from the headspace of living cultures, with the highest isoprene production observed in algae expressing the Ipomoea batatas IspS. Perturbations of the downstream carotenoid pathway through keto carotenoid biosynthesis enhanced isoprene titers, which could be further enhanced by increasing flux towards DMADP through heterologous co-expression of a yeast isopentenyl-DP delta isomerase. Multiplexed controlled-environment testing revealed that cultivation temperature, rather than illumination intensity, was the main factor affecting isoprene yield from the engineered alga. This is the first report of heterologous isoprene production from a eukaryotic alga and sets a foundation for further exploration of carbon conversion to this commodity chemical.

异戊二烯是一种透明、无色、挥发性的5碳碳氢化合物,是所有细胞类异戊二烯中的一种单体,也是一种在工业中具有多种应用的平台化学品。作为细胞耐热机制的一部分,许多植物已经进化出具有从二甲基烯丙基二磷酸(DMADP)中释放异戊二烯的能力的异戊二烯合成酶(IspSs)。异戊二烯具有疏水性和挥发性,能迅速离开植物组织,是全球植被的主要碳排放源之一。类异戊二烯代谢的普遍性允许表达异源IspSs的微生物产生挥发性异戊二烯。在这里,我们比较了来自核基因组的异源过表达和绿色微藻莱茵衣藻中四种植物萜烯合成酶(TP)在质体中的定位。使用密封小瓶混合营养培养,从活培养物的顶部空间实现了异戊二烯生产的直接定量,在表达Ipomoea batatas IspS的藻类中观察到最高的异戊二烯生产。通过酮类胡萝卜素生物合成对下游类胡萝卜素途径的干扰增强了异戊二烯滴度,通过异源共表达酵母异戊烯基DPδ异构酶增加向DMADP的流量可以进一步增强异戊二烯滴度。多重控制环境试验表明,影响工程藻类异戊二烯产量的主要因素是培养温度,而不是光照强度。这是第一份由真核藻类生产异源异戊二烯的报告,为进一步探索碳转化为这种商品化学品奠定了基础。
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引用次数: 0
Improved protein glycosylation enabled heterologous biosynthesis of monoterpenoid indole alkaloids and their unnatural derivatives in yeast 改进的蛋白质糖基化使单萜吲哚生物碱及其非天然衍生物在酵母中异源生物合成成为可能
IF 5.2 Q1 Engineering Pub Date : 2023-06-01 DOI: 10.1016/j.mec.2022.e00215
Mohammadamin Shahsavarani , Joseph Christian Utomo , Rahul Kumar , Melina Paz-Galeano , Jorge Jonathan Oswaldo Garza-García , Zhan Mai , Dae-Kyun Ro , Yang Qu

With over 3000 reported structures, monoterpenoid indole alkaloids (MIAs) constitute one of the largest alkaloid groups in nature, including the clinically important anticancer drug vinblastine and its semi-synthetic derivatives from Catharanthus roseus (Madagascar’s periwinkle). With the elucidation of the complete 28-step biosynthesis for anhydrovinblastine, it is possible to investigate the heterologous production of vinblastine and other medicinal MIAs. In this study, we successfully expressed the flavoenzyme O-acetylstemmadenine oxidase in Saccharomyces cerevisiae (baker’s yeast) by signal peptide modification, which is a vinblastine biosynthetic gene that has not been functionally expressed in this system. We also reported the simultaneous integration of ∼18 kb MIA biosynthetic gene cassettes as single copies into four genomic loci of baker’s yeast by CRISPR-Cas9, which enabled the biosynthesis of vinblastine precursors catharanthine and tabersonine from the feedstocks secologanin and tryptamine. We further demonstrated the biosynthesis of fluorinated and hydroxylated catharanthine and tabersonine derivatives using our yeasts, which showed that the MIA biosynthesis accommodates unnatural substrates, and the system can be further explored to produce other complex MIAs.

单萜类吲哚生物碱(MIAs)具有3000多种已报道的结构,是自然界中最大的生物碱类之一,包括临床上重要的抗癌药物长春碱及其来自长春花(马达加斯加长春花)的半合成衍生物。随着脱水长春碱完整的28步生物合成的阐明,有可能研究长春碱和其他药用MIA的异源生产。在本研究中,我们通过信号肽修饰成功地在酿酒酵母(面包酵母)中表达了风味酶O-乙酰基茎胺氧化酶,这是一种尚未在该系统中功能表达的长春碱生物合成基因。我们还报道了通过CRISPR-Cas9将~18kb MIA生物合成基因盒以单拷贝形式同时整合到面包酵母的四个基因组位点中,这使得长春碱前体长春花碱和色胺能够从饲料中生物合成。我们使用我们的酵母进一步证明了氟化和羟基化的长春花碱和tabersonine衍生物的生物合成,这表明MIA生物合成适应非天然底物,该系统可以进一步探索产生其他复杂的MIA。
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引用次数: 7
Metabolic engineering of low-pH-tolerant non-model yeast, Issatchenkia orientalis, for production of citramalate 用于生产柠檬酸盐的低pH耐受性非模式酵母Issatchenkia orientalis的代谢工程
IF 5.2 Q1 Engineering Pub Date : 2023-06-01 DOI: 10.1016/j.mec.2023.e00220
Zong-Yen Wu , Wan Sun , Yihui Shen , Jimmy Pratas , Patrick F. Suthers , Ping-Hung Hsieh , Sudharsan Dwaraknath , Joshua D. Rabinowitz , Costas D. Maranas , Zengyi Shao , Yasuo Yoshikuni

Methyl methacrylate (MMA) is an important petrochemical with many applications. However, its manufacture has a large environmental footprint. Combined biological and chemical synthesis (semisynthesis) may be a promising alternative to reduce both cost and environmental impact, but strains that can produce the MMA precursor (citramalate) at low pH are required. A non-conventional yeast, Issatchenkia orientalis, may prove ideal, as it can survive extremely low pH. Here, we demonstrate the engineering of I. orientalis for citramalate production. Using sequence similarity network analysis and subsequent DNA synthesis, we selected a more active citramalate synthase gene (cimA) variant for expression in I. orientalis. We then adapted a piggyBac transposon system for I. orientalis that allowed us to simultaneously explore the effects of different cimA gene copy numbers and integration locations. A batch fermentation showed the genome-integrated-cimA strains produced 2.0 g/L citramalate in 48 h and a yield of up to 7% mol citramalate/mol consumed glucose. These results demonstrate the potential of I. orientalis as a chassis for citramalate production.

甲基丙烯酸甲酯(MMA)是一种重要的石油化工产品,有着广泛的应用。然而,它的制造有很大的环境足迹。生物和化学合成(半合成)可能是降低成本和环境影响的一种很有前途的替代方法,但需要能够在低pH下生产MMA前体(柠檬酸盐)的菌株。非传统酵母Issatchenkia orientalis可能被证明是理想的,因为它可以在极低的pH值下存活。在这里,我们展示了I.orientalis用于柠檬酸盐生产的工程。通过序列相似性网络分析和随后的DNA合成,我们选择了一种更具活性的柠檬酸合成酶基因(cimA)变体在东方I.orientalis中表达。然后,我们将piggyBac转座子系统用于东方I.orientalis,使我们能够同时探索不同cimA基因拷贝数和整合位置的影响。分批发酵表明,基因组整合的cimA菌株在48小时内产生2.0g/L的柠檬酸盐,并且产量高达7%摩尔柠檬酸盐/mol消耗的葡萄糖。这些结果证明了东方乳杆菌作为柠檬酸盐生产底盘的潜力。
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引用次数: 2
Novel insights into construct toxicity, strain optimization, and primary sequence design for producing recombinant silk fibroin and elastin-like peptide in E. coli 在大肠杆菌中生产重组丝素蛋白和弹性蛋白样肽的构建毒性、菌株优化和一级序列设计的新见解
IF 5.2 Q1 Engineering Pub Date : 2023-06-01 DOI: 10.1016/j.mec.2023.e00219
Alexander Connor , Caleb Wigham , Yang Bai , Manish Rai , Sebastian Nassif , Mattheos Koffas , R. Helen Zha

Spider silk proteins (spidroins) are a remarkable class of biomaterials that exhibit a unique combination of high-value attributes and can be processed into numerous morphologies for targeted applications in diverse fields. Recombinant production of spidroins represents the most promising route towards establishing the industrial production of the material, however, recombinant spider silk production suffers from fundamental difficulties that includes low titers, plasmid instability, and translational inefficiencies. In this work, we sought to gain a deeper understanding of upstream bottlenecks that exist in the field through the production of a panel of systematically varied spidroin sequences in multiple E. coli strains. A restriction on basal expression and specific genetic mutations related to stress responses were identified as primary factors that facilitated higher titers of the recombinant silk constructs. Using these findings, a novel strain of E. coli was created that produces recombinant silk constructs at levels 4–33 times higher than standard BL21(DE3). However, these findings did not extend to a similar recombinant protein, an elastin-like peptide. It was found that the recombinant silk proteins, but not the elastin-like peptide, exert toxicity on the E. coli host system, possibly through their high degree of intrinsic disorder. Along with strain engineering, a bioprocess design that utilizes longer culturing times and attenuated induction was found to raise recombinant silk titers by seven-fold and mitigate toxicity. Targeted alteration to the primary sequence of the recombinant silk constructs was also found to mitigate toxicity. These findings identify multiple points of focus for future work seeking to further optimize the recombinant production of silk proteins and is the first work to identify the intrinsic disorder and subsequent toxicity of certain spidroin constructs as a primary factor related to the difficulties of production.

蜘蛛丝蛋白(spidroins)是一类引人注目的生物材料,表现出高价值属性的独特组合,可以加工成多种形态,用于不同领域的靶向应用。蜘蛛蛋白的重组生产代表了建立该材料工业生产的最有前途的途径,然而,重组蜘蛛丝的生产存在根本困难,包括低滴度、质粒不稳定和翻译效率低下。在这项工作中,我们试图通过在多个大肠杆菌菌株中生产一组系统变化的蜘蛛蛋白序列,来更深入地了解该领域存在的上游瓶颈。对基础表达的限制和与应激反应相关的特异性遗传突变被确定为促进重组丝构建体更高滴度的主要因素。利用这些发现,产生了一种新的大肠杆菌菌株,其产生的重组丝构建体的水平是标准BL21(DE3)的4-33倍。然而,这些发现并没有延伸到类似的重组蛋白,一种弹性蛋白样肽。研究发现,重组丝蛋白,而不是弹性蛋白样肽,可能通过其高度的内在紊乱对大肠杆菌宿主系统产生毒性。与菌株工程一起,发现利用较长培养时间和减弱诱导的生物工艺设计可以将重组丝滴度提高7倍并减轻毒性。还发现对重组丝构建体的一级序列的靶向改变可以减轻毒性。这些发现为未来寻求进一步优化丝蛋白重组生产的工作确定了多个重点,也是第一项将某些蜘蛛蛋白构建体的内在紊乱和随后的毒性确定为与生产困难相关的主要因素的工作。
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引用次数: 3
Re-evaluation of the impact of BUD21 deletion on xylose utilization by Saccharomyces cerevisiae BUD21缺失对酿酒酵母木糖利用影响的再评价
IF 5.2 Q1 Engineering Pub Date : 2023-06-01 DOI: 10.1016/j.mec.2023.e00218
Venkatachalam Narayanan , Anders G. Sandström , Marie F. Gorwa-Grauslund

Various rational metabolic engineering and random approaches have been applied to introduce and improve xylose utilization and ethanol productivity by Saccharomyces cerevisiae. Among them, the BUD21 gene was identified as an interesting candidate for enhancing xylose consumption as its deletion appeared to be sufficient to improve growth, substrate utilization and ethanol productivity on xylose, even in a laboratory strain lacking a heterologous xylose pathway. The present study aimed at studying the influence of BUD21 deletion in recombinant strains carrying heterologous oxido-reductive xylose utilization pathway. The positive effect of BUD21 gene deletion on aerobic growth and xylose utilization could not be confirmed in two non-engineered laboratory strains (BY4741 and CEN.PK 113-7D) that were grown in YP rich medium with 20 g/L xylose as sole carbon source, despite the fact that effective deletion of BUD21 gene was confirmed using both genotypic (colony PCR) and phenotypic (heat sensitive phenotype of the BUD21 deletion mutant) control experiments. Therefore, the effect of BUD21 deletion on xylose fermentation might be strain- or medium-dependent.

各种合理的代谢工程和随机方法已被应用于引入和提高酿酒酵母对木糖的利用率和乙醇产量。其中,BUD21基因被鉴定为增强木糖消耗的有趣候选者,因为其缺失似乎足以提高木糖的生长、底物利用率和乙醇生产率,即使在缺乏异源木糖途径的实验室菌株中也是如此。本研究旨在研究BUD21缺失对携带异源氧化还原木糖利用途径的重组菌株的影响。BUD21基因缺失对需氧生长和木糖利用的积极作用在两个非工程实验室菌株(BY4741和CEN.PK 113-7D)中不能得到证实,这两个菌株生长在以20g/L木糖作为唯一碳源的富含YP的培养基中,尽管使用基因型(菌落PCR)和表型(BUD21缺失突变体的热敏表型)对照实验证实了BUD21基因的有效缺失。因此,BUD21缺失对木糖发酵的影响可能是菌株或培养基依赖性的。
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引用次数: 0
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Metabolic Engineering Communications
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