Metabolic flux redirection from a central metabolic pathway toward a synthetic pathway using a metabolic toggle switch

doi:10.1016/j.ymben.2014.02.008

Metabolic Engineering 代谢工程

Volume 23, May 2014, Pages 175-184
第 23 卷，2014 年 5 月，第 175-184 页

https://doi.org/10.1016/j.ymben.2014.02.008 Get rights and content 获取权限和内容

Highlights 亮点

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A synthetic genetic circuit named “metabolic toggle switch” for the redirection of metabolic flux was constructed in E. coli.
在 E. coli 中构建了一种名为“代谢切换开关”的合成遗传电路，用于重定向代谢通量。
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Excess carbon flux in the TCA cycle was redirected toward isopropanol production pathway by metabolic toggle switch.
TCA 循环中过量的碳通量通过代谢切换开关被重定向到异丙醇生产途径。
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Using metabolic toggle switch, isopropanol production titer and yield improved up to 3.7 and 3.1 times, respectively.
使用代谢切换开关，异丙醇的产量和收率分别提高了 3.7 倍和 3.1 倍。

Abstract 摘要

Overexpression of genes in production pathways and permanent knockout of genes in competing pathways are often employed to improve production titer and yield in metabolic engineering. However, the deletion of a pathway responsible for growth and cell maintenance has not previously been employed, even if its competition with the production pathway is obvious. In order to optimize intracellular metabolism at each fermentation phase for bacterial growth and production, a methodology employing conditional knockout is required. We constructed a metabolic toggle switch in Escherichia coli as a novel conditional knockout approach and applied it to isopropanol production. The resulting redirection of excess carbon flux caused by interruption of the TCA cycle via switching gltA OFF improved isopropanol production titer and yield up to 3.7 and 3.1 times, respectively. This approach is a useful tool to redirect carbon flux responsible for bacterial growth and/or cell maintenance toward a synthetic production pathway.
在生产途径中过度表达基因并永久敲除竞争途径中的基因，常被用于提高代谢工程中的生产效价和产量。然而，即便与生产途径的竞争显而易见，先前并未采用过删除负责生长和细胞维持的途径的方法。为了在细菌生长和生产的每个发酵阶段优化细胞内代谢，需要一种采用条件性敲除的方法。我们在大肠杆菌中构建了一种代谢切换开关，作为一种新颖的条件性敲除方法，并将其应用于异丙醇生产。通过将 gltA 关闭导致 TCA 循环中断，从而重新定向过量的碳流，使得异丙醇的生产效价和产量分别提高了 3.7 倍和 3.1 倍。此方法是重新定向负责细菌生长和/或细胞维持的碳流向合成生产途径的有用工具。

Keywords 关键词

Synthetic genetic circuit

Metabolic flux redirection

Metabolic toggle switch

Synthetic pathway

合成遗传电路代谢通量重定向代谢切换开关合成途径

1. Introduction 1. 引言

Metabolically engineered microorganisms have been developed for the bio-based production of a variety of chemicals including solvents, fuels, polymers, pharmaceuticals, and perfumes (Keasling, 2013, Rabinovitch-Deere et al., 2013). In order to improve the titer and yield of the target product, both the overexpression of genes responsible for target compound production (Wang et al., 1999, Lee et al., 2007, Lütke-Eversloh and Stephanopoulos, 2008) and the deletion of genes responsible for by-product synthesis are required (Clomburg and Gonzalez, 2010; Qian et al., 2009; Atsumi et al., 2008a, Balzer et al., 2013). To address the latter, permanent knock out of the undesired genes from the chromosome by homologous recombination is usually applied (Jarboe et al., 2010) and generally improves the titer and yield of the target bio-based product (Zhang et al., 2007, Park et al., 2007, Alper et al., 2005, Matjan et al., 1989, Lee et al., 2005). However, some of these genes are responsible for bacterial growth and/or cell maintenance. Deletion of these genes would increase the titer and yield of the desired product per cell, but decrease the growth rate and/or final cell density, and perhaps resulting in cell death. These genes are rarely knocked out in metabolic engineering, since the final cell density is important for the total titer and yield during fermentation. An alternate approach is to keep the expression of these genes high until an adequate cell mass is achieved, then turn these genes off. For example, instead of permanently knocking out a gene from the chromosome, a novel gene expression control system could conditionally inhibit the expression of a specific gene. The Tet-Off system which turns expression of its controlling genes OFF by the addition of tetracycline and its derivative is often used for this purpose and has been applied to several organisms and/or cultured cells from mammals, plants, and insects (Zhu et al., 2002). However, such a gene expression control system has not been applied to the metabolic engineering of microorganisms.
代谢工程微生物已被开发用于生物基生产多种化学品，包括溶剂、燃料、聚合物、药品和香水（Keasling, 2013; Rabinovitch-Deere et al., 2013）。为了提高目标产品的滴度和产量，既需要过表达负责目标化合物生产的基因（Wang et al., 1999; Lee et al., 2007; Lütke-Eversloh and Stephanopoulos, 2008），也需要删除负责副产物合成的基因（Clomburg and Gonzalez, 2010; Qian et al., 2009; Atsumi et al., 2008a; Balzer et al., 2013）。针对后者，通常通过同源重组从染色体中永久敲除不需要的基因（Jarboe et al., 2010），这通常会提高目标生物基产品的滴度和产量（Zhang et al., 2007; Park et al., 2007; Alper et al., 2005; Matjan et al., 1989; Lee et al., 2005）。然而，其中一些基因负责细菌生长和/或细胞维持。删除这些基因将提高每个细胞所需产品的滴度和产量，但会降低生长速率和/或最终细胞密度，并可能导致细胞死亡。这些基因在代谢工程中很少被敲除，因为最终细胞密度对于发酵过程中的总滴度和产量至关重要。另一种方法是保持这些基因的高表达，直到达到足够的细胞量，然后关闭这些基因。例如，不是永久性地从染色体中敲除一个基因，而是一种新型的基因表达控制系统可以有条件地抑制特定基因的表达。Tet-Off 系统通过添加四环素及其衍生物来关闭其控制基因的表达，通常用于此目的，并已应用于来自哺乳动物、植物和昆虫的几种生物和/或培养细胞（Zhu 等，2002）。然而，这种基因表达控制系统尚未应用于微生物的代谢工程。

On the other hand, synthetic biology research has yielded synthetic genetic circuits that imitate natural gene expression systems. Examples are a genetic switch in bacteriophage lambda, and the cyanobacteria circadian oscillator (Gardner et al., 2000, Stricker et al., 2008). Synthetic genetic circuits encoding larger and more complex systems have recently been developed for biotechnological applications (Slusarczyk et al., 2012). Although each synthetic genetic circuit described to date is promising for metabolic engineering, there are only a few examples of their application to bio-production. One of the examples is the production of lycopene using engineered Escherichia coli (Farmer and Liao, 2000). In this report, a dynamic controller sensing the intracellular concentration of acetyl-phosphate as a signal for excess flux to the waste product was developed and this system contributed to improvement of lycopene production. The other example is the production of a fatty acid ester using engineered E. coli. In this report, a dynamic sensor-regulator system was also developed for sensing the intracellular concentration of precursor fatty acyl-CoA and improved its productivity (Zhang et al., 2012). In addition to these two examples, a few synthetic genetic circuits (a dynamic metabolic valve and a metabolic switchboard) have also been developed for biotechnological application, nevertheless they have not been applied to specific bio-production yet (Solomon et al., 2012, Callura et al., 2012).
另一方面，合成生物学研究已产生了模仿自然基因表达系统的合成基因电路。例如，噬菌体λ中的基因开关和蓝藻昼夜节律振荡器（Gardner 等，2000；Stricker 等，2008）。最近，编码更大更复杂系统的合成基因电路已被开发用于生物技术应用（Slusarczyk 等，2012）。尽管迄今为止描述的每个合成基因电路对于代谢工程都颇具前景，但在生物生产中的应用实例却寥寥无几。其中之一是利用工程化大肠杆菌生产番茄红素（Farmer 和 Liao，2000）。在该报告中，开发了一种动态控制器，它感知细胞内乙酰磷酸浓度作为流向废物产物的过量通量信号，这一系统促进了番茄红素生产的改进。另一个例子是利用工程化大肠杆菌生产脂肪酸酯。本报告还开发了一种动态传感器-调节器系统，用于感知前体脂肪酰辅酶 A 的细胞内浓度，并提高了其生产效率（Zhang 等，2012）。除了这两个例子外，还开发了一些合成遗传电路（动态代谢阀和代谢交换板）用于生物技术应用，但它们尚未应用于特定的生物生产（Solomon 等，2012，Callura 等，2012）。

Recently, in order to produce various target products, synthetic metabolic pathways have been designed using a metabolic intermediate as a precursor and have been constructed in appropriate host microorganisms. In particular, E. coli has been employed as promising host microorganism for many synthetic pathways including 1,3-propanediol (Nakamura and Whited, 2003), 1-butanol (Dellomonaco et al., 2011), isobutanol (Atsumi et al., 2008a), 1-propanol (Atsumi and Liao, 2008b), isopropanol (Hanai et al., 2007), acetone (Bermejo et al., 1998), itaconate (Liao and Chang, 2010), cadaverine (Qian et al., 2010), putrescine (Qian et al., 2009), taxadiene (Ajikumar et al., 2010), and isoprenoid (Chang and Keasling, 2006). Most synthetic pathways require endogenous metabolites such as phosphoenolpyruvate, pyruvate, and acetyl-CoA. These three metabolites are also precursors for the TCA cycle. Therefore, the production of these chemicals competes with the TCA cycle. To improve the titer and yield from a desired synthetic pathway, the genes encoding the enzymes for the competing pathways are often knocked out. However, as described above, since deletion of genes associated with the TCA cycle causes negative effect on cell growth and final cell density, these genes are rarely employed for deletion candidate target to increase the titer and yield of the target compound.
最近，为了生产各种目标产品，人们利用代谢中间体作为前体设计了合成代谢途径，并在适当的宿主微生物中构建了这些途径。特别是，大肠杆菌已被用作许多合成途径的有前途的宿主微生物，包括 1,3-丙二醇（Nakamura 和 Whited，2003）、1-丁醇（Dellomonaco 等，2011）、异丁醇（Atsumi 等，2008a）、1-丙醇（Atsumi 和 Liao，2008b）、异丙醇（Hanai 等，2007）、丙酮（Bermejo 等，1998）、衣康酸（Liao 和 Chang，2010）、尸胺（Qian 等，2010）、腐胺（Qian 等，2009）、紫杉二烯（Ajikumar 等，2010）和异戊二烯（Chang 和 Keasling，2006）。大多数合成途径需要内源性代谢物，如磷酸烯醇式丙酮酸、丙酮酸和乙酰辅酶 A。这三种代谢物也是 TCA 循环的前体。因此，这些化学品的生产与 TCA 循环竞争。为了提高所需合成途径的滴度和产量，通常会敲除编码竞争途径酶的基因。然而，如上所述，由于与 TCA 循环相关的基因删除会对细胞生长和最终细胞密度产生负面影响，这些基因很少被用作提高目标化合物滴度和产量的删除候选目标。

We focused on citrate synthase (CS) (EC 2.3.3.1) as a model candidate for the conditional inhibition of gene expression. CS catalyzes the condensation reaction of one molecule of acetyl-CoA and one molecule of oxaloacetate to one molecule of citrate. CS is a pace-making enzyme in the TCA cycle (Davis and Gilvarg, 1956) as the entry reaction for the TCA cycle and is important for bacterial growth. The deletion of gltA, which codes for CS, has not been employed in metabolic engineering because of its negative impact on bacterial growth. E. coli in which gltA is deleted shows no growth in M9 minimal medium, even if there is adequate glucose as a carbon source (Fisher et al., 2011, Mainguet et al., 2013). However, conditional inhibition of gltA expression after sufficient bacterial growth can redirect the flux of the TCA cycle toward a desired bio-production pathway. To this end, synthetic bistable genetic toggle switches look promising. Genetic toggle switches imitate the gene regulatory system used to switch the lytic life cycle and lysogenic life cycle in bacteriophage lambda (Gardner et al., 2000). Thus, genetic toggle switches could be used to switch the expression state between two different genes.
我们聚焦于柠檬酸合酶（CS）（EC 2.3.3.1）作为基因表达条件抑制的模型候选。CS 催化一分子乙酰-CoA 与一分子草酰乙酸缩合生成一分子柠檬酸的反应。作为 TCA 循环的起始反应，CS 是该循环中的关键酶（Davis 和 Gilvarg，1956），对细菌生长至关重要。由于其对细菌生长的负面影响，编码 CS 的 gltA 基因在代谢工程中尚未被利用。删除 gltA 的 E. coli 在 M9 基本培养基中无法生长，即使有充足的葡萄糖作为碳源（Fisher 等，2011；Mainguet 等，2013）。然而，在细菌充分生长后对 gltA 表达的条件抑制，可以将 TCA 循环的通量转向期望的生物生产途径。为此，合成的双稳态基因切换开关显得前景广阔。基因切换开关模仿了用于在噬菌体 lambda 中切换裂解生命周期和溶源生命周期的基因调控系统（Gardner 等，2000）。因此，基因切换开关可用于在两个不同基因之间切换表达状态。

This study aimed to demonstrate a major improvement in titer and yield of a specific bio-production. For this purpose, we developed a metabolic toggle switch based on previously developed synthetic toggle switch (Gardner et al., 2000). This switch was used as a novel metabolic engineering tool for the metabolic flux redirection of a central metabolic pathway in E. coli. The gene expression of gltA was kept high during the growth phase and then was turned off using a metabolic toggle switch. Isopropanol production via a synthetic metabolic pathway in E. coli was chosen as a model target product for bio-production using acetyl-CoA as the starting metabolite (Hanai et al., 2007). Isopropanol is one of the simplest secondary alcohols and is used as a starting material for the production of bio-plastics. This challenging study demonstrates improved titer and yield of a bio-production by application of synthetic genetic circuits.

2. Materials and methods 2. 材料与方法

2.1. Chemicals and regents
2.1. 化学品和试剂

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), Wako Pure Chemical Industries, Ltd. (Osaka, Japan), or MP Biomedicals (Solon, OH) unless otherwise specified.
所有化学品均购自 Sigma-Aldrich（圣路易斯，密苏里州）、和光纯药工业株式会社（大阪，日本）或 MP Biomedicals（索伦，俄亥俄州），除非另有说明。

2.2. Culture medium and conditions
2.2. 培养基和条件

For plasmid preparation, E. coli strains were cultured using 3 mL Luria–Bertani (LB) medium in test tubes by incubation at 37 °C on a rotary shaker (250 rpm). All shake flask cultures were started by 1% (v/v) inoculation from 3 mL modified M9 minimal medium containing 10 g L⁻¹ glucose and 10 ppm thiamin hydrochloride. Overnight cultures were shaken at 37 °C in a rotary shaker (250 rpm). All strains except for the isopropanol-producing strains were cultured with 20 mL M9 minimal medium containing 10 g L⁻¹ glucose in a flask and shaken at 30 °C in a rotary shaker (250 rpm). For isopropanol production cultures, M9 minimal medium containing 20 g L⁻¹ glucose was used and the strains were shaken at 30 °C in a rotary shaker (250 rpm).
对于质粒制备，大肠杆菌菌株在试管中使用 3 mL Luria-Bertani (LB) 培养基，在 37°C 的旋转摇床（250 rpm）中培养。所有摇瓶培养均以 1% (v/v) 的接种量从含有 10 g L ⁻¹ 葡萄糖和 10 ppm 盐酸硫胺素的 3 mL 改良 M9 基本培养基开始。过夜培养在 37°C 的旋转摇床（250 rpm）中震荡。除异丙醇生产菌株外，所有菌株均在含有 10 g L ⁻¹ 葡萄糖的 20 mL M9 基本培养基中培养，并在 30°C 的旋转摇床（250 rpm）中震荡。对于异丙醇生产培养，使用含有 20 g L ⁻¹ 葡萄糖的 M9 基本培养基，菌株在 30°C 的旋转摇床（250 rpm）中震荡。

2.3. Strains and plasmids
2.3. 菌株和质粒

All E. coli strains and plasmids used are listed in Table 1. E. coli DH5alphaZ1 (Lutz and Bujard, 1997) was used to prepare all plasmids. All E. coli strains were constructed based on BW25113 and JW0336 (ΔlacI) and JW0710 (ΔgltA). Strain TA1015 was obtained by removal of the kanamycin marker from JW0336 using plasmid pCP20 (Datsenko and Wanner, 2000). gltA gene knocking out from TA1015 was performed by P1 transduction using JW0710 as the donor strain. The resulting strain was TA1116. TA1184 was obtained by removal of the kanamycin marker from TA1116 using plasmid pCP20. lacI^q was amplified from pZS4INT-laci/tetR (Lutz and Bujard, 1997) using primers T329 (5′-GCCAT CCTCG AGCGT TGACA CCATC GAATG GTGCA AAACC-3′) and T330 (5′-GCCAT CGGAT CCTCA CTGCC CGCTT TCCAG TCG-3′). The amplified PCR product was digested with XhoI and BamHI, and inserted into the XhoI and BamHI site of pZS^⁎24MCS (Lutz and Bujard, 1997). The resulting plasmid was pTA216. pTA216 was introduced into TA1015 and TA1184 for constitutive expression of lacI. The resulting strains were TA1041 and TA1334, respectively. P_LtetO₁ fragment linking tryptophan terminator (T_trp) was amplified from pZA31-luc (Lutz and Bujard, 1997) using primers T417 (5′-GCCAT CAAGC TTAGC CCGCC TAATG AGCGG GCTTT TTTTT TCTAG ATCCC TATCA GTGAT AGAGA TTGAC ATCCC T-3′) and T349 (5′-GCCAT CGGAT CCTTT CTCCT CTTTA ATGAA TTCGG TCAGT GC-3′). The amplified PCR fragment was digested with HindIII and BamHI and inserted into the HindIII and BamHI site of pZE22-MCS (Lutz and Bujard, 1997). The resulting plasmid was pTA646. tetR was amplified from pZS4INT-laci/tetR (Lutz and Bujard, 1997) using primers T939 (5′-GCCAT CGGTA CCATG GCTGG TTCTC GCAGA AAGAA AC-3′) and T940 (5′-GCCAT CGGGC CCTA AGACC CACTT TCACA TTTAA GTTGT TTTTC-3′). The amplified PCR fragment was digested with Acc65I and ApaI and inserted into the Acc65I and ApaI site of pTA646. The resulting plasmid was pTA649. gltA with a LAA degradation tag sequence (Prindle et al., 2011, Keiler et al., 1996) (gltA.LAA) was amplified from BW25113 genome using primer T1042 (5′-GCCAT CGGAT CCATG GCTGA TACAA AAGCA AAACT CACC-3′) and T1045 (5′-GCCAT CACGC GTTTA AGCTG CTAAA GCGTA GTTTT CGTCG TTTGC TGCAC GCTTG ATATC GCTTT TAAAG TCGC-3′). The amplified PCR fragment was digested with BamHI and MluI inserted into the BamHI and MluI site under P_LtetO₁ promoter in pTA649. The resulting plasmid was pTA659. In order to change the origin of replication (and thus copy number) in pTA659 from ColE1 to p15A, pTA669 was produced by inserting an AvrII-SacI fragment of pZA22-MCS (Lutz and Bujard, 1997) into an AvrII-SacI site of pTA659. To construct medium copy vacant switch vector, pTA695 was produced by inserting an AvrII-SacI fragment of pZA22-MCS (Lutz and Bujard, 1997) into an AvrII-SacI site of pTA649. pTA669 and pTA659 were introduced into TA1334 and 1041, respectively. The resulting strains were designed as TA1357 and TA1414, respectively. For isopropanol production, pTA147 (Soma et al., 2012), which contains the isopropanol production pathway genes, was introduced into TA1357 and TA1414. The resulting strains were TA1415 and TA1424, respectively.
所有使用的大肠杆菌菌株和质粒均列于表 1 中。大肠杆菌 DH5alphaZ1（Lutz 和 Bujard，1997）用于制备所有质粒。所有大肠杆菌菌株均基于 BW25113、JW0336（ΔlacI）和 JW0710（ΔgltA）构建。通过使用质粒 pCP20（Datsenko 和 Wanner，2000）从 JW0336 中去除卡那霉素标记，获得了菌株 TA1015。使用 JW0710 作为供体菌株，通过 P1 转导从 TA1015 中敲除了 gltA 基因。所得菌株为 TA1116。通过使用质粒 pCP20 从 TA1116 中去除卡那霉素标记，获得了 TA1184。lacI ^q 使用引物 T329（5′-GCCAT CCTCG AGCGT TGACA CCATC GAATG GTGCA AAACC-3′）和 T330（5′-GCCAT CGGAT CCTCA CTGCC CGCTT TCCAG TCG-3′）从 pZS4INT-laci/tetR（Lutz 和 Bujard，1997）中扩增。扩增的 PCR 产物用 XhoI 和 BamHI 消化，并插入到 pZS ^⁎ 24MCS（Lutz 和 Bujard，1997）的 XhoI 和 BamHI 位点。所得质粒为 pTA216。将 pTA216 引入 TA1015 和 TA1184 中，用于 lacI 的组成型表达。所得菌株分别为 TA1041 和 TA1334。 P _L tetO ₁ 片段连接色氨酸终止子（T _trp ）从 pZA31-luc（Lutz 和 Bujard, 1997）使用引物 T417（5′-GCCAT CAAGC TTAGC CCGCC TAATG AGCGG GCTTT TTTTT TCTAG ATCCC TATCA GTGAT AGAGA TTGAC ATCCC T-3′）和 T349（5′-GCCAT CGGAT CCTTT CTCCT CTTTA ATGAA TTCGG TCAGT GC-3′）扩增。扩增的 PCR 片段用 HindIII 和 BamHI 消化并插入到 pZE22-MCS（Lutz 和 Bujard, 1997）的 HindIII 和 BamHI 位点。得到的质粒为 pTA646。tetR 从 pZS4INT-laci/tetR（Lutz 和 Bujard, 1997）使用引物 T939（5′-GCCAT CGGTA CCATG GCTGG TTCTC GCAGA AAGAA AC-3′）和 T940（5′-GCCAT CGGGC CCTA AGACC CACTT TCACA TTTAA GTTGT TTTTC-3′）扩增。扩增的 PCR 片段用 Acc65I 和 ApaI 消化并插入到 pTA646 的 Acc65I 和 ApaI 位点。得到的质粒为 pTA649。带有 LAA 降解标签序列的 gltA（Prindle 等, 2011, Keiler 等, 1996）（gltA.LAA）从 BW25113 基因组使用引物 T1042（5′-GCCAT CGGAT CCATG GCTGA TACAA AAGCA AAACT CACC-3′）和 T1045（5′-GCCAT CACGC GTTTA AGCTG CTAAA GCGTA GTTTT CGTCG TTTGC TGCAC GCTTG ATATC GCTTT TAAAG TCGC-3′）扩增。扩增的 PCR 片段用 BamHI 和 MluI 消化，并插入到 pTA649 中 P _L tetO ₁ 启动子下的 BamHI 和 MluI 位点。所得质粒为 pTA659。为了将 pTA659 中的复制起点（从而改变拷贝数）从 ColE1 更改为 p15A，通过将 pZA22-MCS（Lutz 和 Bujard，1997）的 AvrII-SacI 片段插入 pTA659 的 AvrII-SacI 位点，产生了 pTA669。为了构建中等拷贝数的空切换载体，通过将 pZA22-MCS（Lutz 和 Bujard，1997）的 AvrII-SacI 片段插入 pTA649 的 AvrII-SacI 位点，产生了 pTA695。pTA669 和 pTA659 分别被引入 TA1334 和 1041。所得菌株分别命名为 TA1357 和 TA1414。为了生产异丙醇，将包含异丙醇生产途径基因的 pTA147（Soma 等，2012）引入 TA1357 和 TA1414。所得菌株分别为 TA1415 和 TA1424。

Table 1. Bacterial strains and plasmids used in this study.
表 1. 本研究中使用的细菌菌株和质粒。

Strains/plasmid 菌株/质粒	Relevant characteristics 相关特性	References/source 参考文献/来源
E. coli strains E. coli 菌株
BW25113	lacI^q rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78	Datsenko and Wanner (2000) Datsenko 和 Wanner（2000）
JW0336	BW25113 ΔlacI785::kan^R	Baba, 2006 爸爸，2006
JW0710	BW25113 ΔgltA770:: kan^R	Baba, 2006 爸爸，2006
TA1015	Same as JW0336 but was removed kan^R 与 JW0336 相同，但删除了 kan ^R
TA1116	TA1015ΔgltA770:: kan^R	This study 本研究
TA1184	Same as TA1116 but was removed kan^R 同 TA1116，但已移除 kan ^R	This study 本研究
TA1041	TA1015/pTA216	This study 本研究
TA1334	TA1184/pTA216	This study 本研究
TA1357	TA1334/pTA669	This study 本研究
TA1414	TA1041/pTA695	This study 本研究
TA1415	TA1357/pTA147	This study 本研究
TA1424	TA1414/pTA147	This study 本研究

Plasmids 质粒
pTA16	pZA31luc (p15A, cm^R, P_LtetO₁::luc)	EXPRESSYS
pTA43	pZE22luc (ColE1, kan^R, P_LlacO₁::luc)	EXPRESSYS
pTA154	pZA21MCS (p15A, kan^R, P_LtetO₁::MCS1)	EXPRESSYS
pTA157	pZS^⁎24MCS (pSC101^⁎, kan^R, P_lac/ara-1::MCS1)	EXPRESSYS
pTA158	pZS4INT-laci/tetR (pSC101, spec^R, P_lacI^q::lacI, PN25::tetR) pZS4INT-laci/tetR (pSC101, 抗性标记 ^R , 启动子 _lacI ^q ::lacI, PN25::tetR)	EXPRESSYS
pTA215	p15A, cm ^R, P_lacI^q::lacI	This study 本研究
pTA216	pSC101^⁎, cm^R, P_lacI^q::lacI	This study 本研究
pTA646	ColE1, kan^R, P_LlacO₁::MCS, P_LtetO₁::MCS	This study 本研究
pTA649	ColE1, kan^R, P_LlacO₁::tetR, P_LtetO₁::MCS	This study 本研究
pTA659	ColE1, kan^R, P_LlacO₁::tetR, P_LtetO₁::gltA.LAA	This study 本研究
pTA695	p15A, kan^R, P_LlacO₁::tetR, P_LtetO₁::MCS	This study 本研究
pTA669	p15A, kan^R, P_LlacO₁::tetR, P_LtetO₁::gltA.LAA	This study 本研究
pTA147	ColE1, amp^R, P_LlacO₁::thlA, atoAD, adc, Cbadh	Soma et al. (2012) Soma 等人（2012）

Abbreviations: Amp, ampicillin; Kan, kanamycin; Spec, spectinomycin; Cm, Chloramphenicol; R, resistance.
缩写：Amp，氨苄青霉素；Kan，卡那霉素；Spec，壮观霉素；Cm，氯霉素；R，抗性。

2.4. Citrate synthase assay
2.4. 柠檬酸合酶测定

Cells from 10 mL culture broth were harvested by centrifugation at 5000×g for 10 min. The pellet was suspended in 2 mL of 120 mM Tris/HCl, pH 8.0, containing 1 mM EDTA, 10 mM MgCl₂ and 0.1 M KCl, and was sonicated on ice. After centrifugation at 25,000×g for 15 min, the supernatant fraction was assayed for enzyme activity (Bloxham et al., 1983). The protein concentrations of the crude extracts were determined by absorbance at 280 nm. The activity of citrate synthase, the gltA gene product, was determined using a citrate synthase assay kit (Sigma-Aldrich, St. Louis, MO). The enzyme reaction was initiated by adding 0.3 mM acetyl-CoA into the reaction mixture (0.5 mM oxaloacetate (OXA) and 0.1 mM 5, 5′-dithiobis (2-nitrobenzoic acid) (DTNB) in 100 mM Tris–HCl (pH 7.5)). The enzyme reaction was monitored by the reaction of coenzyme A with DTNB to form thionitrobenzoic acid (TNB). One unit of citrate synthase was defined as the conversion of 1.0 µmole DTNB into TNB per minute (Bloxham et al., 1983). Absorbance at 412 nm was monitored using an Infinity 200 pro plate reader (TECAN, Männedorf, Switzerland).
从 10 mL 培养液中通过 5000×g 离心 10 分钟收集细胞。将沉淀悬浮于 2 mL 含有 1 mM EDTA、10 mM MgCl ₂ 和 0.1 M KCl 的 120 mM Tris/HCl (pH 8.0)缓冲液中，并在冰上进行超声处理。以 25,000×g 离心 15 分钟后，上清液用于酶活性测定（Bloxham 等，1983）。粗提物的蛋白质浓度通过 280 nm 处的吸光度测定。柠檬酸合酶（gltA 基因产物）的活性使用柠檬酸合酶测定试剂盒（Sigma-Aldrich, St. Louis, MO）进行测定。酶反应通过向反应混合物（0.5 mM 草酰乙酸（OXA）和 0.1 mM 5,5′-二硫代双（2-硝基苯甲酸）（DTNB）在 100 mM Tris–HCl (pH 7.5)中）添加 0.3 mM 乙酰辅酶 A 启动。通过辅酶 A 与 DTNB 反应生成硫代硝基苯甲酸（TNB）来监测酶反应。一个柠檬酸合酶单位定义为每分钟将 1.0 µmole DTNB 转化为 TNB（Bloxham 等，1983）。使用 Infinity 200 pro 酶标仪（TECAN, Männedorf, Switzerland）监测 412 nm 处的吸光度。

2.5. Measurement of extracellular metabolites
2.5. 细胞外代谢物的测量

Optical density at 600 nm (OD₆₀₀) of bacterial culture was measured using a SpectraMax M2 96 well plate reader (Molecular Devices, Union City, CA). Dry cell weight (DCW) was calculated from OD₆₀₀ (OD₆₀₀=0.25 g DCW L⁻¹). To quantitate extracellular metabolites, 1 ml bacterial culture broth was centrifuged for 1 min at 13,000xg in a micro-centrifuge and the supernatant was filtered through a 0.2 µm syringe filter for HPLC and GC analysis. Alcohol compounds produced were quantified using a GC equipped with a flame ionization detector, as previously reported (Hanai et al., 2007). The fermentation by-products (alpha-ketoglutarate, pyruvate, citrate, lactate, acetate, formate, fumalate, malate and succinate) were quantified using a HPLC system for organic acid analysis (Shimadzu Scientific Instruments, Inc., Kyoto, Japan), equipped with an ion-exclusion chromatography column (Showdex RSpak KC-811, SHOWA DENKO K.K., Tokyo, Japan) and a conductivity detector (Shimadzu CCD-10 A vp). The mobile phase was 5.0 mM p-nitorophenylphosphate, the flow rate was 1.0 mL/min, and the column was kept at 40 °C. Extracellular glucose was quantified using a HPLC system for sugar compound analysis (Shimadzu Scientific Instruments, Inc., Kyoto, Japan) equipped with a ligand exchange chromatography column (Showdex SP0810, SHOWA DENKO K.K., Tokyo, Japan) and a differential refractive index detector (Shimadzu RID-10A). The mobile phase was MilliQ water, the flow rate was 1.0 mL/min, and the column was kept at 80 °C. The theoretical maximum acetate yield is defined as 2 mol of acetate produced per 1 mol glucose consumed. The theoretical maximum isopropanol yield is defined as 1 mol of isopropanol produced per 1 mol glucose consumed.
使用 SpectraMax M2 96 孔板阅读器（Molecular Devices，联合城，加利福尼亚州）测量细菌培养物在 600 nm 处的光密度（OD ₆₀₀ ）。从 OD ₆₀₀ 计算干细胞重量（DCW）（OD ₆₀₀ =0.25 g DCW L ⁻¹ ）。为了定量细胞外代谢物，将 1 ml 细菌培养液在微型离心机中以 13,000xg 离心 1 分钟，上清液通过 0.2 µm 注射器过滤器过滤，用于 HPLC 和 GC 分析。产生的醇类化合物使用配备火焰离子化检测器的 GC 进行定量，如先前报道（Hanai 等，2007）。发酵副产物（α-酮戊二酸、丙酮酸、柠檬酸、乳酸、乙酸、甲酸、富马酸、苹果酸和琥珀酸）使用 HPLC 系统进行有机酸分析（Shimadzu Scientific Instruments, Inc.，京都，日本），配备离子排斥色谱柱（Showdex RSpak KC-811，昭和电工株式会社，东京，日本）和电导检测器（Shimadzu CCD-10 A vp）。流动相为 5.0 mM 对硝基苯磷酸盐，流速为 1.0 mL/min，柱温保持在 40°C。使用配备有配体交换色谱柱（Showdex SP0810，昭和电工株式会社，东京，日本）和差示折光检测器（Shimadzu RID-10A）的 HPLC 系统（岛津科学仪器公司，京都，日本）对细胞外葡萄糖进行定量分析。流动相为 MilliQ 水，流速为 1.0 mL/min，色谱柱保持在 80°C。理论最大乙酸产率定义为每消耗 1 摩尔葡萄糖产生 2 摩尔乙酸。理论最大异丙醇产率定义为每消耗 1 摩尔葡萄糖产生 1 摩尔异丙醇。

2.6. Measurement of intracellular metabolites
2.6. 细胞内代谢物的测量

TA1357 strains were cultured in 20 mL M9 medium containing 10 g L⁻¹ glucose and 10 ppm thiamin hydrochloride at 30 °C in a rotary shaker (250 rpm). 0.1 mM IPTG was added at 9 h for switching gltA gene expression OFF in the TA1357 cells. Cells were collected at 3 h and 23 h via rapid vacuum filtration using a membrane filter (Omnipore, 0.45 μM, 25-mm diameter polytetrafluoroethylene, Millipore, Billerica, MA). Each filter was transferred into a 15 mL centrifuge tube then immediately quenched with liquid nitrogen and stored at −80 °C until used for metabolite extraction. Intracellular metabolites were extracted from lyophilized cells using the chloroform–methanol–water method (Hasunuma et al., 2011). The water phase of the extract (300 μL) was dried under vacuum and stored at −80 °C until analysis. Dried extracts were dissolved in 50 μL MilliQ water, then applied to a LC–QqQ–MS system (high-performance liquid chromatography: Agilent 1290 Infinity, MS: Agilent 6460 with Jet Stream Technology) (Agilent Technologies, Waldbronn, Germany) controlled by MassHunter Workstation Data Acquisition software (Agilent Technologies, Waldbronn, Germany). LC–QqQ–MS analysis was performed using conditions described previously (Kato et al., 2012).
TA1357 菌株在含有 10 g/L 葡萄糖和 10 ppm 盐酸硫胺素的 20 mL M9 培养基中，在 30°C 的旋转摇床（250 rpm）中培养。在 9 小时时加入 0.1 mM IPTG 以关闭 TA1357 细胞中的 gltA 基因表达。分别在 3 小时和 23 小时通过快速真空过滤使用膜过滤器（Omnipore，0.45 μM，25 毫米直径聚四氟乙烯，Millipore，Billerica，MA）收集细胞。每个过滤器转移到 15 mL 离心管中，立即用液氮淬灭，并储存在-80°C，直到用于代谢物提取。细胞内代谢物通过冻干细胞使用氯仿-甲醇-水法提取（Hasunuma 等，2011）。提取物的水相（300 μL）在真空下干燥并储存在-80°C，直到分析。干燥的提取物溶解在 50 μL MilliQ 水中，然后应用于 LC-QqQ-MS 系统（高效液相色谱：Agilent 1290 Infinity，质谱：Agilent 6460 with Jet Stream Technology）（安捷伦科技，Waldbronn，德国），由 MassHunter Workstation 数据采集软件（安捷伦科技，Waldbronn，德国）控制。 LC–QqQ–MS 分析采用先前描述的条件进行（Kato 等人，2012 年）。

3. Results 3. 结果

3.1. Construction of the genetic toggle switch and regulation of gltA expression
3.1. 遗传切换开关的构建与 gltA 表达的调控

All bacterial cultures were grown by batch flask fermentation using M9 minimal medium containing 10 or 20 g L⁻¹ glucose. The gltA deleted E. coli strains, JW0710 (BW21553 ΔgltA::kan^R), TA1116 (BW21553 ΔlacI ΔgltA::kan^R), and TA1184 (BW21553 ΔlacI ΔgltA), showed no growth under these culture conditions. However, when M9 medium was supplemented with 1% (w/v) casamino acid or LB medium, these ΔgltA strains showed similar growth curves to wild type and ΔlacI strain JW0336. CS activity was not detected in crude cell extracts of the ΔgltA strains. The synthetic gltA OFF switch was constructed in TA1184 and CS activity was assayed (Fig. 3). Switching gltA OFF is caused by repression of gltA transcription from the P_LtetO₁ promoter (Lutz and Bujard, 1997) by the TetR repressor. TetR repressor encoded by tetR under the P_LlacO₁ promoter and its expression is induced by the addition of 0.1 mM IPTG (Fig. 1-B). In the absence of IPTG, the CS activity of TA1357 (ΔlacI ΔgltA/pTA216, pTA669) (Fig. 2-A) containing a gltA OFF switch on the medium copy plasmid was 2.8 times higher than that of control strain TA1414 (ΔlacI/pTA216, pTA695) (Fig. 2-A) in which CS is expressed from native chromosomal gltA. The final cell density of TA1357 was comparable with that of TA1414 (TA1414=0.27±0.01, TA1357=0.28±0.01 g DCW L⁻¹). When 0.1 mM IPTG was added to TA1357 after 9 h of cultivation, its CS activity decreased by 93% within 4 h (Fig. 3-A). Additionally, in the presence of IPTG, the specific growth rate of TA1357 at logarithmic growth phase (11–13 h) decreased by about 67% compared to TA1414 and TA1357 without IPTG (TA1414=0.12±0.01, TA1357_IPTG (−)=0.13±0.01, TA1357_IPTG 9 h=0.08±0.00 g DCW⁻¹ h⁻¹). In the absence of IPTG, CS activity of TA1357 and of TA1414 were maintained until late logarithmic growth phase (~21 h). These results indicated that the expression of gltA was turned off by the synthetic gltA OFF switch, resulting in the effective conditional disappearance of CS activity.
所有细菌培养物均采用含有 10 或 20 g/L 葡萄糖的 M9 最小培养基，通过批量瓶发酵进行培养。gltA 缺失的大肠杆菌菌株 JW0710（BW21553 ΔgltA::kan）、TA1116（BW21553 ΔlacI ΔgltA::kan）和 TA1184（BW21553 ΔlacI ΔgltA）在这些培养条件下未显示生长。然而，当 M9 培养基补充 1%（w/v）酪蛋白氨基酸或 LB 培养基时，这些ΔgltA 菌株显示出与野生型和ΔlacI 菌株 JW0336 相似的生长曲线。在ΔgltA 菌株的粗细胞提取物中未检测到 CS 活性。合成的 gltA 关闭开关在 TA1184 中构建，并测定了 CS 活性（图 3）。gltA 的关闭是由 TetR 阻遏物抑制 P tetO 启动子（Lutz 和 Bujard，1997）的 gltA 转录引起的。TetR 阻遏物由 tetR 编码，受 P lacO 启动子调控，其表达通过添加 0.1 mM IPTG 诱导（图 1-B）。在没有 IPTG 的情况下，含有 gltA 关闭开关的中等拷贝质粒的 TA1357（ΔlacI ΔgltA/pTA216, pTA669）（图 2-A）的 CS 活性比对照菌株 TA1414（ΔlacI/pTA216, pTA695）（图）高 2.8 倍。 2-A) 其中 CS 由天然染色体 gltA 表达。TA1357 的最终细胞密度与 TA1414 相当（TA1414=0.27±0.01，TA1357=0.28±0.01 g DCW L ⁻¹ ）。当在培养 9 小时后向 TA1357 添加 0.1 mM IPTG 时，其 CS 活性在 4 小时内下降了 93%（图 3-A）。此外，在存在 IPTG 的情况下，TA1357 在对数生长期（11-13 小时）的比生长速率比未添加 IPTG 的 TA1414 和 TA1357 降低了约 67%（TA1414=0.12±0.01，TA1357_IPTG (−)=0.13±0.01，TA1357_IPTG 9 h=0.08±0.00 g DCW ⁻¹ h ⁻¹ ）。在没有 IPTG 的情况下，TA1357 和 TA1414 的 CS 活性一直保持到对数生长后期（约 21 小时）。这些结果表明，gltA 的表达被合成的 gltA 关闭开关关闭，导致 CS 活性有效条件性消失。

3.2. Effect of switching gltA OFF on growth, glucose consumption and acetate production
3.2. 关闭 gltA 对生长、葡萄糖消耗和乙酸生成的影响

To confirm the effect of switching gltA OFF on growth, glucose consumption and acetate production, IPTG was added to TA1357 cultures at various time points (0, 6, 9, 12, and 15 h). Without the addition of IPTG, the growth, glucose consumption and acetate production of TA1357 were similar to those of control strain TA1414 (Fig. 4). On the other hand, the growth of TA1357 was inhibited by the addition of IPTG. Especially, the addition of IPTG at earlier time points (0, 6, and 9 h) effectively inhibited growth (Fig. 4-A, Table 2). The later the addition of IPTG, the smaller the increase in concentration of acetate (Fig. 4-C). Maximum acetate production (67.1 mM) was obtained when the IPTG was added to TA1357 at 0 h; the amount of acetate produced (a yield of 73 mol/mol% (Table 2)) was more than three times that of TA1414. The acetate production rate at logarithmic growth phase was 2.6 times higher than that of TA1414 (Table S-1), and at stationary phase it was 5.1 times higher (Table S-1). Additionally, switching gltA OFF in TA1357 caused 26–50 times higher accumulation of pyruvate than that of TA1414 (TA1357=5.2–10.0 mM, TA1414=~0.2 mM), whereas TA1414 showed 7.5 times higher accumulation of alpha-KG than that of TA1357 (TA1357=~1.3 mM, TA1414=9.7 mM). These results suggested that switching gltA OFF decreased the metabolic flux toward the TCA cycle, resulting in growth inhibition and increased acetate production.
为了确认关闭 gltA 对生长、葡萄糖消耗和乙酸产量的影响，在不同时间点（0、6、9、12 和 15 小时）向 TA1357 培养物中添加了 IPTG。在不添加 IPTG 的情况下，TA1357 的生长、葡萄糖消耗和乙酸产量与对照菌株 TA1414 相似（图 4）。另一方面，添加 IPTG 抑制了 TA1357 的生长。特别是在较早的时间点（0、6 和 9 小时）添加 IPTG 有效地抑制了生长（图 4-A，表 2）。IPTG 添加的时间越晚，乙酸浓度的增加越小（图 4-C）。当在 0 小时向 TA1357 添加 IPTG 时，获得了最大的乙酸产量（67.1 mM）；产生的乙酸量（产率为 73 mol/mol%（表 2））是 TA1414 的三倍多。对数生长期的乙酸产率是 TA1414 的 2.6 倍（表 S-1），而在稳定期则是 5.1 倍（表 S-1）。此外，在 TA1357 中关闭 gltA 导致丙酮酸的积累比 TA1414 高 26-50 倍（TA1357=5.2-10.0 mM，TA1414=~0.2 mM），而 TA1414 显示出 7.α-KG 的积累量比 TA1357 高 5 倍（TA1357≈1.3 mM，TA1414=9.7 mM）。这些结果表明，关闭 gltA 降低了流向 TCA 循环的代谢通量，导致生长抑制和乙酸产量增加。

Table 2. Comparison of fermentation data from different strains for endogenous acetate production.
表 2. 不同菌株内源性乙酸生产发酵数据的比较。

Empty Cell	(g L⁻¹)	(mM)	(mol/mol%)
Empty Cell	Cell density 细胞密度	Acetate 醋酸酯	Yield 产量
TA1357_0 h	1.1±0.1	67.1±1.2	73.1±6.3
TA1357_3 h	1.1±0.0	61.4±6.8	65.3±5.1
TA1357_6 h	1.0±0.1	60.7±2.7	62.7±3.1
TA1357_9 h	1.0±0.0	52.2±3.1	42.8±0.7
TA1357_12 h	1.2±0.0	39.9±0.4	32.3±1.4
TA1357_15 h	1.2±0.0	29.4±1.5	23.9±1.3
TA1357_18 h	1.2±0.0	27.3±2.0	23.6±1.0
TA1357 (−)	1.4±0.0	21.2±1.1	20.7±0.6
TA1414	1.4±0.0	21.4±0.8	17.3±0.7

3.3. Redistribution of intracellular metabolic flux associated with switching gltA OFF
3.3. 与关闭 gltA 相关的细胞内代谢通量重新分配

To confirm the changes in intracellular metabolites associated with switching gltA OFF, IPTG was added to TA1357 at 9 h; cells were collected for LC–MS analysis before (3 h) and after (23 h) the switching. TA1414 and TA1357 cells without the addition of IPTG were also collected at the same time points. As shown in Fig. 5, the intracellular concentrations of intermediate metabolites in the first 5 steps of glycolysis were not significantly changed in both strains and at both sampling points, with or without added IPTG. On the other hand, the concentrations of metabolites at the remaining steps of glycolysis and the TCA cycle changed remarkably along with the progress of culture phase, and differences due to the presence or absence of switching gltA OFF were clearly observed. Intracellular citrate levels of TA1414 and TA1357 without switching gltA OFF (IPTG⁻) increased 3.0 and 6.3 times from 3 h to 23 h, respectively. In contrast, intracellular citrate levels of TA1354 with switching gltA OFF (IPTG⁺) decreased by 85%, which is equivalent to 3.6% intracellular citrate levels in TA1414. Similar trends were observed in these strains for alpha-KG, which is produced from citrate. After switching gltA OFF, the intracellular alpha-KG level of TA1357 decreased to 5.2% that of TA1414 at 23 h. After switching gltA OFF, the acetyl-CoA level of TA1357 increased to 3.2 times that of TA1414, in contrast to the observed significant decrease in intracellular citrate levels. It is likely that over-accumulated acetyl-CoA was used as a precursor for acetate production. Pyruvate, a precursor of acetyl-CoA, increased with the progress of culture phase for all strains and conditions. Pyruvate accumulation by TA1357 with switching gltA OFF was much more than those in other strains and the conditions, and was 15.9 times that of TA1414. These results indicate that the redistribution of carbon flux from the TCA cycle was realized by the gltA switch OFF.
为了确认与关闭 gltA 相关的细胞内代谢物变化，9 小时时向 TA1357 添加了 IPTG；在切换前后（3 小时和 23 小时）收集细胞进行 LC-MS 分析。同时，未添加 IPTG 的 TA1414 和 TA1357 细胞也在相同时间点被收集。如图 5 所示，无论是否添加 IPTG，两种菌株在糖酵解前 5 步的中间代谢物细胞内浓度在采样点均未发生显著变化。另一方面，糖酵解剩余步骤及 TCA 循环中的代谢物浓度随着培养阶段的进展显著变化，且明显观察到是否关闭 gltA 带来的差异。未关闭 gltA 的 TA1414 和 TA1357（IPTG ⁻ ）细胞内柠檬酸水平从 3 小时到 23 小时分别增加了 3.0 倍和 6.3 倍。相比之下，关闭 gltA 的 TA1354（IPTG ⁺ ）细胞内柠檬酸水平下降了 85%，相当于 TA1414 细胞内柠檬酸水平的 3.6%。对于由柠檬酸产生的α-KG，这些菌株中也观察到了类似的趋势。关闭 gltA 后，TA1357 的细胞内α-KG 水平在 23 小时时降至 TA1414 的 5.2%。关闭 gltA 后，TA1357 的乙酰-CoA 水平增加到 TA1414 的 3.2 倍，这与观察到的细胞内柠檬酸水平显著下降形成对比。很可能过度积累的乙酰-CoA 被用作乙酸生产的前体。丙酮酸作为乙酰-CoA 的前体，在所有菌株和条件下随着培养阶段的进展而增加。关闭 gltA 后，TA1357 的丙酮酸积累远高于其他菌株和条件，是 TA1414 的 15.9 倍。这些结果表明，通过关闭 gltA 实现了碳通量从 TCA 循环的重新分配。

3.4. Isopropanol production by switching gltA OFF
3.4. 通过关闭 gltA 生产异丙醇

As described above, we improved acetate production by interrupting the TCA cycle by switching gltA OFF. In order to demonstrate metabolic flux redirection from the central metabolic pathway toward a synthetic pathway, we constructed a metabolic toggle switch which simultaneously enabled gltA expression to be switched OFF and the genes for the isopropanol synthetic pathway to be expressed (Fig. 1, Fig. 2-B). The switching (expression of enzymes in the isopropanol production pathway and switching gltA OFF) in TA1415 (Fig. 2-B) was induced by the addition of 0.1 mM IPTG at 0, 6, 9, 12, and 15 h. TA1424, a wild type strain having a vacant vector (switching plasmid without gltA) (Fig. 2-B) and isopropanol production pathway was used as the control strain. The isopropanol production pathway for TA1424 was induced at OD₆₀₀=0.6 (6 h). Initiating switching gltA OFF in TA1415 at early logarithmic phase (0, 6, and 9 h) inhibited bacterial growth (Fig. 6-A). TA1415 reached stationary phase within 22 h, except when IPTG was added at 0 h; in this case, 49 h was required to reach stationary phase due to a significant decrease in the specific growth rate. Under these fermentation conditions, TA1424 produced 13.7 mM isopropanol (Fig. 6-C). As shown in Fig. 6-C, in the presence of IPTG, isopropanol production by TA1415 improved. In particular, switching gltA OFF in early logarithmic growth phase had a significant positive effect on isopropanol production. When IPTG was added at 0, 6, and 9 h, the final isopropanol concentration was 45.7±1.4, 48.3±0.1, 50.9±1.5 mM, respectively. These production titers correspond to 3.3 to 3.7 times of that produced by TA1424. When IPTG was added at the middle logarithmic growth phase (12 h), isopropanol production titers improved up to 2.3 times that of TA1424 (Table 3). Even when IPTG was added at the late logarithmic growth phase (15 h), the concentration of isopropanol produced by TA1415 (20.5±1.9 mM) was higher than that by TA1424. As shown in Table 3, isopropanol production yield to glucose was also improved by switching in early logarithmic growth phase: up to 2.8–3.1 times that of TA1424. This demonstration proved that the metabolic toggle switch contributed to improvement of isopropanol production titer and yield via metabolic flux redirection caused by the interruption of the TCA cycle. Switching in early logarithmic growth phase had a particularly strong effect on isopropanol production improvement.
如上所述，我们通过关闭 gltA 中断了 TCA 循环，从而提高了乙酸盐的产量。为了展示代谢流从中心代谢途径向合成途径的重新定向，我们构建了一个代谢切换开关，该开关能够同时关闭 gltA 的表达并启动异丙醇合成途径基因的表达（图 1，图 2-B）。在 TA1415 中（图 2-B），通过在 0、6、9、12 和 15 小时添加 0.1 mM IPTG 来诱导切换（异丙醇生产途径中的酶表达和关闭 gltA）。TA1424 作为对照菌株，是一种携带空载体（无 gltA 的切换质粒）的野生型菌株（图 2-B），并具有异丙醇生产途径。TA1424 的异丙醇生产途径在 OD ₆₀₀ =0.6（6 小时）时被诱导。在 TA1415 中对数生长早期（0、6 和 9 小时）启动关闭 gltA 抑制了细菌的生长（图 6-A）。除了在 0 小时添加 IPTG 的情况外，TA1415 在 22 小时内进入稳定期；在此情况下，由于比生长速率显著下降，需要 49 小时才能达到稳定期。在这些发酵条件下，TA1424 产生了 13.7 mM 的异丙醇（图 6-C）。如图 6-C 所示，在 IPTG 存在的情况下，TA1415 的异丙醇产量有所提高。特别是在对数生长早期关闭 gltA 对异丙醇产量有显著的正面影响。当在 0、6 和 9 小时添加 IPTG 时，最终的异丙醇浓度分别为 45.7±1.4、48.3±0.1、50.9±1.5 mM。这些产量相当于 TA1424 产量的 3.3 到 3.7 倍。当在对数生长中期（12 小时）添加 IPTG 时，异丙醇产量提高了至 TA1424 的 2.3 倍（表 3）。即使在对数生长后期（15 小时）添加 IPTG，TA1415 产生的异丙醇浓度（20.5±1.9 mM）也高于 TA1424。如表 3 所示，通过在对数生长早期切换，异丙醇对葡萄糖的产率也有所提高：最高可达 TA1424 的 2.8–3.1 倍。这一证明表明，代谢切换开关通过中断 TCA 循环引起的代谢流重定向，有助于提高异丙醇的产量和产率。在早期对数生长期进行切换对异丙醇生产的改善具有特别强烈的影响。

Table 3. Comparison of fermentation data from different strains for isopropanol production.
表 3. 不同菌株生产异丙醇的发酵数据比较。

Empty Cell	(g L⁻¹)	(mM)	(mol/mol%)
Empty Cell	Cell density 细胞密度	Isopropanol 异丙醇	Yield 产量
TA1415_0 h	1.1±0.0	45.7±1.4	44.7±2.3
TA1415_6 h	1.1±0.0	48.3±0.1	47.3±0.5
TA1415_9 h	1.2±0.0	50.9±1.5	48.5±1.4
TA1415_12 h	1.4±0.0	38.1±4.2	36.2±4.2
TA1415_15 h	1.5±0.0	20.5±1.9	20.5±1.7
TA1424	1.3±0.0	13.7±0.3	15.8±0.1

4. Discussion 4. 讨论

In the bio-based production of chemical compounds, the competitive relationship between bacterial growth and/or cell maintenance pathways and the desired production pathway usually results in decreased titer and yield of the target compound. In particular, carbon flux into the TCA cycle significantly competes with bio-based production pathways requiring precursors important in the TCA cycle such as acetyl-CoA, pyruvate, and phosphoenolpyruvate. In conventional metabolic engineering, these competing pathways, which are required for bacterial growth, cannot be eliminated using the gene knock out strategy. In this study, we developed a synthetic genetic circuit in E. coli as a metabolic engineering tool for designed metabolic flux redirection via conditional interruption of a target metabolic pathway.
在化合物的生物基生产中，细菌生长和/或细胞维持途径与所需生产途径之间的竞争关系通常会导致目标化合物的滴度和产量降低。特别是，进入 TCA 循环的碳通量与需要 TCA 循环中重要前体（如乙酰-CoA、丙酮酸和磷酸烯醇丙酮酸）的生物基生产途径显著竞争。在传统的代谢工程中，这些对细菌生长必需的竞争途径无法通过基因敲除策略消除。在本研究中，我们开发了一种合成基因电路作为代谢工程工具，通过条件性中断目标代谢途径来实现设计的代谢通量重定向。

The enzyme activity of CS which catalyzes the first step of the TCA cycle of TA1357 rapidly decreased in response to the addition of IPTG, resulting in a 67% decrease in specific growth rate compared to TA1414. This result indicates that bacterial growth under aerobic conditions is controllable via TCA cycle modulation by the gltA OFF switch. Nevertheless, CS activity after switching gltA OFF remained at 17.5% of TA1414. In order to completely arrest carbon influx into the TCA cycle, strict regulation of CS activity is required. In this study we utilized results from conventional research on synthetic genetic circuits and added an AANDENYALAA tag for protein degradation at the C-terminus of CS to eliminate residual enzyme activity after switching (Prindle et al., 2011, Keiler et al., 1996). However, the effect of this degradation tag has not been investigated with respect to individual enzymes, including CS, so its effectiveness has to be verified. In addition to the method using C-terminus degradation tag, it has been reported that proteins having a destabilizing amino acid residue at their N-terminus end are degraded by the ClpAP protease in E. coli (Dougan et al., 2012). Further efforts are required to improve the responsiveness and operability of synthetic genetic circuits utilizing such protein engineering modifications.
TA1357 中催化 TCA 循环第一步的 CS 酶活性在添加 IPTG 后迅速下降，导致比 TA1414 相比比生长速率下降了 67%。这一结果表明，在有氧条件下通过 gltA 关闭开关调节 TCA 循环可以控制细菌生长。然而，关闭 gltA 后 CS 活性仍保持在 TA1414 的 17.5%。为了完全阻止碳流入 TCA 循环，需要严格调控 CS 活性。在本研究中，我们利用合成遗传电路的传统研究结果，并在 CS 的 C 端添加了 AANDENYALAA 标签以消除关闭后的残留酶活性（Prindle 等，2011；Keiler 等，1996）。然而，这种降解标签对包括 CS 在内的个别酶的效果尚未得到研究，因此其有效性需要验证。除了使用 C 端降解标签的方法外，据报道，在大肠杆菌中，N 端具有不稳定氨基酸残基的蛋白质会被 ClpAP 蛋白酶降解（Dougan 等，2012）。需要进一步努力，以提高利用此类蛋白质工程修饰的合成基因电路的响应性和可操作性。

In order to achieve the efficient utilization of the carbon source both in the bacterial growth phase and production phase, the TCA cycle was conditionally interrupted via switching gltA OFF using a synthetic toggle switch. As expected, switching gltA OFF inhibited aerobic bacterial growth by interrupting carbon flux to the TCA cycle and the excess carbon flux were redistributed into endogenous acetate production pathway. Accumulated acetyl-CoA pool could thus be utilized for acetate production to compensate for ATP synthesis by the TCA cycle. In addition, alpha-KG was detected in the culture of TA1414 in stationary phase, but not from the culture of TA1357 with switching gltA OFF. This indicated that some TCA cycle activity remained even at stationary phase and competed with acetate production. According to these results, switching gltA OFF effectively up-regulates carbon flux for acetate production by interrupting the TCA cycle in both the logarithmic and stationery phases.
为了实现细菌生长阶段和生产阶段碳源的高效利用，通过使用合成切换开关关闭 gltA，有条件地中断了 TCA 循环。正如预期的那样，关闭 gltA 通过中断碳流向 TCA 循环来抑制需氧细菌的生长，并将多余的碳流重新分配到内源性乙酸生产途径中。因此，积累的乙酰辅酶 A 池可用于乙酸生产，以补偿 TCA 循环的 ATP 合成。此外，在 TA1414 的静止期培养物中检测到了α-KG，但在关闭 gltA 的 TA1357 培养物中未检测到。这表明即使在静止期，TCA 循环的某些活性仍然存在，并与乙酸生产竞争。根据这些结果，关闭 gltA 通过在指数期和静止期中断 TCA 循环，有效上调了乙酸生产的碳流。

CS activity is generally inhibited by intracellular NADH allosterically (Nguyen et al., 2001, Duckworth and Tong, 1976). However, citrate synthesis and subsequent TCA cycle activity remained even at stationary phase in TA1414. This result suggested that intracellular NADH was inadequate to regulate TCA cycle activity. On the other hand, the key enzymes in the glycolytic pathway, such as phosphofructokinase I and pyruvate kinase II, are activated allosterically by a decrease of the ATP/ADP ratio (Kotlarz et al., 1975, Babul, 1978). Although the glucose consumption of TA1414 was decreased during the transition to stationary phase, such changes were not observed in TA1357 by switching gltA OFF (Table S-1). These results indicated that intracellular ATP synthesis decreased in association with conditional TCA cycle interruption. Consequently, ATP synthesis via acetate production was enhanced when switching gltA OFF in TA1357. Improvement in acetate production and glucose consumption have also been observed in ΔatpA strains, which lack the gene encoding the F1-ATPase alpha subunit responsible for ATP production in the electron transport chain (Noda et al., 2006).
CS 活性通常被细胞内 NADH 通过变构作用抑制(Nguyen et al., 2001, Duckworth and Tong, 1976)。然而，在 TA1414 中，即使在静止期，柠檬酸合成和随后的 TCA 循环活性仍然存在。这一结果表明，细胞内 NADH 不足以调节 TCA 循环活性。另一方面，糖酵解途径中的关键酶，如磷酸果糖激酶 I 和丙酮酸激酶 II，通过 ATP/ADP 比率的降低而变构激活(Kotlarz et al., 1975, Babul, 1978)。尽管 TA1414 在进入静止期时葡萄糖消耗减少，但在 TA1357 中通过关闭 gltA 并未观察到这种变化（表 S-1）。这些结果表明，细胞内 ATP 合成随着条件性 TCA 循环中断而减少。因此，在 TA1357 中关闭 gltA 时，通过乙酸生产增强 ATP 合成。在ΔatpA 菌株中也观察到了乙酸生产和葡萄糖消耗的改善，这些菌株缺乏编码电子传递链中负责 ATP 生产的 F1-ATP 酶α亚基的基因(Noda et al., 2006)。

Intracellular metabolites were quantitatively analyzed to confirm the effect of switching gltA OFF on the intracellular metabolic state of glycolysis and the TCA cycle. Without switching gltA OFF, TA1357 showed an intracellular metabolic state similar to that of TA1414. In contrast, when switching gltA OFF was performed with TA1357, citrate and alpha-KG decreased significantly to 3.6% and 5.2% that of TA1414, and pyruvate and acetyl-CoA (which are upstream metabolites of citrate synthesis) increased up to 15.9 times and 3.2 times that of TA1414. These results proved that the switching gltA OFF were working well for interrupting the TCA cycle and redirecting flux from the TCA cycle to the acetate production pathway.
对细胞内代谢物进行了定量分析，以确认关闭 gltA 对糖酵解及 TCA 循环的细胞内代谢状态的影响。未关闭 gltA 时，TA1357 展现出与 TA1414 相似的细胞内代谢状态。相比之下，当对 TA1357 执行关闭 gltA 操作后，柠檬酸和α-酮戊二酸显著下降至 TA1414 的 3.6%和 5.2%，而丙酮酸和乙酰辅酶 A（柠檬酸合成上游代谢物）则分别增加至 TA1414 的 15.9 倍和 3.2 倍。这些结果证明了关闭 gltA 有效地中断了 TCA 循环，并将代谢流从 TCA 循环重定向至乙酸生产途径。

To demonstrate the improvement in bio-production using a metabolic toggle switch, accumulated acetyl-CoA was used as the starting metabolite for isopropanol production using engineered E. coli. As expected, switching improved isopropanol production titer and yield. The improvement was significant when switching was triggered at early and mid-logarithmic growth phase. When the switching was performed at 0 h, bacterial growth was significantly inhibited and isopropanol production was not observed until 22 h. The remarkable growth inhibition and delay of isopropanol production might be caused by the burden of overexpression of isopropanol production enzymes. This burden would arise because bacterial growth and protein biosynthesis are both dependent on amino acid biosynthesis. Since the TCA cycle is responsible for the biosynthesis of amino acids related to bacterial growth, such as glutamate, glutamine, aspartate and asparagine, interrupting the TCA cycle at the beginning of cultivation would slow both growth and isopropanol production. Therefore, in contrast to the case lacking the isopropanol production pathway, the best improvement in isopropanol production titer was observed when the switching was performed at 9 h (Fig. 6-C). When IPTG was added at 6 h, specific glucose consumption rate was 1.5 times faster than the case which IPTG was added at 9 h (Table S2). As the result, the glucose consumption of TA1415_IPTG 6 h was 12 mM higher than that of TA1415_IPTG 9 h at 38 h (Fig. 6-B). This improvement of glucose consumption did not contribute to the isopropanol production of TA1415_IPTG 6 h. Instead of isopropanol, acetate production of TA1415_IPTG 6 h increased 11.3 mM higher than that of TA1415_IPTG 9 h (Data not shown). For the efficient redirection of excess carbon flux, the deletion of competing pathway would be important in addition to the optimization of the timing for the dynamic control of target gene.
为了展示使用代谢切换开关在生物生产中的改进，累积的乙酰-CoA 被用作工程改造的大肠杆菌生产异丙醇的起始代谢物。正如预期的那样，切换提高了异丙醇的产量和产率。当在生长对数期的早期和中期触发切换时，改进尤为显著。如果在 0 小时进行切换，细菌生长会受到显著抑制，直到 22 小时才观察到异丙醇的生产。这种显著的生长抑制和异丙醇生产的延迟可能是由于异丙醇生产酶过度表达的负担所致。这种负担的产生是因为细菌生长和蛋白质生物合成都依赖于氨基酸的生物合成。由于 TCA 循环负责与细菌生长相关的氨基酸（如谷氨酸、谷氨酰胺、天冬氨酸和天冬酰胺）的生物合成，在培养开始时中断 TCA 循环会减缓生长和异丙醇的生产。因此，与缺乏异丙醇生产途径的情况相比，当在 9 小时进行切换时，观察到异丙醇生产滴度的最佳改善（图 6-C）。当在 6 小时添加 IPTG 时，特定葡萄糖消耗速率比在 9 小时添加 IPTG 的情况快 1.5 倍（表 S2）。结果，TA1415_IPTG 6 h 在 38 小时时的葡萄糖消耗量比 TA1415_IPTG 9 h 高 12 mM（图 6-B）。这种葡萄糖消耗的改善并未促进 TA1415_IPTG 6 h 的异丙醇生产。相反，TA1415_IPTG 6 h 的乙酸盐产量比 TA1415_IPTG 9 h 高 11.3 mM（数据未显示）。为了有效重定向多余的碳通量，除了优化目标基因动态控制的时机外，删除竞争途径也很重要。

TA1415 showed growth comparable to TA1424, and its production titer and yield of isopropanol reached 3.7 and 3.1 times that of TA1424, respectively. This result proved that switching improves bio-production titer and yield from acetyl-CoA by redirecting carbon flux from the central metabolic pathway toward the desired synthetic metabolic pathway. Improvement of yield and titer of isopropanol production has been attempted to several host organisms such as Candida and Clostridium (Tamakawa et al., 2013, Dusséaux et al., 2013). In these reports, the production titers were 1.3–1.5 times increased by the up-regulation of genes for isopropanol production and the deletion of competing by-production pathway such as ethanol and acetate. The further improvement of the productivity of isopropanol production would be attained with a combination of these metabolic engineering efforts and gltA OFF switch. (Table 3). To date, as a fermentation engineering efforts, two stage fermentation processes consisting of an aerobic cell growth phase followed by an anaerobic production phase have been employed to redirect carbon flux from the TCA cycle toward the metabolic pathway responsible for producing the target compounds (e.g., alanine, lactate and succinate) (Zhang et al., 2007, Zhou et al., 2012, Jiang et al., 2010). However, these processes cannot be applied to products which are only produced by aerobic fermentation, such as isopropanol.
TA1415 显示出与 TA1424 相当的生长情况，其异丙醇的生产滴度和产量分别达到了 TA1424 的 3.7 倍和 3.1 倍。这一结果证明了通过将碳流从中枢代谢途径转向目标合成代谢途径，开关能够提高乙酰-CoA 的生物生产滴度和产量。提高异丙醇生产的产量和滴度已在多种宿主生物如假丝酵母和梭菌中进行了尝试（Tamakawa 等人，2013；Dusséaux 等人，2013）。在这些报告中，通过上调异丙醇生产基因和删除竞争性副产物途径如乙醇和乙酸盐，生产滴度提高了 1.3-1.5 倍。结合这些代谢工程努力和 gltA OFF 开关，异丙醇生产的效率有望得到进一步提升（表 3）。迄今为止，作为发酵工程的一项努力，已经采用了由需氧细胞生长阶段和厌氧生产阶段组成的两阶段发酵过程，以将碳通量从 TCA 循环转向负责生产目标化合物（如丙氨酸、乳酸和琥珀酸）的代谢途径（Zhang 等，2007；Zhou 等，2012；Jiang 等，2010）。然而，这些过程无法应用于仅通过需氧发酵生产的产品，如异丙醇。

In this study, we constructed a metabolic toggle switch in engineered E. coli as a novel conditional knockout approach for metabolic engineering and applied it to isopropanol production. As a result, carbon influx into the TCA cycle was interrupted and excess carbon flux redirected toward a synthetic isopropanol production pathway following sufficient bacterial growth. Use of this metabolic toggle switch demonstrated a novel approach for intentionally switching intracellular metabolism as appropriate, from bacterial growth phase to bio-production phase, by direct regulation of specific metabolic flux. This is a general strategy which could be applied to other bio-productions in order to enhance metabolic flux to synthetic metabolic pathways while minimizing negative effects on bacterial growth and cell maintenance. In future, additional tuning for sensitive response and optimization of the gltA expression level will allow further improvements in production for titer and yield.
在本研究中，我们构建了一种工程化大肠杆菌中的代谢切换开关，作为一种新颖的条件敲除方法应用于代谢工程，并将其应用于异丙醇的生产。结果发现，在细菌充分生长后，进入 TCA 循环的碳流被中断，多余的碳流被重新导向合成的异丙醇生产途径。使用这种代谢切换开关展示了一种新颖的方法，通过直接调控特定代谢流，有意在细菌生长阶段到生物生产阶段之间适当地切换细胞内代谢。这是一种通用策略，可应用于其他生物生产，以增强向合成代谢途径的代谢流，同时最小化对细菌生长和细胞维持的负面影响。未来，对敏感响应的进一步调整和 gltA 表达水平的优化将有助于在产量和收率方面实现进一步的生产改进。

Acknowledgments 致谢

This research was supported by JSPS Grant-in-Aid for Scientific Research on Innovative Areas Grant number 23119002. We would like to express gratitude to S. Atsumi (University of California, Davis) for helpful advice.
本研究得到了 JSPS 创新领域科学研究补助金（编号 23119002）的支持。我们要感谢加州大学戴维斯分校的 S. Atsumi 提供的有益建议。

Appendix A. Supplementary materials
附录 A. 补充材料

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Supplementary Material

References

Ajikumar et al., 2010
P.K. Ajikumar, W.H. Xiao, K.E. Tyo, Y. Wang, F. Simeon, E. Leonard, O. Mucha, T.H. Phon, B. Pfeifer, G. Stephanopoulos
Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli
Science, 330 (2010), pp. 70-74
Crossref View in Scopus Google Scholar
Alper et al., 2005
H. Alper, K. Miyaoku, G. Stephanopoulos
Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets
Nat. Biotechnol., 5 (2005), pp. 612-616
Crossref View in Scopus Google Scholar
Atsumi et al., 2008a
S. Atsumi, A.F. Cann, M.R. Connor, C.R. Shen, K.M. Smith, M.P. Brynildsen, K.J. Chou, T. Hanai, J.C. Liao
Metabolic engineering of Escherichia coli for 1-butanol production
Metab. Eng., 10 (2008), pp. 305-311
View PDF View article View in Scopus Google Scholar
Atsumi and Liao, 2008b
S. Atsumi, J.C. Liao
Directed evolution of Methanococcus jannaschii citramalate synthase for biosynthesis of 1-propanol and 1-butanol by Escherichia coli
Appl. Environ. Microbiol., 74 (2008), pp. 7802-7808
View in Scopus Google Scholar
Babul, 1978
J. Babul
Phosphofructokinases from Escherichia coli. Purification and characterization of the nonallosteric isozyme
J. Biol. Chem., 25 (1978), pp. 4350-4355
View PDF View article Google Scholar
Balzer et al., 2013
G.J. Balzer, C. Thakker, G.N. Bennett, K.Y. San
Metabolic engineering of Escherichia coli to minimize byproduct formate and improving succinate productivity through increasing NADH availability by heterologous expression of NAD⁺-dependent formate dehydrogenase
Metab. Eng., 20 (2013), pp. 1-8
View PDF View article View in Scopus Google Scholar
Bermejo et al., 1998
L.L. Bermejo, N.E. Welker, E.T. Papoutsakis
Expression of Clostridium acetobutylicum ATCC 824 genes in Escherichia coli for acetone production and acetate detoxification
Appl. Environ. Microbiol., 64 (1998), pp. 1079-1085
Crossref View in Scopus Google Scholar
Bloxham et al., 1983
D.P. Bloxham, C.J. Herbert, S.S. Ner, W.T. Drabble
Citrate synthase activity in Escherichia coli harbouring hybrid plasmids containing the gltA gene
J. Gen. Microbiol., 129 (1983), pp. 1889-1897
Crossref View in Scopus Google Scholar
Callura et al., 2012
J.M. Callura, C.R. Cantor, J.J. Collins
Genetic switchboard for synthetic biology applications
Proc. Natl. Acad. Sci. USA, 10 (2012), pp. 5850-5855
Crossref View in Scopus Google Scholar
Chang and Keasling, 2006
M.C. Chang, J.D. Keasling
Production of isoprenoid pharmaceuticals by engineered microbes
Nat. Chem. Biol., 6 (2006), pp. 674-681
Crossref View in Scopus Google Scholar
Clomburg and Gonzalez, 2010
J.M. Clomburg, R. Gonzalez
Metabolic engineering of Escherichia coli for the production of 1,2-propanediol from glycerol
Biotechnol. Bioeng., 108 (2010), pp. 867-879
Google Scholar
Datsenko and Wanner, 2000
K.A. Datsenko, B.L. Wanner
One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
Proc. Natl. Acad. Sci. USA, 97 (2000), pp. 6640-6645
View in Scopus Google Scholar
Davis and Gilvarg, 1956
B.D. Davis, C. Gilvarg
The role of the tricarboxylic acid cycle in acetate oxidation in Escherichia coli
J. Biol. Chem., 222 (1956), pp. 307-319
View in Scopus Google Scholar
Dellomonaco et al., 2011
C. Dellomonaco, J.M. Clomburg, E.N. Miller, R. Gonzalez
Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals
Nature, 10 (2011), pp. 355-359
Crossref View in Scopus Google Scholar
Dougan et al., 2012
D.A. Dougan, D. Micevski, K.N. Truscott
The N-end rule pathway: from recognition by N-recognins, to destruction by AAA⁺proteases
Biochim. Biophys. Acta, 1823 (2012), pp. 83-91
View PDF View article View in Scopus Google Scholar
Duckworth and Tong, 1976
H.W. Duckworth, E.K. Tong
The binding of reduced nicotinamide adenine dinucleotide to citrate synthase of Escherichia coli K12
Biochemistry, 13 (1976), pp. 108-114
Crossref View in Scopus Google Scholar
Dusséaux et al., 2013
S. Dusséaux, C. Croux, P. Soucaille, I. Meynial-Salles
Metabolic engineering of Clostridium acetobutylicum ATCC 824 for the high-yield production of a biofuel composed of an isopropanol/butanol/ethanol mixture
Metab. Eng., 18 (2013), pp. 1-8
View PDF View article View in Scopus Google Scholar
Farmer and Liao, 2000
W.R. Farmer, J.C. Liao
Improving lycopene production in Escherichia coli by engineering metabolic control
Nat. Biotechnol., 18 (2000), pp. 533-537
View in Scopus Google Scholar
Fisher et al., 2011
M.A. Fisher, K.L. McKinley, L.H. Bradley, S.R. Viola, M.H. Hecht
De novo designed proteins from a library of artificial sequences function in Escherichia coli and enable cell growth
PLoS One, 6 (2011), p. e15364
Crossref View in Scopus Google Scholar
Gardner et al., 2000
T.S. Gardner, C.R. Cantor, J.J. Collins
Construction of a genetic toggle switch in Escherichia coli
Nature, 20 (2000), pp. 339-342
View in Scopus Google Scholar
Hanai et al., 2007
T. Hanai, S. Atsumi, J.C. Liao
Engineered synthetic pathway for isopropanol production in Escherichia coli
Appl. Environ. Microbiol., 73 (2007), pp. 7814-7818
View in Scopus Google Scholar
Hasunuma et al., 2011
T. Hasunuma, T. Sanda, R. Yamada, K. Yoshimura, J. Ishii, A. Kondo
Metabolic pathway engineering based on metabolomics confers acetic and formic
Microb. Cell Factories, 10 (2011), p. 2
View in Scopus Google Scholar
Jarboe et al., 2010
L.R. Jarboe, X. Zhang, X. Wang, J.C. Moore, K.T. Shanmugam, L.O. Ingram
Metabolic engineering for production of biorenewable fuels and chemicals: contributions of synthetic biology
J. Biomed. Biotechnol., 76 (2010), p. 761042
Google Scholar
Jiang et al., 2010
M. Jiang, S.W. Liu, J.F. Ma, K.Q. Chen, L. Yu, F.F. Yue, B. Xu, P. Wei
Effect of growth phase feeding strategies on succinate production by metabolically engineered Escherichia coli
Appl. Environ. Microbiol., 76 (2010), pp. 1298-1300
View in Scopus Google Scholar
Kato et al., 2012
H. Kato, Y. Izumi, T. Hasunuma, F. Matsuda, A. Kondo
Widely targeted metabolic profiling analysis of yeast central metabolites
J. Biosci. Bioeng., 113 (2012), pp. 665-673
View PDF View article View in Scopus Google Scholar
Keasling, 2013
J.D. Keasling
Manufacturing molecules through metabolic engineering
Science, 330 (2013), pp. 1355-1358
Google Scholar
Keiler et al., 1996
K.C. Keiler, P.R. Waller, R.T. Sauer
Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA
Science, 16 (1996), pp. 990-993
Crossref View in Scopus Google Scholar
Kotlarz et al., 1975
D. Kotlarz, H. Garreau, H. Buc
Regulation of the amount and of the activity of phosphofructokinases and pyruvate kinases in Escherichia coli
Biochim. Biophys. Acta, 13 (1975), pp. 257-268
View PDF View article View in Scopus Google Scholar
Lee et al., 2007
K.H. Lee, J.H. Park, T.Y. Kim, H.U. Kim, S.Y. Lee
Systems metabolic engineering of Escherichia coli for l-threonine production
Mol. Syst. Biol., 3 (2007), p. 149
Crossref Google Scholar
Lee et al., 2005
S.J. Lee, D.Y. Lee, T.Y. Kim, B.H. Kim, J. Lee, S.Y. Lee
Metabolic engineering of Escherichia coli for enhanced production of succinic acid, based on genome comparison and in silico gene knockout simulation
Appl. Environ. Microbiol., 71 (2005), pp. 7880-7887
View in Scopus Google Scholar
Liao and Chang, 2010
Liao, J.C., Chang, P., 2010. Genetically modified microorganisms for producing itaconic acid with high yields. Industrial Technology Research Institute, Assignee. United States patent US 2010/0285546 A1.
Google Scholar
Lutz and Bujard, 1997
R. Lutz, H. Bujard
Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements
Nucleic Acids Res., 25 (1997), pp. 1203-1210
View in Scopus Google Scholar
Lütke-Eversloh and Stephanopoulos, 2008
T. Lütke-Eversloh, G. Stephanopoulos
Combinatorial pathway analysis for improved l-tyrosine production in Escherichia coli: identification of enzymatic bottlenecks by systematic gene overexpression
Metab. Eng., 10 (2008), pp. 69-77
View PDF View article View in Scopus Google Scholar
Mainguet et al., 2013
S.E. Mainguet, L.S. Gronenberg, S.S. Wong, J.C. Liao
A reverse glyoxylate shunt to build a non-native route from C4 to C2 in Escherichia coli
Metab. Eng., 19 (2013), pp. 116-127
View PDF View article View in Scopus Google Scholar
Matjan et al., 1989
F. Matjan, K.Y. Alam, D.P. Clark
Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase
J. Bacteriol., 171 (1989), pp. 342-348
Crossref View in Scopus Google Scholar
Nakamura and Whited, 2003
C.E. Nakamura, G.M. Whited
Metabolic engineering for the microbial production of 1,3-propanediol
Curr. Opin. Biotechnol., 14 (2003), pp. 454-459
View PDF View article View in Scopus Google Scholar
Nguyen et al., 2001
N.T. Nguyen, R. Maurus, D.J. Stokell, A. Ayed, H.W. Duckworth, G.D. Brayer
Comparative analysis of folding and substrate binding sites between regulated hexameric type II citrate synthases and unregulated dimeric type I enzymes
Biochemistry, 40 (2001), pp. 13177-13187
View in Scopus Google Scholar
Noda et al., 2006
S. Noda, Y. Takezawa, T. Mizutani, T. Asakura, E. Nishiumi, K. Onoe, M. Wada, F. Tomita, K. Matsushita, A. Yokota
Alterations of cellular physiology in Escherichia coli in response to oxidative phosphorylation impaired by defective F1-ATPase
J. Bacteriol., 188 (2006), pp. 6869-6876
View in Scopus Google Scholar
Park et al., 2007
J.H. Park, K.H. Lee, T.Y. Kim, S.Y. Lee
Metabolic engineering of Escherichia coli for the production of l-valine based on transcriptome analysis and in silico gene knockout simulation
Proc. Natl. Acad. Sci. USA, 104 (2007), pp. 7797-7802
Crossref Google Scholar
Prindle et al., 2011
A. Prindle, P. Samayoa, I. Razinkov, T. Danino, L.S. Tsimring, J. Hasty
A sensing array of radically coupled genetic ‘biopixels’
Nature, 18 (2011), pp. 39-44
Google Scholar
Qian et al., 2009
Z.G. Qian, X.X. Xia, S.Y. Lee
Metabolic engineering of Escherichia coli for the production of putrescine: a four carbon diamine
Biotechnol. Bioeng., 104 (2009), pp. 651-662
View in Scopus Google Scholar
Qian et al., 2010
Z.G. Qian, X.X. Xia, S.Y. Lee
Metabolic engineering of Escherichia coli for the production of cadaverine: a five carbon diamine
Biotechnol. Bioeng., 108 (2010), pp. 93-103
Crossref Google Scholar
Rabinovitch-Deere et al., 2013
C.A. Rabinovitch-Deere, J.W. Oliver, G.M. Rodriguez, S. Atsumi
Synthetic biology and metabolic engineering approaches to produce biofuels
Chem. Rev., 113 (2013), pp. 4611-4632
Crossref View in Scopus Google Scholar
Slusarczyk et al., 2012
A.L. Slusarczyk, A. Lin, R. Weiss
Foundations for the design and implementation of synthetic genetic circuits
Nat. Rev. Genet., 18 (2012), pp. 406-420
Crossref View in Scopus Google Scholar
Solomon et al., 2012
K.V. Solomon, T.M. Sanders, K.L. Prather
A dynamic metabolite valve for the control of central carbon metabolism
Metab. Eng., 14 (2012), pp. 661-671
View PDF View article View in Scopus Google Scholar
Soma et al., 2012
Y. Soma, K. Inokuma, T. Tanaka, C. Ogino, A. Kondo, M. Okamoto, T. Hanai
Direct isopropanol production from cellobiose by engineered Escherichia coli using a synthetic pathway and a cell surface display system
J. Biosci. Bioeng., 114 (2012), pp. 80-85
View PDF View article View in Scopus Google Scholar
Stricker et al., 2008
J. Stricker, S. Cookson, M.R. Bennett, W.H. Mather, L.S. Tsimring, J. Hasty
A fast, robust and tunable synthetic gene oscillator
Nature, 27 (2008), pp. 516-519
Crossref View in Scopus Google Scholar
Tamakawa et al., 2013
H. Tamakawa, T. Mita, A. Yokoyama, S. Ikushima, S. Yoshida
Metabolic engineering of Candida utilis for isopropanol production
Appl. Microbiol. Biotechnol., 97 (2013), pp. 6231-6239
Crossref View in Scopus Google Scholar
Wang et al., 1999
C.W. Wang, M.K. Oh, J.C. Liao
Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli
Biotechnol. Bioeng., 62 (1999), pp. 235-241
View in Scopus Google Scholar
Zhang et al., 2012
F. Zhang, J.M. Carothers, J.D. Keasling
Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids
Nat. Biotechnol., 25 (2012), pp. 354-359
Crossref View in Scopus Google Scholar
Zhang et al., 2007
X. Zhang, K. Jantama, J.C. Moore, K.T. Shanmugam, L.O. Ingram
Production of l-alanine by metabolically engineered Escherichia coli
Appl. Microbiol. Biotechnol., 77 (2007), pp. 355-366
Crossref View in Scopus Google Scholar
Zhou et al., 2012
L. Zhou, K.M. Tian, D.D. Niu, W. Shen, G.Y. Shi, S. Singh, Z.X. Wang
Improvement of d-lactate productivity in recombinant Escherichia coli by coupling production with growth
Biotechnol. Lett., 34 (2012), pp. 1123-1130
Crossref View in Scopus Google Scholar
Zhu et al., 2002
Z. Zhu, T. Zheng, C.G. Lee, R.J. Homer, J.A. Elias
Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling
Semin. Cell. Dev. Biol., 13 (2002), pp. 121-128
View PDF View article View in Scopus Google Scholar

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Engineering metabolism through dynamic control
2015, Current Opinion in Biotechnology
Metabolic engineering has proven crucial for the microbial production of valuable chemicals. Due to the rapid development of tools in synthetic biology, there has been recent interest in the dynamic regulation of flux through metabolic pathways to overcome some of the issues arising from traditional strategies lacking dynamic control. There are many diverse implementations of dynamic control, with a range of metabolite sensors and inducers being used. Furthermore, control has been implemented at the transcriptional, translational and post-translational levels. Each of these levels have unique sets of engineering tools, and allow for control at different dynamic time-scales. In order to extend the applications of dynamic control, new tools are required to improve the dynamics of regulatory circuits. Further study and characterization of circuit robustness is also needed to improve their applicability to industry. The successful implementation of dynamic control, using technologies that are amenable to commercialization, will be a fundamental step in advancing metabolic engineering.
Self-induced metabolic state switching by a tunable cell density sensor for microbial isopropanol production
2015, Metabolic Engineering
Citation Excerpt :
The pTA1145 plasmid was used as the MTS module induced by the synthetic lux system. The original pTA669 has been constructed as a switching plasmid containing tetR and gltA expression modules (Soma et al., 2014). To change tetR expression from IPTG-inducible to AHL-LuxR-inducible, the PLlacO1 promoter of pTA669 was replaced with the PluxlacO fragment from pTA1083 at the AatII and Acc65I sites, resulting in pTA1145.
Chemicals production by engineered microorganisms often requires induction of target gene expression at an appropriate cell density to reduce conflict with cell growth. The lux system in Vibrio fischeri is a well-characterized model for cell density-dependent regulation of gene expression termed quorum sensing (QS). However, there are currently no reports for application of the lux system to microbial chemical production. Here, we constructed a synthetic lux system as a tunable cell density sensor-regulator using a synthetic lux promoter and a positive feedback loop in Escherichia coli. In this system, self-induction of a target gene expression is driven by QS-signal, and its threshold cell density can be changed depending on the concentration of a chemical inducer. We demonstrate auto-redirection of metabolic flux from central metabolic pathways toward a synthetic isopropanol pathway at a desired cell density resulting in a significant increase in isopropanol production.
Metabolic engineering strategies for microbial synthesis of oleochemicals
2015, Metabolic Engineering
Microbial synthesis of oleochemicals has advanced significantly in the last decade. Microbes have been engineered to convert renewable substrates to a wide range of molecules that are ordinarily made from plant oils. This approach is attractive because it can reduce a motivation for converting tropical rainforest into farmland while simultaneously enabling access to molecules that are currently expensive to produce from oil crops. In the last decade, enzymes responsible for producing oleochemicals in nature have been identified, strategies to circumvent native regulation have been developed, and high yielding strains have been designed, built, and successfully demonstrated. This review will describe the metabolic pathways that lead to the diverse molecular features found in natural oleochemicals, highlight successful metabolic engineering strategies, and comment on areas where future work could further advance the field.
Dynamic knockdown of E. coli central metabolism for redirecting fluxes of primary metabolites
2015, Metabolic Engineering
Control of native enzyme levels is important when optimizing strains for overproduction of heterologous compounds. However, for many central metabolic enzymes, static knockdown results in poor growth and protein expression. We have developed a strategy for dynamically modulating the abundance of native enzymes within the host cell and applied this to a model system for myo-inositol production from glucose. This system relies on controlled degradation of a key glycolytic enzyme, phosphofructokinase-I (Pfk-I). Through tuning Pfk-I levels, we have been able to develop an Escherichia coli strain with a growth mode close to wild type and a production mode with an increased glucose-6-phosphate pool available for conversion into myo-inositol. The switch to production mode is trigged by inducer addition, allowing yield, titer, and productivity to be managed through induction time. By varying the time of Pfk-I degradation, we were able to achieve a two-fold improvement in yield and titers of myo-inositol.
Quorum sensing for population-level control of bacteria and potential therapeutic applications
2020, Cellular and Molecular Life Sciences
Dynamic metabolic engineering: New strategies for developing responsive cell factories
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[1] Ajikumar et al., 2010
P.K. Ajikumar, W.H. Xiao, K.E. Tyo, Y. Wang, F. Simeon, E. Leonard, O. Mucha, T.H. Phon, B. Pfeifer, G. Stephanopoulos
Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli
Science, 330 (2010), pp. 70-74
Crossref View in Scopus Google Scholar

[2] Alper et al., 2005
H. Alper, K. Miyaoku, G. Stephanopoulos
Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets
Nat. Biotechnol., 5 (2005), pp. 612-616
Crossref View in Scopus Google Scholar

[3] Atsumi et al., 2008a
S. Atsumi, A.F. Cann, M.R. Connor, C.R. Shen, K.M. Smith, M.P. Brynildsen, K.J. Chou, T. Hanai, J.C. Liao
Metabolic engineering of Escherichia coli for 1-butanol production
Metab. Eng., 10 (2008), pp. 305-311
View PDF View article View in Scopus Google Scholar

[4] Atsumi and Liao, 2008b
S. Atsumi, J.C. Liao
Directed evolution of Methanococcus jannaschii citramalate synthase for biosynthesis of 1-propanol and 1-butanol by Escherichia coli
Appl. Environ. Microbiol., 74 (2008), pp. 7802-7808
View in Scopus Google Scholar

[5] Babul, 1978
J. Babul
Phosphofructokinases from Escherichia coli. Purification and characterization of the nonallosteric isozyme
J. Biol. Chem., 25 (1978), pp. 4350-4355
View PDF View article Google Scholar

[6] Balzer et al., 2013
G.J. Balzer, C. Thakker, G.N. Bennett, K.Y. San
Metabolic engineering of Escherichia coli to minimize byproduct formate and improving succinate productivity through increasing NADH availability by heterologous expression of NAD⁺-dependent formate dehydrogenase
Metab. Eng., 20 (2013), pp. 1-8
View PDF View article View in Scopus Google Scholar

[7] Bermejo et al., 1998
L.L. Bermejo, N.E. Welker, E.T. Papoutsakis
Expression of Clostridium acetobutylicum ATCC 824 genes in Escherichia coli for acetone production and acetate detoxification
Appl. Environ. Microbiol., 64 (1998), pp. 1079-1085
Crossref View in Scopus Google Scholar

[8] Bloxham et al., 1983
D.P. Bloxham, C.J. Herbert, S.S. Ner, W.T. Drabble
Citrate synthase activity in Escherichia coli harbouring hybrid plasmids containing the gltA gene
J. Gen. Microbiol., 129 (1983), pp. 1889-1897
Crossref View in Scopus Google Scholar

[9] Callura et al., 2012
J.M. Callura, C.R. Cantor, J.J. Collins
Genetic switchboard for synthetic biology applications
Proc. Natl. Acad. Sci. USA, 10 (2012), pp. 5850-5855
Crossref View in Scopus Google Scholar

[10] Chang and Keasling, 2006
M.C. Chang, J.D. Keasling
Production of isoprenoid pharmaceuticals by engineered microbes
Nat. Chem. Biol., 6 (2006), pp. 674-681
Crossref View in Scopus Google Scholar

[11] Clomburg and Gonzalez, 2010
J.M. Clomburg, R. Gonzalez
Metabolic engineering of Escherichia coli for the production of 1,2-propanediol from glycerol
Biotechnol. Bioeng., 108 (2010), pp. 867-879
Google Scholar

[12] Datsenko and Wanner, 2000
K.A. Datsenko, B.L. Wanner
One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
Proc. Natl. Acad. Sci. USA, 97 (2000), pp. 6640-6645
View in Scopus Google Scholar

[13] Davis and Gilvarg, 1956
B.D. Davis, C. Gilvarg
The role of the tricarboxylic acid cycle in acetate oxidation in Escherichia coli
J. Biol. Chem., 222 (1956), pp. 307-319
View in Scopus Google Scholar

[14] Dellomonaco et al., 2011
C. Dellomonaco, J.M. Clomburg, E.N. Miller, R. Gonzalez
Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals
Nature, 10 (2011), pp. 355-359
Crossref View in Scopus Google Scholar

[15] Dougan et al., 2012
D.A. Dougan, D. Micevski, K.N. Truscott
The N-end rule pathway: from recognition by N-recognins, to destruction by AAA⁺proteases
Biochim. Biophys. Acta, 1823 (2012), pp. 83-91
View PDF View article View in Scopus Google Scholar

[16] Duckworth and Tong, 1976
H.W. Duckworth, E.K. Tong
The binding of reduced nicotinamide adenine dinucleotide to citrate synthase of Escherichia coli K12
Biochemistry, 13 (1976), pp. 108-114
Crossref View in Scopus Google Scholar

[17] Dusséaux et al., 2013
S. Dusséaux, C. Croux, P. Soucaille, I. Meynial-Salles
Metabolic engineering of Clostridium acetobutylicum ATCC 824 for the high-yield production of a biofuel composed of an isopropanol/butanol/ethanol mixture
Metab. Eng., 18 (2013), pp. 1-8
View PDF View article View in Scopus Google Scholar

[18] Farmer and Liao, 2000
W.R. Farmer, J.C. Liao
Improving lycopene production in Escherichia coli by engineering metabolic control
Nat. Biotechnol., 18 (2000), pp. 533-537
View in Scopus Google Scholar

[19] Fisher et al., 2011
M.A. Fisher, K.L. McKinley, L.H. Bradley, S.R. Viola, M.H. Hecht
De novo designed proteins from a library of artificial sequences function in Escherichia coli and enable cell growth
PLoS One, 6 (2011), p. e15364
Crossref View in Scopus Google Scholar

[20] Gardner et al., 2000
T.S. Gardner, C.R. Cantor, J.J. Collins
Construction of a genetic toggle switch in Escherichia coli
Nature, 20 (2000), pp. 339-342
View in Scopus Google Scholar

[21] Hanai et al., 2007
T. Hanai, S. Atsumi, J.C. Liao
Engineered synthetic pathway for isopropanol production in Escherichia coli
Appl. Environ. Microbiol., 73 (2007), pp. 7814-7818
View in Scopus Google Scholar

[22] Hasunuma et al., 2011
T. Hasunuma, T. Sanda, R. Yamada, K. Yoshimura, J. Ishii, A. Kondo
Metabolic pathway engineering based on metabolomics confers acetic and formic
Microb. Cell Factories, 10 (2011), p. 2
View in Scopus Google Scholar

[23] Jarboe et al., 2010
L.R. Jarboe, X. Zhang, X. Wang, J.C. Moore, K.T. Shanmugam, L.O. Ingram
Metabolic engineering for production of biorenewable fuels and chemicals: contributions of synthetic biology
J. Biomed. Biotechnol., 76 (2010), p. 761042
Google Scholar

[24] Jiang et al., 2010
M. Jiang, S.W. Liu, J.F. Ma, K.Q. Chen, L. Yu, F.F. Yue, B. Xu, P. Wei
Effect of growth phase feeding strategies on succinate production by metabolically engineered Escherichia coli
Appl. Environ. Microbiol., 76 (2010), pp. 1298-1300
View in Scopus Google Scholar

[25] Kato et al., 2012
H. Kato, Y. Izumi, T. Hasunuma, F. Matsuda, A. Kondo
Widely targeted metabolic profiling analysis of yeast central metabolites
J. Biosci. Bioeng., 113 (2012), pp. 665-673
View PDF View article View in Scopus Google Scholar

[26] Keasling, 2013
J.D. Keasling
Manufacturing molecules through metabolic engineering
Science, 330 (2013), pp. 1355-1358
Google Scholar

[27] Keiler et al., 1996
K.C. Keiler, P.R. Waller, R.T. Sauer
Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA
Science, 16 (1996), pp. 990-993
Crossref View in Scopus Google Scholar

[28] Kotlarz et al., 1975
D. Kotlarz, H. Garreau, H. Buc
Regulation of the amount and of the activity of phosphofructokinases and pyruvate kinases in Escherichia coli
Biochim. Biophys. Acta, 13 (1975), pp. 257-268
View PDF View article View in Scopus Google Scholar

[29] Lee et al., 2007
K.H. Lee, J.H. Park, T.Y. Kim, H.U. Kim, S.Y. Lee
Systems metabolic engineering of Escherichia coli for l-threonine production
Mol. Syst. Biol., 3 (2007), p. 149
Crossref Google Scholar

[30] Lee et al., 2005
S.J. Lee, D.Y. Lee, T.Y. Kim, B.H. Kim, J. Lee, S.Y. Lee
Metabolic engineering of Escherichia coli for enhanced production of succinic acid, based on genome comparison and in silico gene knockout simulation
Appl. Environ. Microbiol., 71 (2005), pp. 7880-7887
View in Scopus Google Scholar

[31] Liao and Chang, 2010
Liao, J.C., Chang, P., 2010. Genetically modified microorganisms for producing itaconic acid with high yields. Industrial Technology Research Institute, Assignee. United States patent US 2010/0285546 A1.
Google Scholar

[32] Lutz and Bujard, 1997
R. Lutz, H. Bujard
Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements
Nucleic Acids Res., 25 (1997), pp. 1203-1210
View in Scopus Google Scholar

[33] Lütke-Eversloh and Stephanopoulos, 2008
T. Lütke-Eversloh, G. Stephanopoulos
Combinatorial pathway analysis for improved l-tyrosine production in Escherichia coli: identification of enzymatic bottlenecks by systematic gene overexpression
Metab. Eng., 10 (2008), pp. 69-77
View PDF View article View in Scopus Google Scholar

[34] Mainguet et al., 2013
S.E. Mainguet, L.S. Gronenberg, S.S. Wong, J.C. Liao
A reverse glyoxylate shunt to build a non-native route from C4 to C2 in Escherichia coli
Metab. Eng., 19 (2013), pp. 116-127
View PDF View article View in Scopus Google Scholar

[35] Matjan et al., 1989
F. Matjan, K.Y. Alam, D.P. Clark
Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase
J. Bacteriol., 171 (1989), pp. 342-348
Crossref View in Scopus Google Scholar

[36] Nakamura and Whited, 2003
C.E. Nakamura, G.M. Whited
Metabolic engineering for the microbial production of 1,3-propanediol
Curr. Opin. Biotechnol., 14 (2003), pp. 454-459
View PDF View article View in Scopus Google Scholar

[37] Nguyen et al., 2001
N.T. Nguyen, R. Maurus, D.J. Stokell, A. Ayed, H.W. Duckworth, G.D. Brayer
Comparative analysis of folding and substrate binding sites between regulated hexameric type II citrate synthases and unregulated dimeric type I enzymes
Biochemistry, 40 (2001), pp. 13177-13187
View in Scopus Google Scholar

[38] Noda et al., 2006
S. Noda, Y. Takezawa, T. Mizutani, T. Asakura, E. Nishiumi, K. Onoe, M. Wada, F. Tomita, K. Matsushita, A. Yokota
Alterations of cellular physiology in Escherichia coli in response to oxidative phosphorylation impaired by defective F1-ATPase
J. Bacteriol., 188 (2006), pp. 6869-6876
View in Scopus Google Scholar

[39] Park et al., 2007
J.H. Park, K.H. Lee, T.Y. Kim, S.Y. Lee
Metabolic engineering of Escherichia coli for the production of l-valine based on transcriptome analysis and in silico gene knockout simulation
Proc. Natl. Acad. Sci. USA, 104 (2007), pp. 7797-7802
Crossref Google Scholar

[40] Prindle et al., 2011
A. Prindle, P. Samayoa, I. Razinkov, T. Danino, L.S. Tsimring, J. Hasty
A sensing array of radically coupled genetic ‘biopixels’
Nature, 18 (2011), pp. 39-44
Google Scholar

[41] Qian et al., 2009
Z.G. Qian, X.X. Xia, S.Y. Lee
Metabolic engineering of Escherichia coli for the production of putrescine: a four carbon diamine
Biotechnol. Bioeng., 104 (2009), pp. 651-662
View in Scopus Google Scholar

[42] Qian et al., 2010
Z.G. Qian, X.X. Xia, S.Y. Lee
Metabolic engineering of Escherichia coli for the production of cadaverine: a five carbon diamine
Biotechnol. Bioeng., 108 (2010), pp. 93-103
Crossref Google Scholar

[43] Rabinovitch-Deere et al., 2013
C.A. Rabinovitch-Deere, J.W. Oliver, G.M. Rodriguez, S. Atsumi
Synthetic biology and metabolic engineering approaches to produce biofuels
Chem. Rev., 113 (2013), pp. 4611-4632
Crossref View in Scopus Google Scholar

[44] Slusarczyk et al., 2012
A.L. Slusarczyk, A. Lin, R. Weiss
Foundations for the design and implementation of synthetic genetic circuits
Nat. Rev. Genet., 18 (2012), pp. 406-420
Crossref View in Scopus Google Scholar

[45] Solomon et al., 2012
K.V. Solomon, T.M. Sanders, K.L. Prather
A dynamic metabolite valve for the control of central carbon metabolism
Metab. Eng., 14 (2012), pp. 661-671
View PDF View article View in Scopus Google Scholar

[46] Soma et al., 2012
Y. Soma, K. Inokuma, T. Tanaka, C. Ogino, A. Kondo, M. Okamoto, T. Hanai
Direct isopropanol production from cellobiose by engineered Escherichia coli using a synthetic pathway and a cell surface display system
J. Biosci. Bioeng., 114 (2012), pp. 80-85
View PDF View article View in Scopus Google Scholar

[47] Stricker et al., 2008
J. Stricker, S. Cookson, M.R. Bennett, W.H. Mather, L.S. Tsimring, J. Hasty
A fast, robust and tunable synthetic gene oscillator
Nature, 27 (2008), pp. 516-519
Crossref View in Scopus Google Scholar

[48] Tamakawa et al., 2013
H. Tamakawa, T. Mita, A. Yokoyama, S. Ikushima, S. Yoshida
Metabolic engineering of Candida utilis for isopropanol production
Appl. Microbiol. Biotechnol., 97 (2013), pp. 6231-6239
Crossref View in Scopus Google Scholar

[49] Wang et al., 1999
C.W. Wang, M.K. Oh, J.C. Liao
Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli
Biotechnol. Bioeng., 62 (1999), pp. 235-241
View in Scopus Google Scholar

[50] Zhang et al., 2012
F. Zhang, J.M. Carothers, J.D. Keasling
Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids
Nat. Biotechnol., 25 (2012), pp. 354-359
Crossref View in Scopus Google Scholar

[51] Zhang et al., 2007
X. Zhang, K. Jantama, J.C. Moore, K.T. Shanmugam, L.O. Ingram
Production of l-alanine by metabolically engineered Escherichia coli
Appl. Microbiol. Biotechnol., 77 (2007), pp. 355-366
Crossref View in Scopus Google Scholar

[52] Zhou et al., 2012
L. Zhou, K.M. Tian, D.D. Niu, W. Shen, G.Y. Shi, S. Singh, Z.X. Wang
Improvement of d-lactate productivity in recombinant Escherichia coli by coupling production with growth
Biotechnol. Lett., 34 (2012), pp. 1123-1130
Crossref View in Scopus Google Scholar

[53] Zhu et al., 2002
Z. Zhu, T. Zheng, C.G. Lee, R.J. Homer, J.A. Elias
Tetracycline-controlled transcriptional regulation systems: advances and application in transgenic animal modeling
Semin. Cell. Dev. Biol., 13 (2002), pp. 121-128
View PDF View article View in Scopus Google Scholar

Outline 大纲

Cited by (164) 被引用次数（164）

Figures (6) 图 (6)

Tables (3) 表（3）

Extras (1) 附加内容 (1)

Metabolic Engineering 代谢工程

Highlights 亮点

Abstract 摘要

Keywords 关键词

1. Introduction 1. 引言